The comparison of adipose-derived stromal cells (ADSCs) delivery method in a murine model of hindlimb ischemia.

BACKGROUND: Adipose-derived stromal cells (ADSCs) demonstrate ability to promote tissue healing and down-regulate excessive inflammation. ADSCs have been used to treat critical limb ischemia in preclinical and clinical trials, but still, there is little known about their optimal delivery strategy. To date, no direct analysis of different methods of ADSCs delivery has been performed in the hindlimb ischemia model. Therefore, in this study we focused on the therapeutic efficacy of different ADSCs delivery methods in a murine model of hindlimb ischemia. METHODS: For the hADSCs isolation, we used the subcutaneous adipose tissue collected during the surgery. The murine hindlimb ischemia was used as a model. The unilateral femoral artery ligation was performed on 10-12-week-old male C57BL/6. ADSCs were delivered directly into ischemic muscle, into the contralateral muscle or intravenously. 7 and 14 days after the surgery, the gastrocnemius and quadriceps muscles were collected for the immunohistochemical analysis. The results were analyzed with relevant tests using the Statistica software. RESULTS: Our research revealed that muscle regeneration, angiogenesis, arteriogenesis and macrophage infiltration in murine model of hindlimb ischemia differ depending on ADSCs delivery method. We have demonstrated that intramuscular method (directly into ischemic limb) of ADSCs delivery is more efficient in functional recovery after critical limb ischemia than intravenous or contralateral route. CONCLUSIONS: We have noticed that injection of ADSCs directly into ischemic limb is the optimal delivery strategy because it increases: (1) muscle fiber regeneration, (2) the number of capillaries and (3) the influx of macrophages F4/80+/CD206+.

Antitumor effect of anti-vascular therapy with STING agonist depends on the tumor microenvironment context.

Introduction: Targeting tumor vasculature is an efficient weapon to fight against cancer; however, activation of alternative pathways to rebuild the disrupted vasculature leads to rapid tumor regrowth. Immunotherapy that exploits host immune cells to elicit and sustain potent antitumor response has emerged as one of the most promising tools for cancer treatment, yet many treatments fail due to developed resistance mechanisms. Therefore, our aim was to examine whether combination of immunotherapy and anti-vascular treatment will succeed in poorly immunogenic, difficult-to-treat melanoma and triple-negative breast tumor models. Methods: Our study was performed on B16-F10 melanoma and 4T1 breast tumor murine models. Mice were treated with the stimulator of interferon genes (STING) pathway agonist (cGAMP) and vascular disrupting agent combretastatin A4 phosphate (CA4P). Tumor growth was monitored. The tumor microenvironment (TME) was comprehensively investigated using multiplex immunofluorescence and flow cytometry. We also examined if such designed therapy sensitizes investigated tumor models to an immune checkpoint inhibitor (anti-PD-1). Results: The use of STING agonist cGAMP as monotherapy was insufficient to effectively inhibit tumor growth due to low levels of STING protein in 4T1 tumors. However, when additionally combined with an anti-vascular agent, a significant therapeutic effect was obtained. In this model, the obtained effect was related to the TME polarization and the stimulation of the innate immune response, especially activation of NK cells. Combination therapy was unable to activate CD8+ T cells. Due to the lack of PD-1 upregulation, no improved therapeutic effect was observed when additionally combined with the anti-PD-1 inhibitor. In B16-F10 tumors, highly abundant in STING protein, cGAMP as monotherapy was sufficient to induce potent antitumor response. In this model, the therapeutic effect was due to the infiltration of the TME with activated NK cells. cGAMP also caused the infiltration of CD8+PD-1+ T cells into the TME; hence, additional benefits of using the PD-1 inhibitor were observed. Conclusion: The study provides preclinical evidence for a great influence of the TME on the outcome of applied therapy, including immune cell contribution and ICI responsiveness. We pointed the need of careful TME screening prior to antitumor treatments to achieve satisfactory results.

The Effect of Radiotherapy on Cell Survival and Inflammatory Cytokine and Chemokine Secretion in a Co-Culture Model of Head and Neck Squamous Cell Carcinoma and Normal Cells.

Radiotherapy (RT) is one of the main treatments for head and neck squamous cell carcinomas (HNSCCs). Unfortunately, radioresistance is observed in many cases of HNSCCs. The effectiveness of RT depends on both the direct effect inducing cell death and the indirect effect of changing the tumor microenvironment (TME). Knowledge of interactions between TME components after RT may help to design a new combined treatment with RT. In the study, we investigated the effect of RT on cell survival and cell secretion in a co-culture model of HNSCCs in vitro. We examined changes in cell proliferation, colony formation, cell cycle phases, type of cell death, cell migration and secretion after irradiation. The obtained results suggest that the presence of fibroblasts and endothelial cells in co-culture with HNSCCs inhibits the function of cell cycle checkpoints G1/S and G2/M and allows cells to enter the next phase of the cell cycle. We showed an anti-apoptotic effect in co-culture of HNSCCs with fibroblasts or endothelial cells in relation to the execution phase of apoptosis, although we initially observed increased activation of the early phase of apoptosis in the co-cultures after irradiation. We hypothesize that the anti-apoptotic effect depends on increased secretion of IL-6 and MCP-1.

The Complex Composition of Trans-resveratrol, Quercetin, Vitamin E and Selenium Inhibits the Growth of Colorectal Carcinoma.

BACKGROUND/AIM: Numerous studies have demonstrated an anti-cancer action of plant-derived polyphenols. Their action is mainly related to antioxidant, anti-inflammatory, immunomodulatory and inhibitory properties. It is expected that proper composition of nutrition factors with anti-cancer activity may prevent from cancer incidence or inhibit cancer progression. The aim of the study was to investigate the anti-cancer properties of a standardized composition of compounds: trans-resveratrol, quercetin, vitamin E and selenium (Neoplasmoxan, Vebiot) in a mouse model of CT26 colorectal carcinoma. MATERIALS AND METHODS: Colorectal carcinoma cells (CT26) were introduced subcutaneously (2×105/mouse) on the back of the mice. Neoplasmoxan suspension was administered intragastrically, daily, for 21 consecutive days. In collected tumors, the area occupied by tumor blood vessels and the number of immune cells; macrophages and CD8-positive cytotoxic T lymphocytes were evaluated. RESULTS: It was observed that administration of Neoplasmoxan inhibits the growth of colorectal carcinoma in mice. Tumor volume after Neoplasmoxan administration was 40% smaller than in control groups. No overall toxicity of Neoplasmoxan was observed. The area of blood vessels in tumors of mice that received Neoplasmoxan was reduced by approximately 20%. The area occupied by macrophages increased about 60% compared to the control group. However, no increased number of CD8-positive cytotoxic T lymphocytes was observed in the group that received Neoplasmoxan. CONCLUSION: A tendency of Neoplasmoxan to inhibit the growth of colorectal carcinoma was recorded. It also seems that additional combination of the tested preparation with standard chemotherapy or radiotherapy should bring a synergistic therapeutic effect.

Mesenchymal stromal cells as carriers of IL-12 reduce primary and metastatic tumors of murine melanoma.

Due to immunosuppressive properties and confirmed tropism towards cancer cells mesenchymal stromal cells (MSC) have been used in many trials. In our study we used these cells as carriers of IL-12 in the treatment of mice with primary and metastatic B16-F10 melanomas. IL-12 has confirmed anti-cancer activity, induces a strong immune response against cancer cells and acts as an anti-angiogenic agent. A major limitation of the use of IL-12 in therapy is its systemic toxicity. The aim of the work was to develop a system in which cytokine may be administered intravenously without toxic side effects. In this study MSC were used as carriers of the IL-12. We confirmed antitumor effectiveness of the cells secreting IL-12 (MSC/IL-12) in primary and metastatic murine melanoma models. We observed inhibition of tumor growth and a significant reduction in the number of metastases in mice after MSC/IL-12 administration. MSC/IL-12 decreased vascular density and increased the number of anticancer M1 macrophages and CD8+ cytotoxic T lymphocytes in tumors of treated mice. To summarize, we showed that MSC are an effective, safe carrier of IL-12 cytokine. Administered systemically they exert therapeutic properties of IL-12 cytokine without toxicity. Therapeutic effect may be a result of pleiotropic (proinflammatory and anti-angiogenic) properties of IL-12 released by modified MSC.

The Proper Administration Sequence of Radiotherapy and Anti-Vascular Agent-DMXAA Is Essential to Inhibit the Growth of Melanoma Tumors.

Vascular disrupting agents (VDAs), such as DMXAA, effectively destroy tumor blood vessels and cause the formation of large areas of necrosis in the central parts of the tumors. However, the use of VDAs is associated with hypoxia activation and residues of rim cells on the edge of the tumor that are responsible for tumor regrowth. The aim of the study was to combine DMXAA with radiotherapy (brachytherapy) and find the appropriate administration sequence to obtain the maximum synergistic therapeutic effect. We show that the combination in which tumors were irradiated prior to VDAs administration is more effective in murine melanoma growth inhibition than in either of the agents individually or in reverse combination. For the first time, the significance of immune cells' activation in such a combination is demonstrated. The inhibition of tumor growth is linked to the reduction of tumor blood vessels, the increased infiltration of CD8+ cytotoxic T lymphocytes and NK cells and the polarization of macrophages to the cytotoxic M1 phenotype. The reverse combination of therapeutic agents showed no therapeutic effect and even abolished the effect of DMXAA. The combination of brachytherapy and vascular disrupting agent effectively inhibits the growth of melanoma tumors but requires careful planning of the sequence of administration of the agents.

Brachytherapy in a Single Dose of 10Gy as an "in situ" Vaccination.

Radiotherapy (RT) is one of the major methods of cancer treatment. RT destroys cancer cells, but also affects the tumor microenvironment (TME). The delicate balance between immunomodulation processes in TME is dependent, among other things, on a specific radiation dose. Despite many studies, the optimal dose has not been clearly determined. Here, we demonstrate that brachytherapy (contact radiotherapy) inhibits melanoma tumor growth in a dose-dependent manner. Doses of 10Gy and 15Gy cause the most effective tumor growth inhibition compared to the control group. Brachytherapy, at a single dose of ≥ 5Gy, resulted in reduced tumor blood vessel density. Only a dose of 10Gy had the greatest impact on changes in the levels of tumor-infiltrating immune cells. It most effectively reduced the accumulation of protumorogenic M2 tumor-associated macrophages and increased the infiltration of cytotoxic CD8+ T lymphocytes. To summarize, more knowledge about the effects of irradiation doses in anticancer therapy is needed. It may help in the optimization of RT treatment. Our results indicate that a single dose of 10Gy leads to the development of a robust immune response. It seems that it is able to convert a tumor microenvironment into an "in situ" vaccine and lead to a significant inhibition of tumor growth.

Adipose tissue-derived stromal cells stimulated macrophages-endothelial cells interactions promote effective ischemic muscle neovascularization.

Neovascularization, the process of new blood vessels formation in response to hypoxia induced signals, is an essential step during wound healing or ischemia repair. It follows as a cascade of consecutive events leading to new blood vessels formation and their subsequent remodeling to a mature and functional state, enabling tissue regeneration. Any disruption in consecutive stages of neovascularization can lead to chronic wounds or impairment of tissue repair. In the study we try to explain the biological basis of accelerated blood vessels formation in ischemic tissue after adipose tissue-derived stromal cells (ADSCs) administration. Experiments were performed on mouse models of hindlimb ischemia. We have evaluated the level of immune cells (neutrophils, macrophages) infiltration. The novelty of our work was the assessment of bone marrow-derived stem/progenitor cells (BMDCs) infiltration and their contribution to the neovascularization process in ischemic tissue. We have noticed that ADSCs regulated immune response and affected the kinetics and ratio of macrophages population infiltrating ischemic tissue. Our research revealed that ADSCs promoted changes in the morphology of infiltrating macrophages and their tight association with forming blood vessels. We assume that recruited macrophages may take over the role of pericytes and stabilize the new blood vessel or even differentiate into endothelial cells, which in consequence can accelerate vascular formation upon ADSCs administration. Our findings indicate that administration of ADSCs into ischemic muscle influence spatio-temporal distribution of infiltrating cells (macrophages, neutrophils and BMDCs), which are involved in each step of vascular formation, promoting effective ischemic tissue neovascularization.

Author Correction: Combination of anti-vascular agent - DMXAA and HIF-1α inhibitor - digoxin inhibits the growth of melanoma tumors.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Monitoring of diffusion properties and transverse relaxation time of mouse ischaemic muscle after administration of human mesenchymal stromal cells derived from adipose tissue.

OBJECTIVES: Application of non-invasive imaging methods plays an important role in the assessment of cellular therapy effects in peripheral artery disease. The purpose of this work was to evaluate the kinetics of MRI-derived parameters characterizing ischaemic hindlimb muscle after administration of human mesenchymal stromal cells derived from adipose tissue (hADSC) in mice. MATERIALS AND METHODS: MRI experiments were performed on a 9.4T Bruker system. The measurement protocol included transverse relaxation time mapping and diffusion tensor imaging. The monitoring period encompassed 14 days after femoral artery ligation and subsequent cell administration. The effect of hADSC transplantation was compared with the effect of normal human dermal fibroblasts (NHDFs) and phosphate-buffered saline injection. RESULTS: The most significant differences between the hADSC group and the remaining ones were observed around day 3 after ischaemia induction (increased transverse relaxation time in the hADSC group in comparison with the control group) and around day 7 (increased transverse relaxation time and decreased third eigenvalue of the diffusion tensor in the hADSC group in comparison with the control and NHDF groups) at the site of hADSC injection. Histologically, it was associated with increased macrophage infiltration at days 3-7 and with the presence of small regenerating fibres in the ischaemic tissue at day 7. CONCLUSIONS: Our results underscore the important role of macrophages in mediating the therapeutic effects of hADSCs and confirm the huge potential of magnetic resonance imaging in monitoring of cellular therapy effects.

The effect of culture media on large-scale expansion and characteristic of adipose tissue-derived mesenchymal stromal cells.

BACKGROUND: Adipose tissue-derived mesenchymal stromal cells (ASCs) have been shown to exhibit some promising properties of their use in regenerative medicine as advanced therapy medicinal products (ATMP). However, different sources of their origin, methods of isolation, and expansion procedures cause the laboratory and clinical results difficult to compare. METHODS: ASCs were isolated from lipoaspirates and cultured in three different medium formulations: αMEM and DMEM as a basal medium supplemented with 10% of human platelet lysate (hPL) and DMEM supplemented with 20% fetal bovine serum (FBS) and bFGF as a gold standard medium. Subsequently, the impact of culture media on ASCs growth kinetics, their morphology and immunophenotype, ability to differentiate, clonogenic potential, and secretion profile was evaluated. RESULTS: All cultured ASCs lines showed similar morphology and similar clonogenic potential and have the ability to differentiate into three lines: adipocytes, osteoblasts, and chondroblasts. The immunophenotype of all cultured ASCs was consistent with the guidelines of the International Society for Cell Therapy (ISCT) allowing to define cells as mesenchymal stromal cell (MSC) (≥ 95% CD105, CD73, CD90 and ≤ 2% CD45, CD34, CD14, CD19, HLA-DR). The immunophenotype stabilized after the second passage and did not differ between ASCs cultured in different conditions. The exception was the ASCs grown in the presence of FBS and bFGF, which expressed CD146 antigens. The secretion profile of ASCs cultured in different media was similar. The main secreted cytokine was IL-6, and its level was donor-specific. However, we observed a strong influence of the medium formulation on ASCs growth kinetics. The proliferation rate of ASCs in medium supplemented with hPL was the highest. CONCLUSIONS: Culture media that do not contain animal-derived antigens (xeno-free) can be used to culture cells defined as MSC. Xeno-free medium is a safe alternative for the production of clinical-grade MSC as an advanced therapy medicinal product. Additionally, in such culture conditions, MSC can be easily expanded in accordance with the Good Manufacturing Process (GMP) requirements to a desired amount of cells for clinical applications.

Human ADSC xenograft through IL-6 secretion activates M2 macrophages responsible for the repair of damaged muscle tissue.

BACKGROUND: Adipose-derived mesenchymal stromal cells (ADSCs) are multipotent stromal cells. The cells secrete a number of cytokines and growth factors and show immunoregulatory and proangiogenic properties. Their properties may be used to repair damaged tissues. The aim of our work is to explain the muscle damage repair mechanism with the utilization of the human adipose-derived mesenchymal stromal cells (hADSCs). METHODS: For the hADSCs isolation, we used the subcutaneous adipose tissue collected during the surgery. The murine hind limb ischemia was used as a model. The unilateral femoral artery ligation was performed on 10-12-week-old male C57BL/6NCrl and NOD SCID mice. The mice received PBS- (controls) or 1 × 106 hADSCs. One, 3, 7, 14 and 21 days after the surgery, we collected the gastrocnemius muscles for the immunohistochemical analysis. The results were analyzed with relevant tests using the Statistica software. RESULTS: The retention time of hADSCs in the limb lasted about 14 days. In the mice receiving hADSCs, the improvement in the functionality of the damaged limb occurred faster than in the control mice. More new blood vessels were formed in the limbs of the mice receiving hADSCs than in limbs of the control mice. hADSCs also increased the infiltration of the macrophages with the M2 phenotype (7-AAD-/CD45+/F4/80+/CD206+) into the ischemic limbs. hADSCs introduced into the limb of mice secreted interleukin-6. This cytokine stimulates the emergence of the proangiogenic M2 macrophages, involved, among others, in the repair of a damaged tissue. Both macrophage depletion and IL-6 blockage suppressed the therapeutic effect of hADSCs. In the mice treated with hADSCs and liposomes with clodronate (macrophages depletion), the number of capillaries formed was lower than in the mice treated with hADSCs alone. Administration of hADSCs to the mice that received siltuximab (human IL-6 blocker) did not cause an influx of the M2 macrophages, and the number of capillaries formed was at the level of the control group, as in contrast to the mice that received only the hADSCs. CONCLUSIONS: The proposed mechanism for the repair of the damaged muscle using hADSCs is based on the activity of IL-6. In our opinion, the cytokine, secreted by the hADSCs, stimulates the M2 macrophages responsible for repairing damaged muscle and forming new blood vessels.

Combination of anti-vascular agent - DMXAA and HIF-1α inhibitor - digoxin inhibits the growth of melanoma tumors.

Vascular disrupting agents as DMXAA inhibit tumor growth only for a short period of time followed by rapid tumor regrowth. Among others, hypoxia and presence of transcription factor HIF-1α are responsible for tumors regrowth. The aim of our study was to investigate the inhibition of murine melanoma growth by combining two agents: anti-vascular - DMXAA and the HIF-1α inhibitor - digoxin and explaining the mechanism of action of this combination. After DMXAA treatment tumor size was reduced only for a limited time. After 7 days regrowth of tumors was observed and number of vessels was increased especially in tumor's peripheral areas. DMXAA also induced an influx of immune cells: macrophages, CD8+ cytotoxic lymphocytes, NK cells, CD4+ lymphocytes. Administration of digoxin alone inhibited the growth of tumors. Administration of both agents in the proper sequence significantly inhibited the regrowth of tumors better than either agents alone. Combination therapy reduced number of newly formed vessels. In tumors of mice treated with combination therapy, the number of macrophages M1, CD8+ cytotoxic lymphocytes, NK cells and to a lesser extent CD4+ cells was increased. The combination of anti-vascular agents with HIF-1α inhibitors appears to be an effective therapeutic option.