Use of an injectable liquid containing bioactive molecules released from a neo-synthesized extracelllar matrix for the local treatment of solid cancers

Abstract

An injectable liquid containing bioactive molecules released from a powder comprising a neo-synthesized extracellular matrix for use in the treatment or prevention of cancer, in particular in the local treatment of solid tumors.

Description

[0001] USE OF AN INJECTABLE LIQUID CONTAINING BIOACTIVE MOLECULES RELEASED FROM A NEO-SYNTH ESIZED EXTRACELLLAR MATRIX FOR THE LOCAL TREATMENT OF SOLID CANCERS

[0002] FIELD OF THE INVENTION

[0003] The present invention relates to an injectable liquid containing bioactive molecules released from a powder of a 3-dimensional neo-synthesized extracellular matrix, in particular for the inhibition of the viability, migration and proliferation of tumor cells. Cancer is a critical public health concern and the world's leading cause of death, with annual significant increasing rates. Cancer can be categorized into the following 5 groups according to the type of cells they start in:

[0004] ■ carcinoma - cancer of the skin or of tissues that line or cover internal organs;

[0005] ■ sarcoma - cancer starting in the connective or supportive tissues such as bone, cartilage, fat, muscle or blood vessels;

[0006] ■ leukemia - cancer of the white blood cells appearing in the bone marrow;

[0007] ■ lymphoma and myeloma - cancer of the immune system;

[0008] ■ brain and spinal cord cancers - known as central nervous system cancers.

[0009] Among these, only carcinoma and sarcoma are solid state tumors. The market for solid tumors therapy is significant and continues to grow as advancements in research and treatment options emerge.

[0010] Solid tumors refer to tumors that form in solid tissues such as the lungs, breast, colon, prostate, skin, bone and liver, among others. According to the International Agency for Research on Cancer (www.iarc.who.int), cancer is a leading cause of death worldwide, accounting for nearly 10 million deaths in 2020.

[0011] The treatment of solid cancers typically involves the use of multiple modalities, such as surgery, systemic anti-cancer therapy and radiotherapy, alone or in combination or sequentially (Saini & Twelves, 2021 ). For the systemic approach, drugs approved to treat solid tumors anywhere in the body regardless of tumor origin can be of chemical origin (i.e. Dabrafenib, Doxorubicin) or monoclonal antibodies (immune checkpoint inhibitor; like Dostarlimab). Chemotherapy presents the major disadvantage that the patient could suffer from treatment-related side effects, off-target effects, and drug resistance (Schirmacher, van Gool, & Stuecker, 2019).

[0012] The antibody approach has its own specific limitations: their clinical efficacy is hampered by poor tumor tissue penetration and heterogeneous distribution. (Paresishvili & Kakabadze, 2023) (Rohaan, Wilgenhof, & Haanen, 2018).

[0013] Therefore, an alternate path forward is currently under strenuous investigation: intratumoral (IT) delivery of anti-cancer drugs, which provides a highly localized effect, higher drug concentrations at the tumor, potentially improving treatment efficacy, reduced systemic toxicity and bypassing of potential drug resistance mechanisms present at the systemic level.

[0014] Thus, oncology drug development targets the above challenges by deploying new cancer therapy strategies and is gathering momentum due to recent advances in drug screening technologies targeting selected indications of solid tumors for a local delivery.

[0015] WO2Q21105404A1 to Novadip Biosciences discloses sterile and desiccated biomaterials comprising devitalized differentiated cells having tissue regenerating and / or repairing properties. The biomaterials comprise a particulate material. The cells and the particulate material are embedded in a neo-synthesized extracellular matrix. The particulate material is preferably gelatin, a ceramic material, or a demineralized bone matrix (DBM).

[0016] Among a long list of disorders, also cancer is described, including breast cancer, a skin cancer and a bone cancer.

[0017] However, the inhibition of cancer is only exemplified in respect of the exosome.

[0018] Consequently, only the anti-cancer use of the extracellular extract, i.e. , the extracellular vesicles was exemplified.

[0019] WO2Q22112528A1 to Novadip Biosciences discloses cellular and / or extracellular extracts obtained from a scaffold-free 3-dimensional culture of mature cells and a particulate material for preventing and / or treating cancer. The mature cells secrete the neosynthesized extracellular matrix. Both the mature cells and the particulate material are embedded in the neo-synthesized extracellular matrix. The extracts further comprise a pharmaceutically acceptable carrier. However, the inhibition of cancer is only exemplified in respect of the isolated extracellular vesicles, i.e. the supernatant, also referred to as the exosome.

[0020] Consequently, only the anti-cancer use of the extracellular extract, i.e., the extracellular vesicles was exemplified.

[0021] International patent application PCT / EP2024 / 054107 to Novadip Biosciences discloses a composition comprising:

[0022] ■ devitalized differentiated cells;

[0023] ■ 3-dimensional neo-synthesized extracellular matrix as a vehicle of bioactive compounds selected from the group consisting of proteins, mRNAs, miRNAs, lipids, extracellular vesicles proteins, mRNAs, miRNAs, lipids, extracellular vesicles; and

[0024] ■ a particulate material; for use in the treatment or prevention of cancer; characterized in that:

[0025] ■ the differentiated cells secreted the 3-dimensional neo-synthesized extracellular matrix prior to devitalization;

[0026] ■ the cells and the particulate material are embedded in the 3-dimensional neo- synthesized extracellular matrix;

[0027] ■ the composition is substantially free of extracellular vesicles; and

[0028] ■ the particulate material is gelatin, and even more preferably gelatin beads.

[0029] However, this document does not disclose any concrete release methods nor any examples for its use in the treatment of cancer, in particular in the local treatment of solid tumors.

[0030] SHORT DESCRIPTION OF THE INVENTION

[0031] The present inventors have surprisingly found that the administration of an injectable liquid containing bioactive molecules released from a powder of a desiccated and devitalized neo-synthesized extracellular matrix substantially inhibits the viability, migration and proliferation of cancer cells. The present inventors further found that the injectable liquid containing bioactive molecules released from a desiccated and devitalized neo-synthesized extracellular matrix may act as a carrier for proteins and miRNAs that inhibit the viability, migration and proliferation of the cancer cells. The injectable liquid is, thus, particularly suitable for the local treatment of solid tumors.

[0032] Accordingly, a first aspect of the invention is an injectable liquid containing bioactive molecules released from a powder,

[0033] Wherein preferably the bioactive molecules are released through incubation of the powder in a liquid phase;

[0034] Wherein the powder is obtained or obtainable, preferably through desiccation and / or devitalization, from a biomaterial;

[0035] Wherein the biomaterial comprises: a. Differentiated stem cells, preferably osteogenically differentiated stem cells, even more preferably osteogenically differentiated adipose-tissue derived stem cells (ASCs); b. A 3-dimensional neo-synthesized extracellular matrix as a vehicle of bioactive molecules comprising one or more of mRNAs, miRNAs, lipids, and proteins; and c. A gelatin particulate material or a gelatin-derived hydrogel;

[0036] Wherein the differentiated stem cells secrete the 3-dimensional neo-synthesized extracellular matrix;

[0037] Wherein the gelatin particulate material is added to the differentiated stem cells to induce the secretion of the 3-dimensional neo-synthesized extracellular matrix;

[0038] Wherein the maturation period of the differentiated stem cells after the addition of the gelatin particulate material is from 5 to 15 weeks, preferably from 6 to 10 weeks, even more preferably from 7 to 9 weeks;

[0039] Wherein the differentiated stem cells and the gelatin particulate material are embedded in the 3-dimensional neo-synthesized extracellular matrix;

[0040] Wherein the injectable liquid is characterized by:

[0041] ■ The injectable liquid comprises the bioactive molecules in a therapeutically or cosmetically effective amount,

[0042] ■ The injectable liquid does not contain any viable differentiated stem cells; ■ The injectable liquid does not contain any gelatin particulate material;

[0043] ■ Preferably the injectable liquid comprises extracellular vesicles in an amount of 10 w% or less, preferably 5 w% or less, even more preferably 1 w% or less and even more preferably 0.1 w% and even more preferably 0.01 w% or less as compared to the total weight or the total protein content of the injectable solution; and

[0044] ■ preferably the concentration of total proteins is 0.1 mg / ml to 10 mg / ml, preferably from 0.5 mg / ml to 5 mg / ml, even more preferably from 0.75 mg / ml to 2.5 mg / ml of the injectable liquid; for use in the treatment or prevention of cancer, in particular in the local treatment of solid tumors.

[0045] In another aspect, the injectable liquid is used for:

[0046] ■ inhibiting the viability of cancer cells,

[0047] ■ inhibiting the proliferation of cancer cells;

[0048] ■ inhibiting the migration of cancer cells;

[0049] ■ inhibiting the cell colony formation of cancer cells; or

[0050] ■ any combination thereof.

[0051] In another aspect, the powder is:

[0052] ■ desiccated, preferably by lyophilization;

[0053] ■ size reduced, preferably by grinding to volumetric particle size distribution with a range of 100 to 5000 micrometers as measured by laser diffraction granulometry; and / or

[0054] ■ sterilized, preferably by gamma-irradiation.

[0055] In another aspect, the powder is free of external scaffolds.

[0056] In another aspect, the differentiated stem cells are selected from the group comprising or consisting of osteoblasts, osteocytes, chondroblasts, chondrocytes, keratinocytes, myofibroblasts, epithelial cells, endothelial cells, adipocytes, neural cells, and precursors thereof, and preferably are soft tissue cells, chondroblasts or osteoblasts, even more preferably adipose-derived stem cells differentiated into osteogenic cells.

[0057] In another aspect, the powder comprises the neo-synthesized extracellular matrix in a content of 0.001 w% to 10 w%, preferably from 0.01 w% to 7.5 w%, even more preferably from 0.1 w% to 5 w% as compared to the total weight of the powder. In another aspect, the differentiated stem cells are derived from one or more of:

[0058] ■ pluripotent stem cells (PSCs) such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs);

[0059] ■ adult stem cells such as hematopoietic stem cells (HSCs), skin stem cells (SSCs), neural stem cells (NSCs); and

[0060] ■ mesenchymal stem cells (MSCs), preferably derivable from adipose tissue, peripheral blood or placenta, and preferably are mesenchymal stem cells.

[0061] In another aspect, the stem cells are derived from mesenchymal stromal cells, preferably obtainable from bone marrow, adipose tissue, placenta, or blood.

[0062] In another aspect, the cancer is a solid cancer selected from the group consisting of a bone cancer, a brain cancer, a skin cancer, a breast cancer, a cancer of the central nervous system, a cancer of the cervix, a cancer of the upper aero digestive tract, a colorectal cancer, an endometrial cancer, a germ cell cancer, a bladder cancer, a kidney cancer, a laryngeal cancer, a liver cancer, a lung cancer, a neuroblastoma, an esophageal cancer, an ovarian cancer, a pancreatic cancer, a pleural cancer, a prostate cancer, a retinoblastoma, a small intestine cancer, a soft tissue sarcoma, a stomach cancer, a testicular cancer and a thyroid cancer, preferably, the cancer is

[0063] ■ Osteosarcoma;

[0064] ■ Ewing sarcoma;

[0065] ■ Chondrosarcoma;

[0066] ■ fibrosarcoma;

[0067] ■ melanoma;

[0068] ■ lung cancer;

[0069] ■ breast cancer; or

[0070] ■ glioblastoma. preferably, wherein one or more of the following genes are upregulated:

[0071] ■ HMOX1

[0072] ■ NFkBIA

[0073] ■ PDCD4

[0074] ■ TIMP3

[0075] ■ RhoB ■ PMAIP1

[0076] ■ BNIP3L

[0077] ■ CDKN1A

[0078] ■ BAX

[0079] ■ KLF6

[0080] ■ TIPARP preferably, wherein one or more of the following genes are downregulated:

[0081] ■ MCM5

[0082] ■ CDK4

[0083] ■ LAS1 L

[0084] ■ PLK1

[0085] ■ EBP1

[0086] ■ MKI67

[0087] ■ PTTG1

[0088] ■ CDK1

[0089] ■ CDKN3

[0090] ■ CCNB2

[0091] ■ CKS1 B.

[0092] Another aspect of the present invention is a method for obtaining an injectable liquid, preferably the injectable liquid of the invention, comprising:

[0093] ■ Providing a powder; wherein the powder is obtained or obtainable, preferably through desiccation and / or devitalization, from a biomaterial;

[0094] Wherein the biomaterial comprises: a. Differentiated stem cells, preferably osteogenically differentiated stem cells, even more preferably osteogenically differentiated adipose-tissue derived stem cells (ASCs); b. A 3-dimensional neo-synthesized extracellular matrix as a vehicle of bioactive molecules comprising one or more of mRNAs, miRNAs, lipids, and proteins; and c. A gelatin particulate material or a gelatin-derived hydrogel;

[0095] Wherein the differentiated stem cells secrete the 3-dimensional neosynthesized extracellular matrix; Wherein the gelatin particulate material is added to the differentiated stem cells to induce the secretion of the 3-dimensional neo-synthesized extracellular matrix;

[0096] Wherein the maturation period of the differentiated stem cells after the addition of the gelatin particulate material is from 5 to 15 weeks, preferably from 6 to 10 weeks, even more preferably from 7 to 9 weeks; and

[0097] Wherein the differentiated stem cells and the gelatin particulate material are embedded in the 3-dimensional neo-synthesized extracellular matrix;

[0098] ■ Incubating the powder in a liquid phase to enable the release of the bioactive molecules from the powder into the liquid; wherein the liquid is preferably a water-based liquid preferably comprising physiologically acceptable salts, preferably wherein the liquid is free of organic solvents;

[0099] ■ Subsequently, separating the liquid phase from the powder; and

[0100] ■ Subsequently, collecting the injectable liquid; wherein preferably the injectable liquid comprises extracellular vesicles in an amount of 10 w% or less, preferably 5 w% or less, even more preferably 1 w% or less and even more preferably 0.1 w% and even more preferably 0.01 w% or less as compared to the total weight of the injectable solution; and wherein preferably the concentration of total proteins is 0.1 mg / ml to 10 mg / ml, preferably from 0.5 mg / ml to 5 mg / ml, even more preferably from 0.75 mg / ml to 2.5 mg / ml of the injectable liquid.

[0101] In another aspect, the incubation conditions are one or more of:

[0102] ■ Incubation time: from 10 hours to 120 hours, preferably from 30 hours to 100 hours, even more preferably from 40 hours to 60 hours; and / or

[0103] ■ Incubation temperature: from 10 °C to 60 °C, preferably from 20 °C to 50 °C, even more preferably from 30 °C to 40 ° C or from 15 °C to 25 °C; and / or

[0104] ■ Incubation atmosphere: 1 % to 10 % CO2, preferably, 2.5 % to 7.5 %, even more preferably from 4 % to 6 %; and / or

[0105] ■ Incubation pH: from 5 to 7.5, preferably from 6 to 7, even more preferably from 6.5 to 7. In another aspect, the powder is added to the liquid phase in an amount from 5 mg / ml to 500 mg / ml, preferably from 10 mg / ml to 250 mg / ml, even more preferably from 25 mg / ml to 150 mg / ml, even more preferably 50 mg / ml to 125 mg / ml of the liquid phase.

[0106] In another aspect, the liquid phase comprises physiologically acceptable salts in physiologically acceptable quantities, and preferably is phosphate buffer saline (PBS) or physiological serum.

[0107] Another aspect of the present invention is an injectable liquid obtainable or obtained by the method of the invention.

[0108] Another aspect of the present invention is an injectable liquid obtainable or obtained by the method of the invention for use in the treatment or prevention of cancer, in particular in the local treatment of solid tumors.

[0109] ABBREVIATIONS

[0110] 2D: 2-Dimensional

[0111] 3D: 3-Dimensional

[0112] 8W 8 Weeks

[0113] ATP: Adenosine Triphosphate

[0114] CCK-8: Cell counting Kit-8

[0115] CFU: Colony Forming Unit

[0116] CP: Cell Product

[0117] DB: Development Batches

[0118] ECM: Extracellular Matrix

[0119] FBS: Fetal Bovine Serum hASC: human Adipose-derived Stem Cells hBM-MSCs: primary human Bone Marrow Mesenchymal Stem Cells

[0120] HDFa: primary Human adult Dermal Fibroblasts

[0121] IT: IntraTumoral

[0122] MD: Differentiation Medium MP: Proliferation Medium

[0123] OS cell lines: Osteosarcoma cell lines

[0124] PBS: Phosphate-Buffered Saline

[0125] PS: PhosphatidylSerine

[0126] Px: Passage x

[0127] SVF: Stromal Vascular Fraction

[0128] WST-8: 2- (2- methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H- tetrazolium, monosodium salt

[0129] DETAILED DESCRIPTION OF THE INVENTION

[0130] The invention is now described in further detail.

[0131] Injectable

[0132] The liquid containing bioactive molecules is injectable.

[0133] In one embodiment, injectable means that the injectable liquid can be administered by injection for example by a syringe or any other liquid injection system.

[0134] Release

[0135] The term “release” means any extract. Preferably, the term “release” means an injectable liquid containing bioactive molecules obtained by incubating a powder comprising:

[0136] ■ devitalized differentiated cells;

[0137] ■ 3-dimensional neo-synthesized extracellular matrix as a vehicle of bioactive molecules selected from the group consisting of proteins, mRNAs, miRNAs, lipids, extracellular vesicles proteins, mRNAs, miRNAs, lipids, extracellular vesicles; and

[0138] ■ a gelatin particulate material; in a liquid phase.

[0139] Powder

[0140] Powder means any dried, such as lyophilized, and size-reduced form of a biomaterial comprising 3-dimensional neo-synthesized extracellular matrix.

[0141] The powder may contain water in an amount of 5 w% or less, even more preferably 3 w% or less, even more preferably 1 w% or less. Devitalized differentiated cells

[0142] The devitalized differentiated cells are embedded in the 3-dimensional neo-synthesized extracellular matrix.

[0143] In one embodiment, the differentiated cells are osteo-differentiated, skin-differentiated or chondro-differentiated cells. That means that differentiated cells have the ability to promote bone, skin and / or cartilage formation, and / or to maintain existing bone, skin and / or cartilage in a healthy physiological condition.

[0144] In one embodiment, the devitalized differentiated cells are derived from stem cells, such as pluripotent stem cells (PSCs), for example embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) or adult stem cells such as hematopoietic stem cells (HSCs), skin stem cells (SSCs), neural stem cells (NSCs), and mesenchymal stem cells (MSCs).

[0145] MSCs are present in multiple tissues, including BM, adipose tissue, peripheral blood, and placenta.

[0146] In a preferred embodiment, the devitalized differentiated cells are derived from mesenchymal stem cells, preferably adipose tissue-derived stem cells.

[0147] In one embodiment, the differentiated cells are selected from the group comprising or consisting of osteoblasts, osteocytes, chondroblasts, chondrocytes, keratinocytes, myofibroblasts, epithelial cells, endothelial cells, adipocytes, neural cells, and precursors thereof, and preferably are soft tissue cells, chondroblasts or osteoblasts.

[0148] In one embodiment, the osteogenic differentiation medium comprises or consists of DMEM supplemented with L-alanyl-L-glutamine (Ala-Gin, also called ‘Glutamax®’ or ‘Ultraglutamine®’), hPL, dexamethasone, ascorbic acid and sodium phosphate.

[0149] In one embodiment, the osteogenic differentiation medium comprises or consists of DMEM supplemented with L-alanyl-L-glutamine, hPL, dexamethasone, ascorbic and sodium phosphate, and antibiotics, preferably penicillin, streptomycin, gentamycin and / or amphotericin B.

[0150] In certain embodiments, said cells are selected in a group comprising primary cells, stem cells, genetically modified cells, and a combination thereof.

[0151] After devitalization, usually at most 1 % of said cells are viable, preferably at most 0.1 %, even more preferably at most 0.01 %, even more preferably at most 0.001 % of the cells are viable. The stem cells (examples: pluripotent stem cells (PSCs) such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) or adult stem cells such as hematopoietic stem cells (HSCs), skin stem cells (SSCs), neural stem cells (NSCs), and mesenchymal stem cells (MSCs)) preferably are mesenchymal stromal cells.

[0152] Mesenchymal stromal cells may be obtained from bone marrow, adipose tissue, placenta, and blood. Mesenchymal stromal cells are capable of differentiating into different types of mesenchymal mature cells depending on the differentiation conditions.

[0153] In one embodiment, the differentiated cells are differentiated adipose tissue-derived stem cells (ASCs), preferably ASCs differentiated into cells selected from the group comprising or consisting of osteoblasts, chondrocytes, keratinocytes, myofibroblasts, epithelial, endothelial, connective, or neural cells and adipocytes.

[0154] In some embodiments, ASCs are osteogenic differentiated ACSs, i.e. differentiated into osteogenic cells, in particular into osteoblasts.

[0155] In one embodiment, ASCs are differentiated into chondrogenic cells.

[0156] In a particular embodiment, ASCs are differentiated into chondrocytes.

[0157] In another embodiment, ASCs are keratinic differentiated ACSs. In other words, in one embodiment, ASCs are differentiated into keratinic cells.

[0158] In a particular embodiment, ASCs are differentiated into keratinocytes.

[0159] In another embodiment, ASCs are myofibroblastic differentiated ACSs. In other words, in one embodiment, ASCs are differentiated into myofibroblastic cells. In a particular embodiment, ASCs are differentiated into myofibroblasts.

[0160] In another embodiment, ASCs are endothelial differentiated ACSs. In other words, in one embodiment, ASCs are differentiated into endothelial cells. In a particular embodiment, ASCs are differentiated into endothelial cells.

[0161] In another embodiment, ASCs are epithelial differentiated ACSs. In other words, in one embodiment, ASCs are differentiated into epithelial cells. In a particular embodiment, ASCs are differentiated into epithelial cells.

[0162] In another embodiment, ASCs are adipogenic differentiated ACSs. In other words, in one embodiment, ASCs are differentiated into adipogenic cells. In a particular embodiment, ASCs are differentiated into adipocytes. In another embodiment, ASCs are neural differentiated ACSs. In other words, in one embodiment, ASCs are differentiated into neural cells.

[0163] Lyophilization

[0164] In one embodiment, the powder is desiccated, preferably by lyophilization.

[0165] Size reduction

[0166] After lyophilization, depending on the application, the particle size of the lyophilized powder can be reduced for example by grinding.

[0167] Size distribution

[0168] The particle size distribution after size-reduction can be determined by granulometry. Granulometry allows for the measurement of the size of the particle diameters.

[0169] Laser particle size measurement

[0170] A preferred measurement method is the laser particle size measurement. Laser particle size measurement allows the measurement of sizes between 0.05 and 900 pm.

[0171] The sample can be analyzed in solution (liquid route) or directly after lyophilization and optionally size reduction (dry route).

[0172] Wet laser particle size measurement allows the characterization of dispersions (elementary particle size after chemical dispersion) or suspended solids (“aggregate” particle size).

[0173] Dry laser particle size measurement allows the characterization of powders whose initial aggregation is not destroyed.

[0174] Preferably, the particle size is measured applying the wet method, using a Mastersizer equipment, which determines the particles size through laser diffraction. This technique is based on the measurement of angular intensity variations when the laser beam goes through the sample. Among the most important statistical parameters generated from a particle distribution analysis are the percentiles. These indicate in each case the size x below which a certain quantity of the sample (10% for Dx(10), 50% for Dx(50) and 90% for Dx(90)) by volume lies.

[0175] In one embodiment, the particle size is the Dx(50) by volume.

[0176] For example, a typical particle size may be measured by applying wet leaser measurements using the Malvern Mastersizer. The Dx (10) (pm) indicates the size x below which 10% of total analyzed particles lies. An exemplary particle size distribution according to the invention is:

[0177] Granulometry-liquid route: Dx (10) (pm) 365 Standard deviation: 38

[0178] Dx (50) (pm) 752 Standard deviation: 39

[0179] Dx (90) (pm) 1434 Standard deviation: 144

[0180] Sterilization

[0181] In another embodiment, the powder is sterilized, preferably by gamma-irradiation.

[0182] In some embodiments, the freeze-drying of the biomaterial is performed at a temperature of about -80 °C, preferably of about -50 °C under vacuum.

[0183] In practice, sterilization may be performed by any suitable method known from the state of the art, or a method adapted therefrom. Non-limitative examples of suitable methods include irradiation such as electron beam irradiation, X-ray irradiation, gamma- irradiation, or ultraviolet irradiation.

[0184] In certain embodiments, said sterile biomaterial is obtained by gamma-irradiation, preferably at a dose of about 7 kGy to about 45 kGy, preferably at room temperature. Within the scope of the invention, the expression “about 7 kGy to about 45 KGy” encompasses 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44 and 45 kGy.

[0185] In some embodiments, the biomaterial is obtained by gamma-irradiation at a dose of about 10 kGy to about 40 kGy. Within the scope of the invention, the term “room temperature” is intended to refer to a temperature comprised from about 15°C to 25°C, preferably from 18°C to about 22°C, which encompasses 18°C, 19°C, 20°C, 21 °C and 22°C. In some embodiments, room temperature is a temperature of about 20°C.

[0186] The inventors observed that, in spite of the fact that sample undergoing gamma-irradiation have a general tendency to overheat and to potentially destroy valuable ingredients, gamma-irradiation of the biomaterial of the invention could be performed at room temperature without being substantially affected by overheating.

[0187] In some embodiments, the gamma-irradiation may be performed at a temperature below about 10 °C, preferably on ice (about 0 °C). Wthin the scope of the invention, a temperature below about 10°C encompasses 9.5°C, 8°C, 8.5°C, 8°C, 7.5°C, 7°C, 6.5°C, 6°C, 5°C, 4°C, 3°C, 2°C, 1°C, 0°C, -1°C, -2°C, -3°C, -4°C, -5°C, -10°C, -20°C, -30°C, - 40°C, -50°C, -60°C, -70°C and -80°C.

[0188] In practice, the gamma-irradiation may be performed for a duration that would depend from the size (e.g. expressed in mm3 or cm3 ) and / or the amount (e.g. expressed in mg or g) of biomaterial to be sterilized and / or the dose to be administered.

[0189] In certain embodiments, the gamma-irradiation may be performed from about 10 sec to about 24 h, preferably from about 5 min (300 sec) to about 12h, more preferably, from about 10 min (600 sec) to about 3 h (10,800 sec).

[0190] Within the scope of the invention, the expression “from about 10 sec to about 24 h” encompasses 10 sec, 12 sec, 14 sec, 16 sec, 18 sec 20 sec, 25 sec, 30 sec, 35 sec, 40 sec, 45 sec, 50 sec, 55 sec, 1 min, 1 min 30, 2 min, 2 min 30, 3 min, 3 min 30, 4 min, 4 min 30, 5 min, 6 min, 7 min, 8 min, 9 min, 10 min, 12 min, 14 min, 16 min, 18 min, 20 min, 22 min, 24 min, 26 min, 28 min, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, 1 h, 1 h 30, 2 h, 2 h 30, 3 h, 3 h 30, 4 h, 4 h 30, 5 h, 5 h 30, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h and 24 h.

[0191] 3-dimensional (3D) neo-synthesized extracellular matrix (ECM)

[0192] The 3-dimensional neo-synthesized extracellular matrix is an extracellular matrix that the differentiated cells surprisingly secrete when the particulate material is added.

[0193] The 3-dimensional neo-synthesized extracellular matrix serves as a scaffold. Consequently, no external scaffold has to be added.

[0194] In one embodiment, due to the 3-dimensional neo-synthesized extracellular matrix, the differentiated cells - prior to devitalization - are different from 2-dimensional cell aggregates.

[0195] Thus, the differentiated cells are embedded in the 3-dimensional neo-synthesized extracellular matrix.

[0196] The cells embedded in the 3-dimensional neo-synthesized ECM secret certain proteins and miRNAs with anti-cancer activity.

[0197] After devitalization and optionally size reduction, the 3-dimensional neo-synthesized extracellular matrix is as a carrier for proteins and miRNAs with anti-cancer activity.

[0198] Accordingly, in certain embodiments, the 3-dimensional neo-synthesized extracellular matrix comprises one or more matrisomal proteins specific to soft tissue or calcified tissue. In another embodiment, the 3-dimensional neo-synthesized extracellular matrix comprises one or more matrisomal proteins and miRNA molecules with anti-cancer activity.

[0199] Accordingly, the 3-dimensional neo-synthesized extracellular matrix can be used for the treatment or prevention of cancer, in particular for use in the inhibition of the viability, the migration or the proliferation of cancer cells.

[0200] Secreted proteins with anti-cancer activity

[0201] Examples of proteins with anti-cancer activity secreted by the differentiated cells present in the neo-synthesized extracellular matrix and implicated in the positive regulation of cell death pathways in osteosarcoma (OS) treatment such as apoptosis, autophagy and necroptosis include:

[0202] Accordingly, in another aspect, the injectable liquid of the present invention comprises one or more of the proteins mentioned in the table above. Secreted miRNAs with anti -cancer activity

[0203] Examples of miRNAs with anti-cancer activity secreted by the differentiated cells and present in the neo-synthesized extracellular matrix are: hsa-miR-210-3p, hsa-miR-409-3p, hsa-let-7a-5p, hsa-miR-29b-3p, hsa-miR-30e-3p, hsa-let-7b-5p, hsa-miR-3184-3p, hsa- miR-92a-3p, hsa-miR-320a, hsa-miR-24-3p, hsa-let-7d-5p, hsa-miR-193b-5p, hsa-miR- 361 -3p, hsa-miR-199a-5p, hsa-miR-25-3p, hsa-miR-181a-5p, hsa-miR-151a-3p, hsa- miR-214-3p, hsa-miR-193a-5p, hsa-miR-30c-5p, hsa-miR-154-5p, hsa-let-7f-5p, hsa- miR-199a-3p, hsa-miR-664b-3p, hsa-miR-664a-5p, hsa-miR-3607-5p, hsa-miR-29a-3p, hsa-miR-27a-3p, hsa-miR-92b-3p, hsa-miR-199b-3p, hsa-miR-342-3p, hsa-miR-320b, hsa-miR-1291 , hsa-let-7e-5p, hsa-miR-130a-3p, hsa-miR-3651 , hsa-miR-103b, hsa-miR- 1273g-3p, hsa-miR-30a-3p, hsa-miR-664b-5p, hsa-miR-34a-3p, hsa-miR-125a-5p, hsa- miR-145-5p, hsa-miR-664a-3p, hsa-miR-140-5p, hsa-miR-21 -5p, hsa-miR-28-3p, hsa- miR-98-5p, hsa-miR-3609, hsa-let-7i-5p, hsa-miR-93-5p, hsa-miR-146b-5p, hsa-miR- 374c-3p, hsa-miR-125b-5p, hsa-miR-34a-5p, hsa-miR-337-3p, hsa-miR-10a-5p, hsa-let- 7g-5p, hsa-miR-222-3p, hsa-miR-4449, hsa-miR-22-3p, hsa-miR-191-5p, hsa-miR-3074- 5p, hsa-miR-6516-3p, hsa-miR-4668-5p, hsa-miR-574-3p, hsa-miR-424-5p, hsa-let-7i-3p, hsa-miR-24-2-5p, hsa-miR-199b-5p, hsa-miR-424-3p, hsa-miR-103a-3p, hsa-miR-29b-1- 5p, hsa-miR-423-5p, hsa-miR-328-3p, hsa-miR-324-5p, hsa-miR-335-5p, hsa-miR-574- 5p, hsa-miR-17-5p, hsa-miR-660-5p, hsa-miR-425-5p, hsa-miR-23b-3p, hsa-miR-23a-3p, hsa-miR-185-5p, hsa-miR-4461 , hsa-miR-196a-5p, hsa-let-7d-3p, hsa-miR-374b-5p, hsa- miR-127-3p, hsa-let-7c-5p, hsa-miR-423-3p, hsa-miR-196b-5p, hsa-miR-221-3p, hsa- miR-382-5p, hsa-miR-619-5p, hsa-miR-3613-5p, hsa-miR-3653-5p, hsa-miR-19b-3p, hsa-miR-99b-5p, hsa-miR-376c-3p, hsa-miR-99b-3p, hsa-miR-663b, hsa-miR-495-3p, hsa-miR-454-3p, and a combination thereof.

[0204] Accordingly, in another aspect, the injectable liquid of the present invention comprises one or more of above-mentioned miRNA(s) selected from a group consisting of MiR-140, miR- 199a, miR-34a, miR-335 and miR-505.

[0205] Criteria and conventions for miRNA identification and nomenclature have been described in Ambros et al. (A uniform system for microRNA annotation. RNA 2003 9(3):277-279). The miRNAs sequences may be retrieved from the miRbase database (http: / / www.mirbase.org / ) or the miRDB database (http: / / www.mirdb.org / ).

[0206] In practice, the RNAs profile of the may be assessed by any suitable method known in the art, or any method adapted therefrom. Illustratively, RNA may be extracted, e.g. by means of a commercial kit (such as miRNeasy kit from Qiagen®); and further sequenced, e.g. by meany of a high-throughput sequencing system (such as NextSeq 500 system from Illumina®). Illustratively, one may use the Qiazol lysis reagent (Qiagen®, Hilden, Germany) and a Precellys homogenizer (Bertin® instruments, Montigny-le-Bretonneux, France). RNAs may be purified using Rneasy mini kit (Qiagen®, Hilden, Germany) with an additional on column DNase digestion according to the manufacturer’s instruction.

[0207] Quality and quantity of RNA may be determined by using a spectrophotometer (Spectramax® 190, Molecular Devices®, California, USA). cDNA may be synthesized from 0.5pg of total RNA using RP RNA first strand kit (Qiagen®, Hilden, Germany) for genes expression profiles though customized PCR arrays (Customized Human Osteogenic and angiogenic RP Profiler Assay - Qiagen®, Hilden, Germany). The ABI Quantstudio 5 system (Applied Biosystems®) and SYBR Green ROX Mastermix (Qiagen®, Hilden, Germany) may be used for detection of the amplification product. Quantification may be obtained according to the AACT method. The final result of each sample may be normalized to the means of expression level of housekeeping genes (e.g. ACTB, B2M and GAPDH).

[0208] Scaffold-free

[0209] Consequently, the injectable liquid of the present invention is obtained from a scaffold-free biomaterial. That means that no external, three-dimensional scaffold is used during differentiation.

[0210] Particulate material

[0211] The term “particulate material” as used herein refers to a solid material in the form of particles and preferably is gelatin, even more preferably gelatin beads.

[0212] In one embodiment, the gelatin of the invention is animal gelatin, preferably mammal gelatin, more preferably porcine gelatin.

[0213] As used herein, the term “porcine gelatin” may be replaced by “pork gelatin” or “pig gelatin”. A commercially available example is Cultispher. In one embodiment, the gelatin is porcine skin gelatin.

[0214] In certain embodiments, said gelatin is in the form of particles. The gelatin particles preferably particles have a volumetric mean diameter ranging from about 50 micrometers to about 1 ,000 micrometers as measured by laser diffraction granulometry, preferably with a Malvern Mastersizer. Within the scope of the invention, the expression “from about 50 micrometers to about 1 ,000 micrometers ” encompasses 50 micrometers, 60 micrometers , 70 micrometers , 80 micrometers , 90 micrometers, 100 micrometers, 150 micrometers, 200 micrometers, 250 micrometers, 300 micrometers, 350 micrometers, 400 micrometers, 450 micrometers, 500 micrometers, 550 micrometers, 600 micrometers, 650 micrometers, 700 micrometers, 750 micrometers, 800 micrometers, 850 micrometers, 900 micrometers, 950 micrometers and 1 ,000 micrometers.

[0215] In one embodiment, gelatin is added at a concentration ranging from about 0.1 cm3 to about 5 cm3 for a 150 cm2 vessel, preferably from about 0.5 cm3 to about 4 cm3, more preferably from about 0.75 cm3 to about 3 cm3. In one embodiment, gelatin is added at a concentration ranging from about 1 cm3 to about 2 cm3 for a 150 cm2 vessel. In one embodiment, gelatin is added at a concentration of about 1 cm3, 1 .5 cm3 or 2 cm3 for a 150 cm2 vessel. Within the scope of the invention, the expression “0.1 cm3 to about 5 cm3” encompasses 0.1 cm3, 0.2 cm3, 0.3 cm3, 0.4 cm3, 0.5 cm3, 0.6 cm3, 0.7 cm3, 0.8 cm3, 0.9 cm3, 1 .0 cm3, 1 .5 cm3, 2.0 cm3, 2.5 cm3, 3.0 cm3, 3.5 cm3, 4.0 cm3, 4.5 cm3 and 5.0 cm3.

[0216] In one embodiment, the particulate material is embedded in the secreted neo-synthesized extracellular matrix.

[0217] Matrisome

[0218] “Matrisome” is used to describe the powder comprising, preferably consisting of:

[0219] ■ devitalized differentiated cells;

[0220] ■ 3-dimensional neo-synthesized extracellular matrix as a carrier of bioactive molecules (proteins, mRNAs, miRNAs, lipids, extracellular vesicles);

[0221] ■ a particulate material for use in the treatment or prevention of cancer; characterized in that:

[0222] ■ the differentiated cells secreted the neo-synthesized extracellular matrix prior to devitalization; and

[0223] ■ the cells and the particulate material are embedded in the neo-synthesized extracellular matrix. Extracellular vesicles

[0224] In one embodiment, the injectable liquid of the present invention or the matrisome are substantially free of extracellular vesicles.

[0225] Preferably the injectable liquid comprises extracellular vesicles in an amount of 5 w% or less, even more preferably 1 w% or less and even more preferably 0.1 w% and even more preferably 0.01 w% or less as compared to the total weight or the total protein content of the injectable solution as compared to the total dry weight of the injectable liquid, preferably as compared to the total weight of the protein in the injectable liquid.

[0226] In one embodiment, the extracellular vesicles are measured by nanoparticle tracking analysis (NTA).

[0227] In one embodiment, the concentration of extracellular vesicles is measured by nanoparticle tracking analysis (NTA) as the number of particles per volume of the final product (liquid).

[0228] Pharmaceutically acceptable carrier

[0229] In another aspect, the injectable liquid of the present invention comprises a pharmaceutically acceptable vehicle or carrier. As used herein, “pharmaceutically acceptable carrier” refers to any solvent, dispersion medium, coating, antibacterial and / or antifungal agent, isotonic and absorption delaying agent and the like.

[0230] The pharmaceutically acceptable carrier may comprise one or more ingredient(s) selected in a group of additives polypeptides; amino acids; lipids; and carbohydrates. Among carbohydrates, one may cite sugars, including monosaccharides, di-, tri-, tetra-, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers.

[0231] Examples of suitable pharmaceutically acceptable vehicles may include polypeptides such as, e.g., gelatin, casein, and the like.

[0232] Treatment or prevention of cancer

[0233] The injectable liquid of the present invention is used in the treatment or prevention of cancer, in particular for use in the inhibition of the viability, migration and proliferation of cancer cells

[0234] The terms “treatment”, “treating” or “alleviation” refer to therapeutic treatments wherein the object is to prevent or slow down (lessen) a cancer. A subject is successfully "treated" if, after receiving a therapeutic amount of the to the injectable liquid of the present invention, the subject shows observable and / or measurable reduction in, or absence of cancer.

[0235] The treatment or use can be allogeneic, xenogeneic or autologous. Preferably, the treatment or use of present invention is allogenic.

[0236] “Allogeneic” or “allogenic” therapy means that the donor and the recipient are different individuals of the same species.

[0237] “Autologous” means that the donor and the recipient is the same individual.

[0238] “Xenogeneic” means that the donor is derived from an animal of a different species than the recipient.

[0239] The term “prevention” refers to preventing or avoiding the occurrence of symptom of a tissue disorder, including a skin disorder, bone disorder and / or cartilage disorder. In the present invention, the term “prevention” may refer to a secondary prevention, i.e. to the prevention of the re-occurrence of a symptom or a relapse of a tissue disorder, including a skin disorder, bone disorder and / or cartilage disorder. It may also refer, when the disease is cancer, such as, e.g. , a bone cancer, to the occurrence of metastases after the treatment and / or the removal of a tumor. The term “effective amount” refers to an amount sufficient to effect beneficial or desired results including clinical results. An effective amount can be administered in one or more administrations.

[0240] Cancer

[0241] The injectable liquid of the present inventions is useful in the treatment or prevention of cancer.

[0242] In one embodiment, the injectable liquid of the present invention is useful in one or more of the following treatments:

[0243] ■ inhibiting the viability of cancer cells,

[0244] ■ inhibiting the proliferation of cancer cells;

[0245] ■ inhibiting the migration of cancer cells;

[0246] ■ inhibiting the cell colony formation of cancer cells; or

[0247] ■ any combination thereof.

[0248] In one embodiment, the cancer is a solid cancer selected from the group consisting of a bone cancer, a brain cancer, a skin cancer, a breast cancer, a cancer of the central nervous system, a cancer of the cervix, a cancer of the upper aero digestive tract, a colorectal cancer, an endometrial cancer, a germ cell cancer, a bladder cancer, a kidney cancer, a laryngeal cancer, a liver cancer, a lung cancer, a neuroblastoma, an esophageal cancer, an ovarian cancer, a pancreatic cancer, a pleural cancer, a prostate cancer, a retinoblastoma, a small intestine cancer, a soft tissue sarcoma, a stomach cancer, a testicular cancer and a thyroid cancer, and preferably is bone cancer or any metastases thereof or skin cancer.

[0249] Liquid injection

[0250] In preferred embodiment, the injectable liquid is administered as an injection or in the form of an injectable liquid or an injectable suspension.

[0251] Accordingly, another aspect of the invention is an injectable liquid, wherein the bioactive molecules released from the powder of the invention are suspended or dissolved.

[0252] Accordingly, in this embodiment, the size of the powder comprising the neo-synthesized extracellular matrix, is reduced for example through grinding or any other suitable size reduction method.

[0253] Particle size of the powder

[0254] The volumetric particle size of the powder is between 100 micrometers to 5000 micrometers, preferably, between 100 micrometers to 1000 micrometers, even more preferably from 100 micrometers to 500 micrometers as measured by laser diffraction granulometry, preferably with the Malvern Mastersizer.

[0255] In other embodiments, the volumetric particle size of the powder is between 10 micrometers to 100 micrometers, preferably from 25 micrometers to 75 micrometers as measured by laser diffraction granulometry, preferably with the Malvern Mastersizer.

[0256] Method of treatment

[0257] Another aspect of the present invention is a method of treatment or prevention of cancer, in particular for the local treatment of solid tumors, comprising the administration of an injectable liquid containing bioactive molecules released from a powder,

[0258] Wherein preferably the bioactive molecules are released through incubation of the powder in a liquid phase;

[0259] Wherein the powder is obtained or obtainable, preferably through desiccation and / or devitalization, from a biomaterial;

[0260] Wherein the biomaterial comprises: a. Differentiated stem cells, preferably osteogenically differentiated stem cells, even more preferably osteogenically differentiated adipose-tissue derived stem cells (ASCs); b. A 3-dimensional neo-synthesized extracellular matrix as a vehicle of bioactive molecules comprising one or more of mRNAs, miRNAs, lipids, and proteins; and c. A gelatin particulate material;

[0261] Wherein the differentiated stem cells secrete the 3-dimensional neo-synthesized extracellular matrix;

[0262] Wherein the gelatin particulate material is added to the differentiated stem cells to induce the secretion of the 3-dimensional neo-synthesized extracellular matrix;

[0263] Wherein the maturation period of the differentiated stem cells after the addition of the gelatin particulate material is from 5 to 15 weeks, preferably from 6 to 10 weeks, even more preferably from 7 to 9 weeks;

[0264] Wherein the differentiated stem cells and the gelatin particulate material are embedded in the 3-dimensional neo-synthesized extracellular matrix;

[0265] Wherein the injectable liquid is characterized by:

[0266] ■ The injectable liquid comprises the bioactive molecules in a therapeutically or cosmetically effective amount,

[0267] ■ The injectable liquid does not contain any viable differentiated stem cells;

[0268] ■ The injectable liquid does not contain any gelatin particulate material;

[0269] ■ Preferably the injectable liquid comprises extracellular vesicles in an amount of 5 w% or less, even more preferably 1 w% or less and even more preferably 0.1 w% and even more preferably 0.01 w% or less as compared to the total weight or the total protein content of the injectable solution; and

[0270] ■ Preferably the concentration of total proteins is 0.1 mg / ml to 10 mg / ml, preferably from 0.5 mg / ml to 5 mg / ml, even more preferably from 0.75 mg / ml to 2.5 mg / ml of the injectable liquid Manufacturing process

[0271] The injectable liquid of the invention may be manufactured in a scaffold-free process comprising in the following step order:

[0272] ■ Stem cell collection;

[0273] ■ Stem cell proliferation;

[0274] ■ Osteogenic differentiation;

[0275] ■ Particles sprinkling; and

[0276] ■ 3D-structure formation

[0277] In one embodiment, the osteogenic differentiation medium comprises or consists of DMEM supplemented with L-alanyl-L-glutamine, PL, dexamethasone, ascorbic acid and sodium phosphate.

[0278] In one embodiment, the osteogenic differentiation medium comprises or consists of DMEM supplemented with L-alanyl-L-glutamine, hPL, dexamethasone, ascorbic and sodium phosphate, and antibiotics, preferably penicillin, streptomycin, gentamycin and / or amphotericin B.

[0279] In one embodiment, the osteogenic differentiation medium comprises or consists of DMEM supplemented with L-alanyl-L-glutamine, hPL (about 5%, v / v), dexamethasone (about 1 mM), ascorbic acid (about 0.25 mM) and sodium phosphate (about 2.93 mM). In one embodiment, the osteogenic differentiation medium comprises or consists of DMEM supplemented with L-alanyl-L-glutamine, hPL (about 5%, v / v), dexamethasone (about 1 pM), ascorbic acid (about 0.25 mM) and sodium phosphate (about 2.93 mM), penicillin (about 100 U / mL) and streptomycin (about 100 pg / mL). In one embodiment, the osteogenic differentiation medium further comprises amphotericin B (about 0.1 %).

[0280] In one embodiment, the osteogenic differentiation medium consists of DMEM supplemented with L-alanyl-L-glutamine, hPL (about 5%, v / v), dexamethasone (about 1 pM), ascorbic acid (about 0.25 mM) and sodium phosphate (about 2.93 mM). In one embodiment, the osteogenic differentiation medium comprises or consists of DMEM supplemented with L-alanyl-L-glutamine, hPL (about 5%, v / v), dexamethasone (about 1 mM), ascorbic acid (about 0.25 mM) and sodium phosphate (about 2.93 mM), penicillin (about 100 U / mL), streptomycin (about 100 pg / mL) and amphotericin B (about 0.1 %). In another embodiment, the cells, in particular ASCs, are chondrogenic differentiated. In other words, in a preferred embodiment, the cells, in particular ASCs, are differentiated into chondrogenic cells. In still other words, in a preferred embodiment, the cells, in particular ASCs, are differentiated in chondrogenic medium. In a particular embodiment, the cells, in particular ASCs, are differentiated into chondrocytes.

[0281] In one embodiment, the obtained 3D structures are then desiccated, preferably by lyophilization to obtain a powder (“matrisome”).

[0282] In one embodiment, the desiccated powder is then sterilized, preferably by gamma irradiation.

[0283] SHORT DESCRIPTION OF THE DRAWINGS

[0284] Figure 1 : Downstream manufacturing process of the NVDM2 release-8W (NVDM2R- 8W). Schematic representation of the 2nd phase of the process to obtain NVDM2R-8W from NVDM2-8W powder.

[0285] Figure 2: Principle of Cell Counting Kit-8 (CCK-8): tetrazolium salt (WST-8) is reduced by dehydrogenases in viable cells to give an orange-colored product (formazan), which is soluble in the cell culture medium. The amount of the formazan dye generated by dehydrogenases in cells is directly proportional to the number of living cells:

[0286] Figure 3: Impact of NVDM2R-8W on the viability of bone tumor-related tumor cells: (A) Viability of osteosarcoma cells: 143B and U2-OS cells, (B) Viability of Ewing sarcoma A673 cells, (C) Viablility of chondrosarcoma SW1353 cells and (D)Viabil ity of fibrosarcoma HT1080 cells, upon NVDM2R-8W treatment for 72h. Each NVDM2R-8W treatment was performed in two different doses of the tested NVDM2R-8W samples :“7mg” and “15mg” corresponding to the concentration of the bioactive molecules / factors released from 7mg or 15mg of NVDM2-8W DP powder, respectively, when incubated in 300pl of medium for 48h at 37°C, 5% CO2. The results are shown as % viability measured in treated samples normalized to the values of the untreated. The results of all groups were compared separately with the results of the untreated reference group. Statistical analysis was performed using Prism GraphPad 2. Since the normality wasn’t respected, an Kruskal- Wallis’s test Multiple comparisons with Dunn’s test was applied. Untreated vs test items (*, p-value- O.05; **, p-value- 0.01 ; ***, p-value^ 0.001 and n.s, not significant. Blue dots: values of individual batches (biological replicates). “ NVDM2R-8W*” corresponds to the extract released from the respective amount of non-irradiated NVDM2-8W powder (DS); “Beads” corresponds to the extract released from 15mg of beads, when incubated in 300pl of the medium for 48h at 37°C, 5% CO2. Each chart represents the average value of the data from all batches tested.

[0287] Figure 4: Impact of NVDM2R-8W on the viability of cancer cell lines non related to bone tumors: (A) Viability of melanoma cells (A375 and SK-MEL-28) ; (B) Viability of breast carcinoma cells (Hs-578T); (C) Viability of lung carcinoma (A549); (D) Viability of glioblastoma (U87) upon the respective treatments for 72h. Each NVDM2R-8W treatment was performed in two different doses of the tested NVDM2R-8W samples :“7mg” and “15mg” corresponding to the concentration of the bioactive molecules / factors released from 7mg or 15mg of NVDM2-8W DP powder, respectively, when incubated in 300pl of medium for 48h at 37°C, 5% CO2. The results are shown as % viability measured in treated samples normalized to the values of the untreated. The results of all groups were compared separately with the results of the untreated reference group. Statistical analysis was performed using Prism GraphPad 2. Since the normality was respected, an ordinary one-way Anova Multiple comparisons with Bonferroni’s test was applied.. Untreated vs test items (*, p-value- O.05; **, p-value- 0.01 ; ***, p-value^0.001and n.s, not significant). Blue dots: values of individual batches (biological replicates); “Beads” corresponds to the extract released from 15mg of beads, when incubated in 300pl of the medium for 48h at 37°C, 5% CO2. Each chart represents the average value of the data from all batches tested.

[0288] Figure 5: Impact of NVDM2R-8W on the viability of non-tumor cells. Viability of hBM-MSCs upon the respective treatments for 72h. Each NVDM2R-8W treatment was performed in two different doses of the tested NVDM2R-8W samples :“7mg” and “15mg” corresponding to the concentration of the bioactive molecules / factors released from 7mg or 15mg of NVDM2-8WDP powder, respectively, when incubated in 300pl of medium for48h at37°C, 5% CO2. The results are shown as % viability measured in treated samples normalized to the values of the untreated for each time point Statistical analysis was performed using Prism GraphPad 2. Since the normality was respected, an ordinary one-way Anova Multiple comparisons with Bonferroni’ s test was applied. Untreated vs test items (*, p- value- O.05; **, p-value- 0.01 ; ***, p-value^ 0.001 and n.s, not significant). Blue dots: values of individual batches (biological replicates); “Beads” corresponds to the extract released from 15mg of beads, when incubated in 300pl of the medium for 48h at 37° C, 5% CO2. Each chart represents the average value of the data from all batches tested.

[0289] Figure 6: Principle of RealTime-Glo™ AnnexinV Apoptosis Assay. Phosphatidylserine (PS) is an intra-cytosolic constituent of cytoplasmic membrane. During apoptosis progression, the PS is transferred to the extra-cytosolic part. The apoptosis assay contains equimolar ratios of two annexin V fusion proteins containing complementary subunits of NanoBit® Luciferase (Annexin V-LgBit and Annexin V-smBit). The luminescence increases when the subunits bind into complementary proximity due to the affinity between Annexin V and PS. This increase is correlated with apoptotic progression.

[0290] Figure 7: Apoptosis levels upon the treatment of NVDM2R-8W in OS cell lines (143B, U2- OS) using RealTime-GloTMAnnexinV apoptosis assay. The results are shown as fold induction of apoptosis measured in treated samples normalized to the values of the untreated for each time point. Blue dots: values of individual batches(biological replicates). Each chart represents the average value of the data from all batches tested.

[0291] Figure 8: PCA plot showing morphological (A) and transcriptom ic (B) profiles of 143B cells subjected to different treatment conditions. Every dot data point represents a well-level aggregated morphological or transcriptom ic profiles. All profiles are normalized to untreated conditions using Robust Z-score normalization. Time (3, 6, 12 and 24) is expressed in hours. There are three technical replicates per condition. L, low seeding density (4,000 cells / well); H, high seeding density (7,000 cells / well); Untreated, reference / baseline group; Beads, gelatin bead control group; Dox, doxorubicin (positive control group); NVDM2R-8W-1 , NVDM2R-8W batch DB19; NVDM2R-8W-2, NVDM2R- 8W batch DB29; NVDM2R-8W-3, NVDM2R-8W batch DB30. These structural alterations were accompanied by cell cycle disruption, characterized by a reduction in G1 -phase cells and an accumulation in S / G2 / M or dying / dead phases — indicative of impaired mitotic entry and proliferation arrest (Figure 9).

[0292] Figure 9: Cell cycle phase distributions for all treatments at the 24-hour time point. The graph displays the percentage of cells in each cell cycle phase: subG1 (blue), G1 (orange), S (green), and G2 / M (red). Compositional analyses were performed comparing each treatment to the untreated condition. Data represent mean value from technical replicates for the untreaded, reference / baseline group; doxorubicin, positive control and gelatin beads (control, Beads) conditions, and from both technical and batch (DB19, DB29 and DB30) replicates for NVDM2R-8W. Statistical comparisons were performed using Student t-tests (GraphPad Prism 2), evaluating differences between treated and untreated conditions for each cell cycle phase. *, p-value- O.05; **, p-value- 0.01 ; ***, p-value^ 0.001 , n.s, not significant.

[0293] Figure 10: Heatmap of the top 1 ,000 differentially expressed genes (DEGs) showing changes in expression levels in 143B cells after treatment with NVDM2R-8W and doxorubicin, compared to untreated conditions Seeding conditions: Low seeding density meaning 4,000 cells / well and high seeding density meaning 7,000 cells / well. Timing conditions: T1 means 3 hours, T2 means 6 hours, T3 means 12 hours and T4 means 24 hours. Treatment conditions: Batch 1 means NVDM2R-8W-1 (DB19), Batch 2 means NVDM2R-8W-2 (DB29), Batch 3 means NVDM2R-8W-3 (DB30) and Dox means doxorubicin.

[0294] Figure 11 : NVDM2R-8W induces upregulation of tumor suppressor genes in 143B osteosarcoma cells. Shown are gene upregulated over time (3h, 6h, 12h and 24h), following NVDM2R-8W treatment, known for their tumor suppressive functions. Mean Log2FC represents the average Log2fold change value of triplicates, compared to untreated controls. SD, standard deviation; ARID4A, AT-Rich Interaction Domain 4A; NFKBI, Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor alpha; LINC00472, Long Intergenic Non-Protein Coding RNA 472; HM0X1 , heme oxygenase 1 ; SPEN, Spen Family Transcriptional Repressor; FAM133B, Family With Sequence Similarity 133 Member B; CDH13, Cadherin 13; ZBTB1 , Zinc Finger And BTB Domain Containing 1 ; RCAN1 , Regulator Of Calcineurin 1 ; YPEL5, Yippee Like 5; CYP27C1 , Cytochrome P450 Family 27 Subfamily C Member 1 ; MBNL2, Muscleblind Like Splicing Regulator 2; PDCD4, Programmed Cell Death 4. The highest Mean Log2FC value for each gene is shown in green.

[0295] Figure 11 : NVDM2R-8W treatment suppresses oncogenic gene expression in 143B cells. Shown are genes downregulated over time (3 hours (h), 6h, 12h and 24h), following NVDM2R-8W treatment, known for their oncogenic functions. Mean Log2FC represents the average Log2fold change value of triplicates, compared to untreated controls. SD, standard deviation; PLAU, Plasminogen Activator Urokinase; CCN2, Cellular Communication Network Factor 2; JUN, AP-1 Transcription Factor Subunit; SERPINE1 , serine protease inhibitor clade E member 1 ; FOSL1 , FOS like 1 AP1 transcription Factor Subunit; SPOCD1 , SPOC Domain Containing 1 ; H2AX, H2A.X Variant Histone; PSRC1 , Proline And Serine Rich Coiled-Coil 1 ; ARL4C, ARF Like GTPase 4C; RIN2, Ras And Rab Interactor 2; CDCA4, cell division Cucle-associated protein 4; NCOR2, Nuclear Receptor Corepressor 2; KBTBD6, Kelch Repeat And BTB Domain Containing 6; ID3, Inhibition of DNA Binding 3; PIF1 , DNA Repair and Recombination Helicase PIF1 ; DUSP6, Dual Specificity Phosphatase 6; TWF2, Twinfilin Actin Binding Protein 2; PREX1 , hosphatidylinositol-3,4,5-Trisphosphate Dependent Rac Exchange Factor; PLAUR, Plasm inogene Activator Urokinase Receptor; ESM1 , Endothelial Cell Specific Molecule 1 ; LHX1 , LIM Homeobox 1 , LINC00342, Long intergenic non-protein coding RNA 342; TFAP4, Transcription Factor AP-4; CREB5,CAMP Responsive Element Binding Protein 5. The lowest Mean Log2FC value for each gene is shown in green.

[0296] Figure 13: Hallmark collection GSEA This heatmap shows gene sets from the hallmark collection which showed significant normalized enrichment scores (NES) for all conditions. The colour on the heatmap represents relative expression levels, with higher or lower values indicating significant changes in gene expression in response to the treatments. Seeding conditions: L, low seeding density meaning 4,000 cells per well and H, high seeding density meaning 7,000 cells per well. Timing conditions: T1 means 3 hours, T2 means 6 hours, T3 means 12 hours and T4 means 24 hours. Treatment conditions: Batch 1 means NVDM2R-8W-1 (DB19), Batch 2 means NVDM2R-8W-2 (DB29), Batch 3 means NVDM2R-8W-3 (DB30) and Dox means doxorubicin. Red boxes highlight the biological pathways positively enriched in response to NVDM2R-8W-induced cytotoxicity in 143B cells. Blue boxes highlight the biological pathways negatively enriched in response to NVDM2R-8W-induced cytotoxicity in 143B cells.

[0297] EXAMPLES

[0298] Example 1 : Manufacturing process of NVDM2 release-8W (NVDM2R-8W)

[0299] NVDM2 release-8W (NVDM2R-8W) is an injectable liquid containing the bioactive molecules released from the NVDM2-8W after an incubation step in a liquid phase (phosphate buffer saline). NVDM2-8W is the neosynthesized ECM derived from the «scaffold-free» 3D-biomaterial. NVDM2-8W was produced from osteodifferentiated human adipose-derived stem cells (hASCs) when combined with gelatin particles (Cultispher S from porcine skin), after a lyophilization and an irradiation step.

[0300] 10 different NVDM2R-8W batches were manufactured following the process described below.

[0301] Upstream process: Manufacturing the 3D biomaterial (NVDM2- 8W CP)

[0302] Phase 1 :

[0303] Isolation of the stromal vascular fraction (SVF) cells from the adipose tissue and subsequent expansion of hASCs up to passage P2 / P3 in proliferation medium (MP). The cells are expanded to cell stocks. The cell stock is cryopreserved at different passage levels.

[0304] Phase 2:

[0305] Manufacturing of 3D-CP which includes the following steps:

[0306] ■ Thawing of hASCs: Cells were inoculated into T 150cm2flasks in MP.

[0307] ■ Proliferation phase: The cells proliferate until they reach a confluency of 2^70% and ^100%, before performing the passage P3 / P4.

[0308] ■ Passage P3 / P4 and osteogenic induction phase: At the passage P3 / P4, the cells collected from all the flasks are pooled and then seeded in T 150cm2flasks with re- closable lid (“TPP” flask) in osteogenic differentiation medium (MD). When cells reach a confluency and at least one osteoid nodule (un-mineralized, organic portion of the bone matrix that forms prior to the maturation of bone tissue) is observed in each flask, the addition of Cultispher particles can be launched.

[0309] ■ Addition of gelatin beads commercially available under the trademark Cultispher S: After being exposed to the MD, the culture vessels containing the confluent monolayer of adherent osteogenic cells are sprinkled with Cultispher S particles (1 ,5cc for a 150cm2vessel).

[0310] ■ 3D induction phase: Few days after the addition of the Cultispher S, the osteogenic cells and the particles dispersed become progressively embedded in a neosynthesized extracellular matrix. At this point, the osteogenic cells and the Cultispher S particles start forming a large 3D patch (or few smaller patches) of partially mineralized brownish-yellow malleable and detachable membrane. The formation of the final 3D-CP is obtained at the end of its maturation period which is 8 weeks (8W). After maturation, the 3-dimensional neo-synthesized extracellular matrix is rinsed 3 times with phosphate buffered saline (PBS), placed in newTPPs (2 patches / flask) and incubated in MD without human platelet lysate (hPL) during 24 or 72 hours. This 3-dimensional neosynthesized extracellular matrix is frozen before downstream processing.

[0311] At this point, the 3D-structures are rinsed 3 times with PBS, placed in 15 ml tubes with filtered cap and frozen at -80°C for at least 24h or in dry ice for at least 4h.

[0312] Downstream process: Manufacturing of the injectable liquid

[0313] Phase 1 : Production of NVDM2-8W powder comprising devitalized differentiated cells; 3-dimensional neo-synthesized extracellular matrix and a gelatin particulate material:

[0314] The 3-dimensional neo-synthesized extracellular matrix from the upstream process is lyophilized, grinded and mixed to produce NVDM2-8W powder (DS) (DIN.50.07.03.01 .005 SOP NVDX2 Production). To obtain the lyophilized form of the product, the frozen 3D-neo- synthesized extracellular matrix is freeze-dried using the following parameters:

[0315] ■ Pressure: 0.05 mBar,

[0316] ■ Temperature: -50° C,

[0317] ■ Time: 24h-36h.

[0318] After the freeze-drying process, the dry product is broken with a pestle and mortar to obtain a powder. This powder is terminally sterilized by gamma-irradiation to obtain the NVDM2- 8W powder (DP).

[0319] Phase 2: Release of the bioactive molecules from the NVDM2R-8W powder:

[0320] A defined amount of the NVDM2-8W DP powder is placed in a transwell and incubated in a specific volume of a phosphate buffer saline, as indicated in the Table 1 , for48h at 37°C, 5% CO2. At the end of the incubation time, the transwell is removed and the liquid containing the released extract from NVDM2-8W DP is collected to obtain the NVDM2- release-8W (NVDM2R-8W) product (Figure 1 ).

[0321] Table 1 :

[0322] Indicative volumes of liquid used per transwell to obtain the maximum concentration / dose (15mg / 300ul) of released factors in NVDM2R-8W.

[0323] Overview of the manufacturing process for the tested NVDM2R batches

[0324] For the investigation of NVDM2R-8W anti-tumor activity ten different NVDM2R-8W batches (named as DB1 , DB2, DB3, DB4, DB6, DB15, DB19, DB29, DB30 and DB35) were manufactured in accordance with Table 2:

[0325] Table 2:

[0326] Summary of the main steps of the manufacturing process applied to produce all NVDM2R- 8W batches dedicated to this study. DB: development batches

[0327] Example 2: Overview of the in vitro potency testing approach

[0328] In vitro potency bioassays applied for the assessment of anti-tumor activity:

[0329] Cell-based pharmacogenomics screens are commonly used during the preclinical drug screening process to identify druggable targets by characterizing the biological effects associated with drug response and toxicity. Drug-dose response assays performed in two- dimensional (2D) cell culture are typically used to evaluate drug efficacy and potency in cells exposed to a drug for a defined period of time. To investigate the anti-cancer potential of NVDM2R-8W the following in vitro potency assays have been developed and applied on different tumor and non-tumor cell lines:

[0330] ■ cell counting kit-8 (CCK-8) assay to assess the impact of NVDM2R-8W treatment on cellular proliferation and viability (Figure 2);

[0331] ■ annexin V apoptosis assay to investigate the potential of the product to induce cell death through the positive regulation of the apoptotic pathway (Figure 6).

[0332] Doxorubicin treatment served as a positive control sample. Critical parameters for the development of each assay and cell line (cell seeding density, incubation time for the treatment, medium composition) have been optimized.

[0333] Tumor and non-tumor cell lines tested:

[0334] The impact of NVDM2R-8W treatment was studied in vitro on the following established cell lines:

[0335] ■ Osteosarcoma: 143B, U2-OS;

[0336] ■ Ewing sarcoma: A673;

[0337] ■ Chondrosarcoma: SW1353;

[0338] ■ Fibrosarcoma: HT1080;

[0339] ■ Melanoma: A375, SK-MEL-28;

[0340] ■ Lung cancer: A549;

[0341] ■ Breast cancer: Hs578T;

[0342] ■ Glioblastoma: U87;

[0343] ■ Primary healthy (non-tumor cell lines): hBM-MSCs.

[0344] Summary of the testing plan:

[0345] Table 3: Summary of the potency testing plan applied to the NVDM2R-8W batches shown in Table 2, aiming to evaluate the effect of NVDM2R-8W on cell viability, using CCK-8 assay.

[0346] Table 4: Summary of the testing plan applied to the NVDM2R-8W batches shown in Table

[0347] 2, designed to assess the ability of NVDM2R-8W to induce apoptosis in osteosarcoma cells, using Annexin V apoptosis assay.

[0348] Impact of NVDM2R-8W on cellular viability using CCK-8 viability assay:

[0349] The impact of NVDM2R-8W on cellular viability was assessed by using the CCK-8 viability assay (Sigma- Aldrich, 96992). The CCK-8 assay takes advantage of WST-8 [2-(2-methoxy-4-nitrophenyl)-3-(4- nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt] to directly measure cell viability. Specifically, WST-8 is a water-soluble salt that can be reduced by dehydrogenases in viable cells to a water-soluble formazan dye. The biochemical reaction causes a change in color that can be quantified by measuring absorbance at 450nm. Thus, CCK-8 measures cell viability by correlating the production of colored formazan dye to the amount of living cells in culture (Sanjai, Hakkimane, Guru, & Gaonkar, 2024). Dehydrogenases from viable cells convert WST-8 into a formazan dye, which produces an easily measurable color change that can be quantified in a cell plate reader at 450 nm (Figure 2). The CCK-8 assay was performed on five bone tumor-related cell lines (two OS: 143B, U2O-S; one Ewing sarcoma: A375, one chondrosarcoma: SW1353 and one fibrosarcoma: HT1080), two melanoma cell lines (A375 and SK-MEL-28), one breast cancer cell line (Hs578T) and one lung cancer cell line (A549) in comparison with healthy non-tumor cells such as human bone marrow mesenchymal stem cells (hBMSCs). The cells were seeded in 96-wells plates in the following seeding density:

[0350] ■ Osteosarcoma cells: 143B at 6000 cells / cm2, U2-OS at 12000 cells / cm2;

[0351] ■ Ewing sarcoma: A673 at 18000 cells / cm2;

[0352] ■ Chondrosarcoma: SW1353 at 6000 cells / cm2;

[0353] ■ Fibrosarcoma: HT 1080 at 6000 cells / cm2;

[0354] ■ Melanoma cells : A375 at 6000 cells / cm2and SK-MEL-28 at 12000 cells / cm2;

[0355] ■ Lung cancer: A549 at 10000 cells / cm2;

[0356] ■ Breast cancer: Hs578T at 12000 cells / cm2;

[0357] ■ Glioblastoma: U87 at 6000 cells / cm2;

[0358] ■ Primary healthy (non-tumor cell lines): hBM-MSCs at 10000 cells / cm2. and were cultured in the respective complete culture medium.

[0359] One day after seeding the cells, test products were added to the culture at two different doses for up to 72h. The applied doses of NVDM2R-8W corresponded to the concentration of the bioactive molecules / factors released from 7mg or 15mg of NVDM2- 8W powder, when incubated in 300pl of the respective culture medium (depending on the treated cell line), for48h at 37°C, 5% CO2. The reference compound doxorubicin (0.1 pM) was added to the culture at the same time as the initiation of NVDM2R-8W treatment to demonstrate that the testing system can detect inhibition of cellular viability in the presence of a well-established chemotherapeutic drug. Absorbance was measured after 72h of treatment, as an index of the number of viable cells in culture at this time point.

[0360] This test was performed on samples from nine different NVDM2R-8W batches as shown in Table 3. Each experiment was performed in 96-well plates containing (i) a reference / baseline (untreated) group with cells in complete medium w / o any added compound, (ii) an internal control group, in which the reference compound was added (doxorubicin, 0.1 pM) and (iii) additional groups for two different doses of the tested NVDM2R-8W samples (“7mg” and “15mg” corresponding to the concentration of the bioactive molecules / factors released from 7mg or 15mg of NVDM2-8W powder, respectively, when incubated in 300pl of medium for48h at 37°C, 5% CO2) and one dose of the extract released from gelatine beads alone (“15mg” corresponding to the concentration of the biomolecules released from 15mg of beads when incubated in 300pl of the medium for 48h at 37°C, 5% CO2). All the above groups were tested in three replicates.

[0361] Conclusions:

[0362] A strong inhibitory effect on the viability of the tested bone tumor-related cell lines was induced by NVDM2R-8W (86.2%, 58.2%, 88.6%, 46.4 % and 73.4 % of inhibition on the viability of osteosarcoma 143B and U2-OS, Ewing sarcoma A673, chondrosarcoma SW1353 and fibrosarcoma HT1080 cells, respectively) in a dose-dependent manner, whereas no negative impact on the viability of these cells was observed by the extract released from the beads alone. This suggests that the observed cytotoxic effect induced by NVDM2R-8W on bone tumor cells is due to the presence of the neosynthesized matrix in the product and not due to the gelatin beads (Figure 3).

[0363] Based on the potency data obtained in 143B cells for NVDM2R* (corresponding to the extract released from the lyophilized NVDM2 powder before irradiation) vs NVDM2R (corresponding to the extract released from the lyophilized, grinded and irradiated NVDM2 powder), the irradiation step seems to enhance the anti-tumor activity of the product, by facilitating the release of the bioactive molecules (Figure 3A).

[0364] NVDM2R-8W seems to have the potential to address multiple solid tumors other than bone related sarcomas, by inducing a significant inhibition of 33.7% to 70.1 % on the viability of all tested neoplastic cell lines shown in Figure 4. More specifically, the highest dose of NVDM2R-8W induced 70.1 % and 55.6% of inhibition on the viability of A375 and SK-MEL-28 melanoma cells, respectively; 39.7% of inhibition on the viability of breast carcinoma Hs-578T cells; 47.3% of inhibition on the viability of lung carcinoma A549 cells and 33.7% of inhibition on the viability of glioblastoma U87 cells. However, the highest in vitro potency of the product was observed in case of the sarcoma cells with MSCs origin such as OS, Ewing sarcoma and fibrosarcoma cells (Figure 3; Figure 4).

[0365] As observed in case of bone tumor-related cell lines (Figure 3), in the presence of the released factors from the beads alone, no significant impact was observed on the cell viability of breast carcinoma, lung carcinoma and glioblastoma cells (Figure 4 B,C & D), suggesting that the cytotoxic effect induced by NVDM2R-8W on these cancer cells is due to the presence of bioactive molecules released from the 3D ECM of the product. In contrast, the presence of the beads seemed to have a negative impact on the viability of melanoma but to a lesser degree than NVDM2R-8W (Figure 4A).

[0366] Interestingly, although a significant inhibitory effect was induced on the viability of the tested non-tumor hBM-MSCs in the presence of doxorubicin, a significant boosting effect was observed on their cell viability upon NVDM2R-8W treatment at any applied dose suggesting that NVDM2R-8W cytotoxic effect is specific to cancer cells (Figure 5).

[0367] Impact of NVDM2R-8W on the pro-apoptotic progression of tumor cells

[0368] Chemoresistance is an obstacle in the treatment of cancer. Thus, determining a therapy able to efficiently induce cell death and overcome chemoresistance is important in Oncology. Cell death in its various forms mainly includes cell apoptosis, autophagy and necroptosis (programmed necrosis). Apoptosis has been traditionally thought to be an active form of tumor cell death, and apoptotic signaling pathways have been clearly elucidated in different kinds of cancer cells (Jing Li, 2016).

[0369] Moreover, a successful therapy for cancers such osteosarcoma, requires the selective destruction of cancer cells and it is important to induce cell apoptosis and sensitize resistance of tumor cells (Jun Huang, 2015) (S Bouralexis, 2003).

[0370] Therefore, in the present study we investigated whether the cytotoxic effect of NVDM2R- 8W on the tested tumor cells was induced through the apoptotic pathway.

[0371] A classical assay to assess the ability of a therapeutic compound to induce cell death by apoptosis is the Annexin V Apoptosis assay. Anticancer agent-induced apoptosis or necrosis leads to the loss of membrane integrity before advanced stages of cell death. The loss of cellular asymmetry during apoptosis leads to exposure of phosphatidylserine (PS) on the outer side of the plasma membrane. Annexin V binding is specific for those cells undergoing apoptosis and results in exposure of their PS residues (Chetana Sanjai, 2024). Thus, the RealTime-Glo™ AnnexinV Apoptosis Assay applied in our study detects apoptosis by using a staining technique on PS molecules that have moved beyond the cell membrane. Annexin V is used to detect early apoptosis in cells (Figure 6) (Kevin Kupcho, 2018).

[0372] To assess the ability of NVDM2R-8W to induce apoptosis in cancer cells, the RealTime- Glo™ AnnexinV Apoptosis assay was performed on two osteosarcoma cells lines (143B and U2-OS). For all tested cell lines, cells were seeded in 96-wells plates at seeding density of 31250 cells / cm2and were cultured in complete culture medium. After O / N incubation, test products were added to the culture at two different doses (“7mg” and “15mg” corresponding to the concentration of the bioactive molecules / factors released from 7mg or 15mg of NVDM2-8W powder, respectively, when incubated in 300pl of medium for 48h at 37°C, 5% CO2) in the presence of RealTime-Glo™ AnnexinV Apoptosis reagent for up to 36h. The reference compound doxorubicin (1 pM, 5pM) was added to the culture at the same time of the initiation of NVDM2R-8W treatment to demonstrate that the testing system can detect inhibition of clonogenic activity in the presence of a well-established chemotherapeutic drug.

[0373] A reading was performed after 0, 3, 6, 24, 36 h of co-incubation, to evaluate the apoptotic progression induce on targeted cells. The RealTime-Glo™ AnnexinV Apoptosis Assay is a homogeneous method to determine the magnitude apoptotic progression in culture based on quantitation of luminescence, which signals the presence of apoptotic progression in cells (Figure 6).

[0374] The RealTime-Glo™ AnnexinV apoptosis assay was performed on four NVDM2R-8W batches, as shown in Table 3. Each experiment was performed in 96-well plates containing (i) a reference / baseline group (untreated) with cells in complete medium w / o any added compound, (ii) two internal control groups, in which the reference compound was added (doxorubicin, 1 , 5 pM) and (iii) additional groups for two different doses of the tested NVDM2R-8W samples (“7mg” and “15mg” corresponding to the concentration of the bioactive molecules / factors released from 7mg or 15mg of NVDM2-8W powder, respectively, when incubated in 300pl of medium for48h at 37°C, 5% CO2) and one dose of the extract released from gelatine beads alone (“15mg” corresponding to the concentration of the biomolecules released from 15mg of beads when incubated in 300pl of the medium for 48h at 37°C, 5% CO2). All the above groups were tested in three replicates.

[0375] Conclusions:

[0376] A clear induction of apoptosis was observed in OS cell lines upon the presence of NVDM2R-8W in a dose-dependent manner, whereas this was not the case upon the treatment with the extract released from the beads alone, suggesting that the pro-apoptotic properties of NVDM2R-8W are due to the presence of the neosynthesized matrix in the intermediate product (Figure 7). Example 3: NVDM2R-8W MoA for antitumor activity in osteosarcoma cells

[0377] To explore the transcriptom ic and morphological responses induced by NVDM2R-8W in osteosarcoma (OS) cells, an in vitro time-course study was conducted using the 143B cell line. Cells were treated with either NVDM2R-8W, doxorubicin (as a positive control) or gelatin bead extract and compared to untreated cells (as a reference control). The experimental aim was to evaluate early and late cellular responses to treatment, with particular focus on assessing the reproducibility of biological effects and the transcriptom ic and morphological responses to treatment. This experimental setup was designed to support the mechanistic investigation described above, aligning with the goals of assessing biological reproducibility and consistency of bimodal profiling across treatment conditions.

[0378] Cells were seeded into 96-well plates at two different densities (4,000 and 7,000 cells / well) to account for potential confluency effects on treatment response. After 24 hours, test items were added, and cells were exposed for 3, 6, 12, or 24 hours, enabling a detailed time- resolved analysis of the induced transcriptom ic and morphological changes. Doxorubicin (1 pM) served as a positive control, validating the assay’s ability to detect cellular viability inhibition in the presence of a well-established chemotherapeutic drug. In addition, a gelatin bead extract was also tested to exclude confounding effects from the beads and to ensure that only NVDM2R-8W-specific responses were analyzed. At each time point, plates were collected and processed as shown in Figure 1 for cell cycle, high-content transcriptom ic and morphological analysis. The study included the following treatment groups under both seeding conditions:

[0379] (i) Reference / baseline (reference) group: Untreated cells in complete medium with no added compound.

[0380] (ii) Internal (positive) control group: Cell treated with the doxorubicin (1 pM).

[0381] (iii) Test groups for NVDM2R-8W: Cells treated with three batches of NVDM2R-8W (DB19, DB29, DB30, see table 2) at a dose equivalent to the bioactive molecules / factors released from 15 mg of NVDM2-8W powder after incubation in 300 pL of medium for 48 hours at 37°C, 5% CO2.

[0382] (iv) Gelatin beads control group: Cells treated with extract released from gelatin beads alone, using the same incubation and dose-equivalent conditions as for NVDM2R-8W.

[0383] All conditions were tested in technical triplicate to ensure robustness and reproducibility of the results. This study investigated the morphological and transcriptom ic effects of NVDM2R-8W on 143B osteosarcoma (OS) cells across three production batches, multiple time points and two seeding densities. Consistent cellular responses were observed under all conditions, confirming the robustness and reproducibility of the treatment.

[0384] Workflow

[0385] Workflow used to investigate NVDM2R-8W mechanism of action underlying its cytotoxic (anti-tumor) activity on the 143B cell line. The experiment was conducted in 96-well plates applying two cell seeding density conditions (4,000 cells / well and 7,000 cells / well) where the following groups of samples were included for each condition: (i) untreated group, (ii) doxorubicin-treated group (1 pM) (iii) NVDM2R-8W-treated group (3 batches) (iv) gelatin beads control group. Test items were added 24 hours after seeding and incubated for 3, 6, 12, or 24 hours. Following incubation, plates were collected and processed for transcriptom ic and morphological analysis

[0386] Morphological and Cellular Effects

[0387] NVDM2R-8W induced pronounced and time-dependent morphological changes, with the most significant effects observed after 12 hours. Treated cells displayed:

[0388] ■ Increased cell size and altered endoplasmic reticulum morphology

[0389] ■ Enlarged but fewer nucleoli

[0390] ■ Irregular, less compact nuclear and cell shapes

[0391] Principal component analysis (PCA) was performed to both morphological and transcriptom ic profiles to assess similarities between treatment conditions. The resulting PCA plots are presented in Figure 2.

[0392] In both morphological and transcriptom ic PCA plots, 143B treated under the same conditions generated similar profiles as reflected by their proximity. Across all treatments, samples showed a tendency to cluster by time point, suggesting a temporal response. Importantly, the profiles obtained from the three batches of NVDM2R-8W were highly similar across all time points, supporting the reproducibility of the biological effect.

[0393] Moreover, cell seeding density had a relatively minor influence on clustering patterns, indicating that the treatment-induced effects were robust. As expected, the separation between treatment groups increased over time. For NVDM2R-8W specifically, a marked shift in both morphological and transcriptom ic profiles was observed between 6- and 12- hour timepoints, indicating a time-dependent response.

[0394] Additionally, NVDM2R-8W and doxorubicin treatments resulted in distinct morphological and transcriptom ic signatures. In contrast, cells treated with “Beads” clustered closely with untreated conditions in both PCA plots, indicating that the bead treatment did not induce a strong morphological or transcriptional response in the 143B cells.

[0395] Differential Gene Expression Analysis (DGEA)

[0396] To investigate the transcriptional changes induced by the various treatment conditions, a differential gene expression analysis (DGEA) was performed by comparing gene expression profiles of treated samples to those of untreated controls, considered as baseline.

[0397] When comparing the top 1 ,000 DEGs, distinct patterns of upregulated and downregulated genes were identified in response to NVDM2R-8W compared to doxorubicin (Figure 4). These differences further underscore the unique transcriptional effects of NVDM2R-8W and provide insight into its specific mechanism of action.

[0398] Moreover, to isolate gene expression changes specific to NVDM2R-8W, “upset” plots across time points were generated for NVDM2R-8W and doxorubicin, using genes consistently differentially expressed across all three batches. While some overlap with doxorubicin was observed (as expected), NVDM2R-8W treatment uniquely induced dozens of DEGs, underscoring its distinct mode of action (Figure 4).

[0399] To investigate the biological relevance of the transcriptional response induced by NVDM2R-8W treatment, a literature-based analysis was conducted on the top 100 most significant differentially expressed genes (DEGs), focusing on those that were upregulated (Log2 Fold change(FC)> 1 , adj. p-val< 0.05; Table 1 ) or downregulated (Log2 FC<- 1 , adj. p-val< 0.05; Table 2) over time.

[0400] This approach revealed consistent gene expression patterns indicative of a coordinated anti-tumor response, characterized by the upregulation of tumor suppressor genes and the downregulation of oncogenes (Tables 1 and 2). Specifically, 13 genes (Table 1 ) known to function as tumor suppressors across multiple cancer types were identified as significantly upregulated following treatment, highlighting the potential of NVDM2R-8W to activate tumor-suppressive pathways in 143B cells. In parallel, 24 oncogenes were found to be significantly downregulated (Table 2), further demonstrating the broad impact of NVDM2R-8W treatment on pathways commonly associated with tumor progression.

[0401] Gene set enrichment analysis (GSEA)

[0402] To gain further insight into the biological pathways affected under each treatment condition, Gene Set Enrichment Analysis (GSEA) was also performed by comparing the transcriptom ic profiles of treated samples to those of untreated controls. For each comparison that generated a DEG list, a gene set enrichment analysis was performed on the DEG list ranked according to the Log2FoldChange from highest upregulated to highest downregulated. A gene set or pathway was defined as significant with adjusted p value below 0.05.

[0403] This analysis was conducted using the Hallmark gene set collection from the Molecular Signature Database (MSigDB) (https: / / www.gsea- msigdb.org / gsea / msigdb / collections.jsp). Across all conditions, GSEA identified 31 to 50 significantly enriched Hallmark gene sets (Figure 5). The Hallmark collection comprises well-defined biological states or processes, each represented by genes that exhibit coordinated expression patterns. These gene sets were generated through a computational approach that identifies overlaps among gene sets from various MSigDB collections and retains only those genes with consistent co-expression, thereby reducing noise and redundancy.

[0404] Overall, GSEA revealed that NVDM2R-8W treatment modulates several biologically significant pathways in 143B OS cells, including the activation of inflammatory, metabolic and apoptotic responses, coupled with the suppression of proliferative signalling and EMT. This analysis further confirmed that NVDM2R-8W treatment induces a distinct and specific transcriptom ic signature in OS cells, clearly differentiating them from both untreated cells and those treated with doxorubicin.

[0405] More specifically, GSEA identified pronounced transcriptional responses following NVDM2R-8W treatment. At 24 hours, the Hallmark pathways “TNF-a signaling via NF- KB”, “Apoptosis”, “Heme metabolism” and “Inflammatory response” were positively enriched, indicating activation of inflammatory and pro-apoptotic mechanisms, not observed in doxorubicin- or bead-treated conditions. From these pathways, several key genes were highlighted as major mediators of the treatment response, including HM0X1 , PDCD4, TIMP3, RhoB, BNIP3L, CDKN1A, BAX, NFKBIA, KLF6 and TIPARP (Table 3). In contrast, negatively enriched pathways such as “Oxidative phosphorylation,” “E2F targets,” “MYC targets,” “G2M checkpoint,” and “Epithelial-mesenchymal transition (EMT)” suggested a reduction in metabolic activity, cell cycle progression and cellular plasticity. Within these pathways, five MYC-regulated genes (MCM5, CDK4, LAS1 L, PLK1 , and EBP1) and several G2 / M checkpoint regulators (MKI67, PTTG1 , CDK1,

[0406] CDKN3, CCNB2, CKS1B, and CCNA2) were significantly downregulated, indicating that NVDM2R-8W disrupts proliferative and mitotic control in OS cells(Table 3).

[0407] Table: Key genes highlighted as major mediators of the NVDM2R-8W treatment response, identified through GSEA of Hallmark pathways.

[0408] Conclusion: Collectively, these results provide a comprehensive overview of the molecular mechanisms underlying NVDM2R-8W activity, revealing coordinated pathwaylevel effects that extend beyond individual gene alterations. NVDM2R-8W exerts a robust, reproducible, and multifaceted impact on OS cells, combining morphological, cell cycle and transcriptom ic alterations. It induces a specific and consistent gene expression signature, distinct from both bead and doxorubicin treatments, and targets a broad range of regulatory genes and pathways involved in tumor progression, cell cycle control and apoptosis. Collectively, the data supports a dual mechanism of action involving an early cellular stress response followed by sustained apoptotic activation, positioning NVDM2R-8W as a promising therapeutic candidate for osteosarcoma treatment.

Claims

CLAIMS1 . An injectable liquid containing bioactive molecules released from a powder, Wherein preferably the bioactive molecules are released through incubation of the powder in a liquid phase;Wherein the powder is obtained or obtainable, preferably through desiccation and / or devitalization, from a biomaterial;Wherein the biomaterial comprises: a. Differentiated stem cells, preferably osteogenically differentiated stem cells, even more preferably osteogenically differentiated adipose-tissue derived stem cells (ASCs); b. A 3-dimensional neo-synthesized extracellular matrix as a vehicle of bioactive molecules comprising one or more of mRNAs, miRNAs, lipids, and proteins; and c. A gelatin particulate material or a gelatin-derived hydrogel;Wherein the differentiated stem cells secrete the 3-dimensional neosynthesized extracellular matrix;Wherein the gelatin particulate material is added to the differentiated stem cells to induce the secretion of the 3-dimensional neo-synthesized extracellular matrix;Wherein the maturation period of the differentiated stem cells after the addition of the gelatin particulate material is from 5 to 15 weeks, preferably from 6 to 10 weeks, even more preferably from 7 to 9 weeks;Wherein the differentiated stem cells and the gelatin particulate material are embedded in the 3-dimensional neo-synthesized extracellular matrix;Wherein the injectable liquid is characterized by:■ The injectable liquid comprises the bioactive molecules in a therapeutically or cosmetically effective amount,■ The injectable liquid does not contain any viable differentiated stem cells;■ The injectable liquid does not contain any gelatin particulate material;■ Preferably the injectable liquid comprises extracellular vesicles in an amount of 10 w% or less, preferably 5 w% or less, even more preferably 1 w% or less andeven more preferably 0.1 w% and even more preferably 0.01 w% or less as compared to the total weight or the total protein content of the injectable solution; and■ Preferably the concentration of total proteins is 0.1 mg / ml to 10 mg / ml, preferably from 0.5 mg / ml to 5 mg / ml, even more preferably from 0.75 mg / ml to 2.5 mg / ml of the injectable liquid; for use in the treatment or prevention of cancer, in particular in the local treatment of solid tumors.

2. The injectable liquid for use according to claim 1 , wherein the injectable liquid is used for:■ inhibiting the viability of cancer cells,■ inhibiting the proliferation of cancer cells;■ inhibiting the migration of cancer cells;■ inhibiting the cell colony formation of cancer cells; or■ any combination thereof.

3. The injectable liquid for use according to any one of the preceding claims, wherein the powder is:■ desiccated, preferably by lyophilization;■ size reduced, preferably by grinding to volumetric particle size distribution with a range of 100 to 5000 micrometers as measured by laser diffraction granulometry; and / or■ sterilized, preferably by gamma-irradiation.

4. The injectable liquid for use according to any one of the preceding claims, wherein the powder is free of external scaffolds.

5. The injectable liquid for use according to any one of the preceding claims, wherein the differentiated stem cells are selected from the group comprising or consisting of osteoblasts, osteocytes, chondroblasts, chondrocytes, keratinocytes,myofibroblasts, epithelial cells, endothelial cells, adipocytes, neural cells, and precursors thereof, and preferably are soft tissue cells, chondroblasts or osteoblasts, even more preferably adipose-derived stem cells differentiated into osteogenic cells.

6. The injectable liquid for use according to any one of the preceding claims, wherein the powder comprises the neo-synthesized extracellular matrix in a content of 0.001 w% to 10 w%, preferably from 0.01 w% to 7.5 w%, even more preferably from 0.1 w% to 5 w% as compared to the total weight of the powder.

7. The injectable liquid for use according to any one of the preceding claims, wherein the differentiated stem cells are derived from one or more of:■ pluripotent stem cells (PSCs) such as embryonic stem cells (ESCs) or induced pluripotent stem cells (iPSCs);■ adult stem cells such as hematopoietic stem cells (HSCs), skin stem cells (SSCs), neural stem cells (NSCs); and■ mesenchymal stem cells (MSCs), preferably derivable from adipose tissue, peripheral blood or placenta, and preferably are mesenchymal stem cells.

8. The injectable liquid for use according to any one of the preceding claims, wherein the stem cells are derived from mesenchymal stromal cells, preferably obtainable from bone marrow, adipose tissue, placenta, or blood.

9. The injectable liquid for use according to any one of the preceding claims, wherein the cancer is a solid cancer selected from the group consisting of a bone cancer, a brain cancer, a skin cancer, a breast cancer, a cancer of the central nervous system, a cancer of the cervix, a cancer of the upper aero digestive tract, a colorectal cancer, an endometrial cancer, a germ cell cancer, a bladder cancer, a kidney cancer, a laryngeal cancer, a liver cancer, a lung cancer, a neuroblastoma, an esophageal cancer, an ovarian cancer, a pancreatic cancer, a pleural cancer, a prostate cancer, a retinoblastoma, a small intestine cancer, a soft tissue sarcoma,a stomach cancer, a testicular cancer and a thyroid cancer, preferably, the cancer is■ Osteosarcoma;■ Ewing sarcoma;■ Chondrosarcoma;■ fibrosarcoma;■ melanoma;■ lung cancer;■ breast cancer; or■ glioblastoma, preferably, wherein one or more of the following genes are upregulated:■ HMOX1■ NFkBIA■ PDCD4■ TIMP3■ RhoB■ PMAIP1■ BNIP3L■ CDKN1A■ BAX■ KLF6■ TIPARPPreferably, wherein one or more of the following genes are downregulated:■ MCM5■ CDK4■ LAS1 L■ PLK1■ EBP1■ MKI67■ PTTG1■ CDK1■ CDKN3■ CCNB2CKS1 BCCNA2.

10. Method for obtaining an injectable liquid preferably according to any one of the preceding claims comprising:■ Providing a powder; wherein the powder is obtained or obtainable, preferably through desiccation and / or devitalization, from a biomaterial;Wherein the biomaterial comprises: a. Differentiated stem cells, preferably osteogenically differentiated stem cells, even more preferably osteogenically differentiated adipose-tissue derived stem cells (ASCs); b. A 3-dimensional neo-synthesized extracellular matrix as a vehicle of bioactive molecules comprising one or more of mRNAs, miRNAs, lipids, and proteins; and c. A gelatin particulate material or a gelatin-derived hydrogel;Wherein the differentiated stem cells secrete the 3-dimensional neosynthesized extracellular matrix;Wherein the gelatin particulate material is added to the differentiated stem cells to induce the secretion of the 3-dimensional neo-synthesized extracellular matrix;Wherein the maturation period of the differentiated stem cells after the addition of the gelatin particulate material is from 5 to 15 weeks, preferably from 6 to 10 weeks, even more preferably from 7 to 9 weeks; andWherein the differentiated stem cells and the gelatin particulate material are embedded in the 3-dimensional neo-synthesized extracellular matrix;■ Incubating the powder in a liquid phase to enable the release of the bioactive molecules from the powder into the liquid; wherein the liquid is preferably a water-based liquid preferably comprising physiologically acceptable salts, preferably wherein the liquid is free of organic solvents;■ Subsequently, separating the liquid phase from the powder; and■ Subsequently, collecting the injectable liquid; wherein preferably the injectable liquid comprises extracellular vesicles in an amount of 10 w% or less, 5 w% or less, even more preferably 1 w% or less andeven more preferably 0.1 w% and even more preferably 0.01 w% or less as compared to the total weight of the injectable solution; and wherein preferably the concentration of total proteins is 0.1 mg / ml to 10 mg / ml, preferably from 0.5 mg / ml to 5 mg / ml, even more preferably from 0.75 mg / ml to 2.5 mg / ml of the injectable liquid.11 . Method according to claim 10, wherein the incubation conditions are one or more of:■ Incubation time: from 10 hours to 120 hours, preferably from 30 hours to 100 hours, even more preferably from 40 hours to 60 hours; and / or■ Incubation temperature: from 10 °C to 60 °C, preferably from 20 °C to 50 °C, even more preferably from 30 °C to 40 ° C or from 15 °C to 25 °C; and / or■ Incubation atmosphere: 1 % to 10 % CO2, preferably, 2.5 % to 7.5 %, even more preferably from 4 % to 6 %; and / or■ Incubation pH: from 5 to 7.5, preferably from 6 to 7, even more preferably from 6.5 to 7.

12. Method according to any one of claims 10 or 11 , wherein the powder is added to the liquid phase in an amount from 5 mg / ml to 500 mg / ml, preferably from 10 mg / ml to 250 mg / ml, even more preferably from 25 mg / ml to 150 mg / ml, even more preferably 50 mg / ml to 125 mg / ml of the liquid phase.

13. Method according to any one of claims 10 to 12, wherein the liquid phase comprises physiologically acceptable salts in physiologically acceptable quantities, and preferably is phosphate buffer saline (PBS) or physiological serum.

14. Injectable liquid obtainable or obtained by the method according to any one of claims 10 to 13.

15. Injectable liquid according to claim 14 for use in the treatment or prevention of cancer, in particular in the local treatment of solid tumors.

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