WO2022254075A1 - Cellules souches mésenchymateuses ayant un potentiel thérapeutique accru pour le traitement du cancer - Google Patents

Cellules souches mésenchymateuses ayant un potentiel thérapeutique accru pour le traitement du cancer Download PDF

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WO2022254075A1
WO2022254075A1 PCT/ES2022/070346 ES2022070346W WO2022254075A1 WO 2022254075 A1 WO2022254075 A1 WO 2022254075A1 ES 2022070346 W ES2022070346 W ES 2022070346W WO 2022254075 A1 WO2022254075 A1 WO 2022254075A1
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cells
cell
msc
mscs
melatonin
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Spanish (es)
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Vivian CAPILLA GONZALEZ
Yolanda AGUILERA GARCIA
Laura OLMEDO MORENO
Nuria MELLADO-DAMAS SANZ
Alejandro MARTIN-MONTALVO SANCHEZ
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Fundación Pública Andaluza Progreso Y Salud
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0667—Adipose-derived stem cells [ADSC]; Adipose stromal stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention is within the field of regenerative medicine, and relates to the use of melatonin to enhance the therapeutic effects of mesenchymal stem cells (MSCs) in cancer, particularly glioma.
  • Melatonin pretreatment is generally associated with anti-inflammatory actions, antioxidant effects, and protection of mitochondria, thus promoting MSC survival and preventing MSC senescence.
  • This method aims to provide a cellular (ie MSC) product with superior anticancer properties.
  • MSCs mesenchymal stem cells
  • MSCs mesenchymal stem cells
  • antioxidants such as melatonin could solve this limitation.
  • melatonin exhibits anticancer effects against a variety of tumors. Therefore, the combination of MSC and melatonin could offer a more effective cell therapy for its application in cancer.
  • FIG. 1 Characterization of MT-pretreated human MSCs.
  • A Viability of MSCs after 1 hour exposure to various concentrations of H2O2, as measured by the Alamar Blue assay.
  • B Viability of MSCs after a 1 hour exposure to 1000 pm of H2O2 and various concentrations of MT, as measured by the Alamar Blue assay.
  • C Representative microscope images of MSCs pretreated or not with MT that differentiated into adipocytes (demonstrated by the presence of lipid droplets stained with Oil Red O), osteocytes (demonstrated by the presence of calcium deposits stained with Alizarin Red ) and chondrocytes (demonstrated by the presence of cartilage-specific extracellular matrix components in alcian blue-stained sections of paraffin-embedded chondrocyte spheroids).
  • D Quantification of stainings shown in C.
  • E Representative flow cytometric analysis of cultured MSCs pretreated or not with MT, showing positive expression of MSC-specific markers CD13, CD29, CD73, CD105, and CD90, while there was a negative expression for CD31, CD45, CD34 and HLA II.
  • F Detection of apoptosis using annexin V and the non-vital dye propidium iodide by flow cytometry.
  • G Bar graph representing the number of colonies generated in a colony-forming unit assay.
  • H Bar graph representing the size of colonies generated in a colony-forming unit assay.
  • I Representative diagram of Seahorse analysis of OCR on MSCs pretreated or not with MT. Data are represented as mean ⁇ SEM.
  • FIG. 1 Graph depicting the number of migrated glioma cells via the Transwell assay shown in A.
  • C Metabolic activity determined by the Alamar Blue assay on glioma cells.
  • D Immunocytochemistry against Ki67 (green) and OCT 2/3 (red) in cultured glioma cells. Cell nuclei were stained with Hoechst stain (blue).
  • E Quantification of immunocytochemistry against OCT 2/3 shown in D.
  • F Quantification of immunocytochemistry against Ki67 shown in D.
  • G Analysis of apoptotic glioma cells by flow cytometry after annexin V staining.
  • H Representative Western blot for caspase 3 and Bcl-XL protein levels in glioma cells. GAPDH was used as an internal control. Data are represented as mean ⁇ SEM.
  • FIG. 3 Effects of MSCs pretreated or not with MT on the growth of subcutaneous gliomas in athymic nude mice.
  • Figure 4 Effects of MSCs pretreated or not with MT on the histological phenotype of subcutaneous gliomas in athymic nude mice.
  • A Representative images of tumor sections stained with hematoxylin and eosin, CD34, TUNEL, Ki67, and Sirius Red.
  • B Quantification of the percentage of TUNEL+ cells shown in A.
  • C Correlation between the percentage of TUNEL+ cells and the volume of the tumor.
  • D Quantification of the area with Ki67+ cells shown in A.
  • FIG. 5 Analysis of inflammatory cytokines secreted in the tumor environment in vitro. Cytokine profile in the supernatant of cocultured cells. Bar graphs show the concentration levels of the cytokines.
  • Basic FGF fibroblast growth factor
  • G-CSF granulocyte colony-stimulating factor
  • GM-CSF granulocyte-macrophage colony-stimulating factor
  • IFN-g interferon gamma
  • I L-1a interleukin 1 alpha
  • I L-1b interleukin 1 beta
  • IL-5 interleukin 5
  • IL-6 interleukin 6
  • IL-8 interleukin 8
  • IL-9 interleukin 9
  • MCP-1 monocyte chemoattractant protein 1
  • MIP-1a macrophage inflammatory protein 1 alpha
  • MIP-1 b macrophage inflammatory protein 1 beta
  • TNF-a tumor necrosis factor a
  • VEGF vascular endothelial growth factor
  • FIG. 6 Effects of MSCs pretreated or not with MT on glioma cells (direct system).
  • A Representative images of U87, U87+MSC, and U87+MSCMT spheroids seeded on fibronectin scaffolds and allowed to migrate for 24 h, 48 h, and 72 h.
  • B Quantification of area of migration shown in A.
  • C Representative images of U87, U87+MSC, and U87+MSCMT spheroids seeded on Matrigel scaffolds and allowed to migrate for 24 h, 48 h, 72 h and 96 h.
  • D Quantification of the area of invasion shown in C. Data are represented as mean ⁇ SEM. *p ⁇ 0.05, **p ⁇ 0.01, ***p ⁇ 0.001 compared to U87; One-way ANOVA.
  • Figure 8. Histological analysis of subcutaneous tumor xenografts. Subcutaneous tumors were formed from U87 glioma cells that were implanted in combination or not with MSC or MSCMT.
  • a first aspect of the invention refers to a stem cell obtained or obtainable by a process that comprises placing the cell in contact with melatonin.
  • the stem cell is a mesenchymal stem cell.
  • the stem cell of the invention is an adult stem cell, and more preferably, it is a mesenchymal stem cell.
  • adult stem cell refers to that stem cell that is isolated from a tissue or an organ of an animal in a state of growth subsequent to the embryonic state.
  • the stem cells of the invention are isolated in a postnatal state.
  • they are isolated from a mammal, and more preferably from a human, including neonates, juveniles, adolescents, and adults.
  • ⁇ 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, cardiac muscle, intestine , liver, neural.
  • MSCs may be obtained from, but not limited to, bone marrow, adipose tissue (such as subcutaneous adipose tissue), liver, spleen, testes, menstrual blood, amniotic fluid, pancreas, periosteum, synovium, skeletal muscle, dermis, pericytes, trabecular bone, human umbilical cord, lung, dental pulp, and peripheral blood.
  • adipose tissue such as subcutaneous adipose tissue
  • liver spleen
  • testes menstrual blood
  • amniotic fluid pancreas
  • periosteum synovium
  • skeletal muscle skeletal muscle
  • dermis pericytes
  • trabecular bone human umbilical cord
  • the activated stem cell of the invention can be genetically modified by any conventional method including, by way of illustration, without 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.
  • this aspect of the invention refers to an activated mesenchymal stem cell, hereinafter activated mesenchymal stem cell of the invention, obtained or obtainable by a method comprising contacting the cell with melatonin.
  • activated mesenchymal stem cell is understood to be a cellular product based on MSCs that presents enhanced one or some of its cellular functions (survival, antioxidant activity, mitochondrial function, immunomodulatory capacity, etc.).
  • Another aspect of the invention relates to a population of activated stem cells, hereinafter the activated stem cell population of the invention, comprising at least one activated stem cell of the invention.
  • the stem cell is an MSC.
  • the cell population of the invention comprises at least 20%, preferably 40%, and even more preferably 50%, 60%, 80%, 90%, 95%, or 99% adult stem cells. of the invention.
  • the term "isolated” indicates that the cell or cell population of the invention to which it refers is not found in its 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 xenogeneic cells. In a particular embodiment, said cells are of autologous origin, thus reducing potential complications associated with/or antigenic and immunogenic responses of said cells when administered to the individual.
  • Mesenchymal stem cells activated according to this aspect of the invention will preferably be obtained by pre-treating this cell product with a solution of, preferably 25 mM, melatonin.
  • the pre-treatment will preferably take place during the 24 hours prior to use, but it can also be carried out for a longer period of time (e.g. 48 hours).
  • another preferred embodiment of this aspect of the invention relates to activated mesenchymal stem cells obtainable by a method or methodology for obtaining activated stem cells, which comprises contacting the stem cell with a melatonin solution.
  • the stem cell is a mesenchymal stem cell.
  • the melatonin is in a concentration between 10 pM and 50 pM, more preferably between 15 pM and 40 pM, even more preferably between 20 pM and 30 pM, and most preferably still 25 pM.
  • the pretreatment time is at least 12 hours, preferably at least 16 hours, even more preferably at least 20 hours, and most preferably at least 24 hours.
  • a third aspect of the invention refers to a composition, hereinafter composition of the invention, comprising at least one activated stem cell of the invention.
  • the composition of the invention further comprises a pharmaceutically acceptable carrier.
  • the composition of the invention also comprises another active principle.
  • the stem cells are mesenchymal stem cells.
  • the cell composition of the invention is at least 50%, at least 60%, preferably 70%, more preferably 80%, even more preferably 90%, and still more preferably , 95% of the isolated stem cells activated of the invention.
  • composition of adult cells of the invention may contain a medium in which the cells of the invention are found; said medium must be compatible with said cells.
  • a medium in which the cells of the invention are found; said medium must be compatible with said cells.
  • 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 is preferably a pharmaceutical composition for administration to a subject.
  • pharmaceutically acceptable vehicle means a vehicle that must be approved by a federal or state government regulatory agency or listed in the US Pharmacopeia or European Pharmacopeia, or other generally recognized pharmacopeia for use in animals, and more specifically in humans.
  • vehicle refers to a diluent, adjuvant, excipient or carrier with which the cells or cell population of the invention or said composition comprising stem cells of the invention obtainable according to the process of the invention must be administered; obviously, said vehicle must be compatible with said cells.
  • Illustrative, non-limiting examples of such a vehicle include any physiologically compatible vehicle, for example, isotonic solutions (for example, sterile 0.9% NaCI saline, 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 polyornithine, hydrogels, agarose, silicone dextran sulfate.
  • the support medium may, in specific embodiments, contain growth factors or other agents.
  • the cells can be introduced into a liquid phase of the vehicle which is subsequently treated such that it becomes a more solid phase.
  • the pharmaceutical composition of the invention may also contain, when necessary, additives to increase, control or otherwise direct the desired therapeutic effect of the cells, which said pharmaceutical composition comprises, and/or auxiliary substances or pharmaceutically acceptable substances, such as buffering agents, surfactants, co-solvents, 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 adding additional substances, such as, for example, aspartic acid, glutamic acid, etc.
  • Said 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.
  • active substance means any component that potentially provides a pharmacological activity or other different effect in diagnosis, cure, mitigation, treatment, or prevention of a disease, or that affects the structure or function of the body of man or other animals.
  • active ingredient means any component that potentially provides a pharmacological activity or other different effect in diagnosis, 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 in the drug in an intended modified form that provides the specific activity or effect.
  • Another aspect of the invention relates to an activated stem cell of the invention, the cell population of the invention, or a composition of the invention, for use as a medicament. More preferably the stem cells are mesenchymal stem cells.
  • drug refers to any substance used for the prevention, diagnosis, alleviation, treatment or cure of diseases in humans and animals.
  • the disease is cancer.
  • the pharmaceutical composition of the invention will contain a prophylactically 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! desired therapeutic effect.
  • 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 pursued.
  • the therapeutically effective amount of cells of the invention that must be administered will depend, among other factors, on the characteristics of the subject, the severity of the disease, the form 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 must adjust this dose depending on the factors described above.
  • 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 kilogram of body weight of the recipient, and more preferably between 5x10 5 and 5x10 6 cells of the invention per kilo of the recipient's body weight, in an even more preferred embodiment said composition pharmaceutical will contain approximately between 1x10 6 and 2x10 6 cells of the invention per kilogram of the recipient's body weight, depending on the factors described above.
  • the dose of cells of the invention can be repeated, depending on the state and evolution of the patient, at time intervals of days, weeks or months that must be established by the specialist in each case.
  • Another aspect of the invention relates to an activated stem cell of the invention, the cell population of the invention, or a composition of the invention, to increase, restore or partially or totally replace the functional activity of a diseased tissue or organ. or damaged.
  • Another aspect of the invention relates to an activated stem cell of the invention, the cell population of the invention, or a composition of the invention, for the treatment of cancer.
  • Activated mesenchymal stem cells will be obtained by pretreating this cell product with a solution of, preferably 25 mM, melatonin.
  • the pre-treatment will preferably take place during the 24 hours prior to use, but it can also be carried out for a longer period of time (e.g. 48 hours).
  • another aspect of the invention refers to a procedure or methodology for obtaining activated stem cells, from now on the procedure of the invention, which comprises contacting the stem cell with a melatonin solution.
  • the stem cell is a mesenchymal stem cell.
  • the melatonin is in a concentration between 10 mM and 50 pM, more preferably between 15 pM and 40 pM, even more preferably between 20 pM and 30 pM, and most preferably still 25 pM.
  • the pretreatment time is at least 12 hours, preferably at least 16 hours, even more preferably at least 20 hours, and most preferably at least 24 hours.
  • MSC mesenchymal stem cells
  • MT melatonin
  • EXAMPLE 1 Development of an in vitro coculture model to mimic the in vivo biological interaction between MSCs and glioma cells.
  • MSC culture Adipose tissue-derived MSCs were purchased from ATCC (PCS-500-011). Briefly, MSCs are grown in a growth medium composed of Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Cells were incubated at 37°C in a humidified atmosphere with 20% O2 and 5% C02 until 70-80% confluence. The media were changed every 2-3 days. Passages were performed using a 0.25% trypsin solution. Cells from passages 3 to 6 were used for all experiments.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • penicillin-streptomycin penicillin-streptomycin
  • MT Pretreatment Subconfluent MSC cultures were pretreated with 25 mM MT for 24 hours. The cells were then used for the different experiments proposed here. To determine whether MT pretreatment modifies MSC identity, we evaluated the potential for multiple MSC lineages (adipocytes, osteocytes, and chondrocytes) and the expression of established MSC markers (MSCs express markers CD29, CD13, CD73, CD105 and CD90, while they are negative for the expression of CD31, CD45, CD34 and HLA II) (1-3). We will also determine if MT pretreatment affects cell proliferation (e.g. Alamar Blue), apoptosis (e.g. Annexin V), antioxidant activity (e.g. catalase activity) and mitochondrial function (e.g. Sehorse).
  • cell proliferation e.g. Alamar Blue
  • apoptosis e.g. Annexin V
  • antioxidant activity e.g. catalase activity
  • mitochondrial function e.g. Se
  • Glioma cell culture Glioma cells were obtained from ATCC (U87MG; HTB-14). Briefly, U87 cells are grown in a growth medium composed of Eagle's Minimal Essential Medium (EMEM; ATCC), supplemented with 10% FBS and 1% penicillin-streptomycin. Cells are incubated at 37 °C in a humidified atmosphere with 20% 02 and 5% C02 to 70-80% confluence. The media were changed every 2-3 days. Passaging is done using a 0.25% trypsin solution. Cells from passages 3 to 6 were used for all experiments.
  • EMEM Eagle's Minimal Essential Medium
  • D. Co-cultivation system To study the interaction between glioma cells and MSCs (pretreated or not with MT), the transwell co-cultivation system (0.4 pm pore size) is used. MSCs are seeded on top, while glioma cells are seeded on the bottom. This system allows to easily separate the two cell populations for perform specific studies with glioma cells.
  • the experimental groups are: U87 (U87 cells without MSC), U87 + MSC (U87 cells co-cultured with MSC) and U87 + MSC + MT (U87 cells co-cultured with MSC that were pre-treated with MT).
  • EXAMPLE 2 To study the interactions between MSCs and glioma cells using the in vitro coculture model.
  • glioma cells After 24 hours in the transwell coculture system, glioma cells are used to study proliferation using the Alamar Blue assay and immunocytochemistry.
  • Apoptosis After 24 hours in the transwell coculture system, glioma cells are used to study apoptosis by flow cytometry (Annexin V kit).
  • glioma cells are harvested to investigate the molecular pathways that are modulated by MSCs, using RNA-Seq, RT-PCR, and western blotting.
  • Cytokine profile The cell supernatant generated after cocultivation will be analyzed by multiplex ELISA (Bio-Plex Pro TM Human Cytokine) to identify survival, angiogenic and inflammatory cytokines, among others.
  • EXAMPLE 3 To study the interactions between MSCs and glioma cells using an in vivo model.
  • mice and experimental groups To avoid rejection of the transplanted cells, athymic nude mice (10 weeks old) are used. Animals are randomly assigned to three experimental groups: mice injected with glioma cells (U87 group), mice injected with glioma cells and MSC (U87 + MT group) and control mice (U87 + MSC + MT group).
  • Tumor growth Mice are monitored daily to study tumor evolution throughout the project. Once sacrificed, the tumors are excised for subsequent histological and immunohistochemical analysis.
  • Example 4 Pretreatment of mesenchymal stem cells (MSC) with melatonin (MT) enhances the anticancer properties of the cells.
  • Adipose tissue-derived MSCs were purchased from ATCC (PCS-500-011). Briefly, MSCs were grown in growth medium consisting of Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. Cells were incubated at 37°C in a humidified atmosphere with 20% O2 and 5% CO2 to 70-80% confluence. The media were changed every 2-3 days. Passages were carried out using a 0.25% trypsin solution. For all experiments cells from passages 3-6 were used.
  • DMEM Dulbecco's modified Eagle's medium
  • FBS fetal bovine serum
  • penicillin-streptomycin penicillin-streptomycin
  • MSCs express markers CD29, CD13, CD73, CD105 and CD90, while they are negative for the expression of CD31, CD45, CD34 and HLA II.
  • MSCs express markers CD29, CD13, CD73, CD105 and CD90, while they are negative for the expression of CD31, CD45, CD34 and HLA II.
  • Glioma cell culture Glioma cells were obtained from ATCC (U87MG; HTB-14). Briefly, U87 cells were grown in growth medium consisting of Eagle's minimal essential medium (EMEM; ATCC), supplemented with 10% FBS and 1% penicillin-streptomycin. Cells were incubated at 37°C in a humidified atmosphere with 20% O2 and 5% CO2 to 70-80% confluence. The media were changed every 2-3 days. Passages were carried out using a 0.25% trypsin solution. For all experiments cells from passages 3-6 were used.
  • EMEM Eagle's minimal essential medium
  • Co-culture system To study the interaction between glioma cells and MSCs (pre-treated or not with MT) two approaches were used:
  • the Transwell co-culture system was used (with a pore size of 0.4 pm). MSCs were seeded on top, while glioma cells were seeded on the bottom. This system made it possible to easily separate the two cell populations for specific studies with glioma cells.
  • the experimental groups were: U87 (U87 cells without MSC), U87+MSC (U87 cells cocultured with MSC) and U87+MSC+MT (U87 cells cocultured with MSC that were pretreated with MT).
  • a. Proliferation After 24 hours in the co-culture system, glioma cells were used to study proliferation using the Alamar Blue assay and immunocytochemistry.
  • b. Apoptosis After 24 hours in the co-culture system, glioma cells were used to study apoptosis by flow cytometry (Annexin V kit).
  • c. Migration assay To study how MSCs interfere with the migration ability of U87, the Transwell system (with 0.8 pm pore size) was used. U87 cells were seeded on top with serum-free medium, while MSCs were seeded on the bottom with FBS medium to encourage cell migration through the membrane pores.
  • Tumor beads cells were placed in a 20 ⁇ l hanging drop with growth medium. For each spheroid, 21,000 U87 cells were used, which were cocultivated or not with MSC or MSCMT in a 3:1 ratio.
  • spheroids were transferred to fibronectin precoated wells for migration assays or fibronectin precoated wells. Matrigel for invasion assays. Spheroids were imaged every 24 hours and the area of migration or invasion was analyzed using ImageJ software (version 1.4r; National Institute of Health, Bethesda, MD). and. Molecular study: After 24 hours in the coculture system, glioma cells were harvested to investigate the molecular pathways modulated by MSCs, using RNA-Seq, RT-PCR, and Western blotting. F.
  • Cytokine profiling Cell supernatants generated after cocultivation were analyzed by multiplex ELISA (Bio-Plex ProTM human cytokine) to identify survival, angiogenic, and inflammatory cytokines, among others. Direct and indirect cocultures (Transwell system) were used.
  • mice To avoid rejection of the transplanted cells, athymic nude mice (10 weeks old) were used. Animals were randomly assigned to three experimental groups: mice injected with glioma cells (U87 group), mice injected with glioma cells and MSC (U87+MT group), and control mice (U87+MSC+MT group).
  • U87 group mice injected with glioma cells
  • U87+MT group mice injected with glioma cells and MSC
  • U87+MSC+MT group mice
  • mice were injected subcutaneously into the flank of the right hind paw with 150 ⁇ l of DMEM:Matrigel (1:2) mixture containing T 106 glioma cells, with or without T 106 MSC (pretreated or not with MT). Mice were sacrificed when the tumors reached an average volume of 4000 mm 3 (greater volumes may affect animal welfare).
  • Tumor Growth Mice were monitored daily to study tumor evolution over time, until tumors reached an average volume of 4000 mm 3 . Tumors were measured with a digital caliper every 5 days to estimate their volume using the formula (minor diameter 2 c major diameter)/2. d.
  • mice were subjected to intracardiac perfusion with 0.9% saline solution and 4% paraformaldehyde.
  • Half of each tumor was paraffin-embedded and processed for histological staining (eg, hematoxylin and eosin and Syrian red) as well as immunohistological techniques.
  • Specific markers of proliferation eg, hematoxylin and eosin and Syrian red
  • specific markers of proliferation eg, hematoxylin and eosin and Syrian red

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Abstract

La présente invention concerne des cellules souches mésenchymateuses ayant un potentiel thérapeutique accru pour le traitement du cancer, les cellules souches activées obtenues par un procédé qui consiste à mettre la cellule en contact avec de la mélatonine, le procédé d'obtention et les utilisations correspondantes.
PCT/ES2022/070346 2021-06-02 2022-06-02 Cellules souches mésenchymateuses ayant un potentiel thérapeutique accru pour le traitement du cancer WO2022254075A1 (fr)

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