Olga V. Kurmyshkina

T- and NK-cell populations with regulatory phenotype and markers of apoptosis in circulating lymphocytes of patients with CIN3 or microcarcinoma of the cervix: evidence for potential mechanisms of immune suppression

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.

Caspases as Putative Biomarkers of Cervical Cancer Development

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.

EXPERIMENTAL APPROACHES TO DERIVING PRIMARY TUMOR CELL CULTURES FROM CERVICAL CANCER BIOPSIES: A COMPARATIVE ANALYSIS OF PUBLISHED DATA AND OWN EXPERIENCE

The article provides a detailed description of the procedure of converting primary tumor cells of invasive cervical cancer into a state of a transient in vitro culture, including the description of conditions for tissue dissociation and selection of appropriate cell culture medium. Morphological characteristics of the cell colonies obtained and the results of their phenotyping (based on analysis of cytokeratin expression) are presented. Our own observations are discussed in comparison with the previously published studies aimed at defining optimal conditions to culture primary cells of cervical neoplasia.