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For the last few years Next Generation Sequencing technique and its applications has took the leading position in the arsenal of analytical methods that are used for studying the mechanisms of neoplastic progression. Among various experimental opportunities Next Generation Sequencing provides, RNA-sequencing (RNA-Seq) is of great importance as it makes possible unraveling the highest levels of genome expression regulation, which define the molecular phenotype of cells in composition of a tumor. Considerable amount of current studies carried out with the use of RNA-Seq method are designed as pan-cancer integrated research, in which special attention is payed to virus-associated tumors, including papillomavirus-dependent cervical cancer. This review paper summarizes the results of RNA-Seq studies published world-wide within 2017-2019 years and carried out using clinical samples from cervical cancer patients. New facts concerning such hot topics as genomic and transcriptomic instability, neoantigen load, cellular and molecular heterogeneity, tumor epigenetics, antitumor and antiviral immune response, chronic inflammation, immune exhaustion, phenotypic plasticity and tumor cell resistance, are considered. The whole spectrum of issues that are actively discussed in published literature is systematized according to three levels of organization: «molecular», «cellular» and «organismal». The findings reviewed in the paper convincingly illustrate that wide usage of RNA-Seq technology for profiling primary tumors does facilitate moving to a new level of our understanding of the mechanisms of carcinogenesis and emergence of new directions in cancer treatment, namely targeted and immune-therapy.

Introduction/Background The mechanisms of innate immune response and the immune checkpoint (IC) system-governed mechanisms are the two interconnected areas currently becoming the focus of world research in view of the prospects for therapeutic reactivation of antitumor immunity. This may be especially relevant to virus-associated cancers (as, for example, most cases of cervical cancer), which are known to develop as a result of persistent infection, long-term antigen exposure and are frequently characterized by chronically inflamed environment and local and/or systemic immunosuppression. Studying the immune changes that accompany the earliest stages of tumor progression may provide insight into the mechanisms driving the onset of metastasis; and, in this respect, systemic changes observed in peripheral blood (PB) may possibly reflect local dysfunctions at the tumor site. In case of cervical cancer, these assumptions need more extensive research evidence. Methodology To characterize the changes in the local microenvironment, we compared the transcriptomes of the early invasive squamous cell cervical cancer and its precursor high-grade lesions by performing RNA-sequencing and bioinformatics analysis on a panel of 12 fresh tissue samples comprising HPV(+) cervical intraepithelial neoplasia 3 (CIN3) and carcinoma at FIGO IA1-IIB stages, plus normal epithelium. PB samples were obtained from women with CIN3 and stage IA cancer immediately prior to treatment; PB from healthy women was used as control. Subsets of PB lymphocytes were phenotyped using multicolor flow cytometry. Results Among the differentially expressed genes identified, there were a considerable number of genes that, according to Gene Ontology, are responsible for inflammatory and innate immune responses (including interferon type I and II pathways) and belong to the system of self/non-self DNA/RNA recognition, with multiple anti-inflammatory factors found to be down-regulated, while a spectrum of interferon-stimulated genes, anti-viral/anti-microbial factors, pro-inflammatory cytokines, as well as markers of immune suppression were found up-regulated in invasive cancer. Accordingly, ‘Influenza A’ and ‘Pyrimidine metabolism’ KEGG pathways appeared to be significantly enriched. SPEED enrichment analysis revealed the TLR-, TNF alpha-, and IL1-dependent signalings among the top pathways lying behind the alterations of gene expression patterns observed at the initial stage of invasion. PPI network analysis confirmed close interrelation of differently expressed genes encoding molecular components of inflammatory response and virus recognition system. Among the circulating lymphocyte functional markers that may mirror the described immune alterations, the expression of CD161 in iNKT/NK-like T/NK cells (defined by CD3/CD56, Va24Ja18/Vb11-TCR, and CD4/CD8), the level of CD27 and delta2/delta1 ratio in Tgammadelta subpopulation (defined by CD3/TCRgd), and co-expression pattern of PD1/PDL1/LAG3/TIM3 in 4 subsets of CD4/CD8 T cells defined by the level of CD25/CD127, as well as in NK/NKT cells, were measured, and specific correlated differences between the control and cancer groups and between different lymphocyte populations were detected. Conclusion The findings suggest deep involvement of the inflammation-associated and IC-mediated mechanisms and coordinate contribution of various T cell subsets with innate-like properties in initiation and promotion of invasive growth of cervical carcinoma both at local and systemic levels. Disclosures The study was supported by the state assignment of the Ministry of Science and Higher Education, project No.0752-2020-0007 (AAAA-A20-120070290151-6). The authors declare no conflicts of interest.

Introduction The establishment of a proangiogenic phenotype and epithelial-mesenchymal transition (EMT) are regarded as prerequisites for activation of invasive growth and dissemination of malignant cells in epithelial tumors. Various borderline conditions, as for example a transition between intraepithelial neoplasia and microcarcinoma, can be the source of critical factors that act as driving forces for further tumor spread, but in the case of cervical cancer these issues remain poorly studied. Methods RNA-sequencing and bioinformatics analysis were used to compare transcriptomes and signaling pathways activation profiles in HPV-positive preinvasive neoplastic lesions and early-stage invasive cervical carcinoma samples obtained from patients. Flow cytometry was applied to evaluate the expression of three key lymphangiogenesis and EMT markers (VEGFR3, MET, and SLUG) in epithelial cells derived from enzymatically treated tissue specimens. Results The differentially expressed genes were screened for angiogenesis, lymphangiogenesis, EMT, and invasion regulatory factors and subsequent pathway analysis confirmed enrichment for angiogenesis, epithelial organization, and cell guidance pathways at transition from intraepithelial neoplasia to invasive carcinoma and suggested inflammatory antiviral response-associated pathways to be critically implicated in initiation of invasive growth of cervical cancer. Cell-phenotype-specific expression pattern for VEGFR3, MET, and SLUG was revealed which appeared to be correlated with the amount of tumor-infiltrating lymphocytes at the earliest stages of cancer progression. Conclusion These findings extend the existing knowledge about driving forces of angiogenesis and metastasis in cervical cancer and may be useful for developing new treatments. The study was supported by the state assignment of the Ministry of Science and Higher Education, project No.0752-2020-0007 (AAAA-A20-120070290151-6).

The establishment of a proangiogenic phenotype and epithelial-to-mesenchymal transition (EMT) are considered as critical events that promote the induction of invasive growth in epithelial tumors, and stimulation of lymphangiogenesis is believed to confer the capacity for early dissemination to cancer cells. Recent research has revealed substantial interdependence between these processes at the molecular level as they rely on common signaling networks. Of great interest are the molecular mechanisms of (lymph-)angiogenesis and EMT associated with the earliest stages of transition from intraepithelial development to invasive growth, as they could provide the source of potentially valuable tools for targeting tumor metastasis. However, in the case of early-stage cervical cancer, the players of (lymph-)angiogenesis and EMT processes still remain substantially uncharacterized. In this study, we used RNA sequencing to compare transcriptomes of HPV(+) preinvasive neoplastic lesions and early-stage invasive carcinoma of the cervix and to identify (lymph-)angiogenesis- and EMT-related genes and pathways that may underlie early acquisition of invasive phenotype and metastatic properties by cervical cancer cells. Second, we applied flow cytometric analysis to evaluate the expression of three key lymphangiogenesis/EMT markers (VEGFR3, MET, and SLUG) in epithelial cells derived from enzymatically treated tissue specimens. Overall, among 201 differentially expressed genes, a considerable number of (lymph-)angiogenesis and EMT regulatory factors were identified, including genes encoding cytokines, growth factor receptors, transcription factors, and adhesion molecules. Pathway analysis confirmed enrichment for angiogenesis, epithelial differentiation, and cell guidance pathways at transition from intraepithelial neoplasia to invasive carcinoma and suggested immune-regulatory/inflammatory pathways to be implicated in initiation of invasive growth of cervical cancer. Flow cytometry showed cell phenotype-specific expression pattern for VEGFR3, MET, and SLUG and revealed correlation with the amount of tumor-infiltrating lymphocytes at the early stages of cervical cancer progression. Taken together, these results extend our understanding of driving forces of angiogenesis and metastasis in HPV-associated cervical cancer and may be useful for developing new treatments.

Background Processes and mechanisms responsible for systemic immune suppression in early-stage cervical cancer remain substantially underinvestigated. In this work, we focused on studying the frequencies of circulating regulatory T (CD4 and CD8 Tregs) and NK (NKregs) cells in parallel with assessment of apoptotic markers expression in T cells from patients with preinvasive and microinvasive cervical cancer, with the aim to determine whether up-regulation of apoptosis-associated markers in Т lymphocytes accompanies cervical cancer development and correlates with the change in percentages of regulatory cell populations at systemic level during the initial stages of invasive cervical cancer progression. Methods Fourty two women with histologically confirmed cervical intraepithelial neoplasia grade 3 (CIN3, including carcinoma in situ) or cervical cancer (stage IA) and 30 healthy women (control) were enrolled in the study. Peripheral blood samples were taken immediately before surgery or any treatment and immediately subjected to multicolor flow cytometry. Results Analysis of a combination of CD4/CD8, CD25, CD127, and FoxP3 markers revealed a statistically significant increase in the frequencies of Tregs within both the CD4 and CD8 subsets of circulating lymphocytes in patients with CIN3 and stage IA cancer. In contrast, lower numbers of NKregs (defined as CD16dim/negCD56bright subpopulation) and increased CD56dim/CD56bright NK ratio were found in patients compared to controls, with the percentage of CD16brightCD56dim cells (major subtype of circulating NKs) showing no difference. Patients also exhibited an increased expression of CD95 in total peripheral blood T lymphocytes, along with increased level of Annexin V binding to CD95-positive cells, suggesting higher susceptibility of T cells to apoptosis and potential involvement of CD95-dependent pathway in early-stage cervical cancer. Differential analysis of CD4 and CD8 T cells revealed different trends in the change of CD95 expression, confirming that this change likely has different functional significance for these two subsets. A search for correlations between the phenotypic parameters analyzed in this study was performed to demonstrate that women with early neoplastic lesions of the cervix, such as carcinoma in situ and microinvasive carcinoma, displayed a coordinated increase in expression of Treg markers in circulating lymphocytes, along with more pronounced cross-relationships between Treg numbers, CD95 expression on T cells, and apoptosis, compared to the control group. Conclusions The results of this study suggest that a diversity of immune regulatory mechanisms that provide support for initial stages of invasive growth in cervical cancer patients includes systemic changes in the ratios between the principal regulatory and effector lymphocyte populations both within adaptive and innate immunity.

Introduction/Background Achieving positive outcomes of using immunotherapeutic approaches in cancer treatment implies multifaceted targeting of functionally different mechanisms of immune response, including mechanisms governed by molecules of immune checkpoint system. Functional roles of immune checkpoints in different immune cell subsets of cancer patients are not comprehensively characterized; the existing data are sometimes controversial. Studying the immune checkpoint expression patterns in initial stages of invasive tumor growth and progression, in comparison with non-invasive neoplastic lesions, may help better understand their involvement in regulating tumor-immunity interactions. Little or virtually no data are available for very early (preclinical) cervical cancer. Methodology Fresh cervical epithelial tissue specimens (along with peripheral blood) were collected from women with preclinical early invasive and non-invasive squamous carcinoma of the cervix. Cell suspensions obtained by gentle enzymatic dissociation were subjected to multicolor flow cytometry. Co- expression of PD-1/CD279, PD-L1/CD274, TIM-3/CD366, and LAG-3/CD223 markers was evaluated in major populations of tumor-infiltrating CD4 and CD8 T lymphocytes, including their regulatory subset (Treg), and compared to circulating T cells. Results PD-1/-L1, TIM-3 and LAG-3 were analyzed in total CD4/CD8 T cells, CD4/8CD25(±) T subsets, and CD4/8CD25(+/high)CD127(-/low) Treg. Tumor-infiltrating (TILs) and peripheral blood (PBLs) lymphocytes, either CD4 or CD8, were shown to have different expression levels of immune checkpoint markers with significantly higher levels found on TILs. Early invasive and non-invasive carcinoma samples could also be stratified according to the frequencies of TILs. TIM-3 and LAG-3 were strongly co-expressed with PD-1/-L1 on TILs. The prevalence of CD25(+/high)CD127(-/low) cells among CD4 (CD8) TILs was significantly higher than among PBLs with the highest difference observed for CD8 subset, and the majority of these cells co-expressed PD-1 with TIM-3/LAG-3. All analyzed TILs subsets were found to have exclusively high levels of PD-L1. Conclusion The findings indicate deep involvement of immune checkpoint mechanisms in initiation of invasive growth of cervical carcinoma. Disclosure The work is supported by the Russian Science Foundation (grant No.17-15-01024).

Ability to stimulate angiogenesis/lymphangiogenesis is recognized as an inherent feature of cancer cells providing necessary conditions for their growth and dissemination. "Angiogenic switch" is one of the earliest consequences of malignant transformation that encompasses a great number of genes and triggers a complex set of signaling cascades in endothelial cells. The processes of tumor microvasculature development are closely connected to the steps of carcinogenesis (from benign lesions to invasive forms) and occur through multiple deviations from the norm. Analysis of expression of proangiogenic factors at successive steps of cervical cancer development (intraepithelial neoplasia, cancer in situ, microinvasive, and invasive cancer) enables to reconstruct the regulatory mechanisms of (lymph-)angiogenesis and to discriminate the most important components. This review presents detailed analysis of literature data on expression of the key regulators of angiogenesis in cervical intraepithelial neoplasia and cervical cancer. Their possible involvement in molecular mechanisms of neoplastic transformation of epithelial cells, as well as invasion and tumor metastasis is discussed. Correlation between expression of proangiogenic molecular factors and various clinicopathological parameters is considered, the potential of their use in molecular diagnostics and targeted therapy of cervical cancer is reviewed. Particular attention is paid to relatively poorly studied regulators of lymphangiogenesis and "non-VEGF dependent", or alternative, angiogenic pathways that constitute the prospect of future research in the field.Sposobnost' stimulirovat' angiogenez/limfangiogenez priznana neot"emlemym svoĭstvom rakovykh kletok, obespechivaiushchim ikh neobkhodimymi usloviiami dlia rosta i metastazirovaniia. “Angiogennoe perekliuchenie” – odno iz naibolee rannikh sledstviĭ zlokachestvennoĭ transformatsii, okhvatyvaiushchee bol'shoe chislo genov i zapuskaiushchee slozhnuiu sovokupnost' signal'nykh kaskadov v kletkakh éndoteliia. Protsessy formirovaniia mikrososudistoĭ seti opukholi tesno sopriazheny s étapami kantserogeneza (ot vozniknoveniia dobrokachestvennykh izmeneniĭ do invazivnykh form) i protekaiut s mnogochislennymi otkloneniiami ot normy. Analiz urovnia ékspressii proangiogennykh faktorov pri posledovatel'nykh étapakh razvitiia raka sheĭki matki (intraépitelial'nye neoplazii, rak in situ, mikroinvazivnyĭ i invazivnyĭ rak) daet vozmozhnost' rekonstruirovat' reguliatornye mekhanizmy angiogeneza/limfangiogeneza i vydelit' sredi nikh naibolee vazhnye komponenty. V obzore obobshcheny dannye literatury po ékspressii kliuchevykh reguliatorov angiogeneza pri tservikal'nykh intraépitelial'nykh neoplaziiakh i rake sheĭki matki, obsuzhdaetsia ikh vozmozhnoe uchastie v mekhanizmakh transformatsii épitelial'nykh kletok, invazii i metastazirovanii. Rassmotrena vzaimosviaz' urovnia ékspressii proangiogennykh molekuliarnykh faktorov s razlichnymi kliniko-patologicheskimi parametrami i vozmozhnost' ikh ispol'zovaniia v diagnostike i targetnoĭ terapii raka sheĭki matki. Osoboe vnimanie udeliaetsia sravnitel'no maloizuchennym reguliatoram limfangiogeneza i “ne-VEGF zavisimym”, al'ternativnym, putiam aktivatsii angiogeneza, sostavliaiushchim perspektivu dal'neĭshikh issledovaniĭ v dannoĭ oblasti.

Introduction Achieving positive outcomes of using immunotherapeutic approaches in cancer treatment implies multifaceted targeting of functionally different mechanisms of immune response, including immune exhaustion mechanisms, regulatory cell-mediated pathways, and inflammation. Of particular interest are molecular mechanisms that accompany the initial steps of invasive tumour growth. In the case of virus-associated cancer, particularly cervical cancer, there are still many questions to be answered regarding this issue. Material and methods Transcriptomes of pre-invasive and invasive cervical cancer tissue samples were explored using RNA sequencing (RNAseq) on the Illumina MiSeq platform followed by bioinformatic analysis of immune-related signalling pathways. Flow cytometry was used to phenotype peripheral blood lymphocytes and tumor-infiltrating lymphocytes in enzymatically dissociated tissue samples. Results and discussions As follows from RNAseq data, in early-stage invasive cervical cancer, the expression of a wide set of interferon-induced genes and inflammatory response genes (including a specific group of innate molecular sensors of nucleic acids) was significantly elevated, as compared to pre-invasive carcinoma. In contrast, effector T and NK-cell activation markers were expressed at low levels. Interestingly, many markers of immune exhaustion and inhibitory ‘immune checkpoint’ molecules didn’t show any noticeable increase in mRNA levels, furthermore, some of them were found to have lower expression levels in invasive cancer. Pathway analysis revealed that, among the mechanisms most significantly upregulated in invasive vs. pre-invasive cancer, those mediated by immunosuppressive cytokines and implicated in differentiation of regulatory T cells were prevalent, while IL12-dependent pathways of T and NK-cell activation and differentiation were enriched among the downregulated genes. The results of transcriptome analysis are in agreement with our findings from flow cytometric assessment of regulatory (suppressive) cell populations within tumor-infiltrating lymphocytes and co-expression of PD-1, TIM-3, and LAG-3 in major subsets of peripheral blood lymphocytes in patients with early forms of cervical cancer. Conclusion The observed relationships may be considered a direct consequence of impaired immune cell activation processes and an essential condition for further progression of inflammatory environment required for invasive growth of a tumour. The work is supported by the Russian Science Foundation (project №17-75-10027).

Recognition of microbial components is critical for activation of TLRs, subsequent innate immune signaling, and directing adaptive immune responses. The DNA sensor TLR9 traffics from the endoplasmic reticulum to endolysosomal compartments where it is cleaved by resident proteases to generate a competent receptor. Activation of TLR9 by CpG-motif containing oligodeoxynucleotides (CpG ODNs) is preceded by agonist endocytosis and delivery into the endolysosomes. The events that dictate this process remain largely unknown; furthermore, it is unclear whether the receptors involved in mediating uptake of exogenous DNA are conserved for both naturally derived pathogenic DNA and synthetic ODNs. In this study, we report that peritoneal macrophages from a wild-derived inbred mouse strain, MOLF/Ei, are hyporesponsive to CpG ODN but are fully responsive to bacterial DNA, thus implying that microbial recognition is not fully recapitulated by a synthetic analog. To identify the gene responsible for the CpG ODN defect, we have performed genome-wide linkage analysis. Using N2 backcross mice, we mapped the trait with high resolution to a single locus containing Mrc1 as the gene conferring the trait. We show that mannose receptor 1 (MRC1; CD206) is involved in CpG ODN uptake and trafficking in wild-derived MOLF/Ei peritoneal macrophages. Furthermore, we show that other strains of wild-derived mice also require MRC1 for CpG-induced cytokine responses. These findings reveal novel functions for MRC1 and demonstrate that wild-derived mice are important and indispensable model for understanding naturally occurring regulators of inflammatory responses in innate immune pathways.

Thanks to the numerous studies that have been carried out recently in the field of cytosolic DNA sensing, STING (Stimulator of Interferon Genes) is now recognized as a key mediator of innate immune signaling. A substantial body of evidence derived from in vivo mouse models demonstrates that STING-regulated pathways underlie the pathogenesis of many diseases including infectious diseases and cancers. It has also become evident from these studies that STING is a promising therapeutic target for the treatment of cancer. However, mouse strains commonly used for modelling innate immune response against infections or tumors do not allow investigators to accurately reproduce certain specific characteristics of immune response observed in human cells. In this review, we will discuss recent data demonstrating that the use of wild-derived genetically distinct inbred mice as a model for investigation into the innate immunity signaling networks may provide valuable insight into the STING-regulated pathways specific for human cells. The maximum complexity of STING-mediated mechanisms can probably be seen in case of DNA virus-induced carcinogenesis in which STING may perform unexpected biological activities. Therefore, in another part of this review we will summarize emerging data on the role of STING in human DNA virus-related oncopathologies, with particular attention to HPV-associated cervical cancer, aiming to demonstrate that STING indeed "starts a new chapter" in research on this issue and that wild-derived mouse models of STING-mediated response to infections will probably be helpful in finding out molecular basis for clinical observations.

cFLIP, an inhibitor of apoptosis, is a crucial regulator of cellular death by apoptosis and necroptosis; its importance in development is exemplified by the embryonic lethality in cFLIP-deficient animals. A homolog of caspase 8 (CASP8), cFLIP exists in two main isoforms: cFLIPL (long) and cFLIPR (short). Although both splice variants regulate death receptor (DR)-induced apoptosis by CASP8, the specific role of each isoform is poorly understood. Here, we report a previously unidentified model of resistance to Fas receptor-mediated liver failure in the wild-derived MSM strain, compared with susceptibility in C57BL/6 (B6) mice. Linkage analysis in F2 intercross (B6 x MSM) progeny identified several MSM loci controlling resistance to Fas-mediated death, including the caspase 8- and FADD-like apoptosis regulator (Cflar) locus encoding cFLIP. Furthermore, we identified a 21-bp insertion in the 3' UTR of the fifth exon of Cflar in MSM that influences differential splicing of cFLIP mRNA. Intriguingly, we observed that MSM liver cells predominantly express the FLIPL variant, in contrast to B6 liver cells, which have higher levels of cFLIPR. In keeping with this finding, genome-wide RNA sequencing revealed a relative abundance of FLIPL transcripts in MSM hepatocytes whereas B6 liver cells had significantly more FLIPR mRNA. Importantly, we show that, in the MSM liver, CASP8 is present exclusively as its cleaved p43 product, bound to cFLIPL. Because of partial enzymatic activity of the heterodimer, it might prevent necroptosis. On the other hand, it prevents cleavage of CASP8 to p10/20 necessary for cleavage of caspase 3 and, thus, apoptosis induction. Therefore, MSM hepatocytes are predisposed for protection from DR-mediated cell death.

Resistance to apoptosis is commonly accepted as the principal hallmark of a cancer cell, while caspases are recognized as the key molecular players of the apoptosis regulatory network. Since the level of caspase activity is thought to be directly coupled with aggres‐ sive features of cancer cells (such as ability to withstand immune reactions, invasiveness, drug resistance, etc.), these proteases could serve as objective diagnostic markers espe‐ cially for those types of cancer where early differential diagnosis is needed. Cervical can‐ cer develops through morphologically well-described stages—from intraepithelial lesions of 1/2/3 grade including carcinoma in situ to microinvasive and invasive cancer with pre‐ cancerous lesions known to be potentially reversible. The percentage of cervical neo‐ plasms diagnosed at early stages is relatively high, providing a basis for the use of cervical cancer as an in vivo model to investigate the mechanisms of apoptosis modula‐ tion in malignant cells. The existing diagnostic criteria, despite their usefulness, have sub‐ stantial limitations with respect to cervical cancer and preneoplastic lesions, so caspases may be helpful in improving them, but there is insufficient data regarding the involve‐ ment of these enzymes in cervical cancer development. In this chapter, we report on spe‐ cific patterns of activity of caspases revealed in tissue biopsies and blood lymphocytes in association with different stages of cervical cancer development. The data indicate that caspases are pivotal components of the in vivo molecular “portrait” of cervical cancer and have the potential of being used as biomarkers.

With the stimulator of IFN genes (STING) C terminus being extensively studied, the role of the N-terminal domain (NTD) of STING remains an important subject of investigation. In this article, we identify novel mutations in NTD of Sting of the MOLF strain in response to HSV and Listeria monocytogenes both in vitro and in vivo. These mutations are responsible for low levels of IFN-β caused by failure of MOLF STING to translocate from the endoplasmic reticulum. These data provide evidence that the NTD of STING affects DNA responses via control of trafficking. They also show that the genetic diversity of wild-derived mice resembles the diversity observed in humans. Several human alleles of STING confer attenuated IFN-I production similar to what we observe with the MOLF Sting allele, a crucial functional difference not apparent in classical inbred mice. Thus, understanding the functional significance of polymorphisms in MOLF STING can provide basic mechanistic insights relevant to humans.

In this review we summarize the results of studies employing high-throughput methods of profiling of HPV-associated cervical intraepithelial neoplasia (CIN) and squamous cell cervical cancers at key intracellular regulatory levels to demonstrate the unique identity of the landscape of molecular changes underlying this oncopathology, and to show how these changes are related to the 'natural history' of cervical cancer progression and the formation of clinically significant properties of tumors. A step-wise character of cervical cancer progression is a morphologically well-described fact and, as evidenced by genome-wide screenings, it is indeed the consistent change of the molecular profiles of HPV-infected epithelial cells through which they progressively acquire the phenotypic hallmarks of cancerous cells. In this sense, CIN/cervical cancer is a unique model for studying the driving forces and mechanisms of carcinogenesis. Recent research has allowed definition of the whole-genome spectrum of both random and regular molecular alterations, as well as changes either common to processes of carcinogenesis or specific for cervical cancer. Despite the existence of questions that are still to be investigated, these findings are of great value for the future development of approaches for the diagnostics and treatment of cervical neoplasms.

The cytoplasmic protein Stimulator of Interferon Genes (STING) plays an essential role in sensing cytoplasmic DNA of intracellular pathogens and initiating IFN type I responses in myeloid cells. It can also directly bind cyclic dinucleotides produced by intracellular bacteria or by the DNA-binding cyclic GMP-AMP (cGAMP) synthase. To date, the signaling mechanisms involving STING activation has been studied in macrophages and dendritic cells. Taking into consideration the fact that STING is highly expressed in the thymus and spleen, we decided to investigate its functional role in T cells. We revealed that STING can be activated in T cells by DMXAA (5,6-dimethylxanthenone-4-acetic acid), which binds to the same site in murine STING as cGAMP and triggers phosphorylation of TBK1 and IRF3 resulting in IFNβ production and increased expression of interferon stimulated genes. In contrast to T lymphocytes obtained from C57B6 WT mice, STING−/− T cells displayed no phosphorylation of TBK1/IRF3, and produced no detectable IFNβ upon stimulation. DMXAA treatment also resulted in IFNgamma secretion by WT cells, while DMXAA-treated knockouts produced only a small amount of IFNgamma comparable to unstimulated WT T cells. Both CD4+ and CD8+ subsets of WT T cells responded to DMXAA with phosphorylation of TBK1/IRF3 and IFNβ production, but only CD4+ cells produced IFNgamma. By performing RNA sequencing, numerous differences in gene expression (including interferon stimulated genes) between WT and STING−/− T cells before and after DMXAA stimulation were identified. The work was supported by the Russian Science Foundation (grant no. 115-15-00100 ).

The number of scientific papers devoted to the study of caspases has lately being constantly growing. Caspases are a family of cysteine-dependent aspartate specific proteases. Caspases play an essential role in the apoptosis, necrosis and inflammation processes. Apoptosis is asynchronous programmed cell death, which helps maintain the physiological balance and genetic stability of the organism through self-destruction of genetically modified defect cells. Apoptosis may happen also in normal cells, e.g., during embryogenesis. During apoptosis, activated endogenous nucleases cleave DNA into fragments, but cell membranes and the intracellular matter remain intact, nor is there tissue damage or leukocytic infiltration. In contrast to apoptosis, necrosis is a pathological form of cell death caused by acute damage, rupture of the membrane, release of the cytoplasm content, and the inflammatory process induced thereby [1, 2].

Background The ability to promote an immunosuppressive microenvironment has been recognized as one of the “next generation” cancer hallmarks, being most apparent in case of HPV-induced carcinogenesis, since it is a prerequisite to the virus persistence and proliferation of keratinocytes expressing HPV-antigens. With respect to HPV-associated cervical cancer, it is commonly known that initial pre-cancerous intraepithelial lesions frequently undergo reverse development due to effective recognition and eradication by the immunocompetent cells. Both innate (e.g. NK) and adaptive (helper and cytotoxic O-cells) arms of immunity have been implicated in the regression of neoplastic cells, with NK T-cells considering as a connecting link between two branches. Progression of intraepithelial neoplasia leads to the establishment of pre-invasive cancer, which can be regarded as the result of a completed immunoediting of the tumor locus, because further, with 100% probability, it turns into invasive metastatic form. We assumed that the signs of immune activation, on the one hand, and the signs of immunosuppression, on the other hand, despite the locality of pre-invasive cervical cancer, can be found in the circulation and can be relevant to the development of tolerogenic microenvironment. The aim of the present work was to investigate the number of functionally different subpopulations of T- and NK-lymphocytes in the blood of patients with pre-invasive cervical cancer. Material and methods Blood samples were taken from 35 HPV(+) patients diagnosed with cancer in situ. The control group consisted of 30 healthy HPV(−) women with no pathology of the cervix. Lymphocytes were immunophenotyped by MACSQuant flow cytometer. The percentage of the following populations were measured: CD3+CD95+/high, CD3+CD4+, CD3+CD4+CD95+/high, CD3+CD8+, CD3+CD8+CD95+/high, CD4+CD25+, CD4+CD25high, CD4+CD25+CD127dim/neg, CD4+CD25+FoxP3+ (CD4 Tregs), CD8+CD25+, CD8+CD25+CD127dim/neg, CD8+CD25+FoxP3+ (CD8 Tregs), CD3−CD16+, CD3−CD56+, CD3−(CD16±)CD56bright, CD3−CD16+CD56+, CD3−CD16low/negCD56+, CD3−CD16+CD56- (NK subpopulations), CD3+CD56+, CD3+CD16+CD56+ (NK T-lymphocytes). Results Increase in the number of CD4 Tregs that are known to exert immunosuppressive effect was found in cancer patients’ group, namely the increased quantity of cells with CD4+CD25+ (especially, CD4+CD25high fraction), CD4+CD25+CD127dim/neg, or CD4+CD25+FoxP3+ phenotypes was shown. At the same time, the portion of CD4+FoxP3+ lymphocytes didn’t differ between the studied groups. A rare population of CD8+ Tregs (CD8+CD25+CD127dim/negFoxP3+) also exhibited a tendency to accumulate in the blood of cancer patients compared with the control. Analysis of the major subpopulation of NK cells performing cytotoxic reactions didn’t reveal significant difference between the examined groups. Similar result was obtained in relation to the other functional groups of NK cells, specifically CD3−CD16±CD56bright (cytokine-producing NKs with low cytotoxicity), CD3−CD16dim/−CD56+ (NKs with potential anti-tumor activity), and CD3−CD16+CD56 – (NKs associated with viral infection or NK-precursors). However, an increase of NK T-lymphocyte amounts was established, arguing both activation of anti-tumor response and its inhibition, in view of functional polymorphism of this population. Similarly, the increased number of CD3+CD95+/high cells and CD3+CD4+CD95+/high T-helpers may have dual significance, taking into account that the mid-early T-cell activation marker CD95/Fas presents the key death-receptor (earlier we have revealed the elevated activity of caspases involved in the Fas-mediated signaling in circulating lymphocytes of cervical cancer patients). The total numbers of CD3+CD4+ T-helpers and CD3+CD8+ T-killers were generally comparable between the groups analyzed, with T-killers displaying slightly decreased percentage in the patient group. Conclusion The processes supporting establishment of immunosuppressive microenvironment extend beyond the local level during the earliest steps of cervical cancer progression. The observed changes may reflect the shift of equilibrium between immune activation and suppression, which is the essence of immunoediting. Further phenotypic analysis of cell subpopulations reported here is required for understanding the functional meaning of these changes. The study was supported by the grants NK-1404-32098, 11.G34.31.0052, the Program of strategic development of PetrSU for 2012-2016, and the State task for R&D.