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Background: Epigenetic mechanisms have important roles in the tumour escape from immune responses, such as in MHC class I downregulation or altered expression of other components involved in antigen presentation. Chemotherapy with DNA methyltransferase inhibitors (DNMTi) can thus influence the tumour cell interactions with the immune system and their sensitivity to immunotherapy.Methods: We evaluated the therapeutic effects of the DNMTi 5-azacytidine (5AC) against experimental MHC class I-deficient and -positive tumours. The 5AC therapy was combined with immunotherapy, using a murine model for HPV16-associated tumours.Results: We have demonstrated 5AC additive effects against MHC class I-positive and -deficient tumours when combined with unmethylated CpG oligodeoxynucleotides or with IL-12-producing cellular vaccine. The efficacy of the combined chemoimmunotherapy against originally MHC class I-deficient tumours was partially dependent on the CD8(+)-mediated immune responses. Increased cell surface expression of MHC class I cell molecules, associated with upregulation of the antigen-presenting machinery-related genes, as well as of genes encoding selected components of the IFNγ-signalling pathway in tumours explanted from 5AC-treated animals, were observed.Conclusion: Our data suggest that chemotherapy of MHC class I-deficient tumours with 5AC combined with immunotherapy is an attractive setting in the treatment of MHC class I-deficient tumours. [ABSTRACT FROM AUTHOR]

The combination of photodynamic therapy and radiotherapy has given rise to a modality called radiodynamic therapy (RDT), based on reactive oxygen species-producing radiosensitizers. The production of singlet oxygen, O 2 ( 1 Δ g ), by octahedral molybdenum (Mo 6 ) clusters upon X-ray irradiation allows for simplification of the architecture of radiosensitizing systems. In this context, we prepared a radiosensitizing system using copper-free click chemistry between a Mo 6 cluster bearing azido ligands and the homo-bifunctional linker bis-dPEG 11 -DBCO. The resulting compound formed nanoparticles, which featured production of O 2 ( 1 Δ g ) and efficient cellular uptake, leading to remarkable photo- and radiotoxic effects against the prostatic adenocarcinoma TRAMP-C2 cell line. Spheroids of TRAMP-C2 cells were also used for evaluation of toxicity and phototoxicity. In vivo experiments on a mouse model demonstrated that subcutaneous injection of the nanoparticles is a safe administration mode at a dose of up to 0.08 g kg -1 . The reported results confirm the relevancy of Mo 6 -based radiosensitizing nanosystems for RDT.

X-Ray-induced photodynamic therapy represents a suitable modality for the treatment of various malignancies. It is based on the production of reactive oxygen species by radiosensitizing nanoparticles activated by X-rays. Hence, it allows overcoming the depth-penetration limitations of conventional photodynamic therapy and, at the same time, reducing the dose needed to eradicate cancer in the frame of radiotherapy treatment. The direct production of singlet oxygen by octahedral molybdenum cluster complexes upon X-ray irradiation is a promising avenue in order to simplify the architecture of radiosensitizing systems. One such complex was utilized to prepare water-stable nanoparticles using the solvent displacement method. The nanoparticles displayed intense red luminescence in aqueous media, efficiently quenched by oxygen to produce singlet oxygen, resulting in a substantial photodynamic effect under blue light irradiation. A robust radiosensitizing effect of the nanoparticles was demonstrated in vitro against TRAMP-C2 murine prostatic carcinoma cells at typical therapeutic X-ray doses. Injection of a suspension of the nanoparticles to a mouse model revealed the absence of acute toxicity as evidenced by the invariance of key physiological parameters. This study paves the way for the application of octahedral molybdenum cluster-based radiosensitizers in X-ray-induced photodynamic therapy and its translation to in vivo experiments.

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.

The present study aimed to elucidate the role of cluster of differentiation (CD)8+, CD4+, natural killer (NK), and myeloid (CD11b+) cells in the course of the growth and rejection of experimental major histocompatibility complex (MHC) class I-deficient, HPV16 E6/E7-associated TC-1/A9 tumors in mice. Stable mouse lines (F 30 ) generated by inbreeding of Balb/c and C57BL/6 strains, which were characterized by H-2Db+d-NK1.1neg (B6-neg) and H-2Db-d+NK1.1high (Balb-high) phenotypes, were used for the present study. The novel strains spontaneously regressed tumors in 70-90% of cases. Ex vivo histological analysis of the tumor microenvironment in cryosections showed an indirect correlation between the growth of the transplanted tumor (progressor vs. regressor mice) and the proportion of immunocompetent cell infiltration in the tumors. The regressor mice exhibited a higher infiltration of tumors with CD4+ and CD8+ cells, and in Balb-high with NK cells as well, compared with the progressors. All tumor transplants also indicated a huge infiltration of CD11b+ cells, but this infiltration was not dependent on the stage of the TC-1/A9 tumor development. Depletion of individual cell subpopulations in vivo exhibited different effects on the tumor development in the two strains. Elimination of CD8-positive cells enhanced growth of TC-1/A9 tumor transplants in both hybrid stains, whereas CD4+ cell depletion affected rejection of TC-1/A9 tumors in the B6-neg mice only. Depletion of NK cells with anti-asialo GM1 antibody in the Balb-high strain led to enhancement of tumor growth, which was more pronounced after depletion of the NK1.1+ subpopulation. On the other hand, depletion of NK cells with anti-asialo GM1 in B6-neg mice did not affect the regression of TC-1/A9 tumor transplants, but increased the CD11b+ cell infiltration. In summary, these results indicate that co-operation of particular subsets of immunocompetent cells is essential for the rejection of TC-1/A9 tumor transplants. In B6-neg mice, the co-operative action of CD8+ and CD4+ cells is required, whereas in Balb-high mice, the synergy of CD8+ and NK1.1+ cells is of major importance.

To elucidate the immunological mechanisms critical for tumor progression, we bred novel mouse strains, different in the NKC and H-2D domains. We used inbreeding to generate hybrids of Balb/c and C57BL/6 of stable H-2Db+d-NK1.1neg and H-2Db-d+NK1.1high phenotypes. We analyzed the growth of three established MHC class I-deficient tumor cell lines: TC-1/A9 tumor (HPV-associated) and B16F10 melanoma, both syngeneic to C57BL/6, and the MCB8 (3-methycholanthrene-induced tumor) syngeneic to Balb/c. Furthermore, we induced colorectal carcinoma by azoxymethane-DSS treatment to test the susceptibility to chemically-induced primary cancer. We found that the novel strains spontaneously regressed the tumor transplants syngeneic to both Balb/c (MCB8) and C57BL/6 (B16F10 and TC-1/A9) mice. The H2-Db+d-NK1.1neg, but not the H2-Db-d+NK1.1high strain was also highly resistant to chemically-induced colorectal cancer in comparison to the parental mice. The immune changes during TC-1/A9 cancer development involved an increase of the NK cell distribution in the peripheral blood and spleen along with higher expression of NKG2D activation antigen; this was in correlation with the time-dependent rise of cytotoxic activity in comparison to C57BL/6 mice. The TC-1/A9 cancer regression was accompanied by higher proportion of B cells in the spleen and B220+/CD86+ activated antigen-presenting B cells distributed in the lymphoid organs, as well as in the periphery. The changes in the T-cell population were represented mainly by the prevalence of T helper cells reflected by grown CD4/CD8 ratio, most prominent in the b+d-NK1.1neg strain. The results of the present study imply usefulness of the two novel mouse strains as an experimental model for further studies of tumor resistance mechanisms.

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.

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., (© 2014 Society for Leukocyte Biology.)

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.

Genetically modified tumour cells producing cytokines such as interleukin 12 (IL-12) are potent activators of the antitumour immune responses and represent a promising therapeutic modality when combined with chemotherapy. The objective of this study was to examine whether IL-12-producing cellular vaccines can augment chemotherapy of human papilloma virus (HPV) 16-associated murine tumours with the cytostatic agent gemcitabine (GEM). We found that peritumoral administration of IL-12-producing tumour vaccines enhanced the effect of cytoreductive therapy with GEM both in non-metastasizing murine carcinoma TC-1 and in metastasizing murine carcinoma MK16. The percentage of mice with MK16 metastases and the number of lung metastatic nodules was substantially decreased. In another clinically relevant setting, surgical minimal residual tumour disease, the administration of IL-12-producing tumour vaccine and GEM after the MK16 tumour surgery reduced the percentage of mice with tumour recurrences; similarly, the percentage of metastasis-bearing mice and the number of metastatic nodules was decreased. Tumour inhibitory effects exerted by GEM plus IL-12 were associated with high production of interferon-γ (IFNγ) by splenocytes. Our results suggest that the IL-12-producing vaccine can enhance the effect of GEM chemotherapy in some HPV16-associated murine tumour models.