MDSCs represent one of the key players mediating immunosuppression. These cells accumulate in the TME, lymphoid organs, and blood during tumor growth. Their mobilization was also reported after CY therapy. DNMTi 5AC has been intensively studied as an antitumor agent. In this study, we examined, using two different murine tumor models, the modulatory effects of 5AC on TU-MDSCs and CY-MDSCs tumor growth and CY therapy. Indeed, the percentage of MDSCs in the TME and spleens of 5AC-treated mice bearing TRAMP-C2 or TC-1/A9 tumors was found decreased. The changes in the MDSC percentage were accompanied by a decrease in the Arg-1 gene expression, both in the TME and spleens. CY treatment of the tumors resulted in additional MDSC accumulation in the TME and spleens. This accumulation was subsequently inhibited by 5AC treatment. A combination of CY with 5AC led to the highest tumor growth inhibition. Furthermore, in vitro cultivation of spleen MDSCs in the presence of 5AC reduced the percentage of MDSCs. This reduction was associated with an increased percentage of CD11c⁺ and CD86⁺/MHCII⁺ cells. The observed modulatory effect on MDSCs correlated with a reduction of the Arg-1 gene expression, VEGF production, and loss of suppressive capacity. Similar, albeit weaker effects were observed when MDSCs from the spleens of tumor-bearing animals were cultivated with 5AC. Our findings indicate that beside the direct antitumor effect, 5AC can reduce the percentage of MDSCs accumulating in the TME and spleens during tumor growth and CY chemotherapy, which can be beneficial for the outcome of cancer therapy.

Myeloid-derived suppressor cells (MDSC) play an important role in tumor escape from antitumor immunity. MDSC accumulate in the lymphoid organs and blood during tumor growth and their mobilization was also reported after cyclophosphamide (CY) administration. In this communication, spleen MDSC accumulating after CY therapy (CY-MDSC) were compared with those expanded in mice bearing human papilloma viruses 16-associated TC-1 carcinoma (TU-MDSC). Although both CY-MDSC and TU-MDSC accelerated growth of TC-1 tumors in vivo, their phenotype and immunosuppressive function differed. CY-MDSC consisted of higher percentage of monocyte-like subpopulation and this was accompanied by lower relative expression of immunosuppressive genes and lower suppression of T-cell proliferation. After interferon-γ stimulation, the expression of immunosuppressive genes increased, but the suppressive ability of CY-MDSC did not reach that of TU-MDSC. The phenotype and function of MDSC obtained from mice bearing TC-1 tumors treated with CY was, in general, found to lie between CY-MDSC and TU-MDSC. After in vitro cultivation of MDSC in the presence of interleukin 12 (IL-12), the percentage of CD11b⁺/Gr-1⁺ cells decreased and was accompanied by an increase in the percentage of CD86⁺/MHCII⁺ cells. The strongest modulatory effect was noticed in the group of CY-MDSC. The susceptibility of CY-MDSC to all-trans-retinoic acid (ATRA) was also evaluated. In vitro cultivation with ATRA resulted in MDSC differentiation, and ATRA inhibited MDSC accumulation induced by CY administration. Our findings identified differences between CY-MDSC and TU-MDSC and supported the rationale for utilization of ATRA or IL-12 to alter MDSC accumulation after CY chemotherapy with the aim to improve its antitumor effect.

High hydrostatic pressure (HHP) has been shown to induce immunogenic cell death of cancer cells, facilitating their uptake by dendritic cells (DC) and subsequent presentation of tumor antigens. In the present study, we demonstrated immunogenicity of the HHP-treated tumor cells in mice. HHP was able to induce immunogenic cell death of both TC-1 and TRAMP-C2 tumor cells, representing murine models for human papilloma virus-associated tumors and prostate cancer, respectively. HHP-treated cells induced stronger immune responses in mice immunized with these tumor cells, documented by higher spleen cell cytotoxicity and increased IFNγ production as compared to irradiated tumor cells, accompanied by suppression of tumor growth in vivo in the case of TC-1 tumors, but not TRAMP-C2 tumors. Furthermore, HHP-treated cells were used for DC-based vaccine antigen pulsing. DC co-cultured with HHP-treated tumor cells and matured by a TLR 9 agonist exhibited higher cell surface expression of maturation markers and production of IL-12 and other cytokines, as compared to the DC pulsed with irradiated tumor cells. Immunization with DC cell-based vaccines pulsed with HHP-treated tumor cells induced high immune responses, detected by increased spleen cell cytotoxicity and elevated IFNγ production. The DC-based vaccine pulsed with HHP-treated tumor cells combined with docetaxel chemotherapy significantly inhibited growth of both TC-1 and TRAMP-C2 tumors. Our results indicate that DC-based vaccines pulsed with HHP-inactivated tumor cells can be a suitable tool for chemoimmunotherapy, particularly with regard to the findings that poorly immunogenic TRAMP-C2 tumors were susceptible to this treatment modality.

Cellular senescence is the process of the permanent proliferative arrest of cells in response to various inducers. It is accompanied by typical morphological changes, in addition to the secretion of bioactive molecules, including proinflammatory cytokines and chemokines [known as the senescence-associated secretory phenotype (SASP)]. Thus, senescent cells may affect their local environment and induce a so-called 'bystander' senescence through the state of SASP. The phenotypes of senescent cells are determined by the type of agent inducing cellular stress and the cell lineages. To characterise the phenotypes of senescent cancer cells, two murine cell lines were employed in the present study: TC-1 and B16F10 (B16) cells. Two distinct senescence inductors were used: Chemotherapeutic agent docetaxel (DTX) and a combination of immunomodulatory cytokines, including interferon γ (IFNγ) and tumour necrosis factor α (TNFα). It was demonstrated that DTX induced senescence in TC-1 and B16 tumour cell lines, which was demonstrated by growth arrest, positive β-galactosidase staining, increased p21Waf1 (p21) expression and the typical SASP capable of inducing a ‘bystander’ senescence. By contrast, treatment with a combination of T helper cell 1 cytokines, IFNγ and TNFα, induced proliferation arrest only in B16 cells. Despite the presence of certain characteristic features resembling senescent cells (proliferation arrest, morphological changes and increased p21 expression), these cells were able to form tumours in vivo and started to proliferate upon cytokine withdrawal. In addition, B16 cells were not able to induce a ‘bystander’ senescence. In summary, the present study described cell line- and treatment-associated differences in the phenotypes of senescent cells that may be relevant in optimization of cancer chemo- and immunotherapy.

Chronic inflammation represents a major threat to human health since long‐term systemic inflammation is known to affect distinct tissues and organs. Recently, solid evidence demonstrated that chronic inflammation affects hematopoiesis; however, how chronic inflammation affects hematopoietic stem cells (HSCs) on the mechanistic level is poorly understood. Here, we employ a mouse model of chronic multifocal osteomyelitis (CMO) to assess the effects of a spontaneously developed inflammatory condition on HSCs. We demonstrate that hematopoietic and nonhematopoietic compartments in CMO BM contribute to HSC expansion and impair their function. Remarkably, our results suggest that the typical features of murine multifocal osteomyelitis and the HSC phenotype are mechanistically decoupled. We show that the CMO environment imprints a myeloid gene signature and imposes a pro‐inflammatory profile on HSCs. We identify IL‐6 and the Jak/Stat3 signaling pathway as critical mediators. However, while IL‐6 and Stat3 blockage reduce HSC numbers in CMO mice, only inhibition of Stat3 activity significantly rescues their fitness. Our data emphasize the detrimental effects of chronic inflammation on stem cell function, opening new venues for treatment.

Signal transducer and activator of transcription 3 (STAT3) signalling serves an important role in carcinogenesis and cellular senescence, and its inhibition in tumour cells represents an attractive therapeutic target. Premature cellular senescence, a process of permanent proliferative arrest of cells in response to various inducers, such as cytostatic drugs or ionizing radiation, is accompanied by morphological and secretory changes, and by altered susceptibility to chemotherapeutic agents, which can thereby complicate their eradication by cancer therapies. In the present study, the responsiveness of proliferating and docetaxel (DTX)‑induced senescent cancer cells to small molecule STAT3 inhibitor Stattic and its analogues was evaluated using tumour cell lines. These agents displayed cytotoxic effects in cell viability assays on both proliferating and senescent murine TRAMP‑C2 and TC‑1 cells; however, senescent cells were markedly more resistant. Western blot analysis revealed that Stattic and its analogues effectively inhibited constitutive STAT3 phosphorylation in both proliferating and senescent cells. Furthermore, whether the Stattic‑derived inhibitor K1836 could affect senescence induction or modulate the phenotype of senescent cells was evaluated. K1836 treatment demonstrated no effect on senescence induction by DTX. However, the K1836 compound significantly modulated secretion of certain cytokines (interleukin‑6, growth‑regulated oncogene α and monocyte chemoattractant protein‑1). In summary, the present study demonstrated differences between proliferating and senescent tumour cells in terms of their susceptibility to STAT3 inhibitors and demonstrated the ability of the new STAT3 inhibitor K1836 to affect the secretion of essential components of the senescence‑associated secretory phenotype. The present study may be useful for further development of STAT3 inhibitor‑based therapy of cancer or age‑related diseases.