The present invention relates to a combination of active ingredients for use in the therapeutic treatment of tumor diseases. Over the last decade, the use of tyrosine kinase inhibitors (TKI) in the treatment of solid tumors has become the expected standard of care, particularly in the treatment of tumors considered more oncogenic driven, such as renal cell carcinoma (RCC), gastrointestinal stromal tumor and melanoma. The implementation of TKI therapy as first line treatment has revolutionized the clinical practice and outcomes for metastatic carcinomas.

The anticancer activity of TKIs is related to the inhibition of growth factor receptors overexpressed in several tumors and co-responsible for tumor angiogenesis and cell proliferation. Among the group of tyrosine kinase inhibitors, Sunitinib, and Sorafenib have conferred a good clinical outcome of patients in term of response rate, progression-free survival and overall survival.

Despite the clinical benefits, the use of targeted TKI therapies is not without limitations, including several adverse effects such as hand-foot syndrome, mucosal inflammation, hypothyroidism and fatigue together with hematological adverse events like anemia, leukopenia and thrombocytopenia. Moreover, in the vast majority of cases, TKI long-term anti-tumor effect leads to the development of resistance.

It is now well recognized that the initiation, the maintenance and the recurrence of tumors is sustained from Cancer Stem Cells (CSCs), a small population of cells with stem -like properties, which have been identified in several solid tumors. In particular, in renal cell carcinoma (RCC), a CSC population has been characterized by the expression of surface endoglin (CD105). These renal CSCs display several characteristics typical of cancer stem cells/tumor initiating cells, including 1) clonogenicity, 2) expression of stem cell markers and absence of differentiation markers, 3) in vitro epithelial and endothelial differentiation ability, and 4) generation of in vivo serially transplantable tumors with characteristics similar to the tumor of origin. Hence, targeting cancer stem cells represents an important therapeutic approach to eradicate solid tumors such as RCC, considering their high drug resistance and tumor initiating capability.

Cell-cell interaction is at least in part orchestrated by extracellular vesicles (EVs) that play a key role in cell communication by transferring mRNA, microRNA, lipids and proteins to target cells (Camussi G. et al, Kidney Int., 2010 78(9):838-48). It has been shown that EVs derived from stem cells are able to reprogram tumor cells to a more benign phenotype exerting their anti-tumor effect by blockade of proliferation and induction of apoptosis in vitro and by the regression of ectopic tumors in SCID mice (Bruno S. et al, Stem Cells Dev, 2013 l;22(5):758-7l). This anti-tumor activity is particularly evident for EVs derived from human liver stem cells (HLSC), a stromal cell population isolated from human adult liver that inhibits liver carcinomas as well as gliomas and lymphoblastomas.

W02009050742 discloses the use of microvesicles derived from cells of the endothelial cell lineage, preferably from endothelial progenitor cells, in the treatment of type I or type II diabetes by pancreatic islet transplantation.

WO2011107437 discloses the use of microvesicles derived from adult stem cells for the therapeutic treatment of tumors. In one embodiment, the anti-tumor treatment may additionally comprise the administration of a cytotoxic agent, such as e.g. a TKI compound. However, WO2011107437 does not provide any specific indication on how the combined therapy should be administered in order to be clinically effective. In particular, WO2011107437 is silent whether the micro vescicle and the cytotoxic agent should be administered as a physical mixture or as separate ingredients.

The object of the present invention is to provide a medicament having activity against tumor proliferation and growth, which is effective in the eradication of Cancer Stem Cells (CSCs), thereby achieving a long-lasting clinical response as well as preventing tumor relapse. These and other objects are accomplished by the present inventors, who have found that the administration to cancer stem cells of a TKI compound in combination with extracellular vesicles (EVs) derived from adult stem cells leads to a significant increase in CSCs apoptosis, compared to the TKI compound or the EVs administered alone. In particular, experimental studies carried out by the inventors on renal and breast cancer stem cells have revealed that the pro-apoptotic effect exerted by TKI or EVs alone is dramatically enhanced when these anti-tumor agents are administered in combination either at the same time or in sequential order. As shown in Figure 4, when a sequential administration scheme is applied, a significant increase in the number of apoptotic cancer stem cells is surprisingly achieved by stimulating the assayed cells with EVs after incubation with the TKI compound. Conversely, the pro-apoptotic effect measured when CSCs are pre incubated with EVs before the addition of the TKI agent does not differ substantially from the effect obtained when incubating assayed CSCs with TKI or EVs alone.

Therefore, an aspect of the present invention is a combined pharmaceutical preparation comprising an extracellular vesicle (EV) derived from an adult stem cell and a tyrosine kinase inhibitor (TKI), for simultaneous or sequential use in the therapeutic treatment of a tumor disease and/or in the prevention of tumor relapse in a patient, wherein the sequential use is performed by first administering the tyrosine kinase inhibitor (TKI) and then administering the extracellular vesicle (EV).

Other features and advantages of the combined preparation according to the invention are defined in the appended claims which form an integral part of the description.

According to one embodiment, the extracellular vesicle (EV) is administered at least 40 hours, preferably at least 48 hours, after the administration of the tyrosine kinase inhibitor (TKI).

The results of the apoptosis analysis conducted by the present inventors clearly indicate that the combined preparation of the invention enhances considerably the chemosensitivity of tumor cells to TKI through the pro-apoptotic effect exerted by the EVs. Without wishing to be bound by any theory, the inventors believe that the increased tumor chemosensitivity seen with the combined preparation of the invention may be linked to EV-dependent enhancement of cellular mechanisms induced in target cancer cells upon TKI treatment rather than to epigenetic changes induced by EVs, leading to increased TKI sensitivity. In a preferred embodiment, the adult stem cell is a human liver stem cell or a human mesenchymal stem cell.

A preferred human liver stem cell is the human non-oval liver stem cell (HLSC) expressing both mesenchymal and embryonic stem cell markers. HLSCs are disclosed e.g. in W02006126236.

In another preferred embodiment, the human mesenchymal stem cell is derived from human adult bone marrow (BM-MSC).

Preferably, the extracellular vesicle (EV) expresses a marker selected from CD63 and CD81. According to one preferred embodiment of the invention, the tyrosine kinase inhibitor (TKI) is selected from the group consisting of Gefitinib, Erlotinib, Lapatinib, Vandetanib, Afatinib, Sorafenib, Sunitinib, Pazopanib, Axitinib, Regorafenib, Nintedanib, Levantinib, Cabozantinib, Trametinib, and any combination thereof.

The combined preparation of the invention is suitable for use in the therapeutic treatment of tumor diseases and/or in the prevention of tumor relapse in a patient, preferably for the treatment of solid tumors, more preferably for the treatment of a solid tumor selected from the group consisting of renal cancer, breast cancer, liver cancer and gastrointestinal stromal tumor (GIST).

Further supporting the therapeutic value of the combined preparation of the invention, the present inventors have demonstrated that the co-administration of TKI and EVs, either simultaneous or sequential, is particularly effective against cancer stem cells by inducing activation of cell death.

Therefore, in a preferred embodiment of the invention, the targeted solid tumor comprises one or more cancer stem cells.

It is known that the stem cell markers expressed by CSCs vary according to the type of solid tumor. Consequently, the specific CSC phenotype may assist in the characterization of tumors which are more likely to be responsive to a particular therapeutic treatment as well as in the design of optimal therapeutic regimens.

For example, the combined preparation of the invention has been found to be particularly effective against cancer stem cells expressing at least one stem cell marker selected from the group consisting of CD105, ALDH1, OCT4, SSEA4 and CD24YCD44 ⁺ .

The exact dose of the combined administration of TKI and EVs according to the invention may vary depending on the targeted tumor as well as on the specific components of the combined preparation, i.e. the TKI compound and the type of extracellular vesicle, and on the patient’s characteristics (e.g. sex, age, weight, etc.). Preferably, the daily dosage of the tyrosine kinase inhibitor (TKI) is comprised between 0.5 and 2.0 mg per kilogram body. The extracellular vesicle (EV) may be administered in an amount ranging from 5 x 10 ⁹ to 5 x 10 ¹² EVs per kilogram body per day. In a more preferred embodiment, the therapeutic and/or prophylactic treatment of the invention comprises administering to a patient a dose of the tyrosine kinase inhibitor comprised between 0.7 and 1.5 mg/kg/die of the tyrosine kinase inhibitor (TKI) and a dose of the extracellular vesicles comprised between 1 x l0 ^lo and 1 x l0 ¹² /kg/die of the extracellular vesicles (EV).

As mentioned, the TKI and EVs may also be effectively administered in the form of a pharmaceutical composition, i.e. of a physical mixture of the two active ingredients.

Accordingly, a second aspect of the present invention is a pharmaceutical composition for use in the therapeutic treatment of a tumor disease and/or in the prevention of tumor relapse in a patient, comprising an extracellular vesicle (EV) derived from an adult stem cell, a tyrosine kinase inhibitor (TKI), and optional pharmaceutically acceptable vehicles, excipients and/or diluents.

Several studies have proven the effectiveness of extracellular vesicles (EVs) as drug vehicles, wherein drug loading strategies may involve, for example, direct incorporation of the drug substance into the extracellular vesicle or, alternatively, its binding on the external surface of the EV’s membrane. The present inventors have found that the pro-apoptotic effect exerted on cancer stem cells by extracellular vesicles (EVs) loaded with a tyrosine kinase inhibitor (TKI) is comparable with the rate of cell death measured in the same cellular culture following co-administration of EVs and TKI as separate compounds (see Figure 5).

Therefore, in one embodiment, the pharmaceutical composition comprises TKI-loaded extracellular vesicles, wherein the TKI is incorporated into the extracellular vesicle (EV) or it is bound to the external surface of the EV.

In a preferred embodiment, the extracellular vesicles are loaded with a number of TKI molecules of at least 1 x l0 ³ /EV, preferably with a number of TKI molecules ranging from 1 x l0 ³ /EV to 1 x l0 ⁷ /EV, more preferably with a number of TKI molecules ranging from 1 x l0 ⁴ /EV to 1 x l0 ⁶ /EV.

The pharmaceutical composition of the invention is suitable to be administered as a cancer therapy to any mammal, including human beings.

The pharmaceutical composition of the invention is suitable for administration e.g. via the topical, enteral or parenteral route.

A yet further aspect of the present invention is an in vitro method of promoting apoptosis of cancer stem cells (CSC) in a cell culture, comprising contacting the cell culture first with a tyrosine kinase inhibitor (TKI) and subsequently with extracellular vesicles (EVs) derived from adult stem cells.

According to the method of the invention, the cancer stem cells (CSC) in the cell culture are contacted with extracellular vesicles (EVs) following incubation with a tyrosine kinase inhibitor (TKI).

In one embodiment, the period of incubation in the presence of TKI is of at least 40 hours, preferably of at least 48 hours. In another embodiment, the period of incubation in the presence of EV s is of at least 6 hours, preferably for at least 8 hours.

The following experimental section is provided purely by way of illustration and is not intended to limit the scope of the invention as defined in the appended claims. In the following experimental section reference is made to the appended drawings, wherein:

Figure 1 shows the characterization of G7 renal CSCs. A) Representative FACS analysis of G7 renal CSCs showing the expression of the mesenchymal stem cell markers CD105, CD73 and the embryonic stem cell marker SSEA4, but not of CD133, CD24 and HPCAM. B) Representative micrograph showing the ability of G7 renal CSCs to form spheres when cultured under appropriate culture conditions. C) Representative micrograph showing hematoxylin/eosin staining of tumors generated by G7 renal CSCs recovered from SCID mice. Original magnification: 200x.

Figure 2 shows the characterization of EVs isolated from HFSCs. A) NanoSight size distribution graph showing the quantity and size of HFSC-EVs. B) Representative Western blot analysis of CD63 and CD81 protein expression in HFSC-EVs. The experiments were performed in duplicate and gave similar results. C) Representative electron microscopy of HFSC-EVs (scale bar = 100 nm). D) Representative micrograph showing incorporation of DIF-labelled HFSC-EV s in G7 renal cells after incubation for one hour, detected by confocal microscopy. Original magnification: 630x.

Figure 3 A is a graph showing that incubation of G7 renal CSCs with HFSC-EVs induces a significant dose-dependent apoptotic effect compared to control. Apoptosis was evaluated by Muse Annexin V & Dead Cell Assay as the percentage of apoptotic cells after 48 hours incubation with different doses of HFSC-EVs. Figure 3B is a graph showing that incubation of G7 renal CSCs with ImM Sunitinib (Sun) in combination with different doses of HFSC- EVs (5 x 10 ³ , 10 x 10 ³ , and 50 x 10 ³ EV s/target cells) for 48 hours significantly inhibits proliferation compared to CSCs stimulated with ImM Sunitinib alone. The results are expressed as mean ± SD of five different experiments. Statistical differences were examined by Student’s t-test: *= p < 0.05 vs CTL cells; # = p < 0.05 vs Sunitinib. Figures 3C and 3D show that incubation for 48 hours of G7 renal CSCs (Figure 3C) and C10 breast CSCs (Figure 3D) with HLSC-EVs (50 x 10 ³ EV s/target cells) in combination with ImM Sunitinib (HLSC-EVs+Sun), 5mM Sorafenib (HLSC-EVs+Sor) or 2mM Cabozantinib (HLSC- EVs+Cabo) significantly inhibits cell proliferation compared to controls and CSCs treated with ImM Sunitinib, 5mM Sorafenib or 2mM Cabozantinib alone. The results are expressed as ± SD of three different experiments. Statistical differences were examined by Student’s t-test: *= p < 0.05 vs CTL; # = p < 0.05 vs HLSC-EVs; § = p < 0.05 vs Sunitinib; @ = p < 0.05 vs Sorafenib; ^L = p < 0.05 vs Cabozantinib.

Figure 4A is a schematic representation of the sequential administration of TKIs and EVs. The entire incubation period is of 48 hours. According to the pre-EVs scheme, G7 renal CSCs were first incubated with HLSC-EVs for 8 hours, and then stimulated with Sunitinib (1 mM) or Sorafenib (5 mM) for additional 40 hours. According to the post-EVs scheme, G7 renal CSCs were initially stimulated with Sunitinib (1 mM) or Sorafenib (5 mM) for 40 hours, and then incubated with HLSC-EVs for additional 8 hours. Figure 4B is a graph showing the pro-apoptotic effects on G7 renal CSCs exerted by TKI and EVs administered in pre-EVs or post-EVs sequential order as depicted in figure 4A. A significant increase of the percentage of apoptotic cells is observed following post-EVs sequential administration, compared to pre-EVs. The results are expressed as mean ± SD of three different experiments. Statistical differences were examined by Student’s t-test: *= p < 0.05 vs CTL; # = p < 0.05 vs HLSC- EVs; § = p < 0.05 vs Sunitinib; @ = p < 0.05 vs Sorafenib.

Figure 5 is a graph showing the results of apoptosis analysis on G7 renal CSCs after incubation with HLSC-EVs loaded with Sunitinib (EV-SUN), HLSC-EVs loaded with Sorafenib (EV-SOR), Sunitinib or Sorafenib alone. The supernatants (sum-SUN and surn- SOR) recovered after ultracentrifugation in the loading experiments were used as negative controls. The results are expressed as mean ± SD of three different experiments. Statistical differences were examined by Student’s t-test: *= p < 0.05 vs CTL; # = p < 0.05 vs HLSC- EVs; § = p < 0.05 vs Sunitinib; @ = p < 0.05 vs Sorafenib. Figure 6 shows that co-administration of TKIs and HLSC-EVs on G7 renal CSCs induces a decrease of the phosphorylated form of intracellular proteins. There are shown the results of Western blot (representative micrographs) and densitometric analysis of pAkt/Akt ratio (A), pPTEN/PTEN ratio(B) and pmTOR/mTOR ratio(C) ratio determined in G7 renal CSCs after stimulation for 3 hours with HLSC-EVs (50 x 10 ³ EVs/target cells), Sunitinib (ImM), Sorafenib (5mM) alone or in combination (HLSC-EVs+Sun, HLSC-EVs+Sor). The results shown as arbitrary units, were representative of three different experiments and were normalized to vinculin expression. Statistical differences were examined by Student’s t-test: *= p < 0.001 vs CTL. B, Inset: Western blot micrograph of PTEN expression by HLSC-EVs. Figure 7 shows that the administration of HLSC-EVs alone inhibits the intracellular pCreb/Creb ratio and, in combination with Sorafenib, inhibits also Erkl/2. There are shown the results of Western blot (representative micrographs) and densitometric analysis of pCreb/Creb ratio (A and B) and Erk 1/2 (C) in G7 renal CSCs after stimulation for 3 hours with HLSC-EVs (50 x 10 ³ EVs/target cells), Sunitinib (ImM) or Sorafenib (5mM) alone or in combination (HLSC-EVs+Sun, HLSC-EVs+Sor). The results shown as arbitrary units, were representative, of three different experiments and were normalized to vinculin expression. Statistical differences were examined by Student’s t-test: *= p < 0.001 vs CTL.

1 MATERIAL AND METHODS

1.1 Cancer stem cells isolation and culture

Renal cell carcinoma stem cells (CSCs) were obtained from specimens of renal cell carcinomas from patients undergoing radical nephrectomy according to the Ethics Committee of the S. Giovanni Battista Hospital of Torino, Italy (168/2014). Cells were isolated, using anti-CD 105 Ab coupled to magnetic beads, by magnetic cell sorting using the magnetic- activated cell sorting (MACS) system (Miltenyi Biotec, Auburn, CA, USA) from renal carcinomas (histological types: 3 clear-cell type and 2 undifferentiated carcinomas). Briefly, cells were labelled with the anti-CD 105 mAh for 20 min, washed twice and resuspended in MACS buffer (PBS without Ca ² and Mg ² , supplemented with 1% BSA and 5 mM EDTA) at a concentration of 2xl0 ⁷ cells. After washings, cells were separated on a magnetic stainless steel wool column (Miltenyi Biotec), according to the manufacturer’s recommendations.

Magnetically sorted CDl05 ⁺ CSCs were cultured in the presence of the expansion medium, consisting of DMEM LG (Invitrogen), with insulin-transferrin- selenium, 10 ⁹ M dexamethasone, 100 U penicillin, 1000 U streptomycin, 10 ng/ml EGF (all from Sigma- Aldrich) and 5% fetal calf serum (FCS) (Sigma- Aldrich). For cell cloning, single cells were seeded in 96- well plates in presence of the expansion medium. A G7 CDl05 ⁺ clonal renal cell carcinoma stem cell line was selected and used for all the experiments.

Breast CSCs were isolated from breast lobular-infiltrating carcinoma. Briefly, tumor specimen was finely minced with scissors and digested by incubation for 1 h at 37°C in DMEM containing collagenase II (Sigma Chemical Company, St. Louis, MO, USA). After washings in medium plus 10% FCS (GIBCO, Grand Island, NY, USA), the cell suspension was forced through a graded series of meshes to separate the cell components from stroma and aggregates and, finally, through a 40- pin pore filter (Becton Dickinson, San Jose, CA, USA). Single cells were plated at 1000 cells/ml in serum- free DMEM-F12 (Cambrex BioScience, Venviers, Belgium), supplemented with 10 ng/ml basic fibroblast growth factor (bFGF), 20 ng/ml epidermal growth factor (EGF), 5 (pg/ml insulin and 0.4% bovine serum albumin (all from Sigma).

For cell cloning, single cells were seeded in 96-well plates in presence of the expansion medium. A C10 clonal breast cell carcinoma stem cell line (C10 breast CSCs) was selected and used for all the experiments.

1.2 Human mesenchymal and liver stromal stem cells

HLSC were isolated from human cryopreserved normal hepatocytes obtained from Lonza (Basel, Switzerland, www.lonza.com).

Human hepatocytes were plated in the presence of alfa minimum essential medium/endothelial cell basal medium 1 (expansion media: aMEM/EBM in the ratio 3: 1, Lonza), supplemented with antibiotics (100 U penicillin and l,000U streptomycin; both from Sigma, St. Louis) and 10% Foetal Calf Serum (FCS, Sigma). After 2 week HLSC colonies that were evident were expanded.

Bone marrow-derived mesenchymal stem cells (MSCs) were obtained from Lonza. MSCs were used up to the sixth passage of culture. All of the cell preparations used were positive for the typical MSC markers(CDl05, CD29, CD73, CD44, and CD90 (not shown).

1.3 EVs isolation

The supernatant of HLSCs or MSCs was recovered and centrifuged for 20 min at 3000 g to remove cell debris and apoptotic bodies. An ultracentrifugation at 100,000 g for 2 hours at 4 °C (Beckman Coulter Optima L-90 K, Fullerton, CA, USA) has followed the previous one. Both HLSC-EVs and MSC-EVs were resuspended in RPMI supplemented with 1% dimethyllsulfoxide (DMSO) and frozen at -80 °C for later use.

Concentration and size distribution of EVs were determined by the Nanosight LM10 system(NanoSight, Wiltshire, UK). Briefly, EV preparations were diluted (1:200) in sterile saline solution and analyzed by the Nanoparticle Analysis System using the NTA 1.4 Analytical Software. To evaluate the internalization of EVs in G7 renal CSCs by fluorescent microscopy, EVs were labelled with 1 mM Dil dye (Thermo Fisher Scientific, Waltham, MA, USA). Briefly, purified EVs were resuspended in PBS supplemented with 1 pM Dil dye and ultracentrifuged at 100,000 g for 1 h at 4 °C. Following labelling, the EVs were washed with PBS by ultracentrifugation as mentioned above. The pellet obtained was then resuspended in RPMI with 1% DMSO and frozen for subsequent studies.

For loading experiments, EVs were loaded with 10 pM of Sunitinib or 50 pM of Sorafenib by incubating together for 15 minutes at 37 °C and then ultracentrifuged at 100,000 g for 1 h at 4 °C to remove the unloaded drug. EVs were resuspended in RPMI with 1% DMSO and named EV-SUN those loaded with Sunitinib or EV-SOR those loaded with Sorafenib. The supernatant (sum-SUN and surn-SOR) was recovered and used in the experiments as negative control. The dose of Sunitinib and Sorafenib used was chosen on the base of preliminary experiments showing 8-10% of drug incorporation. Spectrum analysis was used to evaluate the effective drug loading within EVs and it revealed the presence of 1.8 mM Sunitinib and 10 pM for Sorafenib in EV-SUN or EV-SOR.

1.4 Apoptosis assav

Apoptosis was evaluated by Muse™ Annexin V and Dead Cell Assay (Millipore, Merck KGaA, Darmstadt, Germany) according to manufacturer’s instructions.

The assay is based on the detection of phosphatidylserine (PS) on the surface of apoptotic cells, using fluorescently labeled Annexin V in combination with the dead cell marker, 7- AAD. Briefly, G7 renal CSCs or C10 breast CSCs were seeded at the concentration of 2xl0 ³ cells/well and, after cell attachment, were stimulated with HLSC-EVs or Sunitinib or Sorafenib alone or in combination and cultured for 48 hours. At the end of the incubation period, the supernatant containing dead cells and cells were recovered, incubated for 20 minutes with Annexin V/7-AAD reagent and read at Muse. The results were showed as the percentage of total apoptotic cells.

1.5 Phospho-protein array

Intracellular phosphoproteins were evaluated in the lysates of renal G7 CSCs by the magnetic bead-based_immunoassays Bio-Plex Pro cell- signaling assay according to manufacturer’s instruction (BIoRad, Hercules, California, US).

Briefly, cells were treated or not with Sunitinib (1 pM) or Sorafenib (5 pM) or HLSC-EVs (1 x 10 ³ EV/target cell) or with the co-administration of HLSCEVs/Sunitinib or HLSC- EVs/Sorafenib for three hours. Then, cells were lysed and lysates were_incubated with capture antibodies coupled to the beads. Coupled beads react with the sample containing the analyte of interest. After a series of washes to remove unbound protein, a_biotinylated detection antibody was added to create a sandwich complex. The final detection_complex was formed with the addition of streptavidin-phycoerythrin (SA-PE) conjugate and submitted to Bio-Plex system with Bio-Plex Manager software analysis. 1.6 Western blot analysis

G7 renal CSCs were stimulated for 3 hours with HLSC-EVs alone or in combination with Sunitinib_or Sorafenib. At the end of incubation time, cells were lysed in RIPA buffer supplemented with protease and phosphatase inhibitor cocktail and PMSF (Sigma- Aldrich). Aliquots of the cell lysates_containing 30 pg proteins form cells or 10 pg from EVs, as determined by the PierceTM BC A Protein method (Thermo Scientific, Rockford, IL, USA), were run on 4-20% SDS-PAGE under_reducing conditions and blotted onto PVDF membrane filters using the iBLOT system (Life_Technologies). The membranes were blocked in Tris-buffered saline-Tween (TBS-T; 25 mM Tris,_pH 8.0, 150 mM NaCl, and 0.05% Tween-20) containing 5% (w/v) non-fat dried milk for 1 h. After blocking, membranes were probed overnight with primary antibody. Anti-vinculin (Santa Cruz Biotechnology), anti-AKT or anti p-AKT (Ser473), anti-PTEN or anti-pPTEN, anti-mTOR or antip-mTOR, anti-CREB or anti-pCREB and anti-Erk 1/2 (all from Cell Signalling) primary Abs were used.

After extensive washings with TBS-T, the blots were incubated with appropriate peroxidase conjugated secondary antibodies for 1 h at room temperature. Goat anti-Rabbit IgG and goat antimousejgl HRP conjugated secondary antibodies (Thermo Scientific, Rockford, IL, USA) were used. Following incubation, the membranes were washed extensively with TBS- T, probed with_ClarityTM Western ECL substrate (Bio-rad, CA, USA), and detected by the Chemidoc system (Biorad, CA, USA).

2. RESULTS

2.1 Isolation of cancer stem cells from renal carcinoma

Renal CSCs were isolated from renal carcinoma by magnetic cell sorting using selection for the_CDl05 surface antigen. The G7 clone were used for all experiments. Immunophenotypic analysis showed the positivity for CD105, expression of the mesenchymal stem cell marker CD73, SSEA4 and the absence of CD 133 and CD24, known to be marker of normal renal progenitor cells and EPCAM (Figure 1A). When cultured in non-adhesive culture conditions, G7 renal CSCs were able to growth and form spheres that could be propagated for several passages (Figure 1B). In vivo , when implanted subcutaneously in SCID mice, G7 renal CSC were able to generate serial tumors using low dose cells such as lxlO ³ and lxlO ² doses (Figure 1C). Moreover, CDl05 ⁺ cells isolated from tumors were re-injected in SCID mice in order to generate serially transplantable tumors. As shown in Table 1, CDl05 ⁺ cells (lxlO ³ and lxlO ² cells) gave raise to secondary and tertiary tumors (Table 1), confirming their identity as tumor-initiating cells.

Table 1 (Generation of serially transplantable tumors from renal CSC G7 clone)

G7 passage

2.2 HLSC-EVs isolation and their internalization in renal CSCs

HFSC-EVs were isolated by ultracentrifugation from HFSC and analysed in term of size and_distribution by NTA (Figure 2A). EVs were characterized by Western blot analysis for the_expression of their characteristic markers CD63 and CD81 and by electron microscopy for their morphology (Figure 2A).

When incubated with G7 renal CSCs, HFSC-EV s labelled with DIF dye were internalized by tumor cells after 1 hour of_incubation at 37°C, as shown in Figure 2B. These characteristics are similar to those described for EVs derived by mesenchymal stromal cells (MSC-EVs). 2.3 Co-administration of HLSC-EVs and TKIs increase apoptosis of rCSCs

The inventors first evaluated the effect of EVs from bone marrow and from liver mesenchymal stromal cells, at different doses (5, 10, 50 x 10 ³ EV/target cell) on G7 renal CSCs. As shown in Figure 3, HLSCEVs exerted a dose dependent pro-apoptotic effect on G7 renal CSC that was statistically significant at the dose of 50x10 ³ EV/target cell (Figure 3A). Similar results were observed following incubation of G7 renal CSCs with different doses of MSC-EVs (data not shown).

In order to evaluate a possible combinatory effect of EVs with Sunitinib, the inventors first performed dose-response experiments of the TKI alone on G7 renal CSCs. The lower significant dose with an apoptotic effect was shown to be 2 mM Sunitinib. The inventors therefore planned combinatory experiments with a dose minimally affecting renal CSCs (1 mM).

The percentage of G7 renal CSC apoptosis was significantly increased when HLSCEVs were co-administered with 1 pM Sunitinib compared to Sunitinib alone as well as to HLSCEVs alone (Figure 3B). In particular, an increment of about 40% of apoptosis was observed already at the low non-apoptotic dose of 5xl0 ³ HLSC-EV s/target cell in combination with Sunitinib (Figure 3B).

In order to test whether the apoptotic effect was specific for Sunitinib or common to other TKIs used for the treatment of metastatic RCC, the inventors tested also Sorafenib and Cabozantinib alone and in combination with HLSC-EVs. The minimal apoptotic dose of 5 pM was used for Sorafenib and of 2 pM for Cabozantinib.

Figure 3 shows that both the Sorafenib/HLSC-EVs and Cabozantinib/HLSC-EVs co administration induced an enhancement of apoptotic cells, with a similar effect of the Sunitinib/HLSC-EVs coadministration. Moreover, this increment was substantial not only in respect to control cells, but also to cells stimulated with HLSC-EVs or TKIs alone (Figure 3C). Finally, to evaluate whether the observed effect on chemo sensitivity was specific for renal CSCs or could be shared by CSCs of different origin, the effect of HLSC-EVs and Sunitinib/Sorafenib/Cabozantinib co-administration was evaluated also on C10 breast cancer stem cells obtaining a similar apoptotic increment observed for G7 renal CSCs (Figure 3D).

2.4 EV post-incubation but not pre-incubation increase chemosensitivitv

In order to understand the mechanism by which HLSC-EVs in combination with TKIs exert their pro-apoptotic effect, the inventors performed experiments of sequential administration of HLSC-EVs and TKIs. The inventors first incubated G7 renal CSCs with HLSC-EVs for 8 hours and then stimulated with Sunitinib (1 mM) or Sorafenib (5 mM) for additional 40 hours, to reach 48 hour incubation used in co-administration experiments (Figure 4A). On the other hand, the inventors incubated G7 renal CSCs with Sunitinib or Sorafenib for 40 hours and then stimulated with HLSC-EVs for additional 8 hours (Figure 4A).

The results indicated that only the post- incubation and not the pre-incubation of HLSC-EVs enhanced the chemosensitivity of G7 renal CSCs to TKIs (Figure 4B).

This result suggested that the effect of HLSC-EVs/TKI combination on chemosensitivity was probably due to an EV-dependent enhancement of TKI induced mechanisms in target cells rather than to epigenetic changes induced by EV leading to increased TKI sensitivity.

2.5 HLSC-EVs loaded with TKIs has similar pro-apoptotic effect of co-administration

To assess whether the pro-apoptotic effect of EV-TKI co-administration could be further increased using TKI-loaded EVs, the inventors generated HLSC-EVs loaded with Sunitinib or Sorafenib. As these TKIs are lipophilic, EVs were co-incubated with TKIs for 15 minutes followed by ultracentrifugation to wash out the unbound drugs. The EVs obtained were called EV-SUN or EV-SOR to indicate EVs loaded with Sunitinib (10 pM) or Sorafenib (50 pM), respectively. G7 renal CSCs were then incubated with EV-SUN or EV-SOR in the amount needed to reach the same TKI concentration used in experiments above. As shown in Figure 5, the pro- apoptotic effect of HLSC-EVs loaded with TKIs was comparable to that of HLSC-EVs and TKIs co-administration (Figure 5), suggesting that that drug-loaded EVs could be an alternative approach for drug delivery.

2.6 HLSC-EVs co-administered with TKIs inhibited the Akt/mTOR/PTEN pathway

In order to understand what mechanism underlying the pro-apoptotic effect exerted by co administration of TKIs and HLSC-EVs, the inventors performed a Bio-Plex Pro cell signaling assay for the detection of intracellular phosphoproteins. The results obtained were then validated by Western blot analysis. As shown in Figure 6, the inventors found that TKIs and HLSC-EVs co-administration induced a synergistic effect in respect to the use of TKI or EVs alone on specific pathways. In particular, the co-administration of Sunitinib and HLSC-EVs was able to reduce Akt activity and enhance the oncosuppressor PTEN trough decrease of its phosphorylated form in respect to treatments alone (Fig. 6 A and B). The Akt/PTEN pathways was inhibited also by co-administration of Sorafenib and HLSC-EVs, even if the reduction of pPTEN/PTEN ratio did not reach significance (Figure 6 A and B). In addition, the inventors found that the PTEN protein was directly expressed by HLSCEVs (Fig. 6 B, inset). Moreover, HLSC-EVs alone significantly reduced the active phosphorylated form of mTOR (Figure 6 C) and the activation of the Creb transcription factor (Figure 7 A and B).

Finally, the Sorafenib/HLSC-EV co-administration induced a significant decrease of Erk l/2(Figure 7 C) that was specific for this TKI, as no synergistic effect was observed with Sunitinib/HLSC EV (Figure 7 C).

These results show that the combinatory treatment of HLSC-EVs with TKIs may act to inhibit intracellular pathways that are responsible for the effect observed on the overall apoptosis.