Myeloid cells comprise a major component of the tumor microenvironment (TME) that promotes tumor growth and immune evasion. By employing a small-molecule inhibitor of glutamine metabolism, not only were we able to inhibit tumor growth, but we markedly inhibited the generation and recruitment of myeloid-derived suppressor cells (MDSCs). Targeting tumor glutamine metabolism led to a decrease in CSF3 and hence recruitment of MDSCs as well as immunogenic cell death, leading to an increase in inflammatory tumor-associated macrophages (TAMs). Alternatively, inhibiting glutamine metabolism of the MDSCs themselves led to activation-induced cell death and conversion of MDSCs to inflammatory macrophages. Surprisingly, blocking glutamine metabolism also inhibited IDO expression of both the tumor and myeloid-derived cells, leading to a marked decrease in kynurenine levels. This in turn inhibited the development of metastasis and further enhanced antitumor immunity. Indeed, targeting glutamine metabolism rendered checkpoint blockade-resistant tumors susceptible to immunotherapy. Overall, our studies define an intimate interplay between the unique metabolism of tumors and the metabolism of suppressive immune cells.

• Klíčová slova
• Cancer immunotherapy, Immunology, Innate immunity, Oncology,
• MeSH
• buněčná imunita * MeSH
• experimentální nádory imunologie   patologie   terapie   MeSH
• glutamin imunologie   MeSH
• imunoterapie MeSH
• makrofágy imunologie   patologie   MeSH
• myeloidní supresorové buňky imunologie   patologie   MeSH
• myši inbrední BALB C MeSH
• myši knockoutované MeSH
• myši MeSH
• nádorové mikroprostředí imunologie   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural MeSH
• Názvy látek
• glutamin MeSH

Combined immunotherapy constitutes a novel, advanced strategy in cancer treatment. In this study, we investigated immunotherapy in the mouse TC-1/A9 model of human papillomavirus type 16 (HPV16)-associated tumors characterized by major histocompatibility complex class I (MHC-I) downregulation. We found that the induction of a significant anti-tumor response required a combination of DNA vaccination with the administration of an adjuvant, either the synthetic oligodeoxynucleotide ODN1826, carrying immunostimulatory CpG motifs, or α-galactosylceramide (α-GalCer). The most profound anti-tumor effect was achieved when these adjuvants were applied in a mix with a one-week delay relative to DNA immunization. Combined immunotherapy induced tumor infiltration with various subsets of immune cells contributing to tumor regression, of which cluster of differentiation (CD) 8⁺ T cells were the predominant subpopulation. In contrast, the numbers of tumor-associated macrophages (TAMs) were not markedly increased after immunotherapy but in vivo and in vitro results showed that they could be repolarized to an anti-tumor M1 phenotype. A blockade of T cell immunoglobulin and mucin-domain containing-3 (Tim-3) immune checkpoint had a negligible effect on anti-tumor immunity and TAMs repolarization. Our results demonstrate a benefit of combined immunotherapy comprising the activation of both adaptive and innate immunity in the treatment of tumors with reduced MHC-I expression.

• Klíčová slova
• CpG ODN, DNA immunization, MHC-I, cancer immunotherapy, tumor-associated macrophages, α-galactosylceramide,
• MeSH
• adjuvancia imunologická terapeutické užití   MeSH
• CD8-pozitivní T-lymfocyty imunologie   MeSH
• down regulace MeSH
• experimentální nádory terapie   MeSH
• galaktosylceramidy imunologie   MeSH
• imunoterapie metody   MeSH
• makrofágy imunologie   MeSH
• MHC antigeny I. třídy imunologie   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• oligodeoxyribonukleotidy imunologie   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• adjuvancia imunologická MeSH
• galaktosylceramidy MeSH
• MHC antigeny I. třídy MeSH
• oligodeoxyribonukleotidy MeSH

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.

• Klíčová slova
• IL-12, cancer chemotherapy, cell therapy, cellular senescence, docetaxel,
• MeSH
• bystander efekt účinky léků   MeSH
• časové faktory MeSH
• cytokiny genetika   metabolismus   MeSH
• docetaxel MeSH
• experimentální nádory genetika   metabolismus   terapie   MeSH
• imunoterapie adoptivní metody   MeSH
• interleukin-12 biosyntéza   farmakologie   MeSH
• kombinovaná terapie MeSH
• myši inbrední C57BL MeSH
• nádorové buněčné linie MeSH
• protinádorové látky farmakologie   MeSH
• stárnutí buněk účinky léků   MeSH
• taxoidy farmakologie   MeSH
• tumor burden účinky léků   MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• cytokiny MeSH
• docetaxel MeSH
• interleukin-12 MeSH
• protinádorové látky MeSH
• taxoidy MeSH

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.

• MeSH
• antigeny nádorové metabolismus   MeSH
• cytotoxicita imunologická MeSH
• dendritické buňky cytologie   MeSH
• docetaxel MeSH
• experimentální nádory farmakoterapie   terapie   MeSH
• hydrostatický tlak MeSH
• imunitní systém MeSH
• imunoterapie metody   MeSH
• infekce papilomavirem farmakoterapie   terapie   MeSH
• interferon gama metabolismus   MeSH
• interleukin-12 metabolismus   MeSH
• lidé MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• nádory prostaty farmakoterapie   metabolismus   terapie   MeSH
• protinádorové látky aplikace a dávkování   MeSH
• protinádorové vakcíny chemie   MeSH
• slezina imunologie   MeSH
• taxoidy aplikace a dávkování   MeSH
• toll-like receptor 9 metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antigeny nádorové MeSH
• docetaxel MeSH
• interferon gama MeSH
• interleukin-12 MeSH
• protinádorové látky MeSH
• protinádorové vakcíny MeSH
• taxoidy MeSH
• Tlr9 protein, mouse MeSH Prohlížeč
• toll-like receptor 9 MeSH

Shock waves can cause significant cytotoxic effects in tumor cells and tissues both in vitro and in vivo. However, understanding the mechanisms of shock wave interaction with tissues is limited. We have studied in vivo effects of focused shock waves induced in the syngeneic sarcoma tumor model using the TUNEL assay, immunohistochemical detection of caspase-3 and hematoxylin-eosin staining. Shock waves were produced by a multichannel pulsed-electrohydraulic discharge generator with a cylindrical ceramic-coated electrode. In tumors treated with shock waves, a large area of damaged tissue was detected which was clearly differentiated from intact tissue. Localization and a cone-shaped region of tissue damage visualized by TUNEL reaction apparently correlated with the conical shape and direction of shock wave propagation determined by high-speed shadowgraphy. A strong TUNEL reaction of nuclei and nucleus fragments in tissue exposed to shock waves suggested apoptosis in this destroyed tumor area. However, specificity of the TUNEL technique to apoptotic cells is ambiguous and other apoptotic markers (caspase-3) that we used in our study did not confirmed this observation. Thus, the generated fragments of nuclei gave rise to a false TUNEL reaction not associated with apoptosis. Mechanical stress from high overpressure shock wave was likely the dominant pathway of tumor damage.

• Klíčová slova
• Apoptosis, Electrical discharge, Necrosis, Shock waves, Tumor damage,
• MeSH
• design vybavení MeSH
• elektrostimulační terapie přístrojové vybavení   metody   MeSH
• eosin MeSH
• experimentální nádory patologie   terapie   MeSH
• fluorescence MeSH
• hematoxylin MeSH
• imunohistochemie metody   MeSH
• kaspasa 3 metabolismus   MeSH
• koncové značení zlomů DNA in situ MeSH
• potkani inbrední LEW MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• eosin MeSH
• hematoxylin MeSH
• kaspasa 3 MeSH

The asparaginyl endopeptidase legumain that is overexpressed in M2-polarized tumor-associated macrophages has been identified as a suitable target for elimination of these cells supporting tumor progression. To enhance the efficacy of DNA immunization against legumain, we performed several modifications in this protein that could improve induction of immune responses. First, we mutated the RGD motif into GGD or RGG sequences. This alteration resulted in diminished maturation of legumain and impaired cellular localization. Then, as tolerance to self-antigens can be broken by the activation of CD4 T-cell help, we tried to enhance the immunogenicity of legumain by the insertion of a foreign helper epitope, namely the p30 epitope from the tetanus toxin. Finally, the 2 modifications were combined. After gene gun DNA immunization of C57BL/6 mice with these constructs, we identified the Lgmn111-119 CD8 T-cell epitope that binds to H-2D molecules. Furthermore, we showed that mutagenesis in the RGD motif significantly enhanced the immune response against legumain. The addition of the p30 helper epitope induced the specific production of IFN-γ by T cells, but did not significantly increase legumain-specific immunity activated after mutagenesis in the RGD motif which might be caused by simultaneous activation of a Th2 response demonstrated by the production of IL-4. However, the beneficial effect of the helper epitope on legumain-specific response was proved after the depletion of regulatory T cells by antibody against CD25 that preferentially stimulated Th1 immunity. The antitumor effect of the modified legumain gene was shown in the immunization against tumors induced by MK16 cells.

• MeSH
• aminokyselinové motivy genetika   MeSH
• biolistika MeSH
• buňky NIH 3T3 MeSH
• CD8-pozitivní T-lymfocyty imunologie   MeSH
• cysteinové endopeptidasy genetika   metabolismus   MeSH
• DNA vakcíny * MeSH
• epitopy T-lymfocytární genetika   metabolismus   MeSH
• experimentální nádory imunologie   terapie   MeSH
• HEK293 buňky MeSH
• imunoterapie metody   MeSH
• interferon gama metabolismus   MeSH
• lidé MeSH
• makrofágy imunologie   MeSH
• mutace genetika   MeSH
• mutageneze cílená MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nádorové biomarkery genetika   metabolismus   MeSH
• peptidové fragmenty genetika   metabolismus   MeSH
• protinádorové vakcíny * MeSH
• T-lymfocyty pomocné-indukující imunologie   MeSH
• tetanový toxin genetika   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• asparaginylendopeptidase MeSH Prohlížeč
• cysteinové endopeptidasy MeSH
• DNA vakcíny * MeSH
• epitopy T-lymfocytární MeSH
• interferon gama MeSH
• nádorové biomarkery MeSH
• P30 tetanus toxin peptide MeSH Prohlížeč
• peptidové fragmenty MeSH
• protinádorové vakcíny * MeSH
• tetanový toxin MeSH

Therapeutic immunization with double recombinants of vaccinia virus (VACV) co-expressing sTβRII increased rejection of established TC-1 tumors in C57BL/6 mice in comparison with single recombinant expressing SigE7LAMP. Recombinant VACV derived from vaccination strain Praha expressed either the sTβRII (ectodomain) or chimeric protein fused to immunoglobulin Fc fragment (sTβRII-Fc-Jun) under control of two different promotors together with the immunogenic tumor associated antigen HPV16 E7 oncoprotein in a form of SigE7LAMP fusion molecule. The ability of soluble receptors to bind TGF-β in vitro was proved. Immunization of mice with double recombinant viruses and virus expressing SigE7LAMP only led to eliciting similar response of E7 specific CD8+ T cells as detected by IFN-γ ELISPOT.

• MeSH
• experimentální nádory terapie   MeSH
• imunizace MeSH
• lidský papilomavirus 16 imunologie   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• Papillomavirus E7 - proteiny imunologie   MeSH
• protein-serin-threoninkinasy genetika   MeSH
• receptory transformujícího růstového faktoru beta genetika   MeSH
• syntetické vakcíny imunologie   MeSH
• T-lymfocyty imunologie   MeSH
• TGF-beta receptor II. typu MeSH
• vakcíny proti papilomavirům imunologie   MeSH
• virus vakcinie genetika   MeSH
• zvířata MeSH
• Check Tag
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• oncogene protein E7, Human papillomavirus type 16 MeSH Prohlížeč
• Papillomavirus E7 - proteiny MeSH
• protein-serin-threoninkinasy MeSH
• receptory transformujícího růstového faktoru beta MeSH
• syntetické vakcíny MeSH
• TGF-beta receptor II. typu MeSH
• vakcíny proti papilomavirům MeSH

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.

• MeSH
• azacytidin farmakologie   MeSH
• DNA primery MeSH
• ELISA MeSH
• experimentální nádory farmakoterapie   imunologie   terapie   virologie   MeSH
• imunoterapie * MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• lidský papilomavirus 16 izolace a purifikace   MeSH
• metylace MeSH
• MHC antigeny I. třídy imunologie   MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• protinádorové antimetabolity farmakologie   MeSH
• sekvence nukleotidů MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• azacytidin MeSH
• DNA primery MeSH
• MHC antigeny I. třídy MeSH
• protinádorové antimetabolity MeSH

DNA vaccines showed great promise in preclinical models of infectious and malignant diseases, but their potency was insufficient in clinical trials and is needed to be improved. In this study, we tested systemic administration of two conventional adjuvants, synthetic oligodeoxynucleotide carrying immunostimulatory CpG motifs (CpG-ODN) and levamisole (LMS), and evaluated their effect on immune reactions induced by DNA vaccines delivered by a gene gun. DNA vaccination was directed either against the E7 oncoprotein of human papillomavirus type 16 or against the BCR-ABL1 oncoprotein characteristic for chronic myeloid leukemia. High doses of both adjuvants reduced activation of mouse splenic CD8(+) T lymphocytes, but the overall antitumor effect was enhanced in both tumor models. High-dose CpG-ODN exhibited a superior adjuvant effect in comparison with any combination of CpG-ODN with LMS. In summary, our results demonstrate the benefit of combined therapy with gene-gun-delivered antitumor DNA vaccines and systemic administration of CpG-ODN or LMS.

• MeSH
• adjuvancia imunologická aplikace a dávkování   MeSH
• bcr-abl fúzní proteiny genetika   imunologie   metabolismus   MeSH
• biolistika MeSH
• CD8-pozitivní T-lymfocyty účinky léků   metabolismus   patologie   MeSH
• DNA vakcíny MeSH
• experimentální nádory imunologie   patologie   terapie   MeSH
• imunita účinky léků   MeSH
• levamisol aplikace a dávkování   MeSH
• lidé MeSH
• modely nemocí na zvířatech MeSH
• myši inbrední BALB C MeSH
• myši inbrední C57BL MeSH
• myši MeSH
• nádorové buněčné linie MeSH
• oligodeoxyribonukleotidy aplikace a dávkování   MeSH
• Papillomavirus E7 - proteiny genetika   imunologie   metabolismus   MeSH
• protinádorové vakcíny * MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adjuvancia imunologická MeSH
• bcr-abl fúzní proteiny MeSH
• CPG-oligonucleotide MeSH Prohlížeč
• DNA vakcíny MeSH
• levamisol MeSH
• oligodeoxyribonukleotidy MeSH
• Papillomavirus E7 - proteiny MeSH
• protinádorové vakcíny * MeSH

AIM OF STUDY: After the treatment of tumors, we often encounter a minimal residual sickness. However, the elimination of these leftover tumor cells is crucial for the patient. In the past years one of the most discussed options for this treatment is Imunotherapy, mainly by Dendritic cells. Dendritic cells are the most efficient cells out of the antigen presenting cell group. METHODS AND RESULTS: In the first part of the project, we perfected a technique of inducting a tumor on an experimental model. We inducted the tumor by the use of Carcinogenic substances or with the help of the Sarkom line imortalized fibroblasts. Another important part of the project was perfecting the method for the preparation of undeveloped dendritic cells from periphery blood monocytes. After these significant procedures were developed and perfected we moved onto the main part of the study. The Induction of a tumor by the carcenogenic substances Ethylennitrosamin and Phenobarbital was successful only in 20 % of the cases and therefore, was unusable for our experiment. We inducted the tumors with the Sarkom line method. After the application of dendritic cells into the tumor, a decrease in the development of the growth of the tumor was achieved. CONCLUSION: Imunotherapy using dendritic cells as a basis for treatment is a perspective method for treatment of tumors.

• MeSH
• dendritické buňky imunologie   transplantace   MeSH
• experimentální nádory patologie   terapie   MeSH
• imunoterapie * MeSH
• injekce do léze MeSH
• krysa rodu Rattus MeSH
• nádorové buněčné linie MeSH
• potkani inbrední LEW MeSH
• potkani Wistar MeSH
• psi MeSH
• transplantace nádorů MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• psi MeSH
• zvířata MeSH
• Publikační typ
• anglický abstrakt MeSH
• časopisecké články MeSH