MAGNETIC PLATELET PROBES TO DETECT CIRCULATING

TUMOR CELLS

TECHNICAL FIELD

[0001] This disclosure relates to the detection of circulating tumor cells (CTCs) and the diagnosis and treatment of cancer and cancer metastases.

BACKGROUND OF THE INVENTION

[0002] CTC detection techniques can be used in the clinical setting. For example,

detection of high CTC levels or increased CTC enumeration in a biopsy of a cancer patient can be used to adjust the treatment of patients. The STIC CTC METABREAST clinical trial (NCT01710605) is evaluating if CTC enumeration can help determine the first line treatment for metastatic HER2 positive breast cancer patients.

[0003] The CellSearch® system (Veridex, Raritan, NJ, USA) relates to CTC detection in liquid biopsies of patients with metastatic breast, prostate or colorectal cancer. This system is based on a magnetic bead-conjugated antibody that targets CTCs in collected blood specimens through binding to EpCAM (Epithelial Cell Adhesion Molecule). The system is sensitive for tumor cells of epithelial origin and consequently the epithelial-to- mesenchymal transition seen in the process of metastatic spread makes difficult, if not impossible, the detection of subgroups of aggressive and invasive tumor cells.

[0004] Jiang et al. ( Lab Chip , 2017, 17:3498) have disclosed a microfluidic chip with immobilized antibody that targets platelet-covered CTCs. For this system, free platelets (which in the blood stream represent 150,000 to 350,000 platelet/uL blood) must be isolated from the sample so that they do not saturate the surface of antibody. In this system, a microfluidic platform is used first to purify the sample of platelets and red cells before perfusing the sample in a second microfluidic device to capture the platelet- covered CTCs. Jiang et al. also described that CTC adhesion on an EpCAM-coated microfluidic chip was significantly lower than their arrest on a chip with immobilized antibody that targets platelet-covered CTCs. This effect was described as being potentially mediated by the platelet coating shielding EpCAM on the surface of CTC.

[0005] Int'l Pub. No. WO 2015109220 Al and Papa et al. ( Sci . Trans. Med., 2019, 11,

479, eaau5898) discloses platelet decoys that bind to platelet receptor substrates but do not undergo platelet activation. Methods of using these decoys for treating, preventing or inhibiting a disease or disorder in a subject are also disclosed.

[0006] U.S. Appl. Pub. No. 2017/0225166 A1 discloses methods and systems for

isolating platelet-associated nucleated target cells, e.g ., such as circulating epithelial cells, CTCs, circulating endothelial cells (CECs), circulating stem cells (CSCs), neutrophils, and macrophages, from sample fluids, e.g. , biological fluids, such as blood, bone marrow, plural effusions, and ascites fluid.

[0007] In addition, the Massachusetts General Hospital has developed a CD41

microfluidic chip that captures platelet-covered CTCs via the CD41 antibody specific for platelets. The CD41 antibody is immobilized on the chip surface.

BRIEF SUMMARY OF THE INVENTION

[0008] In one aspect, the present disclosure provides a composition comprising a

magnetic probe and a platelet cell, resulting in a functionalized platelet magnetic probe. The platelet supporting the magnetic entity (e.g., nanoparticles or magnetic complexes) can be a platelet cell, a freeze-dried platelet, a modified platelet (e.g., a platelet cell that is substantially free of at least one membrane lipid or molecular component present in an unmodified platelet cell) or a synthetic platelet (e.g. an assembly of platelet receptors in membrane mimicking liposomes).

[0009] In another aspect, the present disclosure provides a method of detecting a binding event, wherein the binding event is the interaction of a mammalian circulating tumor cell with a modified platelet cell, comprising: (a) adding platelet cells containing a magnetic probe to a fluid comprising mammalian circulating tumor cells; (b) applying a magnetic field to concentrate the platelet cells containing a magnetic probe; (c) removing the non magnetic supernatant; (d) collecting the platelet cells containing a magnetic probe and any mammalian circulating tumor cells bound to the platelet cells; (e) detecting the presence of mammalian circulating tumor cells in the collected fluid; and (f) integrating the platelet magnetic probe into a device capable of clearing CTCs from patient circulating blood.

[0010] In another aspect, the present disclosure provides a method for diagnosing cancer and/or cancer metastasis, comprising: (a) adding platelet cells containing a magnetic probe to a blood sample of a patient; (b) applying a magnetic field to concentrate the platelet cells containing a magnetic probe; (c) removing the non-magnetic portion of the sample; (d) collecting the platelet cells containing a magnetic probe and any circulating tumor cells bound to the modified platelet cells; (e) detecting the presence of circulating tumor cells in the collected fluid; and (f) integrating the platelet magnetic probe into a device capable of clearing CTCs from patient circulating blood.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Fig. 1 is an illustration of a magnetic platelet probe having iron oxide

(nano)particles internalized inside a platelet.

[0012] Fig. 2 is an illustration of a magnetic platelet probe having iron oxide

(nano)particles attached to a platelet via a linker, whether the linker is a covalent bond or a ligand such as IgG bound to the Fc receptor on platelets.

[0013] Fig. 3 is a flow cytometry histogram of EpCAM detection on the surface of MDA-

MB-231 breast cancer cells with or without incubation with platelets.

[0014] Fig. 4 is a flow cytometry histogram of EpCAM detection on the surface of SK-

BR-3 breast cancer cells with or without incubation with platelets.

[0015] Fig. 5 is a confocal image of human platelets (red) binding to human MDA-MB-

231 breast cancer cells (green).

[0016] Fig. 6 is a scatter graph showing the enumeration of platelet magnetic probes by flow cytometry after retrieval of the platelets using a magnetic field. The platelet magnetic probes were made of lyophilized and fixed platelets, 20 nm iron oxide nanoparticles at a final concentration of 0.5 or 1 mg/mL and immunoglobulins.

[0017] Fig. 7 is a series of six graphs showing MDA-MB-231 cancer cell populations analyzed by flow cytometry after being retrieved from cancer cell suspensions: dot plots of cancer cell granularity (SSC) versus size (FSC) as well as cancer cell granularity (SSC) versus Hoechst staining for (A) unstained cancer cells, (B) Hoechst positive cancer cells and (C) cancer cells magnetically retrieved from suspensions with the magnetic platelet probes.

[0018] Fig. 8 is a series of six graphs showing SK-BR-3 cancer cell populations analyzed by flow cytometry after being retrieved from cancer cell suspensions: dot plots of cancer cell granularity (SSC) versus size (FSC) as well as cancer cell granularity (SSC) versus Hoechst staining for (A) unstained cancer cells, (B) Hoechst positive cancer cells and (C) cancer cells magnetically retrieved from suspensions with the magnetic platelet probes.

[0019] Fig. 9 is a Transmission Electron Micrograph of SPIONs (black dots depicted by arrows) physically entrapped within a platelet decoy via incubation/mixing and centrifugation cycles.

[0020] Fig. 10 is a histogram presenting the magnetic retrieval of Hoechst positive MDA-

MB-231 human breast cancer cells utilizing magnetic platelet decoys loaded with 20nm SPIONs, by the method of incubation/mixing and centrifugation cycles. The suspensions of magnetic platelet decoys and cancer cells were incubated under mixing for 1 hour at 37°C and then placed on a magnet for 1 hour. The data plot represents 6 independent experiments with n=3 replicates.

DETAILED DESCRIPTION OF THE INVENTION

[0021] In some embodiments, the present disclosure provides a composition comprising a magnetic probe and a platelet cell, modified platelet or platelet mimicking entity. In some embodiments, the platelet probe is a platelet cell, a freeze-dried platelet, a modified platelet (e.g., a platelet cell that is substantially free of at least one membrane lipid or molecular component present in an unmodified platelet cell) or a synthetic platelet (e.g. an assembly of platelet receptors in liposomes).

[0022] In some embodiments, the magnetic probe is one or more magnetic nanoparticles.

In some embodiments, the magnetic nanoparticles are nickel-based, cobalt-based, iron- based, iron oxide-based nanoparticles or microparticles. In some embodiments, the magnetic probe is made of any magnetic chemical element such as gadolinium, manganese, etc.

[0023] In some embodiments, the magnetic probe is located within the modified platelet cell following endocytosis of the probe into the cell.

[0024] In some embodiments, the magnetic probe is physically forced/loaded into porous platelets (e.g.‘pHtelet decoys’ described in WO 2015109220 A1 and Papa et al. ( Sci . Trans. Med., 2019, 11, 479, eaau5898)) or into lyophilized platelets by incubation under mixing followed by centrifugations.

[0025] In some embodiments, the magnetic probe is physically forced/loaded into a

platelet cell, a freeze-dried platelet, a modified platelet (e.g., platelet decoys) or a synthetic platelet (e.g., nanoparticle containing platelet membranes or receptors) using a magnet as described by Mykhaylyk et al. (. Nature protocols , 2007, 2, 10, 2391) in the case of magnetofection of suspensive cells.

[0026] In some embodiments, the magnetic probe is covalently bound to the surface of the platelet cell.

[0027] In some embodiments, the magnetic probe is bound to the surface of platelets via

Van der Waals interactions and/or hydrogen bonds using a ligand/substrate binding strategy (e.g. Immunoglobulins/Fc receptors binding).

[0028] In some embodiments, the composition further comprises a tumor cell. In some embodiments, the tumor cell is a cancer cell of any origin (i.e. breast, glioblastoma, pancreatic, colorectal, etc.). In some embodiments, the tumor cell is a circulating tumor cell. In some embodiments, the tumor cell is a mammalian cell. In some embodiments, the mammalian cell is a human cell. In some embodiments, the tumor cell is bound to the magnetic platelet probe.

[0029] In some embodiments, the collected fluid is blood. In some embodiments, the collected fluid is a Pap Smear sample. In some embodiments, the collected fluid is cerebrospinal fluid or pleural fluid or ascitic fluid. In some embodiments, the collected sample is from tissue biopsies or bone marrow biopsies.

[0030] In some embodiments, the present disclosure provides a method of detecting a binding event comprising a magnetic probe or composition of the invention. In some embodiments, the present disclosure provides a method of detecting a binding event, wherein the binding event is the interaction of a mammalian circulating tumor cell with a modified platelet cell, comprising: (a) adding platelet cells containing a magnetic probe to a fluid comprising mammalian circulating tumor cells; (b) applying a magnetic field to concentrate the platelet cells containing a magnetic probe; (c) removing the non-magnetic supernatant; (d) collecting the platelet cells containing a magnetic probe and any mammalian circulating tumor cells bound to the modified platelet cells; (e) detecting the presence of mammalian circulating tumor cells in the collected fluid; and (f) integrating the platelet magnetic probe into a device capable of clearing CTCs from patient circulating blood.

[0031] In some embodiments, the present disclosure provides a method for diagnosing cancer and cancer dissemination comprising a magnetic probe or composition of the invention. In some embodiments, the present disclosure provides a method for diagnosing cancer and/or cancer metastasis comprising: (a) adding platelet cells containing a magnetic probe to a blood sample of a patient; (b) applying a magnetic field to concentrate the platelet cells containing a magnetic probe; (c) removing the non magnetic portion of the sample; (d) collecting the platelet cells containing a magnetic probe and any circulating tumor cells bound to the modified platelet cells; and (e) detecting the presence of circulating tumor cells in the collected fluid. In some embodiments, the present disclosure provides a method for treatment of metastatic cancer by integrating the platelet magnetic probe into a device capable of clearing CTCs from patient circulating blood.

[0032] In some embodiments, the present disclosure provides a method for targeting and/or retrieving CTCs in biological fluids by using magnetic platelets. In some embodiments, the magnetic platelets are living platelets, detergent extracted platelets, synthetic platelets and/or lyophilized platelets.

[0033] In some embodiments, the present disclosure provides a method for recovering CTCs comprising a magnetic probe or composition of the invention. In some

embodiments, the recovering of CTCs is from a liquid biopsy sample, Pap Smear sample, cerebrospinal fluid, pleural fluid, ascitic fluid or tissue biopsy samples.

[0034] In some embodiments, the present disclosure provides a method for detecting multiple CTC subtypes ( e.g ., epithelial, mesenchymal and/or intermediate stages) comprising a magnetic probe or composition of the invention. In some embodiments, the present disclosure provides a method of optimizing cancer treatment comprising using a magnetic probe or composition of the invention to recover CTCs, and profiling the CTCs to identify cancer drug resistance or cancer drug sensitivity.

EXAMPLES EXAMPLE 1

Platelet Binding to Cancer Cells

[0035] Platelets support the metastatic cascade in many ways. For example, platelets protect CTCs from shear stress and immune cell detection, provide CTCs with pro- angiogenic and pro-oncogenic factors, and assist cancer cell arrest within the vasculature (Gay et al ., Nat Rev Cancer , 2011, 11(2): 123-34). These aspects support cancer cell survival and dissemination. Platelet-cancer cell binding occurs via the platelet GPIIb-IIIa receptors (Kitagawa et al. , Cancer Res. 1989, 49(3):537-41; Lonsdorf et al, J Biol Chem. 2012, 287(3):2168-78, Papa et ah, Sci Trans Med, 2019, 11, 479, eaau5898), as well as the platelet CLEC-2 receptors (Lowe et al. , Thromb Res. 2012, 129 Suppl 1 : S30-7;

Suzuki-Inoue et al. , J Biol Chem. 2007, 282(36):25993-6001). The affinity does not rely on epithelial or mesenchymal specific markers. Hence, the natural binding affinity of platelets for tumor cells can be used to target CTCs in liquid biopsy, regardless of whether the tumor cell subtype is epithelial, mesenchymal or in an intermediate stage (i.e., undergoing the epithelial -to-mesenchymal transition, or EMT). Current assays for CTC screening rely on the detection of EpCAM on the surface of cancer cells, leaving tumor cells with low or no EpCAM expression undetected (Balasubramanian et al., PLoS One. 2017, 12(4):e0175414).

[0036] It has been shown that platelets shield EpCAM detection on CTC (Jiang et al.,

Lab Chip , 2017, 17:3498). Blood contains an elevated concentration of platelets with an average of 150,000 to 350,000 thrombocytes per microliter of blood. Also, cancer cells are capable of inducing coagulation and CTCs, whether they travel in the bloodstream alone or as a cluster of cells, are not only covered by platelets, but also by fibrin, leading to multicellular assemblies.

[0037] Taken together, these observations show that access of antibodies to EpCAM at the surface of the tumor cells is limited. Therefore, the present invention has improved access to CTCs due to their natural affinity for platelets.

EXAMPLE 2

Platelet Modification by Detergent Procedure

[0038] Human platelets were modified by a detergent procedure to obtain platelet decoys that are similar to platelets in their ability to bind tumor cells. The detergent procedure was disclosed in Int'l Pub. No. WO 2015109220 Al, the content of which is herein incorporated by reference in its entirety.

[0039] Modified platelets have fewer receptors as a result of the detergent procedure.

However, this is not detrimental to their ability to bind cancer cells, as modified platelets bound both mesenchymal-like and epithelial tumor cells. EXAMPLE 3

Engineering of Magnetic Platelet Probes

[0040] Magnetic platelet probes, e.g., platelets that carry a magnetic probe (e.g, magnetic iron oxide nanoparticles), are prepared by:

(1) internalization of magnetic nanoparticles inside platelets (Fig. 1). It has been reported that platelets are capable of internalizing iron oxide nanoparticles (Aurich et al, ACS Appl Mater Interfaces. 2017, 9(40):34666-34673),

(2) using covalent conjugation - A second approach of covalent binding of magnetic particles at the surface of platelets via surface chemistry can also be used (Fig. 2). Detergent-treated platelets can be biotinylated on their surface by covalently binding NHS-biotin to primary amines on the surface of the platelets (Sarkar et al, Methods Mol Biol. 2011, 698:505-23). Then, streptavidin-terminated IONs can be used to functionalize the surface of platelets. The chemical binding of nanoparticles to the platelet surface can be done via multiple chemical strategies, including, but not limited to, the above- mentioned reaction. Alternative methods of surface functionalization include a reaction between the primary amines of platelets and carboxylic-terminated IONs or a reaction between the thiols on platelets and maleimide-terminated IONs (Stephan et al ., Nat Med. 2010, 16(9): 1035-41).

(3) using immunoglobulin as a linker between platelets and nanoparticles.

Nanoparticles are known to interact with immunoglobulins that subsequently mediate their clearance by phagocytes (Behzadi et al, Chem. Soc. Rev., 2017, 46, 4218). For example, the Fc portion of the IgG engages with the Fc receptors on the platelet surface and result in iron oxide-loaded platelets. The platelets used in this example are lyophilized and fixed platelets, such lyophilized platelets are used to diagnose Von Willebrand disease. Any form of platelets (i.e. live, dead, fixed, lyophilized, modified, detergent extracted platelets) that still display enough Fc receptors can be used as a platform to produce Magnetic Platelet Probes. If live platelets are used, they can be used as such or treated with platelet inhibitors (e.g., aspirin, clopidogrel, prasugrel, ticagrelor, cangrelor, elinogrel, tirofiban, eptifibatide, abciximab, dipyridamole, or cilostazol) or detergent to make them inert. Using inert platelets is preferable to avoid issues of platelet aggregation as well as initiation of the coagulation cascade, which can affect isolation as well as separation of the cancer cells. Furthermore, inactive platelets are better suited for manipulation and storage.

(4) using an incubation under mixing of magnetic probes and platelet decoys followed by centrifugation to physically force/load the platelets with magnetic nanoparticles (e.g. SPIONs). Centrifugation can be performed at 900g to 2,500g and for 10 minutes to 60 minutes.

(5) using a permanent magnet to physically force/load the platelets (e.g. platelet cell, a freeze-dried platelet, a modified platelet or a synthetic platelet) with magnetic nanoparticles (e.g. SPIONs) after immobilizing the suspensive platelets by centrifugation in the bottom of multi wells plate such as described by Mykhaylyk el al. ( Nature protocols , 2007, 2, 10, 2391) in the context of gene delivery by magnetofection of suspensive cells.

Experimental approach.

[0041] It was demonstrated by flow cytometry that EpCAM detection was significantly impaired when cancer cells were pre-coated with platelets in a dose-dependent manner. Indeed, once a ratio of 1000 platelets per CTC is attained, the signal detection becomes extremely weak in MDA-MB-231 and SK-BR-3 breast cancer cells (Fig. 3 and Fig. 4). However, blood contains a much more elevated concentration of platelets with on average 150,000 to 350,000 platelets per microliter of blood, which would even further lower the detected signal. This does not yet take into account that cancer cells are capable of inducing coagulation and that CTCs, whether they travel in the bloodstream alone or as a cluster of cells, are not only covered/protected by platelets, but also by fibrin, leading to multicellular assemblies (Fig. 5).

[0042] To engineer platelet magnetic probes, immunoglobulin (IgG) was used as a linker between lyophilized platelets and IONs (Iron Oxide Nanoparticles) as one approach. Commercially available IONs (bare particles) and lyophilized/fixed platelets were used. Specifically, 100 pL of 50e6 lyophilized and fixed platelets were incubated with human IgG in Tyrode buffer for 1 hr at 37 °C, next 100 pL of iron oxide nanoparticles were added at a final concentration of 1 or 0.5 mg/mL for 1 hr. Platelets were separated from unbound nanoparticles by 10 min centrifugation cycles at l,000g. Platelet magnetic probes were then placed on a magnet over several hours (e.g., 3 hrs or 15 hrs), the liquid was then discarded while the sample was still on the magnet. Lastly, the sample was removed from the magnet and resuspended in Tyrode buffer. Flow cytometry was used to enumerate magnetic platelet probes retrieved at various time points through the process. Using 20 nm iron oxide nanoparticles at either 1 or 0.5 mg/mL leads to the best retrieval percentages obtained for 15 hrs of contact of the magnetic platelet probes with the magnet: 68.66 ± 6.92 % and 65.68 ± 12.36 %, respectively (Fig. 6).

[0043] The loading of magnetic (nano)particles per platelet was assessed by using a colorimetric ferrozine assay or by fluorescence when using fluorescently tagged nanoparticles. Different sizes of nanoparticles (e.g. 20 nm, 100 nm SPIONs) with different surface charges and at various concentrations are tested and the parameters leading to higher loading of magnetic nanoparticles and/or higher magnetic retrieval and/or greater interaction with cancer cells were selected.

[0044] After the preparation of the magnetic platelet probes, the interaction kinetics of cancer cells (cell lines) and magnetic platelet probes were assessed by flow cytometry (in cancer cell suspensions, whether the cancer cells were dispersed in medium, Figs. 7 and Fig. 8, or in blood) to determine the minimum incubation time required for the magnetic platelet probes to retrieve tumor cells in suspension. Specifically, 100,000 Hoechst positive cancer cells in 100 pL of medium are incubated with an additional 100 pL of the magnetic platelet probes (50e6 magnetic platelets total) for 1 hr at 37 °C and left on a magnet for a few hours to 24 hrs. The liquid was then discarded, the sample was subsequently removed from the magnet and resuspended in cancer cell medium before being evaluated by flow cytometry. Parameters such as agitation and CTC concentration were analyzed to optimize the detection of CTCs in samples devoid of, or containing, residual blood cells.

[0045] Regarding the flow cytometry analysis, the cancer cell population was gated on the granularity (side scatter, SSC) versus size (forward scatter, FSC) dot plot for unstained cancer cell controls (Figs. 7 and 8, column A), Hoechst positive cancer cells alone (Figs. 7 and 8, column B) and cancer cells magnetically retrieved from suspensions with the magnetic platelet probes (Figs. 7 and 8, column C). Next, the granularity (SSC) versus Hoechst dot plot was obtained from the gated populations and the absolute count of Hoechst positive MDA cancer cells is determined in the Hoechst positive gate (Figs. 7 and 8, SSC versus Hoechst panels). This shows that this system was capable of retrieving cancer cells of different phenotypes. [0046] To engineer platelet magnetic probes, magnetic probes (SPIONs) and platelet decoys were incubated with mixing to physically force/load the platelets with magnetic nanoparticles as another approach. Specifically, 1 mg/mL SPIONs with a mean size distribution of 20 nm was incubated overnight (specifically 17 hrs) with 50 x 10 ⁶ platelet decoys in 200 pL of Tyrode buffer. Next, the suspension was centrifuged (l,000g, 30 min) and resuspended in 200 pL Tyrode buffer twice and then subsequently used to capture cancer cells. Magnetic platelet decoys obtained with this method, have been examined by transmission electron microscopy, which enable the visualization of successful entrapment of SPIONs within the platelet decoys (Fig. 9). A slight

modification of production parameters, i.e. (i) magnetic particle size distribution, (ii) magnetic particle concentration, (iii) platelet concentration, (iv) incubation time and (v) mixing method (e.g. vortex, gyroplate mixing), could also be used in order to obtain platelet magnetic probes.

[0047] Following the experimental method described in paragraphs [0044] and [0045] regarding CTC sample preparation and recovery analysis, the formulation described in paragraph [0046] allowed the capture of 77.1 ± 1.9% (n=18) of Hoechst positive MDA- MB-231 cells spiked in blood after 1 hour incubation on a magnet (Fig. 10).

EXAMPLE 4

Recovery and Detection of Tumor Cells in Blood Samples

[0048] A method for diagnosing cancer or cancer metastasis is developed. The method comprises: (a) adding modified platelet cells containing a magnetic probe to a blood sample; (b) applying a magnetic field to concentrate the modified platelet cells containing a magnetic probe; (c) removing the non-magnetic portion of the sample; (d) collecting the modified platelet cells containing a magnetic probe and any circulating tumor cells bound to the modified platelet cells; (e) detecting the presence of circulating tumor cells in the collected fluid; and (f) integrating the platelet magnetic probe into a device capable of clearing CTCs from patient circulating blood.

[0049] The ability of magnetic platelet probes to recover cancer cells from blood samples is tested by first doping various epithelial (e.g., MCF-7) or mesenchymal-like (e.g, MDA-MB-231, SK-BR-3) cancer cells in human blood. Non-breast cancer cell lines with low EpCAM expression, such as human lung carcinoma A549, can also be tested.

Platelets can bind cancer cells of any origin and subtype.

[0050] The efficiency of recovery of cancer cells in blood samples utilizing magnetic platelet probes is assessed and the sensitivity of the technique in the lower detection range (i.e., in samples with low CTC count) is evaluated. Mixtures of blood samples containing various concentrations of cancer cells to replicate liquid biopsies are prepared and the percent recovery efficiency is determined. To make this determination, the minimum excess of magnetic platelet probes to be added to the liquid biopsy to recover tumor cells efficiently is evaluated. Magnetic platelet probes are incubated in blood samples at 37°C under gentle agitation. Magnetic platelets bound to tumor cells are subsequently retrieved (positive selection) via the use of a magnet applied on the tube containing the biopsy, while free floating blood cells are discarded. After collecting the fraction of the sample retained by the magnet, cells are stained with a dye-conjugated mouse anti-human CD45 antibody to exclude any false positives from potential immune cell pollution.

[0051] To evaluate the sensitivity of the technique on the lower range of detection,

mixtures of cancer cells in blood with a very low number of cancer cells per mL of blood are used.

Interpretation of data.

[0052] The collected cells are categorized as CTCs only if they have a nucleus (Hoechst positive) and are CD45 negative (to exclude immune cell capture). Cells are also characterized for cytokeratins 8, 18, 19, EpCAM, E-cadherin, N-cadherin, and vimentin expression levels. Flow cytometry is used to evaluate the captured cells profile regarding these specific parameters of analysis. The magnetic particles associated with the platelets (whether IONs are internalized or covalently bound at the platelet surface) carry a red fluorescent probe which defines the detection signal for platelets, while tumor cells (from cell lines) are stained with Hoechst and dye-conjugated antibodies are used for the detection of CD45 and cytokeratins.

EXAMPLE 5

CTC Detection Techniques and Other Uses of Magnetic Platelet Probes

[0053] A CTC detection method that relies on natural affinity (i.e., platelet-CTC

interaction) to target and detect disseminating tumor cells in a blood sample was developed. Because CTCs are also present in the early stages of cancer development, targeting and detecting multiple subtypes of CTC (i.e., epithelial, mesenchymal and intermediate stages: EMT transition) allows significant improvement in the early detection and management of cancer patients. In addition, the method allows for detection of CTCs derived from heterogeneous cancers and allows for treatment adjustments in real time. The method also allows for retrieval of CTCs from liquid biopsy, and can be used to filter out CTCs directly from systemic circulation to prevent metastasis. Furthermore, diagnostic systems, e.g ., microfluidic based magnetic capture or devices that utilize magnetism to attract magnetic platelet probes bound to cancer cells, and therapeutic devices could be further included as a part of the invention. For example, compositions and magnetic probes of the invention could be infused into patients for therapeutic purposes. After infusion into a patient, probes could separate out from blood along with the systemic CTCs that associate with them. The isolated CTCs could then be analyzed for drug resistance and drug sensitivities.

[0054] It is to be understood that the foregoing embodiments and exemplifications are not intended to be limiting in any respect to the scope of the disclosure, and that the claims presented herein are intended to encompass all embodiments and exemplifications whether or not explicitly presented herein.

[0055] All patents and publications cited herein are fully incorporated by reference in their entireties.