BackgroundProcesses 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.MethodsFourty 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.ResultsAnalysis 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.ConclusionsThe 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.

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.

tumor formation and growth is continuous mutagenesis, microevolution and changes in the tissue microenvironment, its molecular-genetic 'portrait' is dynamic and tightly bound to the stage of the disease.Cervical cancer (CeCa) is an example of oncopathology, for which the whole continuum of carcinogenesis, from the earliest stages-cervical intraepithelial neoplasia (CIN) of

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Аннотация. В настоящее время рак шейки матки остается одной из главных причин смертности женщин от онкологических заболеваний. Применяемые сегодня в клинической практике критерии диагностики рака шейки матки имеют ряд ограничений в отношении точности постановки диагноза, возможности прогнозирования течения заболевания и не позволяют учитывать индивидуальный статус пациентки. Для преодоления этих недостатков необходимы разработка и внедрение в клинику новых методов молекулярно-генетической и биохимической диагностики, основанной на анализе изменений экспрессии тканевых биомаркеров.Ключевые слова: биомаркеры, тераностика, диагностические тест-системы, рак шейки матки.

2014, Т. 4, № 2 Иммунитет и рак шейки матки group) in comparison with a surgical treatment only (the control group) have been conducted. The true numerical normalization of subpopulation composition of peripheral blood lymphocyte has been determined including the expression level of suppressor lymphocytes molecular markers (FOXP3 и TGF-β1) and the expression level of CD95-marker in a study group in comparison with a control group. Moreover, true decrease of HPV (human papillomavirus)-positive patients (1.7%) and the absence of E7 oncoprotein in the study group have been discovered.

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IntroductionAchieving 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 methodsTranscriptomes 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 discussionsAs 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.ConclusionThe 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).