WO2016102735A1

WO2016102735A1 - Stem cells irradiated for cancer treatment

Info

Publication number: WO2016102735A1
Application number: PCT/ES2015/070951
Authority: WO
Inventors: José Mariano RUIZ DE ALMODÓVAR RIVERA; Virginea DE ARAÚJO FARIAS; Jesús Joaquín LÓPEZ PEÑALVER; María del Carmen RUIZ RUIZ; Francisco Javier OLIVER POZO; Borja ALONSO LERMA; Rocío SEGUI JIMÉNEZ; Ana GUERRA-LIBRERO RITE; Beatriz Irene FERNÁNDEZ GIL
Original assignee: Universidad De Granada; Servicio Andaluz De Salud; Consejo Superior De Investigaciones Científicas
Priority date: 2014-12-23
Filing date: 2015-12-23
Publication date: 2016-06-30

Abstract

The invention relates to a method for producing activated stem cells, comprising administering low doses of radiation, and to the uses thereof for producing pharmaceutical compositions that can be used to treat locally advanced tumours and cancer on a systemic level.

Description

IRRADIATED MOTHER CELLS FOR CANCER TREATMENT

FIELD OF THE INVENTION

The present invention is within medicine, and more specifically within cell therapy, and refers to the use of stem cells activated by low doses of radiation for the treatment of cancer.

BACKGROUND OF THE INVENTION

Radiation therapy has consistently remained one of the most effective treatments for cancer and approximately 50% of all cancer patients receive radiation therapy in some form, either isolated or in combination with other treatment modalities, at some time during their management. For many tumors and locations, however, a definitive cure cannot be achieved even though cancer treatments are becoming more effective. Reliable information on the relative roles of surgery, radiotherapy, chemotherapy and new areas of cancer drug development is difficult to obtain, but any improvement in the effectiveness of radiotherapy will undoubtedly benefit a significant number. of patients (Begg et al., 201 1. Nat Rev Cancer 1 1, 239-253). Knowledge of the mechanisms by which radiation induces cell death is based on data on cell survival and radiation-induced cell damage (Peacock et al, 1992. BJR supplement, 24, 57-60; Ruiz de Almodóvar et al, 1994b. International journal of radiation biology 65, 641-649; Steel et al, 1989, International journal of radiation biology 56, 1045-1048) and in the consequences that this damage generates in both tumor cells (McMillan and Peacock, 1994. International journal of radiation biology 65, 49-55) and at the level of normal tissues (Ruiz de Almodóvar et al, 1994a. International journal of radiation biology 65, 641-649; López et al, 2005 Breast cancer resea re h : BCR 7, R690-698; López et al, 2002. Breast cancer research and treatment 73, 127-134).

The models proposed so far to explain the result of the therapy on tumor growth and the adverse effects caused, suggest that in addition to DNA damage (McMillan et al., 2001. International journal of radiation oncology, biology, physics 49 , 373-377) the cellular signaling processes promoted by radiation and reaching locations outside the irradiated region - abscopal and bystander effects (Formenti and Demaria 2009. Lancet Oncol 10, 718-726; Mothersill and Seymour, 1997. International journal of radiation biology 71, 421-427; Mothersill and Seymour, 2004. Nat Rev Cancer 4, 158-164) may be crucial in assessing the overall effect of radiotherapy (Gómez-Millán et al, 2012. Radiotherapy and Oncology , Journal of the European Society for Therapeutic Radiology and Oncology 102, 450-458). These evidences force the development of a new model capable of explaining the effect of radiation on the tumor and healthy tissues simultaneously (Lambin et al., 2013. Nat Rev Clin Oncol, 10, 27-40;). In fact, communication between the sublettally damaged cells and the undamaged ones can lead to the reduction of the population of the residual cancer cells to the treatment (Prise and O'Sullivan, 2009. Nat Rev Cancer é, 351-360).

Radiation therapy is a very effective tool to eradicate tumor cells. As with many other medical procedures, the prescription of a radiotherapy cycle should assess the balance between risk and benefit, whose relative weight will be determinant of therapeutic gain. Today we understand that antineoplastic therapy could have an additional decisive effect: the response of sub-lethally injured tumor cells (Marples et al, 2003; Mothersill and Seymour, 2004; Prise and O'Sullivan, 2009. Nat Rev Cancer 9, 351-360) can be described as an interaction between a ligand and its receptor (Gómez-Millan et al., 2012. Radiotherapy and oncology: journal of the European Society for Therapeutic Radiology and Oncology 102, 450-458). Shortly after each radiotherapy session, the cells included in the irradiation volume are classified, for operational purposes, as follows:

A) Dead cells: Cells that have suffered irreparable injuries; these are those included in the compartment called lethal injury of the Curtís model (Curtís, Radiat Res. 1986, 106 (2): 252-270)

B) Committed cells: These are cells affected by damage to their DNA and whose fate depends on a competitive process between the repair and fixation of the radio-induced lesions. They are those that are included in the compartment of potentially lethal lesions and responsible for the curvature of the cell survival curves to radiation (Peacock, et al. 1992, BJR Supplement 24, 57-60).

C) Activated cells: Cells that have been minimally damaged or that have been able to repair the induced lesions to a level of residual damage compatible with the survival of said cells. These cells have been found to be an active source of cytokines, reactive oxygen species and reactive nitrogen species that, through extracellular space or through blood and / or lymphatic circulation, interact and affect the total tumor mass.

D) Unscathed cells: Cells that survive after each fraction of the dose and that forces the prolongation of the treatment with successive dose fractions to achieve tumor control. The probability of controlling the tumor depends essentially on the nature and extent of the tumor, on therapeutic factors such as total dose, dose per fraction and time, and on biological factors such as radiosensitivity and likelihood of adverse side effects.

The population of cells in compartment C is responsible for: a) the bystander effect of short range (Prise KM &O'Sullivan JM, 2009. Nat Rev Cancer 9 (5): 351-360) that is produced by direct cell-cell contact through the gap-junction and by free radical-mediated communications, and b ) of the distant bystander effect of the tumor - abscopal effect - (Formenti SC & Demaria S, 2009. Lancet Oncol 10 (7): 718-726).

According to this model, the bystander action of radiation should be considered as the overall immune response of the tumor and its metastases, if any, and of healthy peritumoral tissues, to radiation-induced stress on the white volume (López E, et al. (2005) Breast Cancer Res 7 (5): R690-698; Pinar et al. 2007, Radial Res. 168 (4): 415-22. Previous results support the hypothesis that the bystander effect may be a generalized phenomenon which occurs when tumor cells are in contact with the extracellular medium conditioned by irradiation of other cells of the same type, however, normal cells, represented in our previous experiments by mesenchymal stem cells derived from the umbilical cord stroma (UCSSC) (Farías et al., 2011. Placenta, 32 (1): 86-95), although extremely sensitive to ionizing radiation, are resistant to any bystander effect induced by the irradiated conditioned medium of tumor cells (RCM) at doses between 0 and 8 Gray (Gómez-Millán et al., 2012. Journal of the European Society for Therapeutic Radiology and Oncology 102, 450-458).

MSC cells have been investigated for the treatment of cancers due to the fact that these cells are able to preferentially lodge in the stroma of tumors and it is known that the incorporation of MSCs into the tumor is increased with antitumor radiotherapy. There is detailed literature that supports the fact that for almost all blood vessels in the body, mesenchymal cells are observed in perivascular locations (Caplan and Correa, 201 1; Cell Stem Cell. 9, 1 1-15; Farias et al, 201 1, Placenta, 32 (1): 86-95). MSCs can either suppress or promote tumors (Bergfeld et al, 2014; Mol Cancer Ther. 13, 962-75. Yagi and Kitagawa, 2013, Front Genet. 4, 261). The existing information proposes that the sequential activation of the MSCs in response to tissue lesions could have a role in tissue regeneration due to the activation of these mobilized and activated MSCs, they act as "medicine" (MSC *) and secrete factors that organize a microenvironment capable of promoting cell therapy (Caplan and Dennis, 2006, J Cell Biochem, 98, 1076-84). It has been described that the bioactivation of the MSCs can be achieved by different treatments and that the molecules secreted by the activated MSCs (MSCs *) affect various immune cell lineages to establish a potent therapeutic microenvironment (Lee et al., 2012, Cell Stem Cell, 2012, 825-35). On the other hand we also know that low doses of radiation contribute to normalize the aberrant tumor vasculature. It would therefore be important to find alternative methods of activating MSCs so that they secrete the molecules into the environment that favors the creation of a therapeutic microenvironment. BRIEF DESCRIPTION OF THE INVENTION

The present invention shows a new therapy, based on radiotherapy, useful for the treatment of locally advanced tumors and cancer at the systemic level.

A first aspect of the invention relates to a method of obtaining activated stem cells, hereafter referred to as the method of the invention, which comprises administering low doses of radiation to stem cells. In a preferred embodiment of this aspect of the invention the stem cells are irradiated with a dose between 0.03 cGy and 6 Gy, more preferably with a dose between 0.25 Gy and 4Gy, and even more preferably with a dose of approximately 2 Gy.

In another preferred embodiment of this aspect of the invention, the stem cells are mesenchymal stem cells.

A second aspect of the invention relates to an activated stem cell, hereinafter activated stem cell of the invention, obtained or obtainable by the process of the invention. Therefore, an activated stem cell of the invention is understood as a stem cell obtained by the process of the invention. In a more preferred embodiment, this aspect of the invention relates to a population of activated stem cells, hereinafter population of activated stem cells of the invention, comprising at least one activated stem cell of the invention. In another preferred embodiment of this aspect of the invention, the stem cells are mesenchymal stem cells.

A third aspect of the invention relates to a composition, hereinafter composition of the invention, comprising at least one activated stem cell of the invention or a population of activated stem cells of the invention. In a preferred embodiment of this aspect, the composition of the invention further comprises a pharmaceutically acceptable carrier. In a preferred embodiment of this aspect the composition of the invention further comprises another active ingredient. In another more preferred embodiment, the active ingredient is a PARP (poly (ADP-ribose) polymerase) inhibitor.

A fourth aspect of the invention relates to a combined preparation, hereafter combined preparation of the invention, comprising: a) a component A that is at least one activated stem cell or a population of activated stem cells of the invention, or a composition of the invention, and,

b) a component B that is a chemotherapeutic agent and / or an antiantiangiogenic agent.

In a preferred embodiment of this aspect the combined preparation of the invention further comprises at least one PARP inhibitor.

A fifth aspect of the invention relates to the use of an activated stem cell of the invention, of a composition of the invention, or of the combined preparation of the invention, in the manufacture of a medicament.

A sixth aspect of the invention relates to the use of an activated stem cell of the invention, of a composition of the invention, or of the combined preparation of the invention, in the preparation of a medicament for the treatment of cancer. In a preferred embodiment of this aspect of the invention, the cancer is a locally advanced or systemic cancer.

A seventh aspect of the invention relates to a method of treating cancer, hereinafter the first method of treating cancer of the invention, comprising: a) administering the activated stem cells of the invention, and

b) administer radiotherapy at conventional doses following the usual protocols. In a preferred embodiment, step (b) comprises administering to the patient a low dose of radiation on the volume occupied by the tumor in an effective amount (2-6 Gy) to activate the tumor cells and favor the tropism of the stem cells towards the tumor

Preferably the stem cells are mesenchymal stem cells (MSC), and / or the activated stem cells are activated mesenchymal stem cells (MSC *).

In another preferred embodiment, new intravenous administrations can subsequently be made, of activated stem cells, preferably where the stem cells are mesenchymal stem cells, and subsequent administration can be both MSC cells and MSC * cells or cells of the invention, by example, in the days of pause of radiotherapy. In a preferred embodiment of this aspect the first cancer treatment method of the invention further comprises administering at least one PARP inhibitor. In another preferred embodiment of this aspect the first cancer treatment method of the invention further comprises administering at least one chemotherapeutic agent and / or an antiangiogenic agent. In another preferred embodiment of this aspect the cancer is a systemic cancer.

An eighth aspect of the invention relates to a method of treating cancer, hereinafter second method of treating cancer of the invention, comprising:

a) administer untreated stem cells, allow them to bio-distribute and, after 24 hours, b) administer radiotherapy at conventional doses following the usual protocols.

In this procedure it is assumed that the activation of mesenchymal cells will occur in situ in coincidence with the irradiation of the tumor.

In a preferred embodiment of this aspect the second cancer treatment method of the invention further comprises administering at least one PARP inhibitor. In another preferred embodiment of this aspect the second cancer treatment method of the invention further comprises administering at least one chemotherapeutic agent and / or an antiangiogenic agent. In another preferred embodiment of this aspect the cancer is a systemic cancer.

In another preferred embodiment, the stem cells will be injected the next day and after 24 hours of waiting the radiation treatment will continue. New intravenous administrations of activated stem cells may be made on the days of radiation therapy pause. In a more preferred embodiment of the invention, the stem cells are mesenchymal stem cells.

DESCRIPTION OF THE FIGURES

Figure 1. Quantitative RT-PCR for TRAIL, DR5 and DKK3 mRNA in MSC cells of human umbilical cord stroma in basal conditions and after stimulation of cells with 2 Gy doses of low linear energy transfer radiation (X-rays or γ). The evolution of the expression levels of each of the genes studied is followed over time (interval between 0 and 30 hours after stimulation) and the differences with respect to the baseline values are calculated using the method 2 ^ΔΔα . In the Y axis, the values are mean ± ESM of triplicates of 3 independent trials (* P <0.05; ** P <0.01; *** P <0.001 **** P <0.0001) . The results are clearly indicative of upward regulation of TRAIL at 24 and 30 hours, DR5 after 4 hours and DKK3 at 30 hours of irradiation. It is important Understand that the values of DKK3 are of the order of 5 times greater at 30 hours of the stimulus than those found in basal cells.

Figure 2. Quantitative RT-PCR for TRAIL and DR5 and DKK3 mRNA in MSC cells of human umbilical cord stroma under basal conditions and after stimulation of cells with 6 Gy doses of low linear energy transfer radiation (X-rays) or γ). The evolution of the expression levels of each of the genes studied is followed over time (between 0 and 30 hours after stimulation) and the differences with respect to the baseline values are calculated using the 2 ^_ΔΔα method. In the Y axis, the values are mean ± ESM of triplicates of 3 independent trials (* P <0.05; ** P <0.01; *** P <0.001 **** P <0.0001) . After 6 Gy the upward regulation of TRAIL is not evidenced while the increase in cellular component (up-regulation) of DR5 occurs at 4, 24 and 30 hours after treatment. The up-regulation of of DKK3 is evident at 24:30. Figure 3. Quantitative RT-PCR for TRAIL and DR5 and DKK3 mRNA in human melanoma A375 and G361 cell lines and in MCF7 (human breast cancer) at baseline conditions and after stimulation of cells with a dose of 2 Gy of Low linear energy transfer radiation (X-ray or γ). The evolution of the expression levels of each of the genes studied is followed over time (between 0 and 30 hours after stimulation) and the differences with respect to the baseline values are calculated using the 2 ^_ΔΔα method. In the Y axis, the values are mean ± ESM of triplicates of 3 independent trials (* P <0.05; ** P <0.01; *** P <0.001 **** P <0.0001) . The expression of these genes also changes in the cell lines tested. Most relevant is the up-regulation of DR5 and DKK3 in A361 which indicates a possible radiosensitizing effect and the up-regulation of TRAIL in MCF7. Of great interest is the absence of DKK3 in this cell line. Note that the corresponding graph is missing.

Figure 4. Another quantitative RT-PCR for TRAIL and DR5 and DKK3 mRNA in another trial on human melanoma A375 and G361 cell lines and in MCF7 (human breast cancer) at baseline conditions and after dose cell stimulation 2 Gy of low linear energy transfer radiation (X or γ rays). The evolution of the expression levels of each of the genes studied is followed over time (interval between 0 and 30 hours after stimulation) and the differences with respect to the baseline values are calculated using the method 2 ^ΔΔα . In the Y axis, the values are mean ± ESM of triplicates of 3 independent trials (* P <0.05; ** P <0.01; *** P <0.001 **** P <0.0001) . The expression of these genes also changes in the cell lines tested. The induction of TRAIL occurs at 24 and 30 hours for the A375 and MCF7 line and the induction of DR5 is evident for all tumor cell lines studied although there are differences in the expression kinetics of this gene. It was not possible to quantify DKK3 expression in MCF7, either at baseline or after stimulation.

Figure 5. A, B: Levels of the TRAIL and DKK3 proteins measured in the culture medium of MSC derived from the umbilical cord stroma (MSC-UCSSCs) in a kinetics experiment of release of these proteins after stimulation of the cells with a 2 Gy dose of ionizing radiation (X-ray or γ). The times at which protein quantification is performed by enzyme-immunoassay were 0, 24 and 48 after treatment. In C and D, TRAIL and DKK3 levels are represented in whole cells. For this, a method that allows the quantification of proteins by immunoassay in intact cells has been used (Siles et al. British journal of cancer 1996; 73 (5): 581-8). The evolution of the intracellular levels of these proteins was made in the Oh (control) times and 24 and 48 hours after irradiation. The whole cell assay shows the changes in the amount of TRAIL form bound to the cell membrane in control cells and after treatment (0, 24 and 48 h). E shows representative results of the response of the enzyme assay method to measure the evolution of TRAIL expression in the cells in basal conditions and after being irradiated with 2 Gy. The image clearly shows the increase, as a function of time, of the levels of protein in the irradiated cells. The full cell assay, F, allows you to measure the baseline levels of TRAIL and its changes after irradiation for amounts of cells as low as 5000 cells per well.

Figure 6. Levels of TRAIL and DKK3 proteins measured in an MSC stimulation experiment with a dose of 6 Gy using low LET radiation (X-ray or γ). The results obtained after measuring the expression of TRAIL and DKK3 in the culture medium are shown in a and b. The points correspond to the times of Oh (control), 24 and 48 hours after irradiation. The results of the TRAIL measurement by the complete cell assay are shown in c and d. The values are expressed in pg / ml in the enzyme-immunoassay of TRAIL, ng / ml for that of DKK3 and in relative units in the case of the test on whole cells. The induction of the form of the TRAIL protein anchored to the cell membrane (whole cell assay) of mesenchymal cells is evident.

Figure 7. The medium conditioned by irradiation of mesenchymal cells (RCM) used repeatedly as a treatment on A375 and G361 cells produces an effect of cell death that is manifested by the decrease in size colonies formed by these cell lines. In this experiment, A375 and G361 cells were grown for 7 days to form colonies. From this point (beginning of the experiment) three conditions were defined: The first in which the colonies were allowed to grow in normal culture medium (experiment control) daily changing the culture medium with fresh medium. In the other two, the culture medium was replaced by RCM obtained after 24 hours and RCM obtained after 48 hours of irradiation of MSC cells. At the initial time, the total area covered by the colonies of tumor cells was quantified after staining the colonies, obtaining a photographic image that was analyzed by ImageJ software (point 0 in the tumor growth kinetics experiments) and its growth was monitored during 5 additional days. Using the data of the graphical representations it is possible to obtain the percentage of cell loss referred to the control experiment, which causes each treatment. For the G361 and A361 cell lines, comparing the settings of the experimental points to an exponential equation shows that the Control curves, and treatment with RCM 24h and with RCM 48h are statistically different. The doubling time values for each of the experimental situations are: T _D for the growth of the colonies of A375 and G361 under Control conditions: 42.0 and 45.6 hours respectively; T _D for the growth of colonies treated with RCM 24h for A375 and G361: 61, 2 and 79.3 hours respectively. These figures allow to calculate a percentage of cell loss of the order of 50% in both cases. Finally, the value of the slopes of the growth of the tumor cell colonies (Tcg) in the experiments performed in the condition of treatment with RCM 48h turned out to be: 1) a very slow positive slope in the case of the A375 cell line, and 2) a slope of negative value for the G361 cells that indicated the progressive reduction in the size of the surface covered by the colonies of these cells subjected to treatment with RCM 48h. This allows us to conclude that cell loss in both cases exceeds 100%. Figure 8. Representative experiment of the growth of experimental tumors consisting of colonies of melanoma A375 and G361 cell lines grown for 7 days and colonies of MCF-7 breast cancer cells grown for 14 days. The study of the growth of the colonies was done under Control conditions and after several experimental treatments with unenradiated and irradiated mesenchymal cells. Also the colonies of the tumor cells were studied without irradiation and after irradiation. In all cases the irradiation was done with a dose of 2 Gy. The area covered by the colonies of tumor cells in each of the experimental conditions tested was measured at the initial time (point 0 in the growth kinetics experiments) and the evolution of its size was followed for an additional 5 days. The mean values ± SEM of the observed colonies are represented on the Y axis. Three different experiments were done each in triplicate. Figure 9. Live test in mice. Results obtained after the treatment of mice with the irradiation of one of the mouse tumors with 3 Gy once a week (marked on the graph with RT). Mice are divided into two groups: Control: Continuous line and O symbol; Treatment: Continuous lines and symbols □ and The growth kinetics of the irradiated tumor is the line marked with □, RT 3Gy. The line marked with ^ represents the evolution over time of the size of the non-irradiated contralateral tumor, Bystander. On day 19 the MSCs were injected. After this, the points corresponding to the evolution of the volume of the tumors are clearly separated from the prolongation of the lines that represent tumor growth (dotted lines).

Figure 10. Study of the role of MSCs as tumor suppressor agents when used alone or when used in combination with radiotherapy. Tumor growth is quantified in isolation in four different conditions:

A) Without treatment (Control Group, O), Volume doubling time: 6.13 days

B) Treated exclusively with radiotherapy (RT Group, Δ, 8 tumors),

C) Treated only with mesenchymal cells (MSC, ♦, 16 tumors),

D) Treated with mesenchymal and radiotherapy (RT + MSC, O, 8 tumors)

Contralateral tumors in the groups of mice treated with RT or with RT + MSC (8 tumors in each of them) are those that are identified as Bystander (RT), V, and Bystander (RT + MSC), ♦ .

Figure 11.- Set of representative images of mice inoculated with 1-10 ⁶ cells of the G361 human melanoma line. Mice with a tumor of approx. 65 mm ³ were treated in different ways for 4 weeks Tumor growth images are taken on days 0 (beginning of treatment), 7, 14, 21 and 28, both for the control group and for the groups of each type of treatment. The black spots that are visible are marks to facilitate the irradiation of the tumor. In the RT group, the tumor on the right side (marked with an arrow) was irradiated, while in the RT + MSC group the irradiated tumor was that on the left (marked with an arrow). The tumors on the opposite side were protected from irradiation and are only affected by the bystander effect.

DESCRIPTION OF THE INVENTION

The results of the authors of the present invention indicate that radiation activated mesenchymal cells are a source of cytokines and their administration, preferably in conjunction with radiotherapy and PARP inhibitors, will increase cytokine levels released within the tumor, thereby increasing the effectiveness of radiotherapy through the potentiation of cell death by short and long range bystander effect.

To verify this hypothesis, the inventors have investigated the mechanisms that determine cell sensitivity to the bystander effect in more depth, using a set of cancer cell lines and mesenchymal cells derived from umbilical cord stroma (UCSSC), including the activation of UCSSC cells with low dose radiotherapy to increase the expression of TRAIL (TNFS10) and DKK3. The activation of MSC with radiotherapy achieves effects similar to those previously described (Lee et al., 2012, Cell Stem Cell, 2012, 825-35) and avoids the use of highly toxic molecules such as TNFa.

The invention is supported by in vitro results that are consistent enough to demonstrate that:

1. TRAIL and DKK-3 are molecules produced by the tumor and by mesenchymal cells that, as a result of the treatment of cells with low-dose radiation of LET (X-ray or γ) between 0.2 and 6 Gy, are they secrete to the extracellular environment, where they can act as signaling molecules to produce cell death in non-irradiated tumor cells.

2. It is possible to increase the death potential of tumor cells using a combination of: 1) medium conditioned by irradiation of mesenchymal cells and 2) substances that pharmacologically act as inhibitors of the PARP-1 enzyme, which can be applied simultaneously, or successively, on the tumor model.

3. It has been shown that activation of MSC-UCSSC cells with low dose radiotherapy can be a useful strategy that provides cellular elements with tumor-suppressive capacity.

Therefore, as shown in the examples of the invention, the use of activated mesenchymal cells (MSC *) enhances the action of radiotherapy applied to the treatment of human cancer models. In addition, based on these results, it is shown that the administration of mesenchymal cells and radiation, together with PARP inhibitors, could lead to an increase in the effectiveness of radiotherapy due to the sum of: i) the action of cytokines released by the MSC * cells to the extracellular medium, ii) the genotoxic actions of radiation and iii) the deterioration of DNA repair mechanisms derived from PARP inhibition. All this translates into the potentiation of short and long-range neighborhood effects (bystander) and, as a consequence, a greater treatment effect.

Thus, as can be seen in Figs. 1 to 6, the ionizing radiation used at low doses on MSC, and on tumor cells, it induces important changes in the expression of the TRAIL, DR5 and DKK3 genes which is translated, in the MSC, by changes in the levels of the proteins encoded by said genes. The membrane anchored form of TRAIL of the MSCs can be quantified by enzyme-assay on whole cell.

Therefore, a first aspect of the invention relates to a method of obtaining activated stem cells, hereafter referred to as the method of the invention, which comprises administering low doses of radiation to said cells. In a preferred embodiment of this aspect of the invention, the dose of radiation administered is between 0.2 and 6 Gy, more preferably between 0.25 and 6 Gy, between 0.2 and 4 Gy, and between 0.5 and 2 Gy. The radiation is administered according to standard techniques known in the state of the art, and with standard megavoltage equipment, such as, for example, but not limited to, AECL Theratron 80, Vary Clinac 4 or Vary Clinac. Preferably, the maximum radiation field size should not be greater than 300 cm ² .

The radiation therapy employed in the process of the invention may comprise external irradiation administered to a cell or a cell population at a dose of 0.2 to 8 Gy per fraction. A preferred range of irradiation dose is 0.5 to 3 Gy per fraction. In certain embodiments, the total dose of radiation therapy is less than 80 Gy, such as less than 75 Gy, such as less than 70 Gy, such as less than 65 Gy, such as less than 60 Gy, such as less than 55 Gy, such as less than 50 Gy, such as less than 45 Gy. In certain embodiments, the dose or radiation therapy is between about 60 to 80 Gy, such as about 40 to 60 Gy, such as about 30 to 50 Gy, such as about 20 to 30 Gy. In certain embodiments, the irradiation dose is selected from 15 to 30 Gy, such as 10 to 20 Gy.

An external irradiation dose can be administered in 1 to 60 fractional doses, such as 5 to 30 fractional doses. In certain embodiments, the fractionated doses are administered with approximately 1.5 to approximately 2 Gy per fraction, such as approximately 1.5 Gy, such as approximately 1.6 Gy, such as approximately 1.7 Gy, such as approximately 1 , 8 Gy, such as about 1.9 Gy, such as about 2.0 Gy, such as about 2.1 Gy, such as about 2.2 Gy, such as about 2.3 Gy such as about 2.4 Gy , such as about 2.5 Gy per fractional dose.

In another preferred embodiment of this aspect of the invention the stem cell would be a cell. mesenchymal mother.

In other embodiments, radiation therapy is administered in a single dose and not in fractional doses. For example, the single dose can be administered with approximately 10 to 20 Gy per dose. Activated stem cells can be administered with a dose of approximately 1-5 Gy. In certain embodiments, a radiation sensitizer is administered to a patient and the patient undergoes a single dose of radiation therapy within 10 minutes, in 20 minutes, in 30 minutes, in 40 minutes, within 50 minutes. or one hour after administration of the sensitizer.

INVENTION CELLS

A second aspect of the invention relates to an isolated activated stem cell, hereinafter activated isolated stem cell of the invention, obtained by the process of the invention.

The stem cell of the invention is an adult stem cell, and more preferably, it is a mesenchymal stem cell. The term "adult stem cell" refers to that stem cell that is isolated from a tissue or organ of an animal in a state of growth after the embryonic state. Preferably, the stem cells of the invention are isolated in a postnatal state. Preferably they are isolated from a mammal, and more preferably from a human, including neonates, juveniles, adolescents and adults. Adult stem cells can be isolated from a wide variety of tissues and organs, such as bone marrow (mesenchymal stem cells, multipotent adult progenitor cells and hematopoietic stem cells), adipose tissue, cartilage, epidermis, hair follicle, skeletal muscle, heart muscle, intestine , liver, neuronal. The term "mesenchymal stem cell" or "MSC", as used herein, refers to a multipotent stromal cell, originating from the mesodermal germ layer, which can be differentiated into a variety of cell types, including osteocytes (bone cells), chondrocytes (cartilage cells) and adipocytes (fat cells). MSCs can be obtained from, without being limited to, bone marrow, adipose tissue (such as subcutaneous adipose tissue), liver, spleen, testicles, menstrual blood, amniotic fluid, pancreas, periosteum, synovial membrane, skeletal muscle, dermis, pericytes, trabecular bone, human umbilical cord, lung, dental pulp and peripheral blood. MSC according to the invention they can be obtained from any of the foregoing tissues, such as from bone marrow, subcutaneous adipose tissue or umbilical cord.

If desired, the activated stem cell of the invention can be genetically modified by any conventional method including, by way of illustration, not limitation, transgenesis processes, deletions or insertions in its genome that modify the expression of genes that are important for its properties. basic (proliferation, migration, differentiation, etc.), or by inserting nucleotide sequences that encode proteins of interest, such as proteins with therapeutic properties. Therefore, in another preferred embodiment, the activated stem cell of the invention has been genetically modified.

In a particular embodiment of the invention, mesenchymal stem cells are obtained from the umbilical cord, preferably from the human umbilical cord. Another preferred embodiment of this aspect of the invention relates to an isolated cell population, hereinafter cell population of the invention, comprising at least one stem cell of the invention. In a preferred embodiment the cell population of the invention comprises at least 20%, preferably 40%, and even more preferably 50%, 60%, 80%, 90%, 95%, or 99% of adult stem cells. of the invention.

The term "isolated" indicates that the cell or cell population of the invention to which it refers, are not in their natural environment. That is, the cell or cell population has been separated from its surrounding tissue. The activated stem cells of the invention, as well as the cells present in the cell population of the invention, can be autologous, allogeneic or xenogenic cells. In a particular embodiment, said cells are of autologous origin, thereby reducing the potential complications associated with / or antigenic and immunogenic responses of said cells when administered to the individual.

COMPOSITION OF THE INVENTION

The activated isolated stem cell of the invention, or the cell population of the invention, can be part of a composition. Therefore, a third aspect of the invention relates to a composition, hereafter referred to as a composition of the invention, comprising at least one activated stem cell of the invention. In a preferred embodiment of this aspect the composition of the invention It also comprises a pharmaceutically acceptable vehicle. In a preferred embodiment of this aspect the composition of the invention further comprises another active ingredient.

Preferably, the cell composition of the invention has at least 50%, at least 60%, preferably 70%, more preferably 80%, even more preferably, 90%, and, even more preferably , 95% of the activated isolated stem cells of the invention.

Said adult cell composition of the invention may contain a medium in which the cells of the invention are found; said medium must be compatible with said cells. For example, but not limited to, isotonic solutions, optionally supplemented with serum; cell culture media or, alternatively, a solid, semi-solid, gelatinous or viscous support medium. The composition of the invention, preferably, is a pharmaceutical composition for administration to a subject.

The term "pharmaceutically acceptable vehicle" refers to a vehicle that must be approved by a federal government regulatory agency or a state government or listed in the United States Pharmacopoeia or the European Pharmacopoeia, or other pharmacopoeia generally recognized for use in animals, and more specifically in humans.

The term "vehicle" refers to a diluent, adjuvant, excipient or carrier with which the cells or the cell population of the invention or of said composition comprising stem cells of the invention obtainable according to the method of the invention should be administered; obviously, said vehicle must be compatible with said cells. Illustrative, non-limiting examples of said vehicle include any physiologically compatible vehicle, for example, isotonic solutions (for example, 0.9% sterile saline NaCI, phosphate buffered saline (PBS), Ringerlactate solution, etc.), optionally supplemented with serum, preferably with autologous serum; cell culture media (eg, DMEM, etc.); or, alternatively, a solid, semi-solid, gelatinous or viscous support medium, such as collagen, collagen-glycosamino-glycan, fibrin, polyvinyl chloride, polyamino acids, such as polylysine, or polynithine, hydrogels, agarose, silicone dextran sulfate. Also, if desired, the support medium may, in specific embodiments, contain growth factors or other agents.

If the support is solid, semi-solid, or gelatinous, the cells can be introduced into a liquid phase of the vehicle that is subsequently treated in such a way that it becomes a more solid phase.

The pharmaceutical composition of the invention, if desired, may also contain, when necessary, additives to increase, control or otherwise direct the desired therapeutic effect of the cells, which comprise said pharmaceutical composition, and / or auxiliary substances or Pharmaceutically acceptable substances, such as buffering agents, surfactants, cosolvents, preservatives, etc. Also, to stabilize the cell suspension, it is possible to add metal chelators. The stability of the cells in the liquid medium of the pharmaceutical composition of the invention can be improved by the addition of additional substances, such as, for example, aspartic acid, glutamic acid, and so on. Such pharmaceutically acceptable substances that can be used in the pharmaceutical composition of the invention are generally known to those skilled in the art and are normally used in the preparation of cellular compositions.

Examples of suitable pharmaceutical vehicles are described, for example, in "Remington's Pharmaceutical Sciences" by E.W. Martin. Additional information on these vehicles can be found in pharmaceutical technology manuals (Galenic Pharmacy). As used herein, the term "active ingredient", "active substance", "pharmaceutically active substance", "active ingredient" or "pharmaceutically active ingredient" means any component that potentially provides a pharmacological activity or other different diagnostic effect, cure, mitigation, treatment, or prevention of a disease, or that affects the structure or function of the body of man or other animals. The term includes those components that promote a chemical change in the preparation of the drug and are present therein in a modified form intended to provide the specific activity or effect.

In another preferred embodiment, the active ingredient is selected from the list consisting of: a poly (ADP-ribose) polymerase (PARP) inhibitor; a radiosensitizer such as nitroimidazole; a growth factor or GF (from English, "growth factor"); an agent directed at the epidermal growth factor receptor (RFCE); a radiolabeled antibody; a remainder of addressing; chemotherapeutic agents, which include, but are not limited to, compounds that induce apoptosis, compounds that reduce lifespan or compounds that make cells sensitive to stress; bacteria, modified or not genetically; or any of its combinations. In another more preferred embodiment of this aspect of the invention, the active ingredient is a poly (ADP-ribose) polymerase (PARP) inhibitor.

In another more preferred embodiment of this aspect of the invention, the active ingredient is a growth factor. More preferably, the growth factor is a granulocyte colony stimulating factor or G-CSF (Granulocyte colony-stimulating factor), and even more preferably, lenograstim and / or filgrastim.

COMBINED PREPARATION OF THE INVENTION

A fourth aspect of the invention relates to a combined preparation, hereafter combined preparation of the invention, comprising:

a) a component A that is an activated stem cell of the invention

In a preferred embodiment of this aspect the combined preparation of the invention further comprises at least one PARP (poly (ADP-ribose) polymerase) inhibitor. Both component A, as component B, or any other component, may be arranged to be administered to a subject simultaneously, in combination or sequentially in one treatment.

By way of non-limiting example, the following may be part of the components of the invention: an inhibitor of poly (ADP-ribose) polymerase (PARP); a radiosensitizer such as nitroimidazole; a growth factors or GF; an agent directed at the epidermal growth factor receptor (RFCE); a radiolabeled antibody; a remainder of addressing; chemotherapeutic agents, which include, but are not limited to, compounds that induce apoptosis, compounds that reduce lifespan or compounds that make cells sensitive to stress; bacteria, modified or not genetically; or any of the combinations of the active ingredients mentioned.

MEDICAL USES OF THE INVENTION

A fifth aspect of the invention relates to the use of an activated stem cell of the invention, of a composition of the invention, or of the combined preparation of the invention, in the manufacture of a medicament. The term "medication", as used herein, refers to any substance used for prevention, diagnosis, relief, treatment or cure of diseases in man and animals. In the context of the present invention, the disease is a cancer, and more preferably it is a systemic cancer.

The pharmaceutical composition of the invention will contain a prophylactic or therapeutically effective amount of the cells of the invention or of the cell population of the invention, preferably, a substantially homogeneous cell population, to provide the desired therapeutic effect.

As used herein, the term "therapeutically or prophylactically effective amount" refers to the amount of cells of the invention contained in the pharmaceutical composition that is capable of producing the desired therapeutic effect and, in general, will be determined, among other factors, due to the characteristics of the cells and the desired therapeutic effect sought. In general, the therapeutically effective amount of cells of the invention to be administered will depend, among other factors, on the subject's own characteristics, the severity of the disease, the manner of administration, etc. For this reason, the doses mentioned in this invention should be taken into account only as a guide for the person skilled in the art, who should adjust this dose depending on the factors described above. As an illustrative and non-limiting example, the pharmaceutical composition of the invention can be administered as a single dose, containing approximately between 1x10 ⁵ and 10x10 ⁶ cells of the invention per kilo of body weight of the recipient, and more preferably between 5x10 ⁵ and 5x10 ⁶ cells of the invention per kilo of the body weight of the recipient, in a more preferred embodiment even said pharmaceutical composition will contain approximately between 1x10 ⁶ and 2x10 ⁶ cells of the invention per kilo of the body weight of the recipient, depending on the factors described above. . The dose of cells of the invention can be repeated, depending on the condition and evolution of the patient, in temporary intervals of days, weeks or months that the specialist must establish in each case.

A sixth aspect of the invention relates to the use of an activated stem cell of the invention, of a composition of the invention, or of the combined preparation of the invention, in the preparation of a medicament for the treatment of cancer. In a preferred embodiment of this aspect of the invention, the cancer is a systemic cancer.

METHOD OF TREATMENT OF THE INVENTION

A seventh aspect of the invention relates to a method of treating cancer, of hereinafter the first method of cancer treatment of the invention, comprising: a) administering the activated stem cells of the invention, and

b) administer radiotherapy at conventional doses following the usual protocols.

In a preferred embodiment, step (b) comprises administering to the patient a low dose of radiation on the volume occupied by the tumor in an effective amount (2-6 Gy) to activate the tumor cells and favor the tropism of the mesenchymal cells towards the tumor

In another preferred embodiment, the stem cells of the invention are mesenchymal stem cells. In another preferred embodiment, new intravenous administrations can be made subsequently, both of MSC cells and MSC * cells or stem cells of the invention, for example, on the days of radiation therapy pause.

In another preferred embodiment of this aspect, the first method of cancer treatment of the invention further comprises administering an active ingredient such as: a poly (ADP-ribose) polymerase (PARP) inhibitor; a radiosensitizer such as nitroimidazole; a growth factors or GF; an agent directed at the epidermal growth factor receptor (RFCE); a radiolabeled antibody; a remainder of addressing; chemotherapeutic agents, which include, but are not limited to, compounds that induce apoptosis, compounds that reduce lifespan or compounds that make cells sensitive to stress; bacteria, modified or not genetically; or any of the combinations of the active ingredients mentioned.

Even more preferably another preferred embodiment of this aspect, the first method of cancer treatment of the invention further comprises administering at least one PARP (poly (ADP-ribose) polymerase) inhibitor. In another preferred embodiment of this aspect the first cancer treatment method of the invention further comprises administering at least one chemotherapeutic agent and / or an antiangiogenic agent. In another preferred embodiment of this aspect the cancer is a systemic cancer.

a) administer untreated stem cells, and

b) administer radiotherapy at conventional doses following the usual protocols, and more preferably, at low doses, to activate the stem cells inside the patient. In another preferred embodiment, 24 hours pass between step (a) and step (b) of the invention to allow the cells to bio-distribute.

Radiation therapy

The source of energy used for radiation therapy can be selected from X-rays or gamma rays, which are the two forms of electromagnetic radiation. The energy source for radiation therapy can be selected from particle beams, which use fast-moving subatomic particles instead of photons. This type of radiation can be referred to as particle beam radiation therapy or particle radiation.

In the present invention, the term "ionizing radiation" means radiation that comprises particles or photons that have sufficient energy or can produce enough energy through nuclear interactions to produce ionization, that is, the gain or loss of electrons. The amount of ionizing radiation needed to kill a given cell generally depends on the nature of that cell. Means for determining an effective amount of radiation are well known in the art. Used herein, the term "an effective dose" of ionizing radiation means a dose of ionizing radiation that causes an increase in cellular damage. In certain embodiments, radiation therapy comprises ionizing radiation, particularly electron beam radiation. An electron beam can be delivered intraoperatively to the tumor site using an electron beam therapeutic system such as that described in US Pat. No. ⁰ 5,418,372 and 5,321,271 the full disclosure of which is incorporated herein by reference. In particular embodiments, the therapeutic electron beam system of the invention provides adequate protection to healthy tissue for the primary x-rays generated by the system, as well as for scattering radiation. In particular embodiments, particle beam therapy is proton beam therapy. Protons deposit their energy in a very small volume, which is called the Bragg peak. Bragg's beak can be used to target high doses of proton beam therapy to a tumor while doing less damage to normal tissues in front of and behind the tumor. Proton therapy is generally reserved for cancers that are difficult or dangerous to treat with surgery, such as a chondrosarcoma at the base of the skull, or it is combined with other types of radiation.

In some embodiments, the radiation therapy is stereotactic (or stereotactic) radiosurgery that uses a large dose of radiation to destroy the tumor tissue. In certain exemplary embodiments, in which the cancer is in the brain, the patient's head may be placed in a special frame, which is attached or mounted on the patient's skull. The plot is used to target high dose radiation beams directly at the tumor in the patient's head. The dose and radiation reception area are coordinated very precisely resulting in little damage to nearby tissues. In some stereotaxy applications, a head frame is not needed. In certain embodiments, real-time imaging systems are used in conjunction with the throttle movement, allowing computer adjustments of the throttle path to compensate for any movement of the patient's head. When a source of radiation therapy is internal, the energy used in internal radiation can come from a variety of sources. For example, the radioactive isotope can be radioactive iodine, for example, iodine 125 or iodine 131, strontium 89, phosphorus, palladium, cesium, iridium, phosphate, cobalt, or any other isotope known in the art. In certain embodiments, internal radiation is administered as brachytherapy, a radiation treatment based on implanted radioactive seeds emitting radiation from each seed.

In certain aspects, the invention comprises the methods for planning the External radiation therapy in order to target cancer cells and limit exposure to healthy cells. In certain embodiments, radiation treatment planning is carried out in two dimensions (width and height) or three dimensions, for example, with three-dimensional (3-D), conformal radiation therapy. In certain embodiments, 3-D conformal radiotherapy uses computer technology to allow physicians to more accurately determine a tumor with radiation rays (with width, height and depth). A 3-D image of a tumor can be achieved by computed tomography (CT), magnetic resonance imaging (MRI), positron emission tomography (PET), or single photon emission tomography (SPECT). Using the information in the image, special computer programs can design radiation beams that "fit" the shape of the tumor. In certain embodiments, because the healthy tissue surrounding the tumor was largely spared by this technique, higher doses of radiation can be used to treat cancer. In certain particular embodiments, radiation therapy is intensity modulated radiotherapy (IMRT). IMRT is a type of 3-D conformal radiation therapy that uses radiation beams, for example, X-rays of different intensities to deliver different doses of radiation to small areas of tissue at the same time. The technology allows the administration of higher doses of radiation in the tumor and lower doses to nearby healthy tissue. Some techniques provide a higher dose of radiation to the patient each day, potentially shortening the total treatment time and improving the success of the treatment. IMRT can also lead to fewer side effects during treatment. In particular embodiments, the radiation is administered by a linear accelerator that is equipped with a multilayer collimator (a collimator helps shape or sculpt the radiation beams). The equipment can be rotated around the patient so that the radiation beams can be sent from the best angles. The beams conform as closely as possible to the shape of the tumor. In certain embodiments, this technology is used to treat tumors in the brain, head and neck, nasopharynx, breast, liver, lung, prostate, and uterus.

PARP inhibitors

PARP inhibitors are a group of pharmacological inhibitors of the enzyme poly ADP ribose polymerase (PARP). They are developed for multiple indications; The most important is cancer treatment. Several forms of cancer are more dependent on PARP than normal cells, making PARP an attractive target for cancer therapy. Among PARP inhibitors are, but not limited to, iniparib (BSI 201), BMN-673, olaparib (AZD-2281), rucaparib (AG014699, PF-01367338), veliparib (ABT-888), CEP 9722, MK 4827, BGB-290, 3-aminobenzamide, or any combination thereof.

Radiation sensitizers

In some embodiments, the invention provides methods of administering reduced doses of radiation by combining intraoperative radiation with radiation sensitizers. Brown et al. (Int. J. Biol. Radiation Oncology. Phys., 2010, 78 (1): 323-327) provide model data in favor of the use of the ethanidazole sensitizer (ethanidazole radiosensitizer ET), in combination with ablative stereotactic radiotherapy (SABR ). When calculating the expected level of tumor cell death after SABR, Brown et al. indicate that the administration of ET before SABR will reduce the dose and the frequency of irradiation necessary to treat tumors and metastases, especially in tumors with high levels of hypoxia. Therefore, in particular embodiments, the radiation is reduced by up to 1%, up to 5%, up to 10%, up to 15%>, up to 20%>, up to 25%>, up to 30%, up to 35%, up to 40%, up to 45%, up to 50%, up to 55%, up to 60%, up to 65%, up to 70%, up to 75%, up to 80%, up to 85%, up to 90%, up to 95%, and up to 99% compared to intraoperative radiation therapy without sensitizers. On the other hand, the radiation dose in intraoperative radiation therapy can be reduced by 25-50% with respect to the amount of external radiation therapy that may be necessary to treat the disease without the sensitizer. Accordingly, the invention also provides a method for administering reduced doses of radiation to a patient by combining the IORT, SBRT or SRS with a radiation sensitizer selected from nitroimidazoles, in which the radiation is reduced by up to 75 % with respect to radiation therapy administered without sensitizers. Radiation therapy can be used in conjunction with hyperthermia, that is, the use of heat. In certain embodiments, the combination of heat and radiation may increase the response rate of some tumors. A radiosensitizer can be administered in combination with an additional agent. For example, a nitroimidazole can be administered in combination with an additional agent such as a targeting agent, a chemotherapeutic agent or a second radiosensitizer. Addressing agents include any suitable agent to attack cancer cells, such as antibodies. Nitroimidazole can be attached to the targeting agent through covalent or non-covalent attachments. For example, nitroimidazoles, such as 2-nitroimidazoles, can be linked to a targeting agent through a linker such as a biodegradable linker. Alternatively, nitroimidazoles may be linked to a targeting agent through ionic interactions. In certain embodiments, the radiosensitizer and the additional agent may be wrapped within a liposome. Other radiation sensitizers are, but are not limited to, misonidazole, metronidazole, tirapazamine, trans sodium crocetinate, or any combination thereof. Radiolabeled Antibodies and Antibodies

The radiation can be administered directly to the cancer and enhanced by antibodies and / or by the use of radiolabeled antibodies, that is, radioimmunotherapy. Some tumor cells contain specific antigens that trigger the production of tumor-specific antibodies. Large amounts of these antibodies can be produced in the laboratory and bind to radioactive substances, a process known as radiolabeling. Once injected into the body, the antibodies look for cancer cells, which are destroyed by radiation. This approach can reduce or minimize the risk of radiation damage to healthy cells. In certain embodiments, radioimmunotherapy treatments are selected from ibritumomab tiuxetan (Zevalin ®) and tositumomab and iodine 131 tositumomab (Bexxar ®). Radioimmunotherapy can be used in the treatment of advanced non-Hodgkin lymphoma (NHL) adults. In certain embodiments, immunotherapy is used in the treatment of cancers such as leukemia, NHL, colorectal cancer, and liver, lung, brain, prostate, thyroid, breast, ovarian and pancreas cancer.

Addressing elements

In certain embodiments, the cells or other agents of the invention are associated with an addressing element. The addressing element may be covalently bound to nitroimidazole, or associated with nitroimidazole although non-covalent forces, such as ionic bonds, hydrogen bonds, or through encapsulation within a liposome. The addressing element, which assists the cell of the invention or other agent in the location of a particular target region, enters a cell (s) of the target tumor, and / or the location within or proximal to the cell, is You can select based on the particular cell type that is being targeted. The addressing element may further comprise any of a number of different chemical entities. In one embodiment, the addressing element is a small molecule. Molecules that may be suitable for use as targeting moieties in the present invention include haptens, epitopes and dsDNA fragments and analogs and derivatives thereof. Such elements specifically bind to antibodies, fragments or analogs thereof, including mimetics (for haptens and epitopes), and zinc finger proteins (for double stranded DNA fragments). Nutrients believed to trigger endocytosis Mediated by receptor and targeting moieties, therefore useful include biotin, folic acid, riboflavin, carnitine, inositol, lipoic acid, niacin, pantothenic acid, thiamine, pyridoxal, ascorbic acid, and lipid soluble in vitamins A, D, E and K. Another exemplary type of moiety that chooses a small molecule target includes steroid lipids, such as cholesterol and steroid hormones, such as estradiol, testosterone, etc.

Addressing elements may also comprise one or more proteins. Particular types of proteins can be selected based on known characteristics of the target zone or target cells. For example, the probe can be a monoclonal or polyclonal antibody, where a corresponding antigen is shown at the target site. In situations where a particular receptor is expressed by the target cells, the targeting element may comprise a peptidomimetic protein or ligand capable of binding to that receptor. Known cell surface receptor ligand proteins include low density lipoproteins, transferrin, insulin, fibrinolytic enzymes, anti-HER2, platelet-binding proteins such as annexins, and biological response modifiers (including interleukin, interferon, erythropoietin and stimulating factor of colonies). A series of monoclonal antibodies that bind to a specific type of cells have been developed, including specific monoclonal antibodies to tumor associated antigens in humans. Among the many monoclonal antibodies such that can be used are anti-CT, or other interleukin-2 receptor antibodies; 9.2.27 and NR-ML-05 at 250 kilodaltons proteoglycan associated with human melanoma; and NR-LU-10 to a pancarcinoma glycoprotein. An antibody employed in the present invention may be an intact (complete) molecule, a fragment thereof, or a functional equivalent thereof. Examples of antibody fragments are F (ab ') 2, Fab', Fab, and Fv fragments, which can be produced by conventional methods or by genetic or protein engineering.

Other preferred addressing elements include sugars, for example, glucose, fucose, galactose, mannose, which are recognized by specific target receptors. For example, some constructs can be glycosylated with mannose residues, for example, which is attached as C-heterosides to a free nitrogen, to produce specific constructions and with greater affinity for binding to tumors expressing mañosa receptors, for example, glioblastomas and gangliocytomas and bacteria, which are also known to express mañosa receptors (Bertozzi, CR and MD Bednarski Carbohydrate Research 223: 243 (1992); J. Am. Chem. Soc. 1 14: 2242,5543 (1992)), as well as potentially other infectious remains. Certain cells, such as malignant cells and blood cells (for example, A, AB, B, etc.) show particular carbohydrates, for which a corresponding lectin can serve as a targeting element. Chemotherapeutic Agents

In certain embodiments, chemotherapeutic agents may be administered in conjunction with the combined cells, compositions or preparations of the invention. Chemotherapeutic agents include, but are not limited to, those compounds with anti-cancer activity, for example, compounds that induce apoptosis, compounds that reduce lifespan or compounds that make cells sensitive to stress and include: aminoglutethimide, amsacrine, anastrozole , asparaginase, Beg, bicalutamide, bleomycin, buserelin, busulfan, campothecin, capecitabine, carboplatin, carmustine, chlorambucil, cisplatin, cladribine, chlodronate, colchicine, cyclophosphamide, cyproterone, cytarabine, dacarinicorbumin, dacarinicorbumin, dactin-thicorbumin, dactinicorbumin, documin-thicorbumin, dac-thicorbuminin, dactin-thicorbumin, dac-thicorbuminum, dactin-thickenesalbuminase , epirubicin, estradiol, estramustine, etoposide, exemestane, filgrastim, fludarabine, fludrocortisone, fluorouracil, fluoxymesterone, flutamide, gemcitabine, genistein, goserelin, hydroxyurea, idarubicin, ifosfamide, leatinotholcan, iratinotolcan, leatinotholcan, leatinotholcan, leatinotholcan, leatinotholcan, leatinotolcan, leatinotholcan, leatinotholcan, leatinotolcan, leatinotholcan, leatinotholcan, leatinotholcan , leuprolide, levamisole, lomustine, mechlorethamine, medrox iprogesterona, megestrol, melphalan, mercaptopurine, mesna, methotrexate, mitomycin, mitotane, mitoxantrone, nilutamide, nocodazole, octreotide, oxaliplatin, paclitaxel, pamidronate, pentostatin, plicamycin, porfimer, procarbazine, raltitrexed, rituximab, streptozocin, suramin, tamoxifen, temozolomide, teniposide, testosterone, thioguanine, thiotepa, titanocene dichloride, topotecan, trastuzumab, tretinoin, vinblastine, vincristine, vindesine, vinorelbine, or any combination thereof.

These chemotherapeutic agents can be classified by their mechanism of action, but not limited to, for example, the following groups: anti-metabolite / anti-cancer agents, such as pyrimidine analogues (5-fluorouracil, floxuridine, capecitabine, gemcitabine and cytarabine) and purine analogues, folate antagonists and related inhibitors (mercaptopurine, thioguanine, pentostatin and 2-chlorodeoxyadenosine (cladribine)); antiproliferative / antimitotic agents, including natural products such as vinca alkaloids (vinblastine, vincristine, and vinorelbine), microtubule disruptors such as taxanes (paclitaxel, docetaxel), vincristine, vinblastine, nocodazole, epothilones, and navelbine, epidipodophyllotoxydiphospidothodoxydiphospid , DNA damaging agents (actinomycin, amsacrine, anthracyclines, bleomycin, busulfan, camptothecin, carboplatin, chlorambucil, cisplatin, cyclophosphamide, Cytoxan, dactinomycin, daunorubicin, docetaxel, doxorubicin, epirubinamimethalamine, mothylamylamine, hexirchineamimethalamine, ifirimylamine, thymolimamimamimine, thymmethylimamimine, mothimylamine, thymolimamimamylamine, thymolimamimamylamine, thymolimamimamylamine, mothimalchimethalamine, mothimylamotholimamimine, mothimylamylamotholimamimine, mothromylamine, thickechlolamine, michlorochineamylamine, hexopyrimamine, mothromethamimine, thymolimamylamine, mothromethamineamicamimine, michlorochineamine, methicide agent , mitoxantrone, nitrosourea, paclitaxel, plicamycin, procarbazine, teniposide, triethylene thiophosphoramide and etoposide (VP 16)); antibiotics such as dactinomycin (actinomycin D), daunorubicin, doxorubicin (adriamycin), idarubicin, anthracyclines, mitoxantrone, bleomycins, plicamycin, (mitramycin) and mitomycin; enzymes (L-asparaginase that systemically metabolizes L-asparagine and deprives cells that do not have the ability to synthesize their own asparagine); platelet antiaggregants; antiproliferative / antimitotic alkylating agents such as nitrogen mustards (mechlorethamine, cyclophosphamide and the like, melphalan, chlorambucil), ethylenimines and methylmelamines (hexamethylmelamine and thiotepa), alkyl sulphonates (busulfan), nitrosoureas (streptograms) trazenes (for example, dacarbazinine (DTIC)); antiproliferative / antimitotic antimetabolites such as folic acid analogs (methotrexate); coordination complexes of platinum (cisplatin, carboplatin), procarbazine, hydroxyurea, mitotane, aminoglutethimide; hormones, hormone analogues (estrogen, tamoxifen, goserelin, bicalutamide, nilutamide) and aromatase inhibitors (letrozole, anastrozole); anticoagulants (heparin, synthetic heparin salts and other thrombin inhibitors); fibrinolytic agents (such as tissue plasminogen activator, streptokinase and urokinase), aspirin, COX-2 inhibitors, dipyridamole, ticlopidine, clopidogrel, abciximab; antimigratory agents; antisecretory agents (breveldine); immunosuppressants (cyclosporine, tacrolimus (FK-506), sirolimus (rapamycin), azathioprine, mycophenolate mofetil); anti-angiogenic compounds (TNP-470, genistein) and growth factor inhibitors (vascular endothelial growth factor (VEGF) inhibitors, fibroblast growth factor inhibitors (FGF), epidermal growth factor (EGF) inhibitors); angiotensin receptor blocker; donors of nitric oxide; antisense oligonucleotides; antibodies (trastuzumab); cell cycle inhibitors and differentiation inducers (tretinoin); mTOR inhibitors, topoisomerase inhibitors (doxorubicin (adriamycin), amsacrine, camptothecin, daunorubicin, eniposide dactinomycin, epirubicin, etoposide, idarubicin, irinotecan (CPT-1 1) and mitoxantrone, topotecan, irinotecan), corticosteroids (cortisone, dexamethasone , hydrocortisone, methylprednisolone, prednisone and prednisolone); growth factor signal transduction kinase inhibitors; Mitochondrial dysfunction inducers and caspase activators; chromatin disruptors

Radioprotectors

Radioprotectors can also be administered to a patient in combination with the combined cells, compositions or preparations described herein. Radioprotectors are drugs that protect normal (non-cancerous) cells from damage caused by radiation therapy. These agents promote the repair of normal cells that are exposed to radiation. Exemplary radioprotectors include amifostine (trade name Ethyol ®).

In certain embodiments, the methods of the invention further comprise administration. of a bacterium such as salmonella or genetic engineering variants thereof.

Radiation sensitizers can be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients. For example, they can be formulated for administration by, for example, injection (for example subcutaneous, intramuscular, intraparenteral), inhalation or insufflation (either through the mouth or nose) or oral, oral, sublingual, transdermal administration, nasal, parenteral or rectal. In one embodiment, a compound of the invention can be administered locally, at the site where tumor cells are present, that is, in a specific tissue, organ or fluid (e.g., blood, cerebrospinal fluid, etc.)

Typically, the compounds of the invention are administered intravenously. The phrase "pharmaceutically acceptable" or "physiologically acceptable" is used herein to refer to those ligands, materials, compositions, and / or dosage forms that are, within the scope of medical judgment, are suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, in accordance with a reasonable benefit / risk ratio.

Radiation sensitizers of the invention can be formulated for a variety of modes of administration. Techniques and formulations can generally be found at Remington Pharmaceutical Sciences, Meade Publishing Co., Easton, PA

Throughout the description and the claims the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and features of the invention will be derived partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention. The decimal notation used in this document uses the symbol "," to separate units of decimals.

EXAMPLES OF THE INVENTION

An example of the invention is described below with reference to the attached figures. EXAMPLE 1. In vitro tests

The tests carried out show that the activation of tumor and mesenchymal cells with radiation induces the expression of TRAIL and its DR5 receptor and that the soluble form of TRAIL is secreted to the extracellular space in which the cells have been irradiated; In addition, irradiation of MSC cells with a dose of 2 Gy induces DKK3. The effect of radiation on TRAIL expression is clearly superior in MSC-UCSSCs cells than in tumor cells (Figures 1 to 4) and the activity of the culture medium from incubation for 24 or 48 hours of MSC cells irradiated with 2 Gy is obvious.

As a consequence of the induction of TRAIL and DKK3 after irradiation the molecules of these proteins are secreted to the culture medium where they can be quantified as the soluble forms TRAIL (sTRAIL) and DKK3 (Fig. 5 and 6). Our experiments demonstrate the spectacular effect of UCSSCs-RCM used as repeated treatment of colonies of A375 and colonies of G361 (Fig. 7). The quantitative study of the colonies of A375 and G361 in the kinetics experiments of tumor growth under treatment with UCSSCs-RCM shows that in the treated colonies the cell loss is of the order of 50% of what exists in the growth experiments under control conditions.

Furthermore, as it is known that the tumor-suppressing activity of MSC cells, when activated with TNF-α, is mainly due to the expression, in these cells, of high levels of the TRAIL form bound to the cell membrane (Lee et al. 2012, Cell Stem Cell, 2012. 11 (6): p. 825-35) in the experiments it has been proven, through the TRAIL enzyme assay in a complete cell, that radiation induces not only the secretion of TRAIL and DKK3 to the medium but also increases the expression, in relevant amounts, of the molecular form of TRAIL-transmembrane that remains bound in the cell (Fig. 5 and 6). The co-culture experiments of irradiated or non-irradiated tumor and MSC cells have shown us the greatest tumor-suppressor activity of UCSSCs when these cells have been previously irradiated with a low dose of radiation, 2 Gy (Table 1). The analysis of the data summarized in Table 1 allows us to conclude that the treatment of tumor models with MSC causes a delay in the growth of tumor models obtained from any of the three cell lines studied G361, A375 or MCF7. This growth retardation turns out to be a function of the activation of both MSC cells and tumor cells since radiation induces TRAIL in MSCs while also inducing TRAIL receptor (DR5) expression in tumor cells. This mechanism explains the fact that in experiments in which both MSC-UCSSCs cells and cells Tumors have been irradiated the increase in cellular component (or "up-regulation") of both genes lead to increased cell death (Tablal).

Table 1. Growth kinetics parameters of experimental tumors in vitro under control conditions and after different treatments.

In addition, since after irradiation of MSC soluble forms of TRAIL and DKK3 proteins are secreted into the extracellular medium, MSC cells can be considered as "medicinal" cells (Caplan, Al & Correa, D. 201 1. Cell stem cell 9, 1 1-15); In addition, accepting that among the functional characteristics of MSC cells is their tropism - as a determinant to reach the places of the body where there are lesions - and their immunomodulatory capacity (Ranganath, et al., 2012. Cell stem cell 10, 244- 258) We understand that the MSC injected into the body can act as "warehouses of pharmaceutical products", and these cells can be used in cell therapy procedures as a complement to radiotherapy, since, once activated with radiation they become a source of cytokines and cytotoxic tumor-suppressor proteins for tumor cells which can lead to a significant improvement in the biological efficacy of radiation on tumors. These data appear as MSC-ER coefficient in Table 1. In addition, the results of the trials suggest that radiation treatment plus MSC could be used as a strategy to achieve radiosensitization of tumors. From a theoretical point of view, the adjuvant role of MSCs to radiotherapy could be thought of as a tool capable of increasing the weight of the second factor {in square brackets} of our mathematical model of biological action of radiation on tumors, increasing through of the potentiation of the bystander mechanism the probability of cell death (Gómez-Millán et al., 2012. Journal of the European Society for Therapeutic Radiology and Oncology 102, 450-458; Lara et ai, 2013, Cancer Letters, 2015. 356: p. 5-16)

In the previous equation S is the survival fraction, D the dose, α and β the coefficients of the linear-quadratic model, χ represents the cell death produced by bystander effect, xmax the maximum probability of death produced by bystander effect for dose D , and KBy the association constant between the death receptor and its ligand. And this is biologically conceivable because with the MSC cells activated with radiation we manage to release TRAIL and DKK3 molecules that act as pro-apoptotic proteins in situ within the tumor. In addition, radiation applied to the tumor induces increased expression in its cell death receptor cells - such as DR5. In this way from the theoretical point of view, we understand this effect justified. The data shows that it is a therapeutic tool applicable to the systemic treatment of advanced tumors.

The RT + MSC combination can be an excellent combination for the treatment of cancer because:

i) Radiation therapy is one of the two most effective cancer treatments and more than half of patients receive radiotherapy at some time during the evolution of their neoplastic process;

ii) knowledge of the role of MSCs in cancer treatment is continuously increased and it is known that MSCs are incorporated into the tumor stroma and that this ability to integrate into the tumor increases when the neoplastic process is being treated with radiotherapy

iii) the activation of MSC with 2 Gy (dose that fits the most commonly used in radiotherapy oncology) induces the expression of TRAIL and DKK3 and the proteins that these genes encode increase their intracellular concentration and are released into space extracellular where they can be quantitatively identified (Fig. 5 and 6). DKK3 expression is significantly diminished, or absent, in many experimental tumors (tumor cell lines, in our case in the MCF-7 cell line) and clinical (Tsuji et al. 2000. Biochem. Biophys. Res. Commun. 268 , 20-24). The absence of gene expression is associated with hypermethylation of its promoter. It's known that

REIC / DKK3 interferes with the Wnt signaling pathway via Wnt receptors through which they play different roles in the induction of apoptosis and inhibition of the metastatic process and it has been shown that in cells modified to express REIC / DKK3 using adenovirus vector (Ad-REIC) stress occurs in the endoplasmic reticulum system (ER) and specific apoptosis mechanisms are initiated in human prostate and lung cancer cells (Shien, K., et al., 2014. PloS one 9, e879004). It is understood that the release of DKK3 that occurs when MSC-UCCSCs cells are incorporated into the tumor and are activated by radiation could be a useful strategy in the search for the antitumor response of malignant cells.

iv) The examples of the invention have demonstrated the significant increase in the expression of the form of the TRAIL-transmembrane protein when MSC cells are treated with 2 Gy doses of low LET ionizing radiation. Considering the natural tropism of the MSCs cells towards the tumors, an appropriate therapy would be to inject MSCs cells during the breaks in which the daily radiation treatment is interrupted (weekends in the course of a standard cancer radiotherapy treatment). Once injected, the cells would accumulate in the environment of the body volume undergoing treatment (Kim, SM, et al., 2010, Stem cells 28, 2217-2228) so that at the end of the break, between weeks of treatment, the Subsequent irradiation of the tumor would activate the MSC cells integrated in the therapeutic volume, and there converted into sources of cytokines and tumor-suppressor proteins would contribute to increasing the effects of antitumor radiotherapy through the potentiation of short and long-range neighborhood effects. . With this procedure it would be possible to avoid the complexities and dangers of using MSC cells genetically modified with viral vectors to express TRAIL or DKK3 or the activation of these cells with molecules with important side effects, such as TNFa. And this is so since the radiation that would be used would be the same one that is used normally in the treatments in radiotherapeutic oncology and the product that would be added: "MSC activated by radiation", would be achieved well before the therapeutic irradiation of the patient , well during the course of it. We would thus dispose within the tumor of medicinal MSC cells from which, activated with radiation, they would be released molecules with biological activity capable of acting reinforcing the tumor-suppressor immunological functions within the nucleus of the tumor itself. In addition, we cannot fail to underline that the soluble forms of the sTRAIL and DKK3 molecules can, via lymphatic or blood circulation, reach distal locations of tumor cells thus contributing to enhance the systemic effects of radiation therapy if this form of cell therapy is used. jointly and rationally with clinical radiotherapy.

EXAMPLE 2. In vivo test

For in vivo tests, mice with tumors implanted in the upper part of each of the hind legs were used. NOD / SCID mice of 7 to 9 weeks of age that were inoculated with human breast cancer cells (A375, G361 or MCF-7) were used.

Groups of 6 to 8 mice that were treated with: Radiotherapy, Cell Therapy with MSC cells, Radiotherapy plus MSC, and not undergoing any treatment (Control Group) were included in the study.

All experiments have been carried out in accordance with the recommendations of the Care and Usage Guide for laboratory animals of the Bioethics Committee of the University of Granada and the protocols were also approved by the CSIC Ethics Committee for experimental animals.

Cells were inoculated into the folds of the dorsal skin of NOD / SCID mice mice (1-3- 10 ⁶ cells in 0.1 ml of saline on each of the rear legs mouse). Mice were monitored every 2-3 days by measuring two perpendicular diameters of each of the tumors. Tumor volume was calculated using the formula v = (π / 6) (α ² · b) where a and b are the values of the smallest diameter and diameter respectively. Mice with tumors larger than 64 mm ^{3 were selected} for the following experiments:

In vivo test (I) Radiation therapy In this experiment the mice anesthetized with ketamine were treated with a dose of 3 Gy. The radiation was administered by an X-ray tube (YXLON, model Y, Tu 320-D03) using a voltage of 240.4 kV, and working with a current of 13.0 mA, with a filter of 0.32 mm Cu , an irradiation focus of 5 mm in diameter, the tumor being located at a distance of 10 cm, the irradiation field being a circle of 0.78 cm ² , and using a dose administration rate of 1502 ± 0, 3 mGy / min. The mouse tumor was placed in the hole made on an 8 mm thick steel blade that was used as a shield for the rest of the mouse body. The radiation dose that reaches any region of the mouse body located more than 20 mm from the center of the hole that was used to allow the radiation to reach the tumor was equal to or less than 2.0 ± 0.4 mGy / min. This dose rate allows you to calculate that the dose received by the mouse anywhere in your body including the tumor against the side was less than 4.0 mGy.

The treatment was repeated every week until three to five weeks were completed (according to the experiment performed). After the last dose the study of the mice was continued for another 7 days. Mice in the control group did not receive RT or MSC cell therapy.

The loss of tumor cells derived from each treatment is calculated using the equation proposed by (Steel, Eur J Cancer, 1967. 3 (4): p. 381-7). Obviously the fraction of surviving cells is: (S _F = 1 - C _L ). The expected cell survival value for the therapeutic combination RT + MSC is calculated by multiplying the S _F after RT by the S _F after MSC therapy. The observed value for the combination (O) results from applying the Steel equation to the data observed for the experiment. The additive model (Valeriote and Lin, Cancer Chemother Rep, 1975. 59 (5): p. 895-900) predicts that if the combined treatments show additive effects when the O / E ratio = 0.8-1, 2; The effects are subadditive or antagonistic when O / E> 1, 2, and the therapeutic combination is said to have synergistic effects when the O / E ratio <0.8. The results obtained (Figure 9) allow us to conclude that in the three tumor cell lines tested in vivo, tumor growth shows an adjustable pattern to the exponential growth model; that radiotherapy produces a cytoreductive effect that results in slowing tumor growth and that the measured bystander effect on the contralateral (non-irradiated) tumor is also clearly evident in in vivo experiments. In addition, in the same figure it can be seen that, after the injection of mesenchymal cells (day 20 of experiment), there is a noticeable change in the kinetics of tumor growth since the volume measured for them on days 21 and 22 clearly moves away of what the tumor growth function found for them (dashed lines in the figure) predicts. Statistically, the three curves included in Figure 9 are different (P <0.0001). Consequently, the volume doubling times of each of them are known Control T _D = 4.27 (95% Cl: 4, 13 to 4.41); Treatment with RT T _D = 10.55 (95% Cl: 10.21 to 10.92) and growth of irradiated contralateral tumors (Bystander) T _D = 5.07 (95% Cl: 4.82 to 5.33) . With these data it can be calculated that the cell loss for radiotherapy treatment is greater than 55% and that the irradiation of the tumor located in the treatment center produces, in the non-irradiated tumor, a cell loss due to the bystander effect of the 15% order.

In vivo test (II) Radiation therapy + MSCs

The above results have been confirmed by an experiment in which the role of MSCs cells as tumor suppressing agents when used alone or when used in combination with radiotherapy has been studied (Fig. 10). For this we have used a total of 32 mice in which 3-10 ⁶ G361 cells were implanted in the upper part of each of the two hind legs of the animal. When the tumors reached the size of 64 mm ³ the following groups were established: Control Group (8 mice, 16 tumors); MSC Group (16 mice with G361 tumors, 32 tumors) were all treated with intraperitoneal injections of 10 ⁶ MSC administered every seven days for 4 successive weeks. On the day after the first treatment, half of the group (8 mice, 16 tumors, 8 of them irradiated) were randomly selected to be irradiated in the same way as described in the previous paragraph. The therapeutic combination MSC + RT 3 Gy was continued for three more weeks. The other 8 continued to be treated only with MSC for the additional three weeks.

During the intervals between each treatment session all mice included in the groups: Control, Radiotherapy, MSC therapy and Radiotherapy plus MSC, were followed to control the size of the tumor present in each of its flanks and have sufficient data to calculate the Growth kinetics of each tumor as a function of time and as a function of therapeutic variables. The data corresponding to this experiment are shown in Figure 10 and the evolution of the tumors in Figure 11. The values corresponding to the volume doubling times of each of the series of tumors followed have allowed to calculate the cellular losses derived from each experimental condition in relation to the growth of the Control Group. Note that the graph is marked with a blue line on the day of treatment with MSC and with a red line on the day of treatment with RT.

Parameter Control MSC RT 3G and Bys RT + SC

RT RT + MSC Bys

T _D (days) 6.13 6.69 1 1, 55 9.03 22, 13 9.88

C _L (%) 0.0 8.3 46.9 32, 1 72.3 38.0

MSC-ER 1 0.68 1, 5.81

S _F (%) 100.0 91, 7 53, 1 67.9 27.7 62.0

O / E 0.57 Table 2. Representative values of tumor growth kinetics after RT, MSC and RT + MSC

Applying the additive model, the experimental results conclusively demonstrate that the combination of RT + MSC has a synergistic effect (O / E = 0.57) and that it enhances the action of RT by a factor of 1, 5.

It is also clear that, in the model used, mesenchymal cells are tumor suppressors decreasing cell survival by around 10%, and that, by byderder effect, the contralateral tumor to irradiated decreases their proliferative activity around 30%, and that when the combined RT + MSC therapy is applied, this decrease is enhanced to exceed 35%. Representative growth curves show that these differences, although small, are statistically significant. All these results demonstrate, both in vitro (Figs. 7 and 8) and in vivo (Figs. 9, 10 and 11), that the treatment of tumor models with MSC cells results in a tumor-suppressing action and that this activity is potency when cells are pre-activated with radiation therapy at low doses and, more clearly, when cells are activated directly in vivo when treating the tumor with radiation therapy. The prolongation of the effect of distance radiotherapy (bystander and abscopal effects) and its potentiation by the presence of MSC cells both in the volume of the irradiated tumor and located in foci distal to the main tumor, can be of great interest in Radiation Oncology.

Claims

1. A method of obtaining activated stem cells comprising administering low doses of radiation.

2. The method of obtaining activated stem cells according to the preceding claim, wherein the stem cells are irradiated with a dose between 0.03 cGy and 6 Gy.

3. A method of obtaining activated stem cells according to any of claims 1-2, wherein the stem cells are irradiated with a dose of between 0.25 Gy and

4 Gy.

4. A method of obtaining activated stem cells according to any of claims 1-3, wherein the stem cells are irradiated with a dose of about 2 Gy.

5. A method of obtaining activated stem cells according to any of claims 1-4, wherein the stem cells are mesenchymal stem cells.

6. An activated isolated stem cell obtained by a method according to any of claims 1-5.

7. A cell population comprising at least one isolated activated stem cell according to claim 6.

8. A composition comprising at least one activated stem cell according to claim 6, or a cell population according to claim 7.

9. The composition according to the preceding claim, further comprising a pharmaceutically acceptable carrier.

10. The composition according to any of claims 8-9, which further comprises another active ingredient.

The composition according to any of claims 8-10, wherein the additional active ingredient is a PARP (poly (ADP-ribose) polymerase inhibitor

12. A combined preparation comprising:

a) a component A which is an activated stem cell according to claim 6, a cell population according to claim 7, or a composition according to any of claims 8-11,

13. The combined preparation according to the preceding claim, further comprising at least one PARP (poly (ADP-ribose) polymerase) inhibitor.

14. The use of an activated stem cell according to claim 6, of a cell population according to claim 7, of a composition according to any of claims 8-11, or of the combined preparation according to any of claims 12-13, in The preparation of a medicine.

15. The use of a stem cell according to claim 6, of a cell population according to claim 7, of a composition according to any of claims 8-1 1, or of the combined preparation according to any of claims 12-13, in the development of a medicine for the treatment of cancer.

16. The use of an activated stem cell, a cell population, a composition, or the combined preparation according to the preceding claim, wherein the cancer is a systemic cancer.