FIELD OF THE INVENTION

The present invention falls within the field of the therapeutic treatment of tumours. More specifically, the invention relates to a combination anticancer therapy, including the admini tration of the tumour suppressor miRNA miR-126 carried within exosomes, in combination with a further compound having the purpose of increasing the effectiveness of the tumour suppressor miRNA.

BACKGROUND ART

MicroRNAs (miRNAs) are small non-coding RNAs that regulate the expression of genes involved in multiple cellular processes. miRNAs are also involved in cell communication: they are released into the extracellular environment via exosomes (microvesicles) or bound to proteins (Ago-2, lipoproteins, nucleophosmin). Their de-regulation has been associated with several disease states, including tumours. Some miRNAs are referred to as “oncogenes” as they are involved in tumour development, while others are referred to as “tumour suppressors” because of their ability to inhibit tumour growth (1). Due to these characteristics, miRNAs have been proposed in tumour therapy, which consists in silencing miRNAs acting as oncogenes or in administering miRNAs acting as tumour suppressors.

In particular, miR-126 is a known tumour suppressor. It is under-expressed in many types of tumours and its re-introduction into tumour cells has been found to cause tumour growth inhibition in malignant pleural mesothelioma, non-small cell lung cancer (NSCLC), pancreatic adenocarcinoma (PAAD), glioma, colorectal cancer, prostate cancer, and breast cancer (2).

In (3), miR-126 was found to be largely under-expressed in the malignant part of the malignant pleural mesothelioma (MPM) compared to the adjacent healthy part. In (4), miR-126 was found to affect the energy metabolism of mitochondria, resulting in tumour suppression of malignant mesothelioma (MM).

The tumour suppressive action of miR-126 is mainly performed in the modulation of the IGF-1 (Insulin Growth Factor-1) survival pathway, thus inhibiting the insulin receptor substrate 1 (IRS-1). IRS-1 inhibition alters the tumour cell metabolism by inducing an autophagy process that leads to loss of malignancy (5).

In the clinical practice, the main limitation of miRNA-based anticancer therapy is the need for a carrier that is able to protect miRNAs from nuclease degradation and to distribute them at tissue level, all without inducing negative effects. For this purpose, several transport systems have been developed, such as, for example, liposomes (6).

The use of exosomes as carriers for transporting miRNAs to tumours has also been described. Exosomes are vesicles typically derived from cell multivesicular bodies and are approximately 30-150 nm in diameter, e.g., 50-100 nm in diameter. Compared to synthetic formulations they have numerous advantages, including remaining in circulation for a long time, having the intrinsic ability to distribute at tissue level, having low toxicity, low immunogenicity, and high biocompatibility (7).

By taking advantage of the fact that miRNAs are physiologically internalized in and transported by exosomes, the latter can be easily enriched with the miRNA of interest and used as carriers in the therapeutic treatment of tumours. However, in a previous study (8), the present inventors observed that miR-126 carried by exosomes is taken up by malignant mesothelioma tumour cells but then released into the tumour stroma by exosomal secretion. The exosomes released into the extracellular environment are subsequently taken up by the other surrounding cells that make up the stroma, such as mesothelial cells, fibroblasts, and endothelial cells, thus modifying the therapeutic response as a function of the distribution of miR-126 among the stromal components, which results in decreased therapeutic efficacy.

DESCRIPTION OF THE INVENTION In order to overcome this and other drawbacks of the prior art, the present inventors have developed a combination anticancer therapy comprising the use of miR-126-enriched exosomes in combination with an exosome release inhibitor. This allows miR-126 to be accumulated within the tumour cells, which results in growth arrest and death of the tumour cells. Advantageously and unexpectedly, the combination treatment of the invention was found to be effective both against the primary tumour, limiting its growth and development, and against cancer stem cells, inhibiting the occurrence of relapses and metastases.

The combination anticancer therapy of the present invention can be performed in two different ways. A first alternative is to administer the exosome release inhibitor and the miR-126-enriched exosomes as two separate preparations. In this case, the exosome release inhibitor should be administered to the patient prior to the miR-126-enriched exosomes, e.g., 6-24 hours before. A second alternative, which has the advantage of reducing the systemic toxicity of the treatment, is the use of exosomes both enriched in miR-126 and containing the exosome release inhibitor; in this case, therefore, miR-126 and the exosome release inhibitor are administered in the same preparation.

Therefore, a first aspect of the present invention is a kit comprising an exosome release inhibitor and exosomes enriched in miR-126 for use in the therapeutic treatment of a tumour sensitive to the tumour suppressor miR-126, wherein said use comprises the administration of the exosome release inhibitor and the subsequent administration of exosomes enriched in miR-126.

A second aspect of the present invention is an exosome comprising miR-126 and an exosome release inhibitor, for use in the therapeutic treatment of a tumour sensitive to the tumour suppressor miR-126. The term “an exosome comprising miR-126 and an exosome release inhibitor” means that the exosome acts as a pharmaceutically acceptable carrier and that miR-126 and the inhibitor are contained within the exosomal vesicle and/or that they (or one of them) are present (or is present) on or in the exosome membrane (in other words, that miR-126 and/or the inhibitor are/is chemically or physically linked to the membrane of the exosome). Exosome release inhibitors are molecules known per se. They act by altering the vesicle transport system or inhibiting lipid metabolism. The most efficient ones inhibit acid sphingomyelinase (aSMase), an enzyme that catalyses the hydrolysis of sphingomyelin to ceramide, a process involved in the formation of exosomes (9). In both aspects of the present invention, any known exosome release inhibitor may be used. A review thereof is provided in (9). Among the known inhibitors, those already used in the clinical practice are preferable, among which GW4869 (dihydro imidazole amide), pantothenic acid (vitamin B5), imipramine (tricyclic antidepressant), and statins (simvastatin) are mentioned by way of example. The most preferred exosome release inhibitor in the context of the present invention is GW4869.

As indicated above, various types of tumours sensitive to the tumour suppressor miR-126 are known. Among these, malignant mesothelioma, lung cancer, pancreatic cancer, colorectal cancer, gliomas, prostate cancer, and breast cancer are mentioned by way of example. The aforesaid tumours are therefore suitable to be treated with the combination anticancer therapy of the present invention.

Exosomes which can be used within the scope of the present invention as carriers for the transport of miR-126 and optionally of the exosome release inhibitor are, for example, those derived from endothelial cells (in particular, human umbilical vein endothelial cells, HUVEC), mesothelial cells (for example, MET-5A cell line), fibroblasts (in particular, dermal fibroblasts), mesenchymal cells (in particular, mesenchymal stem cells, MSC) and embryonic cell lines (for example, HEK293 cell line from human embryonic kidney), with the exception of human embryonic stem cells. Among these, HUVEC cells are most preferred as they are naturally rich in miR-126.

As will be described in greater detail in the following experimental section, the cells used as exosome donors are enriched in miR-126 by cell transfection, and then the exosomes, which as a result are enriched in miR-126, are isolated therefrom.

The terms “exosomes enriched in miR-126” and “miR-126-enriched exosomes” used within the scope of the present specification therefore refer to exosomes derived from donor cells subjected to a miR- 126-enrichment treatment, for example by cell transfection, which as a result have a content of miR-126 higher than that of the same naturally occurring or native cells.

The combination anticancer therapy of the present invention can be administered to the patient through any suitable route of administration. By way of non-limiting example, systemic administration (for example, by intravenous injection or infusion), local (intratumoral) administration and administration by aerosol are mentioned. Those skilled in the art are able to select the route of administration which is most suited to the specific circumstances of the case. They are also able to prepare pharmaceutical formulations containing, separately or in combination, the exosome release inhibitor and the miR- 126- enriched exosomes, by selecting pharmaceutically acceptable excipients, carriers and/or diluents suitable for the preparation of dosage forms appropriate for the selected route of administration.

The experimental examples that follow are provided for illustration purposes only and do not limit the scope of the invention as defined in the appended claims.

In the examples, reference is made to the accompanying drawings, wherein:

Figure 1 is a diagram illustrating the procedure for isolating exosomes after enrichment with miR-126.

Figure 2 depicts diagrams showing miR-126 levels before (CTRL) and after transfection with miR-126-mimic in HUVEC and MET-5A cells. The comparison between the groups was carried out by Student’s t-test; the symbol * indicates significant differences with respect to the CTRL, p < 0.05.

Figure 3 depicts diagrams showing miR-126 intracellular levels and the effect on tumour growth. MPM cells (H28, MSTO-211H, MPP89) were treated with either miR- 126- enriched exosomes (exo-miR), the exosome release inhibitor (GW4869), or a combination thereof (GW-exo-miR). The intracellular content of miR-126 was quantified by qRT-PCR (A) and the growth was determined by MTT test (B). The comparison between the groups was carried out by ANOVA-Tukey’s posthoc; the symbol * indicates significant differences with respect to the CTRL, p < 0.05.

Figure 4 is a drawing of the test performed in vivo.

Figure 5 depicts graphs showing tumour growth kinetics (A) and the tumour volume distribution at day 24 from inoculum (B). The comparison between the groups was carried out by ANOVA and ANOVA-repeated measurements with Tukey’s posthoc; the symbol * indicates significant differences with respect to the CTRL, p < 0.05.

Figure 6 depicts graphs showing body weight change over time (A), and the serum (left panel) and tumour (right panel) levels of miR-126 post-treatment (B). The comparison between the groups was carried out by ANOVA-Tukey’s posthoc; the symbol * indicates significant differences with respect to the CTRL, p < 0.05.

Figure 7 depicts diagrams and micrographs showing miR-126 expression kinetics in MSTO-211H cancer stem cell spheroids (A), and the morphological assessment (brightfield), cell death with propidium iodide (PI) staining (B) after the various treatments: untreated (CTRL), incubated with miR-scramble exosomes (exo-scr), miR- 126-enriched exosomes (exo-miR), GW4869 (GW), GW4869 and exo-scr (GW-exo-scr), and GW4869 and exo-miR (GW-exo-miR). Dead cells are coloured in red. The bar indicates 200 pm. The comparison between the groups was carried out by ANOVA- Tukey’s posthoc; the symbol * indicates significant differences with respect to the CTRL, p < 0.05.

Figure 8 depicts diagrams and micrographs showing miR-126 expression kinetics in MPP89 cancer stem cell spheroids (A), and the morphological assessment (brightfield), cell death with propidium iodide (PI) staining (B) after the various treatments: untreated (CTRL), incubated with miR-scramble exosomes (exo-scr), miR-126-enriched exosomes (exo-miR), GW4869 (GW), GW4869 and exo-scr (GW-exo-scr), and GW4869 and exo- miR (GW-exo-miR). Dead cells are coloured in red. The bar indicates 200 mht. The comparison between the groups was carried out by ANOVA-Tukey’s posthoc; the symbol * indicates significant differences with respect to the CTRL, p < 0.05.

Figure 9 depicts diagrams showing the results of cell death analysis performed by the Annexin test. Treated and untreated MSTO-211H (A) and MPP89 (B) CSCs were collected at 24 and 48 hours, labelled with Annexin and propidium iodide (PI), and assessed by flow cytometry. Annexin-positive cells were in the early stages of apoptosis (early-apoptosis), those labelled for both Annexin and PI were in the late stages of apoptosis (late-apoptosis), and those positive for PI alone died of necrosis. The comparison between the groups was carried out by ANOVA-Tukey’s posthoc; the symbol * indicates significant differences with respect to the CTRL, p < 0.05.

Figure 10 depicts diagrams showing miR- 126 intracellular levels after incubation with exo- miR. Adherent A549 cells and their correspondent cancer stem cells (CSCs) were incubated with MET-5A exo-miR and the intracellular miR- 126 content was determined by qRT-PCR. The comparison between the groups was carried out by ANOVA-Tukey’s posthoc; the symbol * indicates significant differences with respect to the CTRL, p < 0.05.

Figure 11 depicts diagrams showing the results of cell death analysis performed by the Annexin test. A549 cells were treated with exo-miR and GW, alone or in combination. After 24 hours of incubation, they were collected, labelled with Annexin and propidium iodide (PI), and assessed by flow cytometry. Annexin-positive cells were in the early stages of apoptosis (early-apoptosis), those labelled for both Annexin and PI were in the late stages of apoptosis (late-apoptosis), and those positive for PI alone died of necrosis. The comparison between the groups was carried out by ANOVA-Tukey’s posthoc; the symbol * indicates significant differences with respect to the CTRL, p < 0.05.

Figure 12 depicts diagrams and micrographs showing the results from cell death assessment of A549-CSC cancer stem cells by morphological analysis (A), and Annexin test (B) after the various treatments: untreated (CTRL), incubated with miR-126-enriched exosomes (exo-miR), with GW4869 (GW), and with the GW-exo-miR combination. The bar indicates 200 pm. The comparison between the groups was carried out by ANOVA- Tukey’s posthoc; the symbol * indicates significant differences with respect to the CTRL, p < 0.05.

Figure 13 depicts chromatograms and diagrams showing the results of western blot analysis. MSTO-211H (A) and MPP89 (B) cancer stem cells were treated with miR- scramble exosomes (exo-scr), miR-126-enriched exosomes (exo-miR) alone and in combination with GW4869 (GW, GW-exo-scr, GW-exo-miR), and expression levels of p- mTOR, mTOR, ULK, LC3-I and LC3-II, and p-p70S6K, p70S6K were assessed by western blot, and the densitometric values of the bands compared to Actin are shown in Figures C and D. The comparison between the groups was carried out by ANOVA- Tukey’s posthoc; the symbol * indicates significant differences with respect to the CTRL, p < 0.05.

Figure 14 illustrates the morphological assessment of cancer stem cells under the transmission electron microscope (TEM). MSTO-211H cancer stem cells (A) were treated with exo-scr (B), exo-miR (C), GW4869 (D) and the GW-exo-scr combination (E), GW- exo-miR (F), and the morphology was assessed by electron microscopy. N=nucleus, m= mitochondrion, a=autophagosome, * mass of damaged, undigested organelles.

EXPERIMENTAL EXAMPLES

EXAMPLE 1: Preparation of exosomes from miR-126-enriched HUVECs miR-126-Enrichment ofHUVEC cells

Exosomes were obtained using HUVEC cells as the donor cells, since they are naturally rich in miR-126, enriched in miR-126 by the cell transfection technique. The HUVEC cell enrichment procedure is described below.

Day-1. HUVECs (GIBCO, C-003-5C) were seeded at the concentration of lxlO ⁶ cells in a T75 flask and maintained in medium- 200 (GIBCO) supplemented with LYES (GIBCO) in an incubator at 37°C, 5% CO2.

Day-2. The culture medium was replaced with medium-200 plus LVES depleted of exosomes by ultracentrifugation at 100,000 g for 4 hours at 4°C.

The HUVECs were transfected with MISSION-microRNA mimic (Sigma, cod MI00200) using ‘HiPerFect transfection reagent’ (Qiagen, cod 301705) as the transfecting agent.

Each T75 HUVEC flask was added with the transfection solution obtained as follows: 1.875 ml of LVES-free medium-200 plus 50 pi mimic (20 pM); after gentle pipetting, 56 pi of ‘HiPerfect transfection reagent’ were added, followed by gentle pipetting and incubation for 15 min at room temperature. The HUVECs were kept in an incubator at 37°C, 5% C0 ₂ .

Day-5, the medium was collected and sequentially centrifuged for 10 min at 500 g to remove dead cells and for 10 min at 1400 g to remove apoptotic bodies, all at room temperature (RT). After centrifugation, the supernatant was stored at -20°C.

Isolation of miR-126-enriched exosomes

The supernatant collected and stored at -20°C was filtered with 30 mm-diameter, 0.22 pm- pore size PVDF filters (Eurozone cod EPFPV-2230), concentrated on 100 kDa-cut off columns (VIVASPIN-20, MWCO-PES, Sartorius cod VS2041) by centrifugation at 700 g for 15 min at RT and, finally, centrifuged at 10,000 g for 15 min at RT.

The concentrates were layered on a sucrose gradient (20 mM TRIS/30% sucrose in D2O at pH 7.4) in ultracentrifugation tubes (10 ml) and ultracentrifuged at 100,000 g for 40 min at 4°C.

The gradient at the bottom of the tube was recovered, washed in PBS, and ultracentrifuged at 100,000 g for 70 min at 4°C (Figure 1). All ultracentrifugations were performed with a TLS-55 swinging-bucket rotor ultracentrifuge (BD, Beckton Dickinson). The exosome pellet was resuspended in PBS (500 pi), incubated with RNase A (0.1 mg/ml) for 30 min at 37°C, filtered through 0.22 pm-pore size, and the protein content was quantified by the Bradford method (Sigma). The exosomes thus obtained can be stored at a temperature of -80°C until use.

EXAMPLE 2: Study on Malignant Pleural Mesothelioma (MPM)

Cancer cells are located within an environment called stroma, which consists of several cellular components, including a cancer stem cell (CSC) sub-population. While primary tumour cells are involved in the development and growth of the mass, CSCs are involved in tumour formation and, as they resistant to conventional therapies, are responsible for relapses and distal spread through metastases.

Therefore, cancer therapy has two main objectives: (i) eliminating the primary tumour by acting on cell growth and death, and (ii) eliminating relapses and spread (metastasis) by acting on cancer stem cells (CSCs).

Malignant pleural mesothelioma (MPM) is an aggressive tumour with few therapeutic approaches. Therefore, the effectiveness of the combination therapy of the present invention has been assessed in adherent tumour cells (primary tumours) and in corresponding MPM CSCs (relapses and metastases).

MATERIALS AND METHODS

Cell cultures

Non-malignant mesothelial (MET-5A), malignant sarcomatoid- (H28), biphasic- (MSTO- 211H) and epithelial- (MPP89) histotype mesothelioma, and non-small cell lung cancer (NSCLC, A549) cell lines were obtained from the American Type Culture Collection (ATCC). MET-5A, H28, MSTO-211H and A549 cells were cultured in RPMI medium supplemented with 10% FBS, 1% penicillin and 1% streptomycin (Life Technologies), whereas MPP89 cells were maintained in Ham’s F10 medium with 15% fetal bovine serum (FBS) supplemented with glutamine (2 mM) and antibiotics. The cell cultures were kept in a humidified incubator at 37°C and 5% CO2. The cells were periodically checked for the absence of mycoplasma contamination by using the mycoplasma PCR test. The cellular genetic profile was performed by using a PowerPlex Fusion 6C system (Promega, Fitchburg, WI).

Formation of cancer stem cells (spheroids)

Cancer stem cells (CSCs) in three-dimensional spheroids were obtained by culturing the MSTO-211H, MPP89 (mesospheres) and A549 cell lines in 24- or 96-well Ultra-Low Attachment plates (Coming Life Sciences) at a density of 10 ⁴ cells/ml or 10 ³ cells/ml, respectively, in serum-free DMEM-F12 (Euroclone) supplemented with 1 x B27 (Invitrogen), 20 ng/ml fibroblast growth factor (bFGF; Millipore), 20 ng/ml epidermal growth factor (EGF; Sigma). The plates were incubated at 37°C in a humidified atmosphere with 5% CO2. The culture medium was changed every 3 days and 400-500 m m-diameter spheroids were formed after 6 days of incubation. Spheroid formation was monitored using a lOx Leica microscope (Leitz, Inc.) with a Spot Insight 3.2.0 camera and Spot Advanced software (Spot Imaging).

Treatments

MPM and A549 cell lines, and MPM- and A549- derived spheroids were treated with 20 pg/ml of miR-126-enriched exosomes (exo-miR) or miR-scramble-enriched exosomes (exo-scr), with and without pre-treatment with the GW4869 exosome release inhibitor (20 mM in DMSO, Sigma- Aldrich).

Apoptosis

Apoptosis was quantified using the V-FITC Annexin method and propidium iodide (PI). Briefly, MPM and A549 adherent (10 ⁵ ) or stem (10 ⁴ ) cells were placed in 24-well Ultra- Low Attachment plates. Subsequently, the cells were treated with exo-miR or exo-scr, with and without GW4869 pre-treatment (20 mM). The cells were collected, washed twice with PBS, resuspended in 0.1 ml of buffer (10 mM HEPES, 140 mM NaCl, 5 mM CaCk, pH 7.4), incubated for 20 min at room temperature with 2 pi of V-FITC Annexin and with 10 pi of PI (10 pg/ml). After incubation, the cells were analysed by flow cytometry (Becton Dickinson, Rutherford, NJ, USA).

PI staining was assessed with an AxioCam MRc5 fluorescence microscope (Zeiss Imager Al).

Determination of cell proliferation

Cell proliferation was assessed by the MTT test. MPM cells (10 ⁴ ) were placed in 96-well plates, and after a 24-hour treatment with either miR-126-enriched exosomes (exo-miR), GW4869 (GW), or a combination thereof, 10 pi of MTT (5 mg/ml in PBS; Sigma) were added to each well. After 3 hours of incubation, the crystals formed were dissolved in isopropanol. The absorbance at 550 nm was read with an ELISA plate reader (Sunrise, Tecan). miR-126 quantification by quantitative RT-PCR ( qRT-PCR ) miR-126 tissue expression was determined from the total RNA obtained using the RNeasy kit (Qiagen). Whereas exosomal miR-126 expression was obtained by isolating total RNA with trizol/chloroform (Sigma) followed by miRNA enrichment with Purelink miRNA- isolation kit (Applied Biosystems, Life Technologies) following the manufacturer's protocol.

Single-stranded cDNAs were synthesized from mRNAs with the High capacity cDNA reverse- transcription kit system (Applied Biosystems, Life Technologies). qRT-PCR reactions were performed using TaqMan® Fast Advanced Master Mix (Applied Biosystems, Life Technologies) and U6 as an internal normalizes cel-miR-39 was added to the exosomal solution and used as an external normalizer. miR-126 was detected in serum and cell samples as described above (10,11). Briefly 2.5 pi of serum were added with 2.5 mΐ of a buffer containing: 2.5% Tween 20 (Euroclone), 50 mmol/L Tris (Sigma- Aldrich) and 1 mmol/L EDTA (Sigma- Aldrich).

5 mΐ of RT reagents were added to 5 mΐ of this mixture. Whereas the cell lysate was obtained by lysing 10 ³ -10 ⁴ cells with a lysis solution containing: triton-X (2%), NP40 (2%), DNAse (2 mΐ) in a total volume of 40 mΐ. Incubation for 30 min at 37°C, and finally at 70°C for 10 min. 10 mΐ of RT reagents were added to 5 mΐ of cell lysate.

The qRT-PCR reactions were carried out by implementing the following protocol: a 2-hour incubation at 37°C was followed by enzymatic inactivation for 5 minutes at 95°C. The cDNA obtained was centrifuged at 9000 g for 5 min to remove the protein precipitate. A volume of 1.33 mΐ of the cDNA solution was used as the template for the qPCR. The qPCR conditions were 60°C for 2 min, 95°C for 10 min, in 40 cycles of 95°C for 15 sec and 60°C for 1 min. The qRT-PCR reactions were performed in duplicate using TaqMan® Fast Advanced Master Mix (Applied Biosystems, Life Technologies) and U6 for normalization.

Western blot analysis

The spheroids were lysed in RIPA buffer containing NasVCC (1 mM) and a protease inhibitor mixture (1 pg/ml). Protein concentration was assessed by the Bradford assay. The lysates (10 pg of proteins) were separated using a 4-12% SDS-PAGE gradient gel (Life Technologies), and then transferred onto a nitrocellulose membrane (Protran). After incubation with 5% skimmed milk in PBS-Tween (0.1%), the membranes were incubated overnight at 4°C with primary antibodies against LC3B, phospho-mTOR (p-mTOR, Ser- 2448), mTOR, phospho-p70S6K, P70S6K and ULK1 (Cell Signaling). b-Actin (Cell Signaling) was used as a loading control. After incubation with HRP-conjugated IgG secondary antibody (Cell Signaling), the blots were developed using ECL (Pierce). Band intensities were visualized and quantified with ChemiDoc using the Quantity One software (Bio-Rad Laboratories).

Animal studies All animal experiments were approved by the Institutional Committee for Animal Care and Use of the International Centre for Genetic Engineering and Biotechnology, Trieste. All animal experiments were carried out according to standard guidelines and followed the best practice procedures. Female NOD/SCID/IL2Rynull (NSG) mice (4-6 weeks, 28 ± 3 g) were used in the study. MSTO-211H cells (1.5 x 10 ⁶ ) were injected subcutaneously into the suprascapular area of the animals. A total of 30 animals were randomly divided into 5 groups with 5 mice in each group; the control group (CTRL) treated with PBS, the group treated with miR-scramble exosomes (exo-scr, 0.7 mg/kg), the group treated with miR- 126-enriched exosomes (exo-miR, 0.7 mg/kg), the group treated with GW4869 (GW, 1.5 mg/kg), the group treated with GW4869 and miR-scramble exosomes (GW-exo-scr), and the group treated with GW4869 and miR-126-enriched exosomes (GW-exo-miR). When the tumour reached a volume of 12 ± 4 mm ³ , the mice were treated intratumorally with 50 mΐ of PBS, exo-scr, exo-miR, alone or in combination with a 6-hour pre-treatment with GW (50 pi, 0.3 mg/ml). 3 treatments with 3-day intervals were carried out (day-10, day-13, day- 16). Tumour size and body weight were measured every 3 days from the day of treatment (day-10) to the day of death (day-30). The tumour volume was quantified with a caliper and analysed using the following formula: Tumour volume (mm ³ ) = 0.5 x length x (width ² ) and the tumour growth was expressed as tumour volume/initial tumour volume x 100. No mice died during the study period.

Transmission Electron Microscopy (TEM)

MSTO-211H cancer stem cells treated with miR-scramble exosomes (exo-scr), miR-126 exosomes (exo-miR), GW4869 (GW), GW-exo-scr, GW-exo-miR, and untreated (CTRL) were collected, fixed in 2% paraformaldehyde for 60 min, and centrifuged to form pellets. The pellets were included in 1% low-melting agarose, fixed in 0.5% osmium tetroxide for 60 min at RT, dehydrated in acetone, and embedded in an epoxy mixture. Thin sections were obtained with a Reichert Ultratome (Reichert Technologies, Depew, NY, USA), stained with lead citrate, and examined using the Philips CM 10 transmission electron microscope (Philips, Eindhoven, the Netherlands). Statistical analysis

The data are presented as means + standard deviations (SD). Comparisons between two groups were performed using Student’s t-test. Whereas analysis of variance (ANOVA) and ANOVA for repeated measurements with Tukey’s post-hoc were used for multiple comparisons. A p < 0.05 value indicated statistical significance. All statistical analyses were performed using the SPSS software.

RESULTS

Enrichment of exosomes with miR-126

Transfection of the exosome-donor HUVEC and MET-5A cells, transfected with miR-126- mimic, resulted in an approximately 300-fold increase in the content of miR-126 within the exosomes compared to the control (Figure 2).

Combined GW-exo-miR treatment induces tumour growth arrest

Treatment of the different-histotype H28 (sarcomatoid), MSTO-211H (biphasic) and MMP89 (epithelioid) MPM adherent cells with HUVEC exosomes enriched in miR-126 (exo-miR, 20 pg/ml) in combination with GW4869 (GW-exo-miR, 20 mM) significantly increased miR-126 intracellular content (Figure 3 A). This increase was associated with inhibition of proliferation (Figure 3B).

Tumour growth arrest was also observed in an animal model in vivo (Xerograph-SCID mice). 30 mice were inoculated with adherent MSTO-211H cells, and after 10 days, when the tumour mass had reached a volume of 10-13 mm ³ , the mice were treated intratumorally (i.t.) at day-10, day-13 and day-16 with PBS (CTRL group, n=5), with miR-scramble- enriched exosomes (exo-scr group, n=5), and with miR-126-enriched exosomes (exo-miR group, n=5), alone or after a 6-hour pre-treatment with GW4869 (GW group, n=5; GW- exo-scr group, n=5; GW-exo-miR group, n=5). Tumour volume and animal body weight were monitored every three days for approximately 20 days after the appearance of the tumour mass. A blood sample was taken at times T-0, T-16 and T-30, and after 30 days from inoculation (T-30), the animals were sacrificed, the blood was taken, and tumours and certain organs (liver, lung, kidney) were collected (Figure 4).

As shown in Figure 5A, i.t. administration of exo-miR alone inhibited tumour growth, which was significantly inhibited when exo-miR was combined with GW (32% tumour growth reduction). Tumour growth inhibition was observed 24 days after inoculation (T- 24) even when GW was combined with exo-scr i.t. (Figure 5B), but this effect was lost after an extended time.

The treatments did not affect the body weight of the animals (Figure 6A). By quantifying miR-126 in serum and in the tumour mass, treatment with exo-scr and exo-miR was found to cause increased miR-126 in semm, whereas miR-126 remained at a low concentration at the tumour level (Figure 6B). Interestingly, pre-treatment with GW4869 resulted in miR- 126 accumulation at the tumour level with consequent low release of miR-126 into the circulatory stream (Figure 6B).

Combined GW-exo-miR treatment induces cancer stem cell (CSC) death.

Cancer stem cells (CSCs) are involved in the occurrence of relapses and metastases, so it is of great importance to find a therapy that can induce their death.

In this study, MPM cells (MSTO-211H and MPP89) were transformed into stem cells by growing them in appropriate culture media as described in Materials and Methods, and then treated with HUVEC exosomes as such (exo-scr), with miR-126-enriched exosomes (exo-miR), alone or in combination with the exosome release inhibitor GW4869 (GW). Treatment of MSTO-211F1 (biphasic histotype) CSCs with exo-scr, and even more with exo-miR, resulted in an increase in the intracellular content of miR-126 after 6 hours from treatment, which then decayed over time (Figure 7A). This increase, although transient, induced the formation of dark areas of clustered cells in the central part of the tumour masses (Figure 7B). Pre-incubation with GW4869 resulted in significant accumulation of miR-126 (> 300-fold) in the tumour cell at 6-12 hours after exo-miR treatment, which was associated with tumour cell cleavage and massive cell death (increase in red nuclei) observed after 24 hours of incubation (Figure 7A,B).

Similarly, the GW4869 and exo-miR combination (GW-exo-miR) resulted in intracellular accumulation of miR-126 in MPP89 cells (epithelial histotype) as soon as 24 hours after treatment (Figure 8A). This increase was associated with cell death as evidenced by propidium iodide (PI) staining (Figure 8B).

The Annexin test with PI was performed to assess the type of cell death through which the GW-exo-miR combination induced cytotoxicity. This test is able to distinguish the viable cells from those dead due to early apoptosis, late apoptosis, and necrosis. As shown in Figure 9, the GW4869 with exo-miR combination induced the death of 100% of CSCs after 48 hours from treatment, whether MSTO-211Fl-derived (Figure 9A) or MPP89- derived (Figure 9B). Cancer cells died due to necrotic processes. The cell that undergoes necrosis loses the ionic selectivity of the cell membrane system, which causes the swelling of the cytoplasm and cell organelles (mitochondria, endoplasmic reticulum, lysosomes) with consequent loss of their structural organization.

Example 3 : Lung cancer studies

Lung adenocarcinoma is one of the tumours sensitive to the miR-126 tumour suppressor. Adherent non-small cell lung cancer (NSCLC) A549 cells and their corresponding stem cells (CSCs) were treated with exo-miR alone or in combination with the exosomal inhibitor GW4869 and cell death was assessed morphologically and with the Annexin test.

Exosomes from mesothelial cells (MET-5A) enriched in miR-126 were used in the treatment. As shown in Figure 2, MET-5A exosomes exhibited miR-126 levels comparable to HUVEC-derived exosomes, but unlike the latter resulted in a lower intracellular level of miR-126: 12-20-fold increase with exo-MET-5A (Figure 10) versus 300-400-fold increase with exo-HUVEC (Figures 7-8).

As shown in Figure 11, GW4869 treatment induced death of adherent A549 cells (50%) by necrosis, the combination further increased cell death (60-70%).

Less efficiently, GW4869 treatment induced death of A549 cancer stem cells (CSCs), and the combination slightly increased death (Figure 12).

Example 4: Autophagy is involved in death induced by the combination of GW4869 and exo-miR

Autophagy is a process of adaptation to stress that protects against cell death; at other times, however, it becomes an alternative path to cell death, called “Type P cell death” or “autophagic death”. In some circumstances, therefore, apoptosis and autophagy can exert synergistic effects, whereas in other situations autophagy can only be induced when apoptosis is suppressed.

In this study, treatment with GW4869, and even more in combination with exo-scr and exo-miR, was found to decrease LC3-I and LC3-II expression, increase mTOR and p70S6K, with consequent inhibition of the autophagy process in both MSTO-211H (Figure 13A,C) and MPP89 (Figure 13B,D) CSCs.

CSCs use autophagy processes to maintain their homeostasis and enhance resilience under stress, such as lack of nutrients and hypoxia, thus promoting their survival.

Inhibition of the aSMase enzyme by GW4869, in addition to inhibiting the release of exosomes into the extracellular space, results in inhibition of the autophagosome-lysosome fusion, thereby inhibiting the autophagy process. In conclusion, the GW-exo-miR combination induces cell death through inhibition of autophagy mediated by activation of the mTOR pathway.

The autophagy mechanism is also supported by morphological assessment by electron microscopy. Treatment with exo-miR caused an increase in the autophagy flow, with the formation of numerous phagosomes and autophagosomes (a). In contrast, treatment with GW4869 inhibited the formation of autophagosomes, and its combination with exo-scr and even more with exo-miR resulted in accumulation of unrepaired and undigested damaged organelles responsible for cell death (Figure 14).

Example 5: Preparation of exo-miR- 126-GW4869

Isolated exo-miR-126 (20 pg/ml, 4xl0 ¹⁰ exosomes) were incubated with GW4869 (10 mM) in 1 ml of PBS and sonicated using a Model 505 Sonic sonicator under the following conditions: 6 cycles of 30 sec for 3 min with 2-min cooling intervals between cycles. After sonication, the exo-miR- 126-GW4869 were incubated at 37°C for 60 min to allow recovery. The exosomes thus obtained were washed in PBS and ultracentrifuged at 100,000 g for 70 min at 4°C.

The combined effect of miR-126 and the drug GW4869 contributes to the accumulation of the miRNA within the cell by blocking the formation and release of exosomes.

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