EFFICACIOUS THERAPIES

FIELD OF THE INVENTION

[0001] The present invention relates to methods and kits for assessing efficacy of anti-cancer therapies, identifying anti-cancer agents, identifying and quantifying immunogenic cell death and identifying subjects who may benefit from particular anti-cancer therapies.

BACKGROUND OF THE INVENTION

Tumors exhibit immunosuppressive features

[0002] Established tumors are typified by an immunosuppresive microenvironment. Countering this naturally occurring phenomenon, emerging evidence suggests that radiation promotes a proimmunogenic milieu within the tumor capable of stimulating host cancer-specific immune responses. Clinically apparent tumors often exhibit immunosuppressive features conducive to unchecked growth of the primary tumor, as well as metastatic spread of the disease. Fortuitously, however, scientific evidence suggests that radiation may promote a type of tumor cell death that stimulates host antitumor immune responses attempting to gain parity with the

immunosuppressive effects prevalent within the non-treated tumor microenvironment. (Kroemer, et al, Annu Rev Immunol, 2013; 31 :51-72; Kroemer, et al, Oncoimmunology, 2012; 1 :407-8; Golden, et al, Front Oncol, "2012; 2:88) This immunogenic type of cancer cell death effectively contributes to priming the host's immune system to recognize and eliminate radiation exposed tumor cells, particularly after immune checkpoint blockade. Importantly, radiotherapy combined with immune checkpoint inhibitors, often stimulates an immune response that positively contributes to local tumor control and can result in memory that confers a selective ability to recognize and eliminate residual malignant cells, either localized outside the radiotherapy site or that emerge from dormancy at a later point in time. Formenti, et al, Lancet Oncol, 2009; 10: 718-26; Formenti, et al, J Natl Cancer Inst, 2013; 105:256-65; Golden, et al, Cancer Immunol Res., 2013; 1 :365-72) [0003] Three distinct ICD components essential for dendritic cell (DC) activation and immune priming have emerged. These include: 1) the cell surface translocation of calreticulin (CALR, better known as CRT), an endoplasmic reticulum (ER) resident chaperone protein and potent DC "eat me" signal; 2) the extracellular release of high mobility group box 1 (HMGB1), a DNA binding protein and toll-like receptor 4 (TLR-4) mediated DC activator; and 3) the liberation of adenosine-5 '-triphosphate (ATP), a cell-cell signaling factor in the extracellular matrix (ECM) that serves to activate P2X7 purinergic receptors on DCs, triggering DC inflammasome activation, secretion of IL-Ιβ, and subsequent priming of interferon-γ (IFNy) producing CD8 ⁺ T cells. (Ma, et al., Semin Immunol, 2010; 22: 113-24; Kroemer, et al, Bull Mem Acad R Med Belg, 2011; 166: 130-8, discussion 139-40) The cumulative effects of all 3 arms act to promote DC phagocytosis of tumor cells, thus facilitating DC processing of tumor-derived antigens and subsequent DC-associated cross-priming of CD8 ⁺ cytotoxic T lymphocytes. (Ma, et al, Semin Immunol, 2010; 22: 113-24; Kroemer, et al, Bull Mem Acad R Med Belg, 2011; 166: 130-8, discussion 139-40; Ma, et al, Cancer Metastasis, 2011; 30:71-82; Locher, et al, Ann NY Acad Sci, 2010; 1209:99-108) Using these phenotypic features of immunogenic death, some chemotherapeutic compounds have been demonstrated to induce ICD, particularly oxaliplatin. (Tesniere, et al, Oncogene, 2010; 29:482-91; Obeid, et al, Cell Death Differ, 2007; 14: 1848-50)

Cancer therapy

[0004] The use of platinum compounds to treat cancer emerged in the 1970s after the

serendipitous discovery of the anti-tumoral activity of cisplatin. (Kelland, Nat Rev Cancer, 2007; 7: 573-84; Monneret, Annales Pharmaceutiques Francaises, 2011; 69: 286-95) Early effort in platinum drug design, thereafter, was aimed at establishing similar compounds with an improved efficacy and therapeutic ratio. (Kelland, Nat Rev Cancer, 2007; 7: 573-84; Monneret, Annales Pharmaceutiques Francaises, 2011; 69: 286-95) Thus, other platinum compounds, such as carboplatin and oxaliplatin, were subsequently developed and tested in the clinic. Oncologists rapidly discovered that a number of disease sites responded exceptionally well to platinum-based chemotherapy (CT). (Muggia, et al, Journal of Chemotherapy, 2004; 16 Suppl 4: 77-82) These observations led to the belief that a platinum-based induction chemotherapy approach, given before radiotherapy (RT) or surgery might improve loco-regional control (LRC) and lower the risk of distant metastases. (Glynne- Jones, et al, J Clin Oncol, 2007; 25: 5281-6) However, as pointed out in a provocative review of >50 individual randomized trials and several meta- analyses, the patients treated by platinum-based chemotherapy given prior to definitive radiation therapy or surgery fared worse than those exposed to initial concomitant chemoradiation, in terms of disease-free survival (DFS) and overall survival (OS). (Glynne- Jones, et al, J Clin Oncol, 2007; 25: 5281-6) Evidence in head and neck, esophageal, cervical, anal,

nasopharyngeal, non-small-cell lung cancer demonstrated a clear advantage for concurrent chemotherapy and radiation therapy as opposed to a sequential approach. (Glynne- Jones, et al, J Clin Oncol, 2007; 25: 5281-6) Multiple mechanisms were advocated to explain the lack of benefit seen with sequential treatment including the prolonged overall treatment time, inadequate activity of chemotherapy alone, and/or the selection of radiation resistant clones by

chemotherapy given prior to definitive treatment with chemoradiation, radiation, or surgery. (Glynne- Jones, et al, J Clin Oncol, 2007; 25: 5281-6)

[0005] Scientific evidence suggests that both platinum-based chemotherapy and radiation therapy may promote a type of tumor cell death that can stimulate a host anti-tumor immune response. (Golden, et al, Frontieers in Oncology, 2012; 2: 88; Kroemer, et al, Annual Review of Immunology, 2012; Kroemer, et al, Oncoimmunology, 2012; 1 : 407-8 ) This immunogenic type of cell death effectively primes the host's immune system to recognize and eliminate tumor cells directly exposed to chemotherapy or radiation therapy. Importantly, at least in some patients, an immune memory may develop with the ability to recognize and eliminate treatment-naive tumor cells either outside the local radiation therapy site or that emerge from dormancy. (Formenti, et al, Journal of the National Cancer Institute, 2013; Formenti, et al, The Lancet Oncology, 2009; 10: 718-26; Formenti, et al, Breast Cancer Research: BCR, 2008; 10: 215 )

[0006] Some suggested that concurrent, as opposed to sequential chemoradiation is more likely to elicit a host anti-tumor immune response, explaining the superior long-term results. (Formenti, et al, The Lancet Oncology, 2009; 10: 718-26; Formenti, et al, Journal of Clinical Oncology: Official Jouranal of the American Society of Clinical Oncology, 2008; 26: 1562-3; author reply 3) However to date, the contributions of immunogenic cell death from concurrent chemotherapy and radiation therapy have yet to be established.

Three distinct ICD components required for immune priming

[0007] Three distinct ICD components required for dendritic cell (DC) activation and immune priming have emerged, including: (1) the cell surface translocation of calreticulin (CRT, an endoplasmic reticulum residing protein chaperone and potent dendritic cell "eat me" signal), and the extracellular release of (2) (HMGB 1 a DNA binding protein and TLR-4 mediated DC activator) and (3) adenosine-5 '-triphosphate (ATP, an activator of the DC P2X7 purinergic receptor that triggers dendritic cell inflammasome activation, secretion of IL-Ιβ, and subsequent priming of IFNy producing CD8 ⁺ T cells). (Ma, et al, Seminars in Immunology, 2010; 22: 113- 24; Kroemer, et al, Bulletin et memoires de I'Academie Royale de Medecine de Belgique, 2011 ; 166: 130-8; discussion 9-40) The net effects of all three arms act to promote dendritic cell phagocytosis of tumor cells, dendritic cell processing of tumor-derived antigens, and dendritic cell-associated cross-priming of CD8 ⁺ cytotoxic T lymphocytes. (Ma, et al, Seminars in

Immunology, 2010; 22: 113-24; Kroemer, et al, Bulletin et memoires de I'Academie Royale de Medecine de Belgique, 2011; 166: 130-8; discussion 9-40; Ma, et al, Cancer Metastasis Reviews, 2011; 30: 71-82; Locher, et al, Annals of the New York Academy of Sciences, 2010; 1209: 99-108) Others demonstrated a differential immunogenic cell death response of platinum compounds, cisplatin and oxaliplatin, and RT on colorectal cancer cells. (Tesniere, et al, Oncogene, 2010; 29: 482-91; Obeid, et al, Cell Death and Differntiation, 2007; 14: 1848-50) It would be useful to determine whether platinum-based concurrent chemotherapy and radiation therapy can effectively enhance immunogenic cell death in dying tumor cells compared to either treatment alone.

HMGB-1

[0008] High- mobility group protein Bl, also known as high- mobility group protein 1 (HMGB-1) and amphoterin, is a protein that in humans is encoded by the HMGBl gene. Like the histones, HMGBl is among the most important chromatin proteins. In the nucleus HMGBl interacts with nucleosomes, transcription factors and histones. This nuclear protein organizes the DNA and regulates transcription. After binding HMGB 1 bends the DNA, which facilitates the binding of other proteins. HMGBl supports transcription of many genes in interactions with many transcription factors or cooperation nucleosoms looses packed DNA and increases the chromatin remodeling, contact with core histones changes the structure of nucleosoms. The presence of HMGBl in the nucleus depends on posttranslational modifications. When the protein is not acetylated, stays in the nucleus, but hyperacetylation on lysine residues occurs to translocation into the cytosol. HMGBl is secreted by immune cells (like macrophages, monocytes and dendritic cells) through leaderless secretory pathways. Activated macrophages and monocytes secrete HMGBl as a cytokine mediator of Inflammation. Antibodies that neutralize HMGBl confer protection against damage and tissue injury during arthritis, colitis, ischemia, sepsis, endotoxemia, and systemic lupus erythematosis. The mechanism of inflammation and damage is binding to TLR4, which mediates HMGB1 -dependent activation of macrophage cytokine release. This positions HMGB1 at the intersection of sterile and infectious inflammatory responses.

Calreticulin

[0009] Calreticulin also known as calregulin, CRP55, CaBP3, calsequestrin-like protein, and endoplasmic reticulum resident protein 60 (ERp60) is a protein that in humans is encoded by the CALR gene. Calreticulin is a multifunctional protein that binds Ca ²⁺ ions (a second messenger in signal transduction), rendering it inactive. The Ca ²⁺ is bound with low affinity, but high capacity, and can be released on a signal. Calreticulin is located in storage compartments associated with the endoplasmic reticulum. The term "Mobilferrin" is considered to be the same as calreticulin by some sources. Calreticulin binds to misfolded proteins and prevents them from being exported from the endoplasmic reticulum to the Golgi apparatus. A similar quality- control chaperone, calnexin, performs the same service for soluble proteins as does calreticulin. Both proteins, calnexin and calreticulin, have the function of binding to oligosaccharides containing terminal glucose residues, thereby targeting them for degradation. In normal cellular function, trimming of glucose residues off the core oligosaccharide added during N-linked glycosylation is a part of protein processing. If "overseer" enzymes note that residues are misfolded, proteins within the RER will re-add glucose residues so that other calreticulin/calnexin can bind to these proteins and prevent them from proceeding to the Golgi. This leads these aberrantly folded proteins down a path whereby they are targeted for degradation.

[0010] Calreticulin (CRT) is expressed in many cancer cells and plays a role to promote macrophages to engulf hazardous cancerous cells. The reason why most of the cells are not destroyed is the presence of another molecule with signal CD47, which blocks CRT. Hence antibodies that block CD47 might be useful as a cancer treatment. In mice models of myeloid leukemia and non-Hodgkin's lymphoma, anti-CD47 were effective in clearing cancer cells while normal cells were unaffected. SUMMARY OF THE INVENTION

[0011] The present invention is based in part on the discovery of a means for detecting, identifying, measuring, quantifying or assessing immunogenic cell death (ICD) including detecting, identifying, measuring, quantifying or assessing one, two, three or more components involved in or necessary for dendritic cell (DC) activation or immune priming. The components involved in or necessary for dendritic cell (DC) activation or immune priming may be one or more of (a) the cell surface translocation of calreticulin (CRT), (b) the extracellular release of HMGB1, and (c) the extracellular release of adenosine-5'-triphosphate (ATP) that in turn promotes dendritic cell (DC) inflammasome activation, secretion of interleukin 1β (IL-Ιβ), and subsequent priming of interferon-γ (IFN-γ) producing CD8 ⁺ T cells.

[0012] In a first aspect, the invention provides methods for identifying, assessing or quantifying immunogenic cell death by detecting or measuring or quantifying one, two or three of the following:

(a) cell surface translocation of calreticulin (CRT),

(b) extracellular release of HMGB1, and

(c) extracellular release of adenosine-5'-triphosphate (ATP).

[0013] In some embodiments, this aspect features identifying, assessing or quantifying the response of a normal or non-neoplastic or non-cancerous cell to an anti-cancer agent, such as, for instance, ionizing radiation or a chemotherapeutic such as a platinum agent, for example, oxaliplatin, cisplatin, or carboplatin, or paclitaxel. In some embodiments, only one of the three is detected or measured, in other embodiments two of the three are detected or measured, and in still other embodiments, all three are detected or measured. Of course, additional factors, proteins, peptides, molecules, markers, values, times, and rates may be detected or measured as well either independently or in conjunction with these three, either concurrently or sequentially. The methods feature first obtaining a biological sample. The biological sample may contain a normal, a neoplastic or a cancerous cell. The methods may be performed in vitro. An increase in one, two or three of the above where the increase is at least about 25%, 50%, 75%, 100%, 150%, two times, three times, four times, five times or more greater than a baseline, a control or a measure made or value obtained prior to exposure to one or more anti-cancer agents may be used to confirm the presence of immunogenic cell death. [0014] The (a) detecting or measuring or quantifying cell surface translocation of calreticulin (CRT) may be performed by transfecting a cell, such as a normal, a neoplastic or a cancerous cell, with an expression system capable of expressing a labeled calreticulin. The labeled calreticulin may be, for instance, calreticulin, HaloTag ^® , and the expression system may be for instance, a pEZ-M02 plasmid containing sequences encoding a fusion CRT -HaloTag protein. The labeled calreticulin may be detectable by any suitable means, such as, for instance, via radiation or fluorescence such as by using fluoroscopy or flow cytometry. In one embodiment, a fluorescent ligand such as the HaloTag ^® alexa fluor 488 ligand may be used to bind and detect HaloTag ^® hydrolase. The fluorescent properties may be detected for instance, by fluoroscopy or flow cytometry.

[0015] The (a) detecting or measuring or quantifying cell surface translocation of calreticulin (CRT) may further feature exposing the normal cell, the neoplastic cell, or the cancerous cell to one or more anti-cancer agents. The exposing the normal, neoplastic or cancerous cell to more than one anti-cancer agent may be performed substantially concurrently or in series, and the anticancer agent may be, for instance, ionizing radiation or a chemotherapeutic such as a platinum agent, for instance, oxaliplatin, cisplatin, or carboplatin, or paclitaxel.

[0016] The (a) detecting or measuring or quantifying cell surface translocation of calreticulin (CRT) may further feature detecting the relative amount of fluorescence of a labeled calreticulin or fluorescent ligand capable of binding to the labeled calreticulin. The relative amount of fluorescence may be detected after 1, 2, 3, 4, 6, 10, 12, 16, 18, 20, 24, 30, 36, 48, 60, 72 or more hours after the normal, neoplastic or cancerous cell is exposed to the anti-cancer agent. The relative amount of fluorescence in the normal, neoplastic or cancerous cell exposed to the anticancer agent may be compared to the amount of fluorescence in a normal, neoplastic or cancerous cell not exposed to the anti-cancer agent. In some instances, the relative amount of fluorescence in the normal, neoplastic or cancerous cell exposed to the anti-cancer agent may be 10%, 25%, 50%, 100%, 150%, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more greater than the fluorescence in a normal, neoplastic or cancerous cell not exposed to the anti-cancer agent. [0017] Increased cell surface translocation of calreticulin (CRT) in a cell after it is exposed to one or more anti-cancer agents is indicative of immunogenic cell death. Increased cell surface translocation of calreticulin (CRT) after a cell is exposed to one or more anti-cancer agents is indicative of immunogenic cell death. In some embodiments, 25%, 50%, 75%, 100%, 200%, 250%), 300%), 400%), 500%) or more increased cell surface translocation of calreticulin (CRT) after a cell is exposed to one or more anti-cancer agents compared to the amount of cell surface translocation of calreticulin (CRT) before the same cell is exposed to one or more anti-cancer agents or compared to a similar cell that is not exposed to one or more anti-cancer agents is used to identify or quantify immunogenic cell death.

[0018] The (b) detecting or measuring or quantifying extracellular release of HMGB1 may be performed by transfecting a normal, neoplastic or cancerous cell with an expression system capable of expressing a labeled HMGB1. The labeled HMGB1 may be, for instance, fused HMGB1-RFP protein, and the expression system may be for instance, a pCMV6-AN-RFP plasmid containing an HMGB1 containing sequences encoding a fusion HMGB1-RFP protein. The labeled HMGB 1 may be detectable by any suitable means, such as, for instance, via radiation or fluorescence such as by using fluoroscopy or flow cytometry. In some

embodiments, the fused HMGB1-RFP protein may be detected in the nucleus of a cell via fluoroscopy. In other embodiments, HMGB1-RFP protein may be detected in media

extracellularly via fluorometry.

[0019] The (b) detecting or measuring or quantifying extracellular release of HMGB 1 may further feature exposing the normal, neoplastic or cancerous cell to one or more anti-cancer agents. The exposing the normal, neoplastic or cancerous cell to more than one anti-cancer agent may be performed substantially concurrently or in series, and the anti-cancer agent may be, for instance, ionizing radiation or a chemotherapeutic such as a platinum agent, for instance, oxaliplatin, cisplatin, or carboplatin, or paclitaxel.

[0020] The (b) detecting or measuring or quantifying extracellular release of HMGB 1 may further feature detecting the relative amount of fluorescence of a labeled HMGBl or ligand capable of binding to the labeled HMGB 1. The relative amount of labeled HMGB 1 may be detected after 1, 2, 3, 4, 6, 10, 12, 16, 18, 20, 24, 30, 36, 48, 60, 72 or more hours after the normal, neoplastic or cancerous cell is exposed to the anti-cancer agent. The relative amount of labeled HMGBl in the a media extracellular to the normal, neoplastic or cancerous cell exposed to the anti-cancer agent may be compared to the amount of labeled HMGBl in a media extracellular to a normal, neoplastic or cancerous cell not exposed to the anti-cancer agent. In some instances, the relative amount of labeled HMGBl in the media extracellular to the neoplastic or cancerous cell exposed to the anti-cancer agent may be 10%, 25%, 50%, 100%, 150%), 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more greater than the amount of labeled HMGB 1 in a media extracellular to the normal, neoplastic or cancerous cell not exposed to the anti-cancer agent.

[0021] Increased extracellular release of HMGBl after a cell is exposed to one or more anticancer agents is indicative of immunogenic cell death. In some embodiments, 25%, 50%, 75%, 100%, 200%, 250%, 300%, 400%, 500% or more increased extracellular release of HMGBl after a cell is exposed to one or more anti-cancer agents compared to the amount of extracellular release of HMGBl before the same cell is exposed to one or more anti-cancer agents or compared to a similar cell that is not exposed to one or more anti-cancer agents is used to identify or quantify immunogenic cell death.

[0022] The (c) detecting or measuring or quantifying extracellular release of adenosine-5'- triphosphate (ATP) may be performed by transfecting a normal, neoplastic or cancerous cell with an expression system capable of expressing a biomarker that may be detected based upon its light emission. The biomarker may become detectable or become more luminescent after or upon exposure to ATP. The biomarker may be, for example, a luciferase such as a firefly luciferase. The biomarker such as luciferase may be detected by, for instance, a luminometer. The expression system may feature a firefly luciferase sequence, and this sequence may be flanked by a folate receptor leader sequence and a folate receptor GPI anchor sequence. An exemplary expression system is demonstrated in Figure 3. The (c) detecting or measuring or quantifying extracellular release of adenosine-5'-triphosphate (ATP) may further feature exposing the normal, neoplastic or cancerous cell to one or more anti-cancer agents. The exposing the normal, neoplastic or cancerous cell to more than one anti-cancer agent may be performed substantially concurrently or in series, and the anti-cancer agent may be ionizing radiation or a

chemotherapeutic such as a platinum agent, for instance, oxaliplatin, cisplatin, or carboplatin, or paclitaxel. [0023] The (c) detecting or measuring or quantifying extracellular release of adenosine-5'- triphosphate (ATP) may further feature detecting the relative amount of luminescence. The relative amount of luminescence may be expressed in relative luminescent units, RLUs, and the relative amount of luminescence may be detected after 1, 2, 3, 4, 6, 10, 12, 16, 18, 20, 24, 30, 36, 48, 60 or 72 or more hours after the normal, neoplastic or cancerous cell is exposed to the anticancer agent. The relative amount of luminescence of the biomarker such as, for instance, luciferase, in the normal, neoplastic or cancerous cell exposed to the anti-cancer agent may be compared to the amount of luminescence of the biomarker such as, for instance, luciferase, in a normal, neoplastic or cancerous cell not exposed to the anti-cancer agent. In some instances, the relative amount of luminescence of the biomarker such as, for instance, luciferase, in the normal, neoplastic or cancerous cell exposed to the anti-cancer agent may be 10%, 25%, 50%, 100%, 150%), 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more greater than the luminescence of the biomarker such as, for instance, luciferase, in a normal, neoplastic or cancerous cell not exposed to the anti-cancer agent.

[0024] Increased extracellular release of adenosine-5'-triphosphate (ATP) after a cell is exposed to one or more anti-cancer agents is indicative of immunogenic cell death. In some

embodiments, 25%, 50%, 75%, 100%, 200%, 250%, 300%, 400%, 500% or more increase in extracellular release of adenosine-5'-triphosphate (ATP) after a cell is exposed to one or more anti-cancer agents compared to the amount of extracellular release of adenosine-5 '-triphosphate (ATP) before the same cell is exposed to one or more anti-cancer agents or compared to a similar cell that is not exposed to one or more anti-cancer agents is used to identify or quantify immunogenic cell death.

[0025] In a second aspect, the invention provides methods of assessing or determining the efficacy of an anti-cancer therapy by detecting or measuring or quantifying one, two or three of the following:

(a) cell surface translocation of calreticulin (CRT),

(b) extracellular release of HMGB1, and

(c) extracellular release of adenosine-5'-triphosphate (ATP).

[0026] In some embodiments, only one of the three is detected or measured, in other

embodiments two of the three are detected or measured, and in still other embodiments, all three are detected or measured. Of course, additional factors, proteins, peptides, molecules, markers, values, times, and rates may be detected or measured as well, either concurrently or sequentially. The methods feature first obtaining a biological sample containing a normal, neoplastic or cancerous cell. The methods may be performed in vitro. An increase in one, two or three of the above where the increase is at least about 25%, 50%, 75%, 100%, 150%, two times, three times, four times, five times or more greater than a baseline, a control or a measure made or value obtained prior to exposure to the anti-cancer therapy may be used to determine that the anticancer therapy is efficacious.

[0027] The (a) detecting or measuring or quantifying cell surface translocation of calreticulin (CRT) may be performed by transfecting a normal, neoplastic or cancerous cell with an expression system capable of expressing a labeled calreticulin. The labeled calrticulin may be, for instance, calreticulin, HaloTag ^® , and the expression system may be for instance, a pEZ-M02 plasmid containing sequences encoding a fusion CRT-HaloTag protein. The labeled calreticulin may be detectable by any suitable means, such as, for instance, via radiation or fluorescence such as by using fluoroscopy or flow cytometry. In one embodiment, a fluorescent ligand such as the HaloTag ^® alexa fluor 488 ligand may be used to bind and detect HaloTag ^® hydrolase. The fluorescent properties may be detected for instance, by fluoroscopy or flow cytometry.

[0028] The (a) detecting or measuring or quantifying cell surface translocation of calreticulin (CRT) may further feature exposing the normal, neoplastic or cancerous cell to one or more anticancer agents. The exposing the normal, neoplastic or cancerous cell to more than one anticancer agent may be performed substantially concurrently or in series, and the anti-cancer agent may be, for instance, ionizing radiation or a chemotherapeutic such as a platinum agent, for instance, oxaliplatin, cisplatin, or carboplatin, or paclitaxel.

[0029] The (a) detecting or measuring or quantifying cell surface translocation of calreticulin (CRT) may further feature detecting the relative amount of fluorescence of a labeled calreticulin or fluorescent ligand capable of binding to the labeled calreticulin. The relative amount of fluorescence may be detected after 1, 2, 3, 4, 6, 10, 12, 16, 18, 20, 24, 30, 36, 48, 60, 72 or more hours after the normal, neoplastic or cancerous cell is exposed to the anti-cancer agent. The relative amount of fluorescence in the normal, neoplastic or cancerous cell exposed to the anticancer agent may be compared to the amount of fluorescence in a normal, neoplastic or cancerous cell not exposed to the anti-cancer agent. In some instances, the relative amount of fluorescence in the normal, neoplastic or cancerous cell exposed to the anti-cancer agent may be 10%, 25%, 50%, 100%, 150%, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more greater than the fluorescence in a normal, neoplastic or cancerous cell not exposed to the anti-cancer agent.

[0030] Increased cell surface translocation of calreticulin (CRT) in a cell after it is exposed to one or more anti-cancer agents is indicative of immunogenic cell death. Increased cell surface translocation of calreticulin (CRT) after a cell is exposed to one or more anti-cancer agents is indicative of immunogenic cell death. In some embodiments, 25%, 50%, 75%, 100%, 200%, 250%), 300%), 400%), 500%) or more increased cell surface translocation of calreticulin (CRT) after a cell is exposed to one or more anti-cancer agents compared to the amount of cell surface translocation of calreticulin (CRT) before the same cell is exposed to one or more anti-cancer agents or compared to a similar cell that is not exposed to one or more anti-cancer agents is used to identify or assess efficacy of an anti-cancer therapy including the one or more anti-cancer agents.

[0031] The (b) detecting or measuring or quantifying extracellular release of HMGB1 may be performed by transfecting a normal, neoplastic or cancerous cell with an expression system capable of expressing a labeled HMGB1. The labeled HMGB1 may be, for instance, fused HMGB1-RFP protein, and the expression system may be for instance, a pCMV6-AN-RFP plasmid containing an HMGB1 containing sequences encoding a fusion HMGB1-RFP protein. The labeled HMGB 1 may be detectable by any suitable means, such as, for instance, via radiation or fluorescence such as by using fluoroscopy or flow cytometry. In some

embodiments, the fused HMGB1-RFP protein may be detected in the nucleus of a cell via fluoroscopy. In other embodiments, HMGB1-RFP protein may be detected in media

extracellularly via fluorometry.

[0032] The (b) detecting or measuring or quantifying extracellular release of HMGB 1 may further feature exposing the normal, neoplastic or cancerous cell to one or more anti-cancer agents. The exposing the normal, neoplastic or cancerous cell to more than one anti-cancer agent may be performed substantially concurrently or in series, and the anti-cancer agent may be, for instance, ionizing radiation or a chemotherapeutic such as a platinum agent, for instance, oxaliplatin, cisplatin, or carboplatin, or paclitaxel.

[0033] The (b) detecting or measuring or quantifying extracellular release of HMGBl may further feature detecting the relative amount of fluorescence of a labeled HMGBl or ligand capable of binding to the labeled HMGB 1. The relative amount of labeled HMGB 1 may be detected after 1, 2, 3, 4, 6, 10, 12, 16, 18, 20, 24, 30, 36, 48, 60, 72 or more hours after the neoplastic or cancerous cell is exposed to the anti-cancer agent. The relative amount of labeled HMGBl in the a media extracellular to the normal, neoplastic or cancerous cell exposed to the anti-cancer agent may be compared to the amount of labeled HMGB 1 in a media extracellular to a normal, neoplastic or cancerous cell not exposed to the anti-cancer agent. In some instances, the relative amount of labeled HMGBl in the media extracellular to the normal, neoplastic or cancerous cell exposed to the anti-cancer agent may be 10%, 25%, 50%, 100%, 150%, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more greater than the amount of labeled HMGB 1 in a media extracellular to the normal, neoplastic or cancerous cell not exposed to the anti-cancer agent.

[0034] Increased extracellular release of HMGBl after a cell is exposed to one or more anticancer agents is indicative of immunogenic cell death. In some embodiments, 25%, 50%, 75%, 100%, 200%, 250%, 300%, 400%, 500% or more increased extracellular release of HMGBl after a cell is exposed to one or more anti-cancer agents compared to the amount of extracellular release of HMGBl before the same cell is exposed to one or more anti-cancer agents or compared to a similar cell that is not exposed to one or more anti-cancer agents is used to assess efficacy of an anti-cancer therapy including the one or more anti-cancer agents.

[0035] The (c) detecting or measuring or quantifying extracellular release of adenosine-5'- triphosphate (ATP) may be performed by transfecting a normal, neoplastic or cancerous cell with an expression system capable of expressing a biomarker that may be detected based upon its light emission. The biomarker may become detectable or become more luminescent after or upon exposure to ATP. The biomarker may be, for example, a luciferase such as a firefly luciferase. The biomarker such as luciferase may be detected by, for instance, a luminometer. The expression system may feature a firefly luciferase sequence, and this sequence may be flanked by a folate receptor leader sequence and a folate receptor GPI anchor sequence. An exemplary expression system is demonstrated in Figure 3. The (c) detecting or measuring or quantifying extracellular release of adenosine-5'-triphosphate (ATP) may further feature exposing the normal, neoplastic or cancerous cell to one or more anti-cancer agents. The exposing the normal, neoplastic or cancerous cell to more than one anti-cancer agent may be performed substantially concurrently or in series, and the anti-cancer agent may be ionizing radiation or a

chemotherapeutic such as a platinum agent, for instance, oxaliplatin, cisplatin, or carboplatin, or paclitaxel.

[0036] The (c) detecting or measuring or quantifying extracellular release of adenosine-5'- triphosphate (ATP) may further feature detecting the relative amount of luminescence. The relative amount of luminescence may be expressed in relative luminescent units, RLUs, and the relative amount of luminescence may be detected after 1, 2, 3, 4, 6, 10, 12, 16, 18, 20, 24, 30, 36, 48 or more hours after the normal, neoplastic or cancerous cell is exposed to the anti-cancer agent. The relative amount of luminescence of the biomarker such as, for instance, luciferase, in the normal, neoplastic or cancerous cell exposed to the anti-cancer agent may be compared to the amount of luminescence of the biomarker such as, for instance, luciferase, in a normal, neoplastic or cancerous cell not exposed to the anti-cancer agent. In some instances, the relative amount of luminescence of the biomarker such as, for instance, luciferase, in the normal, neoplastic or cancerous cell exposed to the anti-cancer agent may be 10%, 25%, 50%, 100%, 150%), 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more greater than the luminescence of the biomarker such as, for instance, luciferase, in a normal, neoplastic or cancerous cell not exposed to the anti-cancer agent.

[0037] Increased extracellular release of adenosine-5'-triphosphate (ATP) after a cell is exposed to one or more anti-cancer agents is indicative of immunogenic cell death. Therefore, increased extracellular release of adenosine-5'-triphosphate (ATP) after a cell is exposed to one or more anti-cancer agents is indicative that the anti-cancer therapy is efficacious. In some embodiments, 25%, 50%, 75%, 100%, 200%, 250%, 300%, 400%, 500% or more increase in extracellular release of adenosine-5 '-triphosphate (ATP) after a cell is exposed to one or more anti-cancer agents compared to the amount of extracellular release of adenosine-5'-triphosphate (ATP) before the same cell is exposed to one or more anti-cancer agents or compared to a similar cell that is not exposed to one or more anti-cancer agents is used to assess or determine the efficacy of an anti-cancer therapy containing the one or more anti-cancer agents. [0038] In a third aspect, the invention provides methods of identifying a potentially efficacious anti-cancer therapy by detecting or measuring or quantifying one, two or three of the following:

(a) cell surface translocation of calreticulin (CRT),

(b) extracellular release of HMGB1, and

(c) extracellular release of adenosine-5'-triphosphate (ATP).

[0039] In some embodiments, only one of the three is detected or measured, in other

embodiments two of the three are detected or measured, and in still other embodiments, all three are detected or measured. Of course, additional factors, proteins, peptides, molecules, markers, values, times, and rates may be detected or measured as well, either concurrently or sequentially. The methods feature first obtaining a biological sample containing a cell such as a neoplastic or cancerous cell. The methods may be performed in vitro. An increase in one, two or three of the above where the increase is at least about 25%, 50%, 75%, 100%, 150%, two times, three times, four times, five times or more greater than a baseline, a control or a measure made or value obtained prior to exposure to one or more anti-cancer agents may be used to identify an anticancer therapy as potentially efficacious.

[0040] The (a) detecting or measuring or quantifying cell surface translocation of calreticulin (CRT) may be performed by transfecting a cell such as a neoplastic or cancerous cell with an expression system capable of expressing a labeled calreticulin. The labeled calrticulin may be, for instance, calreticulin, HaloTag ^® , and the expression system may be for instance, a pEZ-M02 plasmid containing sequences encoding a fusion CRT -HaloTag protein. The labeled calreticulin may be detectable by any suitable means, such as, for instance, via radiation or fluorescence such as by using fluoroscopy or flow cytometry. In one embodiment, a fluorescent ligand such as the HaloTag ^® alexa fluor 488 ligand may be used to bind and detect HaloTag ^® hydrolase. The fluorescent properties may be detected for instance, by fluoroscopy or flow cytometry.

[0041] The (a) detecting or measuring or quantifying cell surface translocation of calreticulin (CRT) may further feature exposing the cell such as a neoplastic or cancerous cell to one or more anti-cancer agents. The exposing the cell such as a neoplastic or cancerous cell to more than one anti-cancer agent may be performed substantially concurrently or in series, and the anti-cancer agent may be, for instance, ionizing radiation or a chemotherapeutic such as a platinum agent, for instance, oxaliplatin, cisplatin, or carboplatin, or paclitaxel.

[0042] The (a) detecting or measuring or quantifying cell surface translocation of calreticulin (CRT) may further feature detecting the relative amount of fluorescence of a labeled calreticulin or fluorescent ligand capable of binding to the labeled calreticulin. The relative amount of fluorescence may be detected after 1, 2, 3, 4, 6, 10, 12, 16, 18, 20, 24, 30, 36, 48, 60, 72 or more hours after the cell such as a neoplastic or cancerous cell is exposed to the anti-cancer agent. The relative amount of fluorescence in the cell such as a neoplastic or cancerous cell exposed to the anti-cancer agent may be compared to the amount of fluorescence in a cell such as a neoplastic or cancerous cell not exposed to the anti-cancer agent. In some instances, the relative amount of fluorescence in the cell such as a neoplastic or cancerous cell exposed to the anticancer agent may be 10%, 25%, 50%, 100%, 150%, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more greater than the fluorescence in a cell such as a neoplastic or cancerous cell not exposed to the anti-cancer agent.

[0043] Increased cell surface translocation of calreticulin (CRT) in a cell after it is exposed to one or more anti-cancer agents is indicative of immunogenic cell death. In some embodiments, 25%, 50%, 75%, 100%, 200%, 250%, 300%, 400%, 500% or more increased cell surface translocation of calreticulin (CRT) after a cell is exposed to one or more anti-cancer agents compared to the amount of cell surface translocation of calreticulin (CRT) before the same cell is exposed to one or more anti-cancer agents or compared to a similar cell that is not exposed to one or more anti-cancer agents is used to identify a potentially efficacious anti-cancer therapy the one or more anti-cancer agents.

[0044] The (b) detecting or measuring or quantifying extracellular release of HMGB 1 may be performed by transfecting a cell such as a neoplastic or cancerous cell with an expression system capable of expressing a labeled HMGB 1. The labeled HMGB 1 may be, for instance, fused HMGB 1-RFP protein, and the expression system may be for instance, a pCMV6-AN-RFP plasmid containing an HMGB 1 containing sequences encoding a fusion HMGB 1-RFP protein. The labeled HMGB 1 may be detectable by any suitable means, such as, for instance, via radiation or fluorescence such as by using fluoroscopy or flow cytometry. In some

embodiments, the fused HMGB 1-RFP protein may be detected in the nucleus of a cell via fluoroscopy. In other embodiments, HMGBl -RFP protein may be detected in media extracellularly via fluorometry.

[0045] The (b) detecting or measuring or quantifying extracellular release of HMGBl may further feature exposing the cell such as a neoplastic or cancerous cell to one or more anti-cancer agents. The exposing the cell such as a neoplastic or cancerous cell to more than one anti-cancer agent may be performed substantially concurrently or in series, and the anti-cancer agent may be, for instance, ionizing radiation or a chemotherapeutic such as a platinum agent, for instance, oxaliplatin, cisplatin, or carboplatin, or paclitaxel.

[0046] The (b) detecting or measuring or quantifying extracellular release of HMGBl may further feature detecting the relative amount of fluorescence of a labeled HMGBl or ligand capable of binding to the labeled HMGB 1. The relative amount of labeled HMGB 1 may be detected after 1, 2, 3, 4, 6, 10, 12, 16, 18, 20, 24, 30, 36, 48, 60, 72 or more hours after the cell such as a neoplastic or cancerous cell is exposed to the anti-cancer agent. The relative amount of labeled HMGB 1 in the a media extracellular to the cell such as a neoplastic or cancerous cell exposed to the anti-cancer agent may be compared to the amount of labeled HMGBl in a media extracellular to a cell such as a neoplastic or cancerous cell not exposed to the anti-cancer agent. In some instances, the relative amount of labeled HMGBl in the media extracellular to the cell such as a neoplastic or cancerous cell exposed to the anti-cancer agent may be 10%, 25%, 50%, 100%), 150%), 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more greater than the amount of labeled HMGB 1 in a media extracellular to the cell such as a neoplastic or cancerous cell not exposed to the anti-cancer agent.

[0047] Increased extracellular release of HMGBl after a cell is exposed to one or more anticancer agents is indicative of immunogenic cell death. In some embodiments, 25%, 50%, 75%, 100%, 200%, 250%, 300%, 400%, 500% or more increased extracellular release of HMGBl after a cell is exposed to one or more anti-cancer agents compared to the amount of extracellular release of HMGBl before the same cell is exposed to one or more anti-cancer agents or compared to a similar cell that is not exposed to one or more anti-cancer agents is used to identify an anti-cancer therapy including the one or more anti-cancer agents that may be efficacious. [0048] The (c) detecting or measuring or quantifying extracellular release of adenosine-5'- triphosphate (ATP) may be performed by transfecting a cell such as a neoplastic or cancerous cell with an expression system capable of expressing a biomarker that may be detected based upon its light emission. The biomarker may become detectable or become more luminescent after or upon exposure to ATP. The biomarker may be, for example, a luciferase such as a firefly luciferase. The biomarker such as luciferase may be detected by, for instance, a luminometer. The expression system may feature a firefly luciferase sequence, and this sequence may be flanked by a folate receptor leader sequence and a folate receptor GPI anchor sequence. An exemplary expression system is demonstrated in Figure 3. The (c) detecting or measuring or quantifying extracellular release of adenosine-5'-triphosphate (ATP) may further feature exposing the cell such as a neoplastic or cancerous cell to one or more anti-cancer agents. The exposing the cell such as a neoplastic or cancerous cell to more than one anti-cancer agent may be performed substantially concurrently or in series, and the anti-cancer agent may be ionizing radiation or a chemotherapeutic such as a platinum agent, for instance, oxaliplatin, cisplatin, or carboplatin, or paclitaxel.

[0049] The (c) detecting or measuring or quantifying extracellular release of adenosine-5'- triphosphate (ATP) may further feature detecting the relative amount of luminescence. The relative amount of luminescence may be expressed in relative luminescent units, RLUs, and the relative amount of luminescence may be detected after 1, 2, 3, 4, 6, 10, 12, 16, 18, 20, 24, 30, 36, 48 or more hours after the cell such as a neoplastic or cancerous cell is exposed to the anti-cancer agent. The relative amount of luminescence of the biomarker such as, for instance, luciferase, in the cell such as a neoplastic or cancerous cell exposed to the anti-cancer agent may be compared to the amount of luminescence of the biomarker such as, for instance, luciferase, in a cell such as a neoplastic or cancerous cell not exposed to the anti-cancer agent. In some instances, the relative amount of luminescence of the biomarker such as, for instance, luciferase, in the cell such as a neoplastic or cancerous cell exposed to the anti-cancer agent may be 10%, 25%, 50%, 100%, 150%, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more greater than the luminescence of the biomarker such as, for instance, luciferase, in a cell such as a neoplastic or cancerous cell not exposed to the anti-cancer agent.

[0050] Increased extracellular release of adenosine-5'-triphosphate (ATP) after a cell is exposed to one or more anti-cancer agents is indicative of immunogenic cell death. Therefore, increased extracellular release of adenosine-5'-triphosphate (ATP) after a cell is exposed to one or more anti-cancer agents is indicative that the anti-cancer therapy is potentially efficacious. In some embodiments, 25%, 50%, 75%, 100%, 200%, 250%, 300%, 400%, 500% or more increase in extracellular release of adenosine-5'-triphosphate (ATP) after a cell is exposed to one or more anti-cancer agents compared to the amount of extracellular release of adenosine-5 '-triphosphate (ATP) before the same cell is exposed to one or more anti-cancer agents or compared to a similar cell that is not exposed to one or more anti-cancer agents is used to determine that an anti-cancer therapy including the one or more anti-cancer agents containing the one or more anti-cancer agents is potentially efficacious.

[0051] In a fourth aspect, the invention provides methods for identifying a subject who may benefit from an anti-cancer therapy by detecting or measuring or quantifying one, two or three of the following:

(a) cell surface translocation of calreticulin (CRT),

(b) extracellular release of HMGB1, and

(c) extracellular release of adenosine-5'-triphosphate (ATP).

[0052] In some embodiments, only one of the three is detected or measured, in other

embodiments two of the three are detected or measured, and in still other embodiments, all three are detected or measured. Of course, additional factors, proteins, peptides, molecules, markers, values, times, and rates may be detected or measured as well, either concurrently or sequentially. The methods feature first obtaining a biological sample containing a neoplastic or cancerous cell. The methods may be performed in vitro. An increase in one, two or three of the above where the increase is at least about 25%, 50%, 75%, 100%, 150%, two times, three times, four times, five times or more greater than a baseline, a control or a measure made or value obtained prior to exposure to one or more anti-cancer agents may be used to identify a subject who may benefit from or respond positively to the anti-cancer therapy. The anti-cancer therapy may feature one or more anti-cancer agents, and the anti-cancer therapy may be radiation therapy or chemotherapy or both either alone, concurrent or sequential.

[0053] The (a) detecting or measuring or quantifying cell surface translocation of calreticulin (CRT) may be performed by transfecting a cell such as a neoplastic or cancerous cell with an expression system capable of expressing a labeled calreticulin. The labeled calrticulin may be, for instance, calreticulin, HaloTag " , and the expression system may be for instance, a pEZ-M02 plasmid containing sequences encoding a fusion CRT-HaloTag protein. The labeled calreticulin may be detectable by any suitable means, such as, for instance, via radiation or fluorescence such as by using fluoroscopy or flow cytometry. In one embodiment, a fluorescent ligand such as the HaloTag ^® alexa fluor 488 ligand may be used to bind and detect HaloTag ^® hydrolase. The fluorescent properties may be detected for instance, by fluoroscopy or flow cytometry.

[0054] The (a) detecting or measuring or quantifying cell surface translocation of calreticulin (CRT) may further feature exposing the cell such as a neoplastic or cancerous cell to one or more anti-cancer agents. The exposing the cell such as a neoplastic or cancerous cell to more than one anti-cancer agent may be performed substantially concurrently or in series, and the anti-cancer agent may be, for instance, ionizing radiation or a chemotherapeutic such as a platinum agent, for instance, oxaliplatin, cisplatin, or carboplatin, or paclitaxel.

[0055] The (a) detecting or measuring or quantifying cell surface translocation of calreticulin (CRT) may further feature detecting the relative amount of fluorescence of a labeled calreticulin or fluorescent ligand capable of binding to the labeled calreticulin. The relative amount of fluorescence may be detected after 1, 2, 3, 4, 6, 10, 12, 16, 18, 20, 24, 30, 36, 48, 60, 72 or more hours after the cell such as a neoplastic or cancerous cell is exposed to the anti-cancer agent. The relative amount of fluorescence in the cell such as a neoplastic or cancerous cell exposed to the anti-cancer agent may be compared to the amount of fluorescence in a cell such as a neoplastic or cancerous cell not exposed to the anti-cancer agent. In some instances, the relative amount of fluorescence in the cell such as a neoplastic or cancerous cell exposed to the anticancer agent may be 10%, 25%, 50%, 100%, 150%, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more greater than the fluorescence in a cell such as a neoplastic or cancerous cell not exposed to the anti-cancer agent.

[0056] Increased cell surface translocation of calreticulin (CRT) in a cell after it is exposed to one or more anti-cancer agents is indicative of immunogenic cell death. In some embodiments, 25%, 50%, 75%, 100%, 200%, 250%, 300%, 400%, 500% or more increased cell surface translocation of calreticulin (CRT) after a cell is exposed to one or more anti-cancer agents compared to the amount of cell surface translocation of calreticulin (CRT) before the same cell is exposed to one or more anti-cancer agents or compared to a similar cell that is not exposed to one or more anti-cancer agents is used to identify a potentially efficacious anti-cancer therapy the one or more anti-cancer agents. As such, such increased cell surface translocation of calreticulin (CRT) is indicative that the anti-cancer therapy is potentially efficacious, and a subject suffering from a cancer involving the subject cells may benefit from the anti-cancer therapy containing the one or more anti-cancer agents.

[0057] The (b) detecting or measuring or quantifying extracellular release of HMGB1 may be performed by transfecting a cell such as a neoplastic or cancerous cell with an expression system capable of expressing a labeled HMGB1. The labeled HMGB1 may be, for instance, fused HMGB1-RFP protein, and the expression system may be for instance, a pCMV6-AN-RFP plasmid containing an HMGB1 containing sequences encoding a fusion HMGB1-RFP protein. The labeled HMGB 1 may be detectable by any suitable means, such as, for instance, via radiation or fluorescence such as by using fluoroscopy or flow cytometry. In some

embodiments, the fused HMGB1-RFP protein may be detected in the nucleus of a cell via fluoroscopy. In other embodiments, HMGB1-RFP protein may be detected in media

extracellularly via fluorometry.

[0058] The (b) detecting or measuring or quantifying extracellular release of HMGB 1 may further feature exposing the cell such as a neoplastic or cancerous cell to one or more anti-cancer agents. The exposing the cell such as a neoplastic or cancerous cell to more than one anti-cancer agent may be performed substantially concurrently or in series, and the anti-cancer agent may be, for instance, ionizing radiation or a chemotherapeutic such as a platinum agent, for instance, oxaliplatin, cisplatin, or carboplatin, or paclitaxel.

[0059] The (b) detecting or measuring or quantifying extracellular release of HMGB 1 may further feature detecting the relative amount of fluorescence of a labeled HMGBl or ligand capable of binding to the labeled HMGB 1. The relative amount of labeled HMGB 1 may be detected after 1, 2, 3, 4, 6, 10, 12, 16, 18, 20, 24, 30, 36, 48, 60, 72 or more hours after the cell such as a neoplastic or cancerous cell is exposed to the anti-cancer agent. The relative amount of labeled HMGB 1 in the a media extracellular to the cell such as a neoplastic or cancerous cell exposed to the anti-cancer agent may be compared to the amount of labeled HMGBl in a media extracellular to a cell such as a neoplastic or cancerous cell not exposed to the anti-cancer agent. In some instances, the relative amount of labeled HMGBl in the media extracellular to the cell such as a neoplastic or cancerous cell exposed to the anti-cancer agent may be 10%, 25%, 50%, 100%), 150%), 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more greater than the amount of labeled HMGB 1 in a media extracellular to the cell such as a neoplastic or cancerous cell not exposed to the anti-cancer agent.

[0060] Increased extracellular release of HMGBl after a cell is exposed to one or more anticancer agents is indicative of immunogenic cell death. In some embodiments, 25%, 50%, 75%, 100%, 200%, 250%, 300%, 400%, 500% or more increased extracellular release of HMGBl after a cell is exposed to one or more anti-cancer agents compared to the amount of extracellular release of HMGBl before the same cell is exposed to one or more anti-cancer agents or compared to a similar cell that is not exposed to one or more anti-cancer agents is used to identify a subject who may benefit from an anti-cancer therapy including the one or more anti-cancer agents.

[0061] The (c) detecting or measuring or quantifying extracellular release of adenosine-5'- triphosphate (ATP) may be performed by transfecting a cell such as a neoplastic or cancerous cell with an expression system capable of expressing a biomarker that may be detected based upon its light emission. The biomarker may become detectable or become more luminescent after or upon exposure to ATP. The biomarker may be, for example, a luciferase such as a firefly luciferase. The biomarker such as luciferase may be detected by, for instance, a luminometer. The expression system may feature a firefly luciferase sequence, and this sequence may be flanked by a folate receptor leader sequence and a folate receptor GPI anchor sequence. An exemplary expression system is demonstrated in Figure 1. The (c) detecting or measuring or quantifying extracellular release of adenosine-5'-triphosphate (ATP) may further feature exposing the cell such as a neoplastic or cancerous cell to one or more anti-cancer agents. The exposing the cell such as a neoplastic or cancerous cell to more than one anti-cancer agent may be performed substantially concurrently or in series, and the anti-cancer agent may be ionizing radiation or a chemotherapeutic such as a platinum agent, for instance, oxaliplatin, cisplatin, or carboplatin, or paclitaxel.

[0062] The (c) detecting or measuring or quantifying extracellular release of adenosine-5'- triphosphate (ATP) may further feature detecting the relative amount of luminescence. The relative amount of luminescence may be expressed in relative luminescent units, RLUs, and the relative amount of luminescence may be detected after 1, 2, 3, 4, 6, 10, 12, 16, 18, 20, 24, 30, 36, 48 or more hours after the cell such as a neoplastic or cancerous cell is exposed to the anti-cancer agent. The relative amount of luminescence of the biomarker such as, for instance, luciferase, in the cell such as a neoplastic or cancerous cell exposed to the anti-cancer agent may be compared to the amount of luminescence of the biomarker such as, for instance, luciferase, in a cell such as a neoplastic or cancerous cell not exposed to the anti-cancer agent. In some instances, the relative amount of luminescence of the biomarker such as, for instance, luciferase, in the cell such as a neoplastic or cancerous cell exposed to the anti-cancer agent may be 10%, 25%, 50%, 100%), 150%), 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more greater than the luminescence of the biomarker such as, for instance, luciferase, in a cell such as a neoplastic or cancerous cell not exposed to the anti-cancer agent.

[0063] Increased extracellular release of adenosine-5'-triphosphate (ATP) after a cell is exposed to one or more anti-cancer agents is indicative of immunogenic cell death. Therefore, increased extracellular release of adenosine-5'-triphosphate (ATP) after a cell is exposed to one or more anti-cancer agents is indicative that the anti-cancer therapy is potentially efficacious, and a subject suffering from a cancer involving the subject cells may benefit from the anti-cancer therapy. In some embodiments, 25%, 50%, 75%, 100%, 200%, 250%, 300%, 400%, 500% or more increase in extracellular release of adenosine-5'-triphosphate (ATP) after a cell is exposed to one or more anti-cancer agents compared to the amount of extracellular release of adenosine- 5'-triphosphate (ATP) before the same cell is exposed to one or more anti-cancer agents or compared to a similar cell that is not exposed to one or more anti-cancer agents is used to identify a subject who may benefit from an anti-cancer therapy including the one or more anticancer agents.

[0064] In a fifth aspect, the invention provides a kit containing means for detecting or measuring or quantifying one, two or three of the following:

(a) cell surface translocation of calreticulin (CRT),

(b) extracellular release of HMGB1, and

(c) extracellular release of adenosine-5'-triphosphate (ATP)

along with instructions for detecting or measuring or quantifying one, two or three of these. The kit may include a labeled calreticulin, an expression system suitable for expressing a labeled calreticulin, and a ligand for a labeled calreticulin such as, for instance, a fluorophore. The kit may also include a labeled HMGB1 or an expression system suitable for expressing a labeled HMGB1. The labeled HMGB1 may be an HMGB 1-RFP fusion protein. The kit may further include a biomarker or reporter molecule whose detection is linked to adenosine-5' -triphosphate (ATP) levels. The biomarker or reporter molecule may be detectable based upon its light emission, for instance, by fluoroscopy. One such suitable biomarker or reporter molecule is a luciferase such as firefly luciferase, known to those skilled in the art.

BRIEF DESCRIPTION OF THE FIGURES

[0065] Figure 1 demonstrates that ionizing radiation induces ATP release into the ECM. (A and

B) TSA cells were stably transfected with a pGEN2.1 plasmid encoding a firefly luciferase reporter sequence flanked by a folate receptor (FR) leader sequence and

glycophosphatidylinositol (GPI) anchor sequence (A, top panel). ATP remains intracellular when tumor cells are in their native condition, correlating with minimal luciferase activity. ATP release upon immunogenic cell death increases pericellular ATP that, in the presence of oxygen, magnesium, and D-luciferin, reacts with the external membrane-bound luciferase and produces photons that can be detected by luminometery (A, bottom panel). (B) The amount of

luminescence detected from 2 x 10 ⁴ cells per well (96-well plate) pGEN2.1-pMe-Luc transfected TSA cells after 24 hours of exposure to increasing doses of ionizing radiation (IR) ranging from 0-20 Gray (Gy) reported as fold-change in relative luminescent units (RLU) in comparison to the luminescent signal detected from non-irradiated cells, normalized to 1. Shown are the mean RLU (n = 8 wells/group) ± SD.

[0066] Figure 2 demonstrates that radiotherapy promotes calreticulin surface translocation. (A-

C) TSA cells were stably transfected with a pEZ-M02 plasmid encoding a calreticulin (CRT) fusion protein with the modular HaloTag ^® reporter, and endoplasmic reticulum targeting KDEL sequences (A, top panel) corresponding to the translation of a fusion CRT-HaloTag-KDEL protein (A, middle panel). CRT remains in the ER in non-stressed cells, whereas upon immunogenic cell death (ICD) CRT translocates to the cell surface (A, bottom panel). Externally localized CRT-HaloTag-KDEL is irreversible bound by membrane impermeable HaloTag ^® Alexa Fluor 488 ligand activating its fluorescent properties that can then be detected via fluorescence microscopy or flow cytometry (A, middle and bottom panels). (B and C) pEZ-M02- CRT-HaloTag-KDEL transfected TSA cells were treated for 24 hours with the indicated dosage of ionizing radiation (IR, delivered at time 0 h) were exposed to the impermeable HaloTag ^® Alexa Fluor 488 ligand. The amount of green fluorescence indicative of cell surface CRT was detected via cytoflourimetric analysis. (B) Representative histograms at each dose of IR. (C) Mean fluorescent intensity (MFI) detected from cells irradiated with the indicated IR dosage versus non-irradiated cells normalized to 1. Shown are the MFI (n = 10,000 cells/group) ± SD.

[0067] Figure 3 demonstrates that radiation therapy promotes HMGBl release. (A-C) TSA cells were stably transfected with a pCMV6-AN-RFP plasmid (A, top panel) comprising the high mobility group box 1 (HMGBl) protein coding open-reading frame sequence fused with a C- terminal red fluorescent protein (RFP) tag (A, middle panel). Under native conditions, HMGBl remains in the nucleus, whereas HMGBl is released from cells undergoing ICD, detectable in the conditioned medium of cultured HMGBl -RFP TSA cells via fluorimetry (A, bottom panel). (B) Chimeric HMGBl -RFP protein can be detected in the nucleus of untreated cells via confocal fluorescence microscopy. (C) Transfected cells were exposed to ionizing radiation (IR) ranging from 0-20 Gray (Gy), as indicated. Released HMGBl -RFP was detected in the conditioned medium 72 hours after treatment via cytofluorimetric analysis and reported as fold change in relative fluorescent units (RFU) in comparison to untreated control cells, normalized to 1. Shown are the MFI (n =6 wells/group) ± SD.

[0068] Figure 4 demonstrates cytotoxicity induced by platinum and ionizing radiation combinatorial treatment in TSA cells. (A and B) The differential cytotoxic effects of platinum compounds ± ionizing radiation (IR) at a dosage of 2 Gray (Gy) was evaluated via colony forming assay. TSA cells (200 cells/well in a 6-well plate; n = 6) were exposed to increasing doses of the indicated platinum agent [0-5 μΜ] for 48 h ± IR [2 Gy], delivered at time 0 hours. After incubating the cells for 10 days, the colonies formed were fixed, stained, and subsequently counted. (A) The percent colonies formed are displayed, normalized to untreated cells (100% colony formation). (B) Corresponding images of crystal violet stained colonies. Shown are the mean colony formation ± SD. Statistical analyses were performed using a paired Student's t test; P values < 0.05 were considered statistically significant.

[0069] Figure 5 demonstrates pericellular ATP in platinum and ionizing radiation treated TSA cells. (A and B) TSA cells transfected with pGEN2.1 vector encoding plasma membrane localized luciferase (pMe-Luc) were used to assay the effects of platinum ± ionizing radiation (IR) on pericellular ATP concentrations. The amount of luminescence detected from 2 x 10 ⁴ pGEN2.1-pMe-Luc transfected TSA cells/well (96-well plate) in the presence of D-luciferin. (A) Luminescence detected after 24 hours of exposure to increasing doses of oxaliplatin (0-10 μΜ). (B) Luminescence detected after 24 hours of exposure to increasing doses of the indicated platinum agent [0-5 μΜ] ± increasing doses of IR, ranging from 0-20 Gray (Gy) delivered at time 0 hours. Values are reported as fold-change in relative luminescent units (RLU) in comparison to the luminescent signal detected from non-irradiated cells, normalized to 1. Shown are the means (n =8 wells/group) ± SD. Statistical analyses were performed by paired Student's t test; P values < 0.05 were considered statistically significant.

[0070] Figure 6 demonstrates calreticulin translocation to the cell surface in platinum and ionizing radiation treated TSA cells. (A and B) Externalization of calreticulin (CRT) was monitored using pEZ-M02-CRT-Halotag-KDEL stably transfected TSA breast cancer cells treated for 24 hours with platinum ± the indicated dosage of ionizing radiation (IR, delivered at time 0 hours) exposed to the impermeable HaloTag ^® Alexa Fluor 488 ligand. The amount of green fluorescence indicative of cell surface CRT was detected via fluorescence cytometry. (A) Representative histograms from the indicated treatment. (B) Mean fluorescent intensity (MFI) detected from cells irradiated with the indicated platinum agent ± IR vs. non-irradiated cells normalized to 1. Shown are the MFI (n =10,000 cells/group) ± SD. Statistical analysis was performed by paired Student's t test; P values < 0.05 were considered statistically significant.

[0071] Figure 7 demonstrates HMGBl release from platinum and ionizing radiation treated TSA cells. (A and B) Red-fluorescence protein (RFP) tagged high mobility group box 1 (HMGBl) expressing TSA cells were used to analyze the combined effect of platinum and ionizing radiation (IR) on the release of HMGBl from TSA mammary carcinoma cells. (A) HMGBl-RFP stably transfected TSA cells were exposed to oxaliplatin (0-100 μΜ) or IR (0-20 Gray) delivered at time 0 hours. Released HMGBl-RFP was detected in the conditioned medium 24-72 hours (as indicated) after treatment via fluorimetry and reported as a fold change in relative fluorescent units (RFUs) in comparison to untreated cells (24 hours). (B) HMGBl-RFP was detected in the conditioned medium of transfected cells exposed for 72 hours with increasing doses of platinum (0-20 μΜ) ± radiation therapy (RT) at a dosage of 2 Gray (Gy) delivered at time 0 hours. The RFU is plotted relative to untreated control cells, normalized to 1. Shown are the mean (n = 6/group) ± SD. Statistical analyses were performed by paired Student's t test: P values < 0.05 were considered statistically significant.

[0072] Figure 8 demonstrates that immunogenic cell death is enhanced in paclitaxel and ionizing radiation treated TSA cells. (A-D) The cytotoxic effects of paclitaxel (PTX) ± ionizing radiation (IR) were evaluated via colony forming assay and molecular markers of immunogenic cell death. (A) TSA cells were plated at 200 cells/well in a 6-well plate. After adherence, cells were exposed to 50 nM paclitaxel ± radiation therapy (RT) at a dosage of 2 Gray (Gy) delivered at time 0 hours and evaluated via colony forming assay. After incubating the cells for 10 days, the colonies formed were fixed, stained, and counted. The percent colonies formed are displayed are displayed, normalized to untreated cells (100% colony formation). (B) To assay release of ATP, TSA cells stably transfected with a plasma membrane localized luciferase (pMe-Luc) plated at 2 x 10 ⁴ cells per well (96-well plate) were exposed to 1 μΜ paclitaxel ± 2 Gy IR, delivered at time 0 hours. The relative luminescent units (RLUs) detected 24 hours later are shown in comparison to untreated cells, normalized to 1. Shown are the mean RLU (n =8 wells/group) ± SD (C) To assay calreticulin (CRT) externalization, CRT-Halotag-KDEL stably transfected TSA cells were treated for 24 hours with 100 nM paclitaxel ± 10 Gy IR delivered at time 0 hours were exposed to the impermeable HaloTag ^® Alexa Fluor 488 ligand. The amount of green fluorescence indicative of cell surface CRT was detected via fluorescence cytometry and reported as fold change in mean fluorescent intensity (MFI) vs. untreated cell levels, normalized to 1. Shown are the MFI (n =10,000 cells/group) ± SD (D) The release of high mobility group box 1 (HMGB1) protein was assayed using red fluorescence protein (RFP)-tagged HMGB1. TSA cells stably transfected with HMGBl-RFP stably were exposed to 1 μΜ paclitaxel ± 2 Gy IR, delivered at time 0 hours. Released HMGBl-RFP was detected in the conditioned medium 24-72 hours after treatment via fluorimetry and reported as fold change in relative fluorescent units (RFU) vs. untreated cell levels, normalized to 1. Shown are the mean RLU (n =6 wells/group) ± SD.

DETAILED DESCRIPTION OF THE INVENTION

[0073] Before the present methods are described, it is to be understood that this invention is not limited to particular methods and experimental conditions described, as such methods and conditions may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise. Thus, for example, references to "the method" includes one or more methods, and/or steps of the type described herein and/or which will become apparent to those persons skilled in the art upon reading this disclosure and so forth in their entirety.

[0074] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference in their entireties.

Definitions

[0075] The terms used herein have the meanings recognized and known to those of skill in the art, however, for convenience and completeness, particular terms and their meanings are set forth below.

[0076] "Subject" or "patient" refers to a mammal, preferably a human, in need of enzyme replacement therapy.

[0077] By "fragment thereof is meant a portion of a full length protein or peptide, for instance, about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more as many amino acids as the full length naturally occurring protein or peptide. By "variant thereof is meant a fragment or full length protein or peptide, having about 50%, 60%, 70%, 80%, 90%, or 95% or more sequence homology to a corresponding naturally occurring protein or peptide. Those of skill in the art readily understand that some amino acids may be substituted without substantially impacting biological activity of a protein or peptide. Fragments and variants of the full length proteins and peptides of the present invention, such as, for instance calreticulin (CRT), HMGB1, and adenosine-5'-triphosphate (ATP) are within the scope of the invention.

[0078] "Treatment" or "treating" refers to therapy, prevention and prophylaxis and particularly refers to the administration of medicine or performing medical procedures with respect to a patient, for either prophylaxis (prevention) or to cure or reduce the extent of or likelihood of occurrence of the infirmity or malady or condition or event. In the present invention, the treatments using the agents described may be provided to treat a cancer or neoplasm. Most preferably, treatment is for the purpose of reducing or diminishing the symptoms or progression of the disease, inhibiting or preventing unwanted cell reproduction or growth or killing or eliminating unwanted cells. Treating as used herein also means the administration of the compounds for preventing the development of a cancer or neoplasm. Furthermore, in treating a subject, the compounds of the invention may be administered to a subject already suffering from the disease.

[0079] In a specific embodiment, the term "about" means within 20%, preferably within 10%, and more preferably within 5% and in some instances within 1% or less.

[0080] An "effective amount" or a "therapeutically effective amount" is an amount sufficient to decrease or prevent the symptoms associated with the conditions disclosed herein as well as an amount sufficient to slow or prevent further pathological damage or unwanted cell growth or expansion. For example, an "effective amount" for therapeutic uses is the amount of the composition comprising an active compound herein required to provide a clinically significant increase in healing rates or reduction in symptoms or to reduce unwanted cell growth or expansion.

[0081] The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset, dizziness and the like, when administered to a human. Preferably, as used herein, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the compound is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water or aqueous solution saline solutions and aqueous dextrose and glycerol solutions are preferably employed as carriers, particularly for injectable solutions.

Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin.

[0082] An individual "at risk" may or may not have detectable disease, and may or may not have displayed detectable disease prior to the treatment methods described herein. "At risk" denotes that an individual who is determined to be more likely to develop a symptom based on conventional risk assessment methods or has one or more risk factors that correlate with development of a disease or condition characterized by unwanted cell growth or expansion. An individual having one or more of these risk factors has a higher probability of developing a disease than an individual without these risk factors.

[0083] "Prophylactic" or "therapeutic" treatment refers to administration to the host of one or more of the subject compositions. If it is administered prior to clinical manifestation of the unwanted condition (e.g., disease or other unwanted state of the host animal) then the treatment is prophylactic, i.e., it protects the host against developing the unwanted condition, whereas if administered after manifestation of the unwanted condition, the treatment is therapeutic (i.e., it is intended to diminish, ameliorate or maintain the existing unwanted condition or side effects therefrom).

[0084] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference in their entireties.

[0085] In accordance with the present invention there may be employed conventional molecular biology, microbiology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature. See, e.g., Sambrook et al, "Molecular Cloning: A Laboratory Manual" (1989); "Current Protocols in Molecular Biology" Volumes I-III [Ausubel, R. M., ed. (1994)]; "Cell Biology: A Laboratory Handbook" Volumes I-III [J. E. Celis, ed.

(1994))]; "Current Protocols in Immunology" Volumes I-III [Coligan, J. E., ed. (1994)];

"Oligonucleotide Synthesis" (M. J. Gait ed. 1984); "Nucleic Acid Hybridization" [B. D. Hames & S. J. Higgins eds. (1985)]; "Transcription And Translation" [B. D. Hames & S. J. Higgins, eds. (1984)]; "Animal Cell Culture" [R. I. Freshney, ed. (1986)]; "Immobilized Cells And Enzymes" [IRL Press, (1986)]; B. Perbal, "A Practical Guide To Molecular Cloning" (1984).

[0086] Conventional radiobiologic models fail to describe the dose-response effects of radiotherapy (RT) regimens on tumor cells in the context of the tumor microenvironment, specifically host anti-tumor immune responses. That local radiation treatment contributes to the success of cancer immunotherapies in preclinical models was previously demonstrated.

Radiation treatment induces immunogenic cell death (ICD) in exposed tumor cells thereby leading to local and systemic immune-mediated cytoreduction. (Formenti, et al, Journal of the national Cancer Institute; 2013)

[0087] The present invention provides an in vitro assay to test the effects of radiotherapy at clinically relevant doses alone or in combination with chemotherapeutic agents on each individual component of ICD. Previously, using the TSA syngeneic murine model of mammary carcinoma, it was reported that radiotherapy in combination with cytotoxic T lymphocyte associated protein 4 (CTLA-4) blockade induces an immune-mediated tumor-inhibitory effect outside the field of radiation, in other words an abscopal effect. (Dewan, et al, Clin Cancer Res, 2009; 15:5379-88; Demaria, et al, Int J Radiat Oncol Biol Phys, 2004; 58:862-70) TSA cells were chosen for developing an in vitro model to determine whether the hallmark features of ICD upon exposing the cells to radiation in a dose-dependent manner (i.e., with increasing doses of radiation) alone or in combination with chemotherapy may be observed. In vitro models are particularly advantageous due to the low cost and feasibility, permitting expedited screening of chemoradiotherapy regimens prior to validating their role in an in vivo setting. The present invention describes the immunogenic death signature displayed by TSA mammary cancer cells exposed to radiotherapy alone or in combination with the platinum agents oxaliplatin and carboplatin, or the taxane paclitaxel.

[0088] In fact, the present invention provides an in vitro radiation associated immunogenic cell death assay (RAICD A) to examine the three hallmark features of immunogenic cell death with radiation treatment with or without chemotherapy. Various platinum (Pt) compounds, i.e.

oxaliplatin (oxali), cisplatin (cis), and carboplatin (carbo) were analyzed. [0089] In the framework of treatment induced immunogenic cell death (ICD), the intensity of immunogenic cell death achieved with concurrent, as opposed to sequential, chemotherapy and radiation treatment is more effective at eliciting a host anti-tumor immune response. (Formenti, et al, The Lancet Oncology, 2009; 10: 718-26; Formenti, et al, Journal of Clinical Oncology, 2008; 26: 1562-3) However, the contributions of immunogenic cell death from concurrent chemotherapy and radiation treatment have yet to be established.

[0090] Three distinct immunogenic cell death components required for dendritic cell (DC) activation and immune priming have emerged, including:

(1) the cell surface translocation of calreticulin (CRT), an endoplasmic reticulum residing protein chaperone and potent DC phagocytosis signal;

(2) the extracellular release of HMGB1, a DNA binding protein and TLR-4 mediated

DC activator; and

(3) adenosine-5 '-triphosphate (ATP) an activator of the DC P2X7 purinergic receptor that triggers DC inflammasome activation, secretion of IL-Ιβ, and subsequent priming of IFNy producing CD8 ⁺ T cells.

{See, Ma, et al, Seminars in Immunology, 2010; 22: 113-24; Kroemer, et al, Bulletin et memoires de VAcademie royale de medecine de Belgique, 2011; 166: 130-8; discussion 9-40) The net effect of all three arms acts to promote dendritic cell phagocytosis of tumor cells, dendritic cell processing of tumor-derived antigens, and dendritic cell -associated cross-priming of CD8 ⁺ cytotoxic T lymphocytes. (Ma, et al, Seminars in Immunology, 2010; 22: 113-24; Kroemer, et al, Bulletin et memoires de VAcademie royale de medecine de Belgique, 2011; 166: 130-8;

discussion 9-40; Ma, et al, Cancer Metastasis Reviews, 2011; 30: 71-82; Locher, et al, Annals of the New York Academy of Sciences, 2010; 1209: 99-108) A differential immunogenic cell death response of platinum compounds, cisplatin and oxaliplatin, and ionizing radiation on colorectal cancer cells has been demonstrated. (Tesniere, et al, Oncogene, 2010; 29: 482-91; Obeid, et al, Cell Death and Differntiation, 2007; 14: 1848-50) However, combining both chemotherapy and radiation in attempts to further improve local control and survival is normal. So, there remains a challenge in understanding the combined effects of platinum and radiation- induced immunogenic cell death and whether or not manipulation of this subroutine of cell death has significant clinical implications. Thus, the present methods provide a means to determine whether platinum-based concurrent chemotherapy and radiation treatment can effectively enhance immunogenic cell death in dying tumor cells better than either treatment alone.

[0091] A central dogma of traditional radiobiology is that the cytotoxic effects of ionizing radiation on tumor cells are primarily due to the production of DNA double strand breaks followed by cell death, either via apoptosis, necrosis, autophagy, mitotic catastrophy, or replicative senescence. (Golden, et al, Frontieers in Oncology, 2012; 2: 88) However, this traditional view of radiation-induced cell death is rapidly evolving to take into account the contribution of the tumor microenvironment and the host's tumor-immunity, particularly in the modern era following the advent of immune-checkpoint inhibitors applied as a means to overcome the immunosuppressive effects of established tumors. (Kroemer, et al, Annu Rev Immunol., 2013; 31 :51-72; Golden, et al, Front Oncol, 2012; 2:88; Formenti, et al, J Natl Cancer Inst, 2013; 105:256-65; Barcellos-Hoff, et al, Int J Radiat Biol, 2009; 85:920-2) Importantly, the recruitment of the host's immune system as a contributor of the "in field" response to radiotherapy can result in immune memory, an advantageous systemic effect that transcends the localized nature of this treatment modality. (Formenti, et al, Lancet Oncol, 2009; 10:718-26)

[0092] Immunogenic cell death is a form of tumor cell death that articulates the effects of ionizing radiation on tumor cells and its impact on host tumor-immunity involved in not only local tumor control but abscopal, as well. (Postow, et al, The New England Journal of Medicine, 2012; 366: 925-31) In the present in vitro model, the data demonstrate that clinically relevant doses of ionizing radiation alone effectively induce the signals for each arm of immunogenic cell death in a dose dependent manner (Figures 1-3). While cancer radiotherapy is associated with pro-immunogenic effects, it also elicits immunosuppressive signaling. (Formenti, et al, Journal of the National Cancer Institute, 2013; 105: 256-65) However, combinations with immune checkpoint inhibitors (such as anti-CTLA4 or anti-PDl) or chemotherapies are likely to overcome these barriers as demonstrated preclinically and in anedoctal clinical cases. (Dewan, et al, Clinical Cancer Research: An Official Journal of the American Associate for Cancer

Research, 2009; 15: 5379-88; Demaria, et al, International Journal of Radiation Oncology, Biology, Physics, 2004; 58: 862-70; Postow, et al, The New England Journal of Medicine, 2012; 366: 925-31; Sullivan, et al, The New England Journal of Medicine, 2013; 369: 173-83) [0093] Ionizing radiation may be combined with selective chemotherapeutic agents capable of enhancing ionizing radiation associated immunogenic cell death. (Formenti, et al, The Lancet Oncology, 2009; 10: 718-26) In the present in vitro model, the hallmark features of immunogenic cell death were examined upon exposing TSA cells to various combinations of platinums with ionizing radiation as a means of selecting the best combinations capable of eliciting

immunogenic cell death. The data demonstrate that platinums differentially exert their repositioning effects on ionizing radiation. Table 1 demonstrates the immunogenic signature of platinum and radiation treatment in this in vitro model. When combined with ionizing radiation, oxaliplatin > carboplatin enhanced the immunogenic signature of TSA cells. These compounds are lead candidates for further testing in in vivo experiments with TSA tumors.

[0094] Cisplatin was less effective at enhancing ionizing radiation induced immunogenic cell death. Cisplatin was less potent than oxaliplatin and carboplatin in TSA cells. High dose cisplatin is required to enhance the therapeutic efficacy of radiation therapy in the clinic, and low dose cisplatin has been shown to be immunosuppressive in mouse models. (Formenti, et al, Journal of Clinical Oncology: Official Jouranal of the American Society of Clinical Oncology, 2008; 26: 1562-3) Oxaliplatin was most effective at enhancing ionizing radiation induced immunogenic cell death. This may partially be explained by the fact that oxaliplatin is more cytotoxic than cisplatin in TSA cells. (Sharma, et al, Journal of Molecular Biology, 2007; 373 : 1123-40).

[0095] An optimized repertoire of immunogenic signals from dying tumor cells may facilitate host anti-tumor immune responses. The present immunogenic cell death assay provides a fast preliminary method to determine the immunogenic signature of various radiation regimens. This assay provides a quick and rational method for selecting potentially immunogenic radiation therapy-based regimens for pre-clinical studies.

[0096] Table 1. immunogenic cell death signature in platinum and IR treated cells

[0097] The relative amount of immunogenic cell death is displayed in Table 1 from the lowest to the highest amount (left to right) in a corresponding color gradient (light to dark) (top panel). The immunogenic cell death signature of ionizing radiation with platinum is also displayed, using the same color gradient for quick visual analysis (bottom panel). The percent colonies formed was determined in untreated or TSA cells treated with 1 μΜ platinum ± ionizing radiation 2 Gy and standardized to the percent colonies formed in untreated cells. The amount of ATP release was determined in untreated or pMe-Luciferease stably expressing TSA cells treated with 5 μΜ platinum ± IR 20 Gy, where the RLUs were standardized to the untreated cells. The amount of CRT translocation was determined in untreated or stably expressing HaloTag-CRT-KDEL TSA cells treated with 500 μΜ platinum ± ionizing radiation 10 Gy, where the MFIs were

standardized to the untreated cells. The amount of HMGB1 release was determined in untreated or stably expressing HMGB1-RFP TSA cells treated with 20 μΜ platinum ± ionizing radiation 2 Gy, where the RFUs were standardized to the untreated cells.

[0098] The results of a multicenter trial of neoadjuvant paclitaxel and radiotherapy given concurrently to treat locally advanced breast cancer patients were previously reported. A 34% pathologic complete response rate, measured by histological examination of the breast and resected lymph nodes from the surgical specimens after mastectomy, was demonstrated in a cohort of 105 locally advanced breast cancer patients. Formenti, et al, J Clin Oncol, 2003; 21 : 864-70) Since pathological response to chemo-radiation was significantly associated with 5 year survival probability, ICD may at least in part contribute to the successful outcomes seen in these patients. Likewise, ICD may partially contribute to the success of concurrent platinum-based chemo-radiation regimens, as witnessed in numerous clinical trials. (Formenti, et al, J Clin Oncol, 2008; 26: 1562-3, author reply 1563) Therefore, IR in combination with platinums or paclitaxel was tested to mimic these clinical conditions and test mechanistically whether the single versus combined therapies manifest differences in ICD. The in vitro assay described herein confirmed enhanced features of ICD when TSA cells were treated concurrently with IR and chemotherapy.

[0099] The hallmark features of ICD were demonstrated upon exposing TSA cells to various combinations of IR with platinums or paclitaxel. This demonstrates that IR in a therapeutic dose- range effectively induces ICD. In addition, when combined with drugs commonly used in the clinic (platinums and taxanes), IR stimulates an optimized repertoire of pro-immunogenic signals from dying tumor cells that may facilitate host anticancer immune responses and may explain some enhanced local and systemic clinical benefits of combinatorial regimens. The in vitro assays described herein provide a convenient screening tool for elucidating optimal therapeutic combinations suitable for clinical testing and therapy.

EXAMPLES

[0100] The following examples are set forth to provide those of ordinary skill in the art with a description of how to make and use the methods and compositions of the invention, and are not intended to limit the scope thereof. Efforts have been made to insure accuracy of numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

EXAMPLE 1

Materials and Methods

[0101] Reagents and materials: Halotag ^® Rl lODirect™ cell permeable ligand, Halotag ^® alexa fluor 488 cell impermeable ligand, and firefly luciferin potassium salt were purchased from Promega (Fitchburg, WI). Oxaliplatin, Cisplatin, and Carboplatin were purchased from Sigma- Aldrich (St. Louis, MO) and dissolved in DMSO at 10 mM, 100 mM, and 25 mM concentrations, respectively.

[0102] Cell culturing: TSA cells were propagated in DMEM GlutaMAX™ (Gibco, Carlsbad, CA) containing 4.5 g/L D-glucose and supplemented with 10% (v/v) fetal bovine serum

(HyClone Laboratories, Logan, UT). Prior to transfection or experimentation, TSA cells tested negative for Mycoplasma (Cellshipper Mycoplasma test; Bionique, Saranac Lake, NY). All TSA derived clones were propagated and assayed in phenol free DMEM supplemented with 10% (v/v) fetal bovine serum (HyClone Laboratories, Logan, UT), 4.5 g/L D-glucose, L-glutamine, and 25 mM HEPEs (Gibco, Carlsbad, CA). Additionally, all TSA derived clones were selected and maintained in 400 μg/ml G418 (Gibco, Carlsbad, CA). All cell lines were grown in a humidified incubator (ThermoScientific, Asheville, NC) at 37°C and a 5% C0 ₂ atmosphere. [0103] Radiation treatment: Cells were exposed to ionizing radiation with a Varian 2300 C/D linear accelerator (Varian Medical Systems, Palo Alto, CA) on a bolus (1.5 cm tissue equivalent material), at 6 MV energy, and a 600 cGy/minute dose rate.

[0104] Colony-formation assay: TSA cells were seeded into 6-well plates at 200 cells per well with DMEM GlutaMAX™ (Gibco, Carlsbad, CA) supplemented with 10% (v/v) fetal bovine serum (HyClone Laboratories, Logan, UT). After complete cell adherence, TSA cells were exposed to ionizing radiation with or without 50 nM paclitaxel in replicates of 6. After 48 hours, the medium was removed, fresh growth medium was added, and the cells were kept in culture undisturbed for 10-12 days, during which time the surviving cells spawned colonies. Formed colonies were fixed with 70% ethanol for 10 minutes and subsequently washed with PBS.

Colonies with > 50 cells were visualized and counted after staining for 20 minutes with 0.1% crystal violet (Sigma-Aldrich, St. Louis, MO) dissolved in distilled water.

[0105] Establishment of a TSA derived clone that stably expresses a plasma membrane targeted luciferase: For pericellular ATP detection, TSA cells were transfected using TurboFectin 8.0 (Origene, Rockville, MD) with a pGEN2.1 CMV expression vector (GenScript, Piscataway, NJ) containing an engineered firefly luciferase-folate receptor chimeric construct (S3) (GenScript, Piscataway, NJ), in accordance to the manufacturer's instructions. Stable clones were selected by means of G418. Pericellular ATP detection was determined by measuring light production on a Lumistar Galaxy luminometer (BMG LABTECH GmbH, Ortenburg, Germany) from the enzymatic reaction of the plasma membrane luciferase in the presence of luciferin substrate solution composed of 3 mM D-luciferin (Sigma-Aldrich, St Louis, MO), 15 mM MgS0 ₄ , (Sigma-Aldrich, St. Louis, MO), and 30 mM HEPES buffer (Gibco, Carlsbad, CA).

[0106] Establishment of a TSA derived clone that stably expresses a HaloTag-CRT-KDEL chimeric protien: For CRT analysis, Turbofectin 8.0 (Origene, Rockville, MD) was used to transfect TSA cells with a pEZ-M02 expression vector containing the Mus musculus CRT- HaloTag-KDEL fusion construct (SI) (GeneCopoeia, Rockville, MD) following the

manufacturer's instructions. Stable clones were selected by means of G418 selection (Gibco, Carlsbad, CA). To validate and detect CRT-HaloTag-KDEL endoplasmic reticulum localization, cloned cells were incubated overnight with 5 μΜ Rl lODirect™ ligand (Promega, Fitchburg, WI), diluted in phenol free medium, according to the manufacturers instructions. CRT-HaloTag- KDEL bound Rl lODirect™ ligand, a small molecule that crosses the cell membrane and labels total HaloTag ^® fusion protein, was visualized on an EVOS fl microscope (Advanced Microscopy Group, Bothell, WA) at _εχ λ470 nm/ _en A525 nm. Positive clones were chosen for subsequent experiments. To visualize CRT-HaloTag-KDEL membranous localization, cloned cells were incubated with the 5 μΜ of the cell membrane impermeable HaloTag ^® alexa fluor 488 ligand (Promega, Fitchburg, WI) for 30 minutes and processed according to the manufacturers instructions. CRT-HaloTag-KDEL bound HaloTag ^® alexa fluor 488 ligand was visualized on an EVOS fl microscope (Advanced Microscopy Group, Bothell, WA) at _εχ λ470 nm/ _en A525 nm.

[0107] Establishment of a TSA derived clone that stably expresses red fluorescence protein (RFP) tagged HMGB 1 : For HMGB l analysis, TSA cells were transfected using TurboFectin 8.0 (Origene, Rockville, MD) with a PrecisionShuttle mammalian pCMV6-AC-RFP expression vector containing a Mus musculus HMGB l construct (S2) and a c-terminal tRFP tag (Origene, Rockville, MD) following the manufacturers instructions. Stable clones were selected by means of G418 selection (Gibco, Carlsbad, CA). To validate and detect HMGB l -RFP nuclear localization, cloned cells were visualized on an EVOS fl microscope (Advanced Microscopy Group, Bothell, WA) at _εχ λ531 nm/ _en A593 nm. Positive clones were chosen for subsequent experiments.

[0108] Luminescence-based detection of pericellular ATP via analysis of pMe-Luciferease expressing TSA cells: TSA cells stably transfected with pGEN2.1-pMe-Luc were seeded in 96- well plates, at a density of 2 x 10 ⁴ cells per well in 50 iL of DMEM phenol free medium. The following day, cells were unexposed or exposed to ionizing radiation and were treated with or without drug, to a total of 100 of medium per well, and allowed to incubate for 24 hours. ATP dependent luminescence was detected on a LUMIstar Galaxy microplate luminometer (BMG LAB TECH GmbH, Ortenberg, Germany) upon the addition of 100 of luciferin substrate solution containing PBS dissolved D-luciferin, HEPEs buffer (Gibco, Carlsbad, CA), and MgS0 ₄ (Sigma-Aldrich, St. Louis, MS). Luminescence was detected for 10s at a gain of 127 immediately after addition of luciferin substrate solution. The luminescence detected upon analysis of untreated cells was normalized to 1 for all experiments and the relative luminescence units for experimental groups are shown. [0109] Cytofluorimetric detection of cell surface CRT via analysis of CRT-HaloTag- KDEL expressing TSA cells: TSA cells stably transfected with CRT-HaloTag-KDEL were seeded in 6- well plates, at a density of 2.5 x 10 ⁵ cells per well. The following day cells were treated and allowed to incubate for 24 hours. CRT cell surface expression was detected with the

impermeable Halotag® Alexa488 according to the manufacturer's instructions. Transfected cells (10 ⁴ cells per treatment) were analyzed on the FACSCalibur (BD Biosciences, San Diego, CA, USA) with CellQuest software (Becton Dickinson, Oakville, Ontario, Canada).

[0110] Detection of extracellular HMGB1 from stably expressing HMGBl-RFP TSA cells: Stably transfected HMGBl-RFP cells were seeded in replicates in 6-well plates, at a density of 1 x 10 ⁵ cells per well. The following day, cells were treated and allowed to incubate for the indicated length of times (24, 48, or 72 hours). Conditioned media was collected, floating cells were removed via centrifugation, and released HMGBl-RFP was detected on the Flexstation 3 spectrophotometer (Molecular Devices, Sunnyvale, CA, USA) at excitation λ530 nM and emission λ593 nM. Fluorescence signal detected in the medium of untreated cells was normalized to 1 for all experiments and relative fluorescence units for experimental groups are shown.

[0111] Statistical analysis: The standard deviations are listed as ± values for each data point. For the analysis of the experimental data, data comparison was analyzed using a paired Student's t- test. P-values <0.05 were considered statistically significant.

Results

[0112[ Radiotherapy promotes ATP release: To rapidly quantify radiation-induced ATP release, TSA cells were transfected with a pGEN2.1 plasmid encoding firefly luciferase bound to the plasma membrane by virtue of a flanking folate receptor (FR) leader sequence and a

glycophosphatidylinositol (GPI) anchor sequence, as previously described by Francesco di Virgilio (Fig. 1A, top panel). (Pellegatti, et al, Mol Biol Cell, 2005; 16: 3659-65; Pellegatti, et al, PLoS One, 2008; 3 :e2599; Michaud, et al, Science, 2011; 334: 1573-7) ATP is an

intracellular molecule under homeostatic conditions in treatment-naive TSA cells. However, ATP is readily released from stressed cells undergoing ICD. (Golden, et al, Front Oncol, 2012; 2:88; Michaud, et al, Science, 2011; 334: 1573-7) In the present in vitro model, pericellular ATP, in the presence of oxygen, magnesium, and D-luciferin substrate, reacts with the extracellular localized, plasma membrane-bound luciferase (pMe-Luc) to produce photons, readily detectable and quantified with a luminometer, thereby serving as a surrogate for the presence of pericellular ATP (Fig. 1 A, bottom panel).

[0113] The amount of luminescence detected and reported as fold change in relative luminescent units (RLUs) after 24 hours of exposure to increasing doses of ionizing radiation (IR) is shown (Fig. IB). Aa radiation dose-dependent increase in RLUs from pGEN2.1-pMe-Luc transfected TSA cells exposed to IR reflecting higher levels of ATP in the ECM of irradiated cells was observed. Specifically, irradiation with 0, 2, 5, 10, and 20 Gray (Gy) induced luciferase-reporter RLUs of 1 ± 0.10, 1.12 ± 0.11, 1.73 ± 0.2, 2.28 ± 0.25, and 2.81 ± 0.38, respectively.

[0114] Radiotherapy promotes CRT translocation: CRT is an ER resident chaperone protein that sequesters calcium and prevents misfolded proteins from leaving the ER. (Michalak, et al, Biochem Cell Biol, 1998; 76:779-85) However, CRT is also well known to serve as an "eat me" signal for DC phagocytosis upon translocation to the surface of dying tumor cells undergoing ICD. (Tesniere, et al, Cell Death Differ, 2008; 15:3-12; Chaput, et al, JMolMed (Berl), 2007; 85: 1069-76; Obeid, et al, NatMed, 2007; 13 :54-61; Zitvogel, et al, Clin Cancer Res, 2010; 16:3100-4) To gain insight into the amount of CRT cell surface translocation among dying TSA cells in response to IR, TSA cells were stably transfected with an engineered DNA construct from the pEZ-M02 vector that stably expresses CRT-HaloTag-KDEL fusion protein, a fluorescence-based reporter of cell surface localized CRT (Fig. 2A, top panel). (Tesniere, et al, Oncogene, 2010; 29:482-91)

[0115] HaloTag ^® is a genetically engineered haloalkane dehalogenase designed to irreversibly bind to synthetic fluorescent ligands. (Obeid, et al, Cell Death Differ, 2007; 14: 1848-50) Under normal (i.e., non-stressed) conditions, the CRT-HaloTag-KDEL fusion protein, akin to native CRT, resides in the ER (Fig. 2A). However, when cells undergo ICD, ER resident CRT, including CRT -HaloTag is translocated to the cell surface, whereby HaloTag ^® Alexa fluor 488, a cell membrane impermeable HaloTag ^® ligand, can selectively label ECM exposed CRT -HaloTag fusion protein (Fig. 2A, bottom panel). (Dewan, et al, Clin Cancer Res, 2009; 15:5379-88) The fluorescent signal from the irreversibly bound ligand can then be detected via flow cytometry, whereby changes in the mean fluorescence intensity (MFI) under various conditions be ascertained. [0116] To confirm the specificity of the impermeable HaloTag Alexa Fluor 488 ligand, untreated cells were exposed to either the 5μΜ impermeable HaloTag ^® Alexa Fluor 488 ligand, or as a control, the cell permeable HaloTag ^® R110 direct ligand, and subject to fluorescence cytometric analysis. This control experiment documented that the system could detect compartmentalized CRT, as untreated TSA cells expressing CRT-Halotag-KDEL labeled with either HaloTag ^® Alexa fluor 488 (baseline control) or HaloTag ^® R110 direct was 1 ± 0.01 and 2.94 ± 0.10, respectively.

[0117] Before testing IR, oxaliplatin was tested as it has previously been shown to cause CRT surface-translocation tumor cells undergoing ICD, as a positive control for the assay. (Martins, et al, Oncogene, 2011; 30: 1147-58) Thus, to confirm the presence of CRT cell surface translocation in our system, CRT-HaloTag-KDEL engineered TSA cells were either untreated or treated with 500 μΜ oxaliplatin for 24 hours. (Martins, et al, Oncogene, 2011; 30: 1147-58) Thereafter, the cells were exposed to 5 μΜ of HaloTag ^® Alexa Fluor 488 ligand and visualized via confocal microscopy. Similarly to previous reports, the oxaliplatin treated cells demonstrated significant fluorescence via confocal microscopy, whereas minimal fluorescence was observed in untreated cells (data not shown). (Martins, et al., Oncogene, 2011; 30: 1147-58) This pilot experiment confirmed that our system could selectively detect cell-surface exposed CRT.

[0118] To quantitatively determine cell surface CRT translocation in response to IR, TSA cells transfected with the CRT-HaloTag-KDEL reporter construct were first evaluated untreated (as a negative control) or treated for 24 hours with 500 μΜ oxaliplatin (as a positive control) or 20 Gy of IR. Transfectants were subsequently stained with the cell membrane impermeable HaloTag ^® Alexa Fluor 488 ligand and subjected to flow cytometry. The MFI fold change in the MFI of untreated cells relative to cells treated for 24 hours with 500 μΜ oxaliplatin or 20 Gy IR increased from 1.17 ± 0.01 to 2.36 ± 0.03 and 2.07 ± 0.04, respectively. Of note, a small MFI fold difference was observed between untreated cells that were unexposed or exposed to 5 μΜ of HaloTag ^® Alexa Fluor 488 ligand, in which case the MFI was 1 ± 0.01 and 1.17 ± 0.01, respectively.

[0119] Interestingly, in a dose-dependent manner IR alone was capable of inducing CRT translocation to the cell surface. The amount of CRT externalization, as indicated by cell surface fluorescence detected via flow cytometry and reported as fold-change in MFI after 24 hours of exposure to increasing doses of IR is shown in Figure 2B and C. The MFI detected from CRT- Halotag transfected TSA cells exposed to IR 0, 2, 5, 10, and 20 Gy was 1 ± 0.01, 1.34 ± 0.01, 1.73 ± 0.02, 2.40 ± 0.03, and 3.18 ± 0.05, respectively. Taken together, these data indicate that the majority of CRT is normally intracellular under homeostatic conditions. However, when cells are exposed to increasing doses of IR, significant amounts of CRT are translocated to the cell surface.

[0120] Radiotherapy promotes HMGB1 release: To rapidly determine the degree to which IR triggers another ICD hallmark, HMGBl release from dying tumor cells, TSA cells were transfected with a different fluorescent reporter, a pCMV6- AN-RFP plasmid encoding a HMGB 1 with a red fluorescent protein (RFP) C-terminal tag (Fig. 3 A, top panel, refer to Fig. S3).

HMGBl has been previously shown to reside in the nucleus under native conditions. (Apetoh et al, Immunol Rev, "The interaction between HMGBl and TLR4 dictates the outcome of anticancer chemotherapy and radiotherapy", 2007; 220: 47-59) Thus, in HMGBl -RFP

transfected TSA cells, we anticipated the presence of fused HMGBl -RFP protein in the nucleus of untreated cells (Fig. 3 A, bottom panel), a subcellular localization verified via confocal microscopy (Fig. 3B). Conversely, released HMGBl -RFP protein was anticipated in the conditioned medium of cells exposed to IR (Fig. 3 A, bottom panel) validated via fluorescence cytometry ( _εχ λ ₅ 3ο nM and enAs93 nm) and reported as fold-change in relative fluorescent units (RFUs) in comparison to untreated controls (Fig. 3C). After 72 h of exposure to increasing IR doses, increasing fluorescence was detected in the conditioned media of irradiated cells (relative to untreated controls) reflecting higher levels of HMGBl -RFP release. Specifically, irradiation with 0, 2, 5, 10, and 20 Gy resulted in RFUs of 1 ± 0.01, 1.16 ± 0.02, 1.56 ± 0.02, 2.20 ± 0.03, and 2.33 ± 0.02, respectively.

[0121] Platinum and radiotherapy enhance TSA cytotoxicity: The combined effects of platinums and radiation on markers of ICD were addressed. To this end, oxaliplatin (a known inducer of ICD) and carboplatin, both drugs that are currently used in the clinic, were selected. Due to cell line specific pharmacodynamic differences, the cytotoxic profiles of the platinum compounds on TSA cells were investigated. [0122] To ascertain the short term cytotoxic effects of the platinum compounds, TSA cells were treated with the platinums for 48 hours and cell viability was determined via a methylthiazolyldiphenyl-tetrazolium bromide (MTT) colorimetric assay. (Mosmann, J Immunol Methods, 1983; 65:55-63; Alley, et al, Cancer Res, 1988; 48:589-601) MTT powder is a yellow water-soluble tetrazolium salt that is catalyzed to a purple formazan chromogen in the presence of dehydrogenases and reductases. This reaction readily occurs in living cells, whereby the absorbance of this purple solution directly corresponds to cell viability and can be measured with a spectrophotometer at λ ₄₉₀ nm. Utilizing this cell viability assay, 100 μΜ of oxaliplatin or carboplatin was found to reduce the percent cell viability (relative to untreated controls) from 100 ± 10.73% to 16.19 ± 0.99% and 10.16 ± 1.21%, respectively.

[0123] The persistent growth-inhibiting effects of platinum therapy on TSA cells following 48hours of treatment by colony forming assay was investigated to further assess the long-term survival (2 week) of individual cells and their ability to spawn clonal descendants following treatment. (Henriksson, et al, In Vitro Cell Dev Biol Anim, "2006; 42:320-3) In cells treated with 1 μΜ of oxaliplatin or carboplatin, the percent of colonies formed after 2 week incubation was reduced (relative to the untreated control) from 100 ± 2.63% to 63.49 ± 6.69% and 71.75 ± 4.79%), respectively (Fig. 4A and B). Taken together, these results indicate that TSA cells are comparably sensitive to oxaliplatin and carboplatin as measured by both the short-term MTT viability assay and the long-term clonal growth assay.

[0124] Since IR therapeutic activity on solid tumors is a cumulative effect of cancer cell death occurring over several rounds of cell division following irradiation, the combined growth- inhibiting effects of platinum and radiotherapy was determined via the long-term colony forming assay, the gold- standard for determining radiation induced cell death. (Golden, et al, Front Oncol, 2012; 2:88; Henriksson, et al, In Vitro Cell Dev Biol Anim, 2006; 42:320-3; Puck, et al, J Exp Med, 1956; 103 : 653-66) In TSA cells the fraction of surviving (SF) colonies after IR 2 Gy (SF ₂ , a measurement of radio sensitivity) was reduced from 100 ± 2.63%> in the control unexposed cells, to 58.41 ± 5.74% (Fig. 4A and B). Of particular interest, the SF ₂ in TSA cells was further reduced when either platinum was added. For instance, when TSA cells were irradiated with 2 Gy and exposed to 1 μΜ of oxaliplatin or carboplatin for 48 hours, the percent of colonies formed was further reduced from 58.41 ± 5.74% to 29.84 ± 2.90% and 24.76 ± 10.08%, respectively (Fig. 4A and B). Collectively, these results indicate that the combination of IR and platinum have an enhanced cytotoxic effect on TSA cells.

[0125] Platinum and radiotherapy induce ATP release from dying tumor cells: To address the degree to which chemotherapy and radiotherapy combined to elicit the ICD hallmark of ATP release, the amount of luminescence from the extracellular anchored luciferase reporter (pMe- Luc) is shown in Figure 5A after 24 hours of exposure to increasing doses of oxaliplatin, a known inducer of ATP release. (Martins, et al, Cell Cycle, 2009; 8:3723-8) pMe-Luc expressing TSA cells exposed to 1, 5, and 10 μΜ oxaliplatin, resulted in an RLU fold increase (relative to untreated control levels) from 1 ± 0.09 to 1.27 ± 0.12, 1.87 ± 0.19, and 1.56 ± 0.18, respectively.

[0126] Interestingly, ATP release was observedfrom cells exposed to IR when combined with oxaliplatin and parity of ATP release when IR was combined with carboplatin (Fig. 5B). For example, untreated vs. either 5 μΜ oxaliplatin or carboplatin treated cells resulted in an RLU fold increase from 1 ± 0.08 to 1.43 ± 0.15 or 1.05 ± 0.15 respectively. Additionally, untreated or IR 20 Gy treated cells resulted in an RLU fold increase from 1 ± 0.08 to 2.61 ± 0.30. However, IR 20 Gy combined with 5 μΜ oxaliplatin significantly stimulated ATP release, as measured by a fold-change in RLU to 4.49 ± 0.68 (P value < 0.001). In contrast, carboplatin treatment, only modestly increased ATP release as shown by an insignificant RLU fold change of 2.47 ± 0.36. In summary, these results indicate that, at the doses of radiation tested IR-induced ATP-release is enhanced by oxaliplatin and conserved in the presence of carboplatin.

[0127] Platinum and radiotherapy cause CRT translocation in dying tumor cells: In order to delineate whether CRT translocation is platinum dose-dependent, TSA CRT-HaloTag-KDEL cells were treated with increasing doses of platinum and assayed 24h later. Interestingly, the degree of CRT translocation was dose-dependent in response to oxaliplatin, a known inducer of CRT translocation, whereas CRT translocation was not dose-dependent in response to

carboplatin treatment. For example, the fold-change in MFI detected on CRT-HaloTag-KDEL TSA cells treated with ΙΟΟμΜ oxaliplatin or carboplatin increased to 1.60 ± 0.04 and 1.47 ± 0.03, respectively from untreated controls levels of 1 ± 0.02. Additionally, the MFI fold-change in cells treated with 500 μΜ oxaliplatin further increased to 2.36 ± 0.03, while the MFI fold- change in cells treated with 500 μΜ carboplatin remained relatively stable at 1.23 ± 0.03

(Fig. 6A and B). [0128] To determine whether concurrent platinum and radiotherapy could synergize to enhance CRT translocation in mammary carcinoma cells, CRT-HaloTag-KDEL transfected TSA cells were treated with various platinum and radiotherapy regimens. Upon the addition of IR, the amount of CRT translocation induced by the platinum agents remained elevated, but did not appear to significantly increase further. For instance, the MFI fold change in untreated vs. IR 10 Gy treated cells increased from 1 ± 0.02 to 2.34 ± 0.06. However, when IR 10 Gy was added to 100 μΜ of oxaliplatin or carboplatin the MFI fold change was 1.95 ± 0.07 and 1.89 ± 0.08, respectively, values only marginally higher than the chemotherapeutic agent alone (Fig. 6A and B). Additionally, when IR 10 Gy was added to 500 μΜ of oxaliplatin or carboplatin the MFI fold change was 2.48 ± 0.17 and 1.75 ± 0.05, respectively, lower or nearly equivalent levels to the same dosage of IR alone and similar to that of the platinum agent only (Fig. 6A and B). In summary, radiotherapy and oxaliplatin monotherapy induced CRT translocation at the dosages tested in a dose-dependent manner. IR did not further enhance platinum-induced CRT translocation, such that upon the addition of IR, the amount of CRT translocation in platinum treated cells remained relatively stable, albeit elevated.

[0129] Platinum and radiotherapy cause HMGB1 release from dying tumor cells: Considering that the kinetics of the reaction could impact the magnitude of the measured response, the ideal timing of HMGB1 release in IR or platinum exposed tumor cells was investigated. To

accomplish this, the RFP -tagged HMGB1 fusion protein was used to detect HMGB1 release into the surrounding media of dying cancer cells after treatment with IR or oxaliplatin, a known inducer of HMGB1 release, in a time and dose-dependent manner (Fig. 7A). (Tesniere, et al, Oncogene, 2010; 29:482-91) The RFUs detected in the conditioned media of untreated controls barely changed over the time course of 24, 48, and 72 hours, from 1 ± 0.16 to 1.16 ± 0.05, and 1.23 ± 0.04 fold respectively. Fluorescence from HMGB1-RFP TSA cells treated with 10 μΜ oxaliplatin similarly minimally changed over 24, 48, and 72 hours, exhibiting slight increases in RFUs from 1.06 ± 0.03 to 1.37 ± 0.03, and 1.49 ± 0.02-fold, respectively, whereas cells treated with 100 μΜ oxaliplatin and incubated for 24, 48, and 72 h following treatment, the RFUs increased more substantially from 1 ± 0.16 in the controls to 1.51 ± 0.05, 2.49 ± 0.04, and 2.76 ± 0.21 fold, respectively. Similarly, HMGB1-RFP TSA cells treated with IR 2 Gy for 24, 48, and 72 hours the fold change in RFUs increased from 1 ± 0.03 (untreated control) to 1.11 ± 0.04, 1.15 ± 0.03, and 1.5 ± 0.17, respectively, whereas cells treated with higher IR at 20 Gy for 24, 48, and 72 h exhibited a more robust increase in the RFUs at the later time points, from 1 ± 0.16 (untreated control) to 1.18 ± 0.05, 1.77 ± 0.03, and 2.95 ± 0.06-fold, respectively. In summary, both IR and oxaliplatin optimally induced HMGB 1 release 72 h after treatment.

[0130] Whether other platinums could mediate HMGB l release was investigated along with whether combinatorial therapy with IR might further enhance liberation of HMGBl . To this end, HMGBl -RFP transfected cells were treated with oxaliplatin or carboplatin and assayed for HMGBl release into the conditioned media 72 hours later. As shown in Figure 7B, in TSA cells treated with 20 μΜ oxaliplatin or carboplatin, the fold change in RFUs increased from 1 ± 0.07 (untreated cells) to 1.44 ± 0.09 and 1.23 ± 0.08, respectively. Treatment with IR 2 Gy alone only affected a minor increase in RFUs from 1 ± 0.07 to 1.18 ± 0.10. However, when IR 2 Gy was added to oxaliplatin and carboplatin, enhanced HMGBl release was observed as reflected by an increase in RFUs detected from 1.18 ± 0.1 (IR alone) to 1.62 ± 0.13 and 1.51 ± 0.09 in cells treated in combination with 20 μΜ oxaliplatin or carboplatin, respectively. Taken together, these results indicate that IR, oxaliplatin, and carboplatin could, in a dose-dependent fashion significantly increase HMGBl release 72 hours after treatment in the mammary carcinoma model. Moreover, IR combined with oxaliplatin or carboplatin may potentially enhance HMGB 1 release from dying TSA cells.

[0131] Paclitaxel and radiotherapy elicit features of ICD from dying tumor cells: Whether concurrent IR and paclitaxel were capable of eliciting features of ICD from dying tumor cells and determine whether the combined regimen could enhance TSA therapeutic cytotoxicity was investigated. For example, the clonal growth measure of radio sensitivity, SF ₂ in TSA cells was reduced from 100 ± 6.03% to 69.57 ± 3.33% in response to IR alone (Fig. 8A). Nevertheless, the SF ₂ in TSA cells was further reduced when paclitaxel was added. For instance, when TSA cells were irradiated with 2 Gy and exposed to 50 nM of paclitaxel for 48 hours, the percent of colonies formed was reduced further from 69.57 ± 3.33% to 31.36 ± 3.07% (Fig. 8A). Of note, paclitaxel alone reduced the surviving fraction to 59.67 ± 3.42%. Hence, these results indicate that IR and paclitaxel enhance the cytotoxic effects on TSA cells.

[0132] To determine whether IR and paclitaxel could intensify hallmarks of ICD, the combined treatment regimen in an in vitro breast cancer model was used. Accordingly, the amount ATP release from dying pMe-Luc expressing TSA cells treated with the combined regimen and observed enhanced ATP release, as measured by increased luciferase activity (Fig. 8B) were determined. For example, 1 μΜ paclitaxel treatment versus untreated cells did not alter luciferase luminescence, as shown by a RLU fold-change from 1 ± 0.06 to 0.98 ± 0.02. IR 2 Gy treated cells exhibited a modest RLU fold increase from 1 ± 0.08 to 1.63 ± 0.07. However, IR 2 Gy plus 1 μΜ paclitaxel stimulated the highest RLU fold-change to 1.95 ± 0.05, indicating that IR and paclitaxel enhanced the release of ATP. The amount of CRT translocation to the cell surface was quantified using the CRT-HaloTag-KDEL transfected TSA cells. The combination of IR and paclitaxel enhanced CRT externalization. For instance, 100 nM paclitaxel only treated cells exhibited a MFI increase from 1 ± 0.01 (untreated cells) to 2.06 ± 0.02-fold and IR 10 Gy only affected a MFI fold-increase to 1.98 ± 0.07. However, the combination exerted an enhanced effect, displaying a MFI fold-increase to 2.36 ± 0.02. Finally, using the HMGBl-RFP transfected TSA cells, an enhanced effect was observed with the combinatorial regimen of paclitaxel and IR with respect to HMGB1 release. For example, 1 μΜ paclitaxel caused a MFI fold-change from 1 ± 0.025 (untreated cells) to 1.73 ± 0.07, whereas, IR 2 Gy barely altered the RFUs detected to 1.03 ± 0.03. Nevertheless, the combined regimen markedly increased the RFUs to 2 ± 0.10-fold control levels, reflecting higher levels of HMGB1 release. Taken together, these results indicate that paclitaxel and IR combinatorial therapies have the potential to enhance each arm of ICD.

EXAMPLE 2

Background

[0133] Multiple prospective randomized trials demonstrate a clear advantage for concurrent radiation (RT) and chemotherapeutic regimens in solid tumors, as opposed to a sequential approach. The present data demonstrate that the intensity of immunogenic cell death (ICD) is greater when RT and chemotherapy are combined thereby inciting a host anti-tumor immune response that contributes to the observed clinical results.

[0134] Three distinct components (calreticulin [CRT] cell surface translocation and HMGB1 and ATP release) are required to induce immunogenic cell death (ICD) in dying tumor cells. The net effects intensify dendritic cell (DC) phagocytosis of tumor cells, dendritic cell processing of tumor-derived antigens, and dendritic cell-associated cross-priming of CD8 ⁺ cytotoxic T lymphocytes (Ma, et al, Seminars in Immunology, 2010; 22: 113-24). [0135] The methods described herein were developed to examine the hallmark features of immunogenic cell death (ICD) with radiation therapy (RT)-based regimens.

Materials/Methods

[0136] TSA (a Balb/c syngeneic murine mammary cancer) derived clones that stably express a plasma membrane targeted luciferase (pMe-Luc), a HaloTag-CRT-KDEL chimeric protein, or a red fluorescence protein tagged HMGB1 chimeric protein were established. Their respective pericellular detected relative luminescent units (RLUs, via luminometry), cell surface mean fluorescent intensity (MFI, via flow cytometry) and media detected relative fluorescent units (RFUs, via fluoremetry) were used as surrogates for ATP release, CRT translocation, and HMGB1 release.

Results

[0137] The assay demonstrated a radiation treatment dose-dependent increase in immunogenic cell death. pMeLuc TSA Cells were exposed to radiation treatment 0, 2, 5, 10, and 20 Gy and pericellular ATP was detected 24 hours later and reported as RLU fold change. HaloTag-CRT- KDEL TSA cells were treated with radiation treatment 0, 2, 5, 10, and 20 Gy, and cell surface CRT was detected 24 hours later and reported as MFI fold change. Finally HMGBl-RFP TSA cells were treated with radiation treatment 0, 2, 5, 10, and 20 Gy and HMGBl-RFP was detected in the media 72 hours later and reported as RFU fold change.

[0138] A radiation treatment dose-dependent increase in immunogenic cell death was observed (Table 2).

[0139] Table 2.

[0140] The assay demonstrated that platinum (Pt) agents differentially enhance the immunogenic signature of radiation treatment. [0141] The presently described methods were used to determine the immunogenic signature of radiation treatment and Pt agents (oxaliplatin, cisplatin, and carboplatin). TSA cells were treated with 1 μΜ Pt (48 hrs) and 2 Gy radiation treatment and TSA colony forming capacity was determined 2 weeks later and reported as percent colonies formed. pMe-Luc TSA cells were treated with 5 μΜ Pt and 20 Gy radiation treatment, and pericellular ATP was detected 24 hours later and reported as RLU fold change. HaloTag-CRT-KDEL TSA cells were treated with 500 μΜ Pt and 10 Gy radiation treatment and cell surface CRT was detected 24 hours later and reported as MFI fold change. HMGB1-RFP TSA cells were treated with 20 μΜ Pt and 2 Gy radiation treatment and HMGB 1 in the media was detected 72 hours later and reported as RFU fold change.

[0142] Platinum agents differentially enhance the immunogenic signature of radiation treatment (Table 3).

[0143] Table 3.

Conclusions

[0144] The assay described herein demonstrated a radiation treatment dose-dependent induction of immunogenic cell death. Additionally, the assay demonstrated that platinum agents differentially enhance the immunogenic signature of radiation treatment in TSA cells.

Oxaliplatin and carboplatin were most effective, but, cisplatin was less so. The assay described herein is capable of testing a wide range of radiation treatment -based regimens, including those combined with platinums, topoisomerase inhibitors, taxanes, vinca alkaloids, and novel targeted agents. The assay can also be used in cell lines other than TSA cells, including proprietary, commercial, and primary cell lines.