PURPOSE: Aspartate β-hydroxylase (ASPH) contributes to carcinogenesis by promoting tumor cell proliferation, migration, and invasion. The enzymatic activity of ASPH can be inhibited by small molecule inhibitors that have been shown to have anti-metastatic activity in rodent models. ASPH has also been shown to inhibit the activation of natural killer (NK) cells. Therefore, this study aimed to investigate the effect of ASPH inhibition on the induction of anti-tumor immunity and to analyze the immune cells involved. METHODS: In the mouse TC-1/A9 model characterized by reversible downregulation of major histocompatibility class I (MHC-I) molecules, ASPH inhibition was combined with stimulation of innate and/or adaptive immunity, and the anti-tumor response was analyzed by evaluation of tumor growth, in vivo depletion of immune cell subpopulations, and ELISPOT assay. Characteristics of immune cells in the spleen and tumor were determined by flow cytometry and single-cell RNA sequencing (scRNA-seq). RESULTS: ASPH inhibition did not reduce tumor growth or promote the anti-tumor effect of innate immunity stimulation with the synthetic oligonucleotide ODN1826, but it significantly enhanced tumor growth reduction induced by DNA vaccination. In vivo immune cell depletion suggested that CD8+ T cells played a critical role in this immunity stimulated by combined treatment with ASPH inhibition and DNA vaccination. ASPH inhibition also significantly enhanced the specific response of CD8+ T cells induced by DNA vaccination in splenocytes, as detected by ELISPOT assay, and reduced the number of regulatory T cells in tumors. scRNA-seq confirmed the improved activation of CD8+ T cells in tumor-infiltrating cells after combined therapy with DNA vaccination and ASPH inhibition. It also showed activation of NK cells, macrophages, and dendritic cells in tumors. CONCLUSION: ASPH inhibition stimulated T-cell-mediated adaptive immunity induced by DNA vaccination. Different types of lymphoid and myeloid cells were likely involved in the activated immune response that was efficient against tumors with MHC-I downregulation, which are often resistant to T-cell-based therapies. Due to different types of activated immune cells, ASPH inhibition could improve immunotherapy for tumors with various MHC-I expression levels.

• Klíčová slova
• ASPH, adaptive immunity, cancer immunotherapy, scRNA-seq, tumor microenvironment,
• Publikační typ
• časopisecké články MeSH

Cancer immunotherapy is increasingly used in clinical practice, but its success rate is reduced by tumor escape from the immune system. This may be due to the genetic instability of tumor cells, which allows them to adapt to the immune response and leads to intratumoral immune heterogeneity. The study investigated spatial immune heterogeneity in the tumor microenvironment and its possible drivers in a mouse model of tumors induced by human papillomaviruses (HPV) following immunotherapy. Gene expression was determined by RNA sequencing and mutations by whole exome sequencing. A comparison of different tumor areas revealed heterogeneity in immune cell infiltration, gene expression, and mutation composition. While the mean numbers of mutations with every impact on gene expression or protein function were comparable in treated and control tumors, mutations with high or moderate impact were increased after immunotherapy. The genes mutated in treated tumors were significantly enriched in genes associated with ECM metabolism, degradation, and interactions, HPV infection and carcinogenesis, and immune processes such as antigen processing and presentation, Toll-like receptor signaling, and cytokine production. Gene expression analysis of DNA damage and repair factors revealed that immunotherapy upregulated Apobec1 and Apobec3 genes and downregulated genes related to homologous recombination and translesion synthesis. In conclusion, this study describes the intratumoral immune heterogeneity, that could lead to tumor immune escape, and suggests the potential mechanisms involved.

• Klíčová slova
• DNA repair, cancer immunotherapy, intratumoral heterogeneity, mutation, tumor microenvironment,
• MeSH
• imunoterapie * metody   MeSH
• infekce papilomavirem imunologie   virologie   MeSH
• lidé MeSH
• modely nemocí na zvířatech MeSH
• mutace MeSH
• myši MeSH
• nádorové mikroprostředí imunologie   genetika   MeSH
• nádory * imunologie   terapie   genetika   MeSH
• regulace genové exprese u nádorů MeSH
• sekvenování exomu MeSH
• únik nádoru z imunitní kontroly genetika   imunologie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• myši MeSH
• ženské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Tumor-associated macrophages (TAMs) plentifully infiltrate the tumor microenvironment (TME), but their role in anti-tumor immunity is controversial. Depending on the acquired polarization, they can either support tumor growth or participate in the elimination of neoplastic cells. In this study, we analyzed the TME by RNA-seq and flow cytometry and examined TAMs after ex vivo activation. Tumors with normal and either reversibly or irreversibly decreased expression of major histocompatibility complex class I (MHC-I) molecules were induced with TC-1, TC-1/A9, and TC-1/dB2m cells, respectively. We found that combined immunotherapy (IT), composed of DNA immunization and the CpG oligodeoxynucleotide (ODN) ODN1826, evoked immune reactions in the TME of TC-1- and TC-1/A9-induced tumors, while the TME of TC-1/dB2m tumors was mostly immunologically unresponsive. TAMs infiltrated both tumor types with MHC-I downregulation, but only TAMs from TC-1/A9 tumors acquired the M1 phenotype upon IT and were cytotoxic in in vitro assay. The anti-tumor effect of combined IT was markedly enhanced by a blockade of the colony-stimulating factor-1 receptor (CSF-1R), but only against TC-1/A9 tumors. Overall, TAMs from tumors with irreversible MHC-I downregulation were resistant to the stimulation of cytotoxic activity. These data suggest the dissimilarity of TAMs from different tumor types, which should be considered when utilizing TAMs in cancer IT.

• Klíčová slova
• colony-stimulating factor-1, immunotherapy, macrophages, major histocompatibility complex, repolarization, tumor,
• Publikační typ
• časopisecké články MeSH