Radiotherapy (RT), besides cancer cells, also affects the tumor microenvironment (TME): tumor blood vessels and cells of the immune system. It damages endothelial cells and causes radiation-induced inflammation. Damaged vessels inhibit the infiltration of CD8+ T lymphocytes into tumors, and immunosuppressive pathways are activated. They lead to the accumulation of radioresistant suppressor cells, including tumor-associated macrophages (TAMs) with the M2 phenotype, myeloid-derived suppressor cells (MDSCs), and regulatory T cells (Tregs). The area of tumor hypoxia increases. Hypoxia reduces oxygen-dependent DNA damage and weakens the anti-cancer RT effect. It activates the formation of new blood vessels and leads to cancer relapse after irradiation. Irradiation may also activate the immune response through immunogenic cell death induction. This leads to the “in situ” vaccination effect. In this article, we review how changes in the TME affect radiation-induced anticancer efficacy. There is a very delicate balance between the activation of the immune system and the immunosuppression induced by RT. The effects of RT doses on immune system reactions and also on tumor vascularization remain unclear. A better understanding of these interactions will contribute to the optimization of RT treatment, which may prevent the recurrence of cancer.

Tumor-associated macrophages (TAMs) play a significant role in at least two key processes underlying neoplastic progression: angiogenesis and immune surveillance. TAMs phenotypic changes play important role in tumor vessel abnormalization/ normalization. M2-like TAMs stimulate immunosuppression and formation of defective tumor blood vessels leading to tumor progression. In contrast M1-like TAMs trigger immune response and normalize irregular tumor vascular network which should sensitize cancer cells to chemo- and radiotherapy and lead to tumor growth regression. Here, we demonstrated that combination of endoglin-based DNA vaccine with interleukin 12 repolarizes TAMs from tumor growth-promoting M2-like phenotype to tumor growth-inhibiting M1-like phenotype. Combined therapy enhances tumor infiltration by CD4⁺, CD8⁺ lymphocytes and NK cells. Depletion of TAMs as well as CD8⁺ lymphocytes and NK cells, but not CD4⁺ lymphocytes, reduces the effect of combined therapy. Furthermore, combined therapy improves tumor vessel maturation, perfusion and reduces hypoxia. It caused that suboptimal doses of doxorubicin reduced the growth of tumors in mice treated with combined therapy. To summarize, combination of antiangiogenic drug and immunostimulatory agent repolarizes TAMs phenotype from M2-like (pro-tumor) into M1-like (anti-tumor) which affects the structure of tumor blood vessels, improves the effect of chemotherapy and leads to tumor growth regression.

Certain anticancer drugs, such as the peptide CAMEL (aa sequence KWKLFKKIGAULKVL) induce necrotic type of cell death. During this process, a protein termed high mobility group box 1 (HMGB1) is released from cell nucleus into cytoplasm and then into extracellular milieu. Outside of cells, it becomes a proinflammatory cytokine. Its effects range from stimulation of cancer as well as endothelial cell proliferation, to activation of angiogenesis, cell motility and induction of inflammatory conditions. Release of HMGB1 cytokine during the course of anticancer therapy has negative effects upon the therapy itself, since it leads to tumor relapse. We assumed that the inhibition of HMGB1 activity may be conducive towards better therapeutic results in case of drugs inducing necrotic cell death. In this context we studied glycyrrhizin (GR), a triterpenoid saponin glycoside of glycyrrhizic acid and a well-known inhibitor of HMGB1. We have shown that GR inhibits proliferation and migration of cells stimulated by HMGB1 cytokine, as well as HMGB1-induced formation of blood vessels and reduces inflammatory condition (lowering tumor necrosis factor α levels). GR-mediated inhibition of HMGB1 activity (CAMEL-induced release) impedes, in turn, tumor regrowth in mice. As expected, inhibited tumor regrowth is linked to diminished tumor levels of the released HMGB1 and reduced inflammatory condition. To conclude, the use of GR significantly improved anticancer effectiveness of the CAMEL peptide.

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. 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. 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. 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.

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.