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// Jana Simova 1 , Olena Sapega 1 , Terezie Imrichova 2 , Ivan Stepanek 1 , Lenka Kyjacova 2 , Romana Mikyskova 1 , Marie Indrova 1 , Jana Bieblova 1 , Jan Bubenik 3 , Jiri Bartek 2, 4, 5 , Zdenek Hodny 2 , Milan Reinis 1 1 Immunology Unit, Czech Centre for Phenogenomics, BIOCEV and Department of Transgenic Models of Diseases, Institute of Molecular Genetics of the ASCR, v.v.i., Prague 14220, Czech Republic 2 Department of Genome Integrity, Institute of Molecular Genetics, v.v.i., Academy of Sciences of the Czech Republic, Prague 14220, Czech Republic 3 First Faculty of Medicine, Charles University in Prague, Prague 12000, Czech Republic 4 Danish Cancer Society Research Center, Copenhagen DK-2100, Denmark 5 Department of Medical Biochemistry and Biophysics, Science For Life Laboratory, Division of Translational Medicine and Chemical Biology, Karolinska Institute, 17121 Solna, Sweden Correspondence to: Zdenek Hodny, email: hodny@img.cas.cz Milan Reinis, email: reinis@img.cas.cz Keywords: cellular senescence, cancer chemotherapy, docetaxel, IL-12, cell therapy Received: April 18, 2016 Accepted: May 29, 2016 Published: July 19, 2016 ABSTRACT Standard-of-care chemo- or radio-therapy can induce, besides tumor cell death, also tumor cell senescence. While senescence is considered to be a principal barrier against tumorigenesis, senescent cells can survive in the organism for protracted periods of time and they can promote tumor development. Based on this emerging concept, we hypothesized that elimination of such potentially cancer-promoting senescent cells could offer a therapeutic benefit. To assess this possibility, here we first show that tumor growth of proliferating mouse TC-1 HPV-16-associated cancer cells in syngeneic mice becomes accelerated by co-administration of TC-1 or TRAMP-C2 prostate cancer cells made senescent by pre-treatment with the anti-cancer drug docetaxel, or lethally irradiated. Phenotypic analyses of tumor-explanted cells indicated that the observed acceleration of tumor growth was attributable to a protumorigenic environment created by the co-injected senescent and proliferating cancer cells rather than to escape of the docetaxel-treated cells from senescence. Notably, accelerated tumor growth was effectively inhibited by cell immunotherapy using irradiated TC-1 cells engineered to produce interleukin IL-12. Collectively, our data document that immunotherapy, such as the IL-12 treatment, can provide an effective strategy for elimination of the detrimental effects caused by bystander senescent tumor cells in vivo .

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 (F30) 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.

Dendritic cell (DC)-based vaccines pulsed with high hydrostatic pressure (HHP)-inactivated tumor cells have recently been shown to be a promising tool for prostate cancer chemoimmunotherapy. In this study, DC-based vaccines, both pulsed and unpulsed, were as effective as docetaxel (DTX) in reducing prostate tumors in the orthotopic transgenic adenocarcinoma of the mouse prostate (TRAMP) model. However, we did not observe any additive or synergic effects of chemoimmunotherapy on the tumor growth, while only the combination of DTX and pulsed dendritic cells resulted in significantly lower proliferation detected by Ki67 staining in histological samples. The DC-based vaccine pulsed with HHP-treated tumor cells was also combined with another type of cytostatic, cyclophosphamide, with similar results. In another clinically relevant setting, minimal residual tumor disease after surgery, administration of DC-based vaccines after the surgery of poorly immunogenic transplanted TRAMP-C2, as well as in immunogenic TC-1 tumors, reduced the growth of tumor recurrences. To identify the effector cell populations after DC vaccine application, mice were twice immunized with both pulsed and unpulsed DC vaccine, and the cytotoxicity of the spleen cells populations was tested. The effector cell subpopulations were defined as CD4+ and NK1.1+, which suggests rather unspecific therapeutic effects of the DC-based vaccines in our settings. Taken together, our data demonstrate that DC-based vaccines represent a rational tool for the treatment of human prostate cancer.

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.

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.

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.

Our study was designed to examine the effects of IL-2 gene therapy in a surgical minimal residual tumour disease (SMRTD). Mice were inoculated s.c. with methylcholanthrene (MC)-induced MC12 sarcoma cells. When the tumours reached 8 to 12 mm in diameter, they were excised, either completely (“microscopic SMRTD”) or incompletely (“macroscopic SMRTD”). On day 90 after surgery, the tumour recurrence rate in untreated mice with microscopic SMRTD was approximately 30%, whereas in those with macroscopic SMRTD it was 75%. After surgery, experimental mice were treated with 2 types of irradiated, IL-2 gene-modified, IL-2-producing tumour cell vaccine. One type of vaccine was derived from the MC12 sarcoma cells (MC12-IL2/IV-3); the other type was derived from an unrelated X63-Ag8.653 plasmacytoma (X63-m-IL-2). Both types of vaccine failed to cure the macroscopic SMRTD. Whereas the X63-m-IL-2 vaccine was also ineffective in the microscopic SMRTD, the MC12-IL2/IV-3 vaccine was capable of preventing growth in all but one mouse (1/64) with microscopic SMRTD when administered 2 to 5 days after surgery. If the vaccination took place 2 days before surgery or later than 5 days after surgery, the therapeutic activity was lost. Vaccination with irradiated parental MC12 cells did not produce any significant benefit compared to the operated-only mice. The protective effect of the MC12-IL2/IV-3 vaccine was specific and comparatively long-lasting. Vaccinated mice, which had rejected the MC12 tumour residuum, were capable of rejecting a second inoculum of the MC12 sarcoma cells injected on days 35 to 110 after surgery but succumbed to the growth of 2 other unrelated murine sarcomas carrying different tumour-rejection antigens. Int. J. Cancer 76:115–119, 1998.© 1998 Wiley-Liss, Inc.

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.

Loss or downregulation of MHC class I molecules on tumour cells is a common mechanism by which tumours can escape T-cell mediated immune responses. In this study, we examined the role of different immune cell lineages in the development of immunity against tumours of the same aetiology but with different MHC class I expression. In vivo depletion of CD8+ cells, but not of CD4+ or NK1.1+ cells in the immunization period resulted in complete elimination of the protective effects of immunization with irradiated TC-1 cells (MHC class I-positive cell line) against the TC-1 tumour challenge. After immunization with irradiated TC-1/A9 or with MK16 tumour cells (MHC class I-deficient sublines) a remarkable dependence on the presence of NK1.1+ cells was observed, while the tumour growth inhibition after CD4+ or CD8+ depletion was not efficient. Cytotoxic activity induced by TC-1 cell immunization was significantly abrogated in the CD8+ and CD4+ but not NK1.1+ cell-depleted mice, as compared to the immunized only controls. After MK16 or TC-1/A9 cell immunization, NK1.1+ but not CD8+ and CD4+ cell-depleted mice displayed significant reduction of specific cytotoxicity. Mice immunized with TC-1 cells showed similar percentage of IFNγ producing cells in CD8+, CD4+ and NK1.1+ cell populations. On the other hand, the highest proportion of IFNγ producing cells after immunization with TC-1/A9 or MK16 cells was concentrated into the NK1.1-positive spleen cell population. Our data demonstrate that the development of immunity against MHC class I-deficient tumours is highly dependent on the activity NK1.1+ cell population.

Natural killer T (NKT) cells are potent modulators of antitumor immunity. Their protective effects can be achieved upon their activation by glycolipid ligands presented in the context of the CD1d molecule. These CD1d-binding glycolipid antigens have been described as potent therapeutic agents against tumors, infections, as well as autoimmune diseases. Immunoregulatory and therapeutic effects of glycolipid ligands depend on their structure and modes of administration. Therefore, more studies are needed for optimization of the particular therapeutic settings. This study was focused on the tumor-inhibitory effects of 12 carbon acyl chain beta-galactosyl ceramide (C12 beta-D-Galactosyl Ceramide; beta-GalCer(C12)) on the growth of human papillomavirus type 16 (HPV16)-associated neoplasms transplanted in syngeneic mice. Treatment of tumor-bearing mice with beta-GalCer(C12) 3-14 days after tumor cell transplantation significantly inhibited the growth of the major histocompatibility complex (MHC) Class I-positive (TC-1), as well as MHC Class I-deficient (TC-1/A9) HPV16-associated tumors. Moreover, administration of beta-GalCer(C12) after surgical removal of TC-1 tumors inhibited the growth of tumor recurrences. Similar results were obtained in the treatment of tumors after chemotherapy. beta-GalCer(C12) treatment turned out to be also synergistic with immunotherapy based on administration of IL-12-producing cellular vaccines. These results suggest that beta-GalCer(C12), whose antitumor effects have so far not been studied in detail, can be effective for the treatment of minimal residual tumor disease as well as an adjuvant for cancer immunotherapy.

We have established animal models of HPV16-associated tumours with distinct levels of MHC class I expression. This model was used for examination of immune responses, production of cytokines and kinetics of immune cell subsets after IL-12 therapy of minimal residual tumour disease induced by CBA-4A (cyclophosphamide derivative) treatment. Upregulation of cytokine production was detected, compared to control animals without tumours. No differences in Th1/Th2 polarization of the immune responses after immunotherapy in animals bearing tumours with different surface expression of MHC class I molecules were observed. In the spleens of TC-1 (MHC class I+) but not of TC-1/A9 (MHC class I-) treated tumour-bearing animals, the cytotoxic CD8+ cells detectable in 51Cr microcytotoxicity assay, were found. In the spleens of TC-1/A9 but not of TC-1 tumour-treated animals, the NK activity measured as the lysis of NK-sensitive YAC-1 targets was detected. Down-regulation of the CD4+ and CD8+ subpopulations in spleens of tumour-bearing animals were not restored after therapy. The percentage of CD25+/CD4+ T regulatory (Treg) cells in lymph nodes remained unchanged. The cytoreductive chemotherapy led to strong upregulation and accumulation of immunosuppressive immature myeloid Gr-1+/CD11b+ cells (IMC) in the spleens of treated animals. The accumulation of Gr-1+/CD11b+ cells was significantly decreased after subsequent IL-12 immunotherapy. These data suggest that elimination of IMC after IL-12 immunotherapy may be responsible for the improvement of antitumour responses after adjuvant IL-12 vaccination for the treatment of CMRTD.

Oligodeoxynucleotides containing guanine-cytidine dimers (CpG ODN) are potent inducers of anti-tumour immune responses. In this study, we analyzed the capacity of CpG ODN to inhibit the growth of both MHC class I-positive and -deficient tumours after debulking the tumour mass by chemotherapy or surgery. We employed an animal model resembling human papillomavirus (HPV) 16-associated tumours. Tumour cell lines with distinct cell surface expression of the MHC class I molecules were injected into syngeneic C57BL/6 mice, and the growing tumours were either subjected to cytoreductive chemotherapy with ifosfamide derivative, CBM-4A, or surgically removed. Subsequent treatment with synthetic CpG ODN significantly blocked the growth of the recurrent tumours. Our results indicate that the therapy with CpG ODN can be effective for the treatment of minimal residual tumour disease of the tumours that have escaped from the immune surveillance by downmodulating the MHC class I expression.

Loss or downregulation of MHC class I molecules on tumour cells is a common mechanism by which tumours can escape from T-cell mediated immune responses. In this study we have investigated the immunologic crossreactivity between murine tumour cell lines expressing human papilloma virus (HPV) 16-derived E6/E7 oncoproteins with distinct surface expression of MHC class I molecules. The aims of this study were to demonstrate whether immune responses capable of coping with MHC class I-positive tumours can also be effective against their MHC class I-deficient derivatives and whether it is possible to induce immunity against MHC class I-deficient tumours by cellular vaccines based on MHC class I-deficient tumour cell lines. Our data showed that immunization with MHC class I-deficient but not with MHC class I positive tumour cells inhibited the growth of MHC class I-deficient tumours. In vivo depletion studies revealed that the mechanisms underlying effective immune responses against MHC class I-negative tumours in animals immunized with MHC class I-deficient tumour cells involved natural killer cells. The presented findings are of particular clinical relevance in the sense of construction of vaccines directed against a broad spectrum of HPV-associated tumours.

Murine carcinoma induced by MK 16 cells expressing HPV 16 E6/E7 oncogenes was utilized to examine the therapeutic effect of dendritic cell-based tumour vaccines. Mice carrying 5-day MK 16 tumours were injected peritumorally with either dendritic cells (DC) or DC pulsed with MK 16 tumour lysate. Both the unpulsed and MK 16 lysate-pulsed DC vaccines inhibited growth of the MK 16 transplants, the pulsed DC being more efficient than the unpulsed vaccines. In vitro priming of the effector cell-mediated anti-MK 16 responses by DC pulsed with MK 16 tumour lysate and a synthetic HPV 16 E7(49-57) peptide RAHYNIVTF was compared. The priming activity of the lysate was substantially higher than that of the HPV 16 E7(49-57) peptide; the priming activity was similar to that of a standard moderately immunogenic chemically-induced sarcoma. Taken collectively, these results suggest that DC vaccines pulsed with HPV 16-associated tumour lysates represent a prospective modality for treatment of HPV 16-associated carcinomas.

We have examined the effect of IL-12-producing cellular vaccines on the cytotoxicity and proliferative potential of CD45+ tumour-infiltrating cells (TIL) in mice carrying syngeneic TC-1 and TC-1/A9 HPV 16-associated tumours after chemotherapy with CBM-4A ifosfamide derivative. The chemotherapy resulted in the decrease of the CD4+ and CD8+ TIL, increase of the Gr-1+/CD11b+ TIL, no changes in the infiltration with CD4+/CD25+ Treg TIL, and decrease of the cytolytic and proliferative potential of the CD45+ TIL. Subsequent immunotherapy with the IL-12-producing, genetically modified TC-1 (TC-1-IL-12) cells increased tumour infiltration with CD8+ and CD4+ cells, decreased the Gr-1+/CD11b+ cells, and increased the cytolytic and proliferative potential of the CD45+ TIL. Taken together, these findings suggest that peritumoral administration of the IL-12-producing cellular vaccine can restore the cytolytic potential and inhibit immunosuppressive TIL-dependent mechanisms in the individuals bearing HPV 16-associated tumours, and explain our previously described tumour-inhibitory effects of the vaccine in mice with minimal residual disease after the tumour chemotherapy.

Downregulation of MHC class I expression on tumour cells, a common mechanism by which tumour cells can escape from specific immune responses, can be associated with coordinated silencing of antigen-presenting machinery genes. The expression of these genes can be restored by IFNγ. In this study we documented association of DNA demethylation of selected antigen-presenting machinery genes located in the MHC genomic locus (TAP-1, TAP-2, LMP-2, LMP-7) upon IFNγ treatment with MHC class I upregulation on tumour cells in several MHC class I-deficient murine tumour cell lines (TC-1/A9, TRAMP-C2, MK16 and MC15). Our data also documented higher methylation levels in these genes in TC-1/A9 cells, as compared to their parental MHC class I-positive TC-1 cells. IFNγ-mediated DNA demethylation was relatively fast in comparison with demethylation induced by DNA methyltransferase inhibitor 5-azacytidine, and associated with increased histone H3 acetylation in the promoter regions of APM genes. Comparative transcriptome analysis in distinct MHC class I-deficient cell lines upon their treatment with either IFNγ or epigenetic agents revealed that a set of genes, significantly enriched for the antigen presentation pathway, was regulated in the same manner. Our data demonstrate that IFNγ acts as an epigenetic modifier when upregulating the expression of antigen-presenting machinery genes.