Integrins are cell adhesion receptors, which play a role in breast cancer invasion, angiogenesis, and metastasis. Moreover, it has been shown that exosomal integrins provide organotropic metastasis in a mouse model. In our study, we aimed to investigate the expression of integrins β3, β4, and αVβ5 on exosomes and tumor cells (circulating tumor cells and primary tumor) and their association with the localization of distant metastasis. We confirmed the association of exosomal integrin β4 with lung metastasis in breast cancer patients. However, we were unable to evaluate the role of integrin β3 in brain metastasis due to the rarity of this localization. We established no association of exosomal integrin αVβ5 with liver metastasis in our cohort of breast cancer patients. The further evaluation of β3, β4, and αVβ5 integrin expression on CTCs revealed an association of integrin β4 and αVβ5 with liver, but not the lung metastases. Integrin β4 in the primary tumor was associated with liver metastasis. Furthermore, an in-depth analysis of phenotypic characteristics of β4+ tumor cells revealed a significantly increased proportion of E-cadherin+ and CD44+CD24- cells in patients with liver metastases compared to patients with lung or no distant metastases.

The presence of stem and epithelial–mesenchymal-transition (EMT) features in circulating tumor cells (CTCs) determines their invasiveness, adaptability to the microenvironment, and resistance to proapoptotic signals and chemotherapy. It also allows them to fulfil the role of metastatic “seeds”. We evaluated the heterogeneity of stem CTCs by their CD44, ALDH1, and CD133 expression depending on N-cadherin expression in breast-cancer patients. A total of 38 female patients were selected for this study. CTC phenotypes were determined by flow cytometry before any type of treatment. Multiplex immunofluorescence was used for the evaluation of tumor-cell heterogeneity in primary lesions. In patients who had CD44-CD24- CTCs, a subset of cells with the expression of other stem-cell markers (CD133 and ALDH1) were detected. Expression of CD133 and/or ALDH1 may be associated with expression of N-cadherin: all populations of N-cadherin+ CTCs demonstrate stem features; in the absence of N-cadherin expression, true nonstem (CD44-CD24-CD133-ALDH1-) cells are found. The heterogeneity of stem marker expression in CTCs was observed regardless of N-cadherin expression. In our study, stromal cell-derived factor-1 (SDF-1) receptor expression in CTCs did not depend on stemlike traits, but was instead associated with N-cadherin expression. Subpopulations of tumor cells, detected both in tumors and blood, were identified. Breast cancer was characterized by pronounced interpersonal and intrapersonal heterogeneity of CTCs by the presence and combination of various stem features and N-cadherin expression. To complete the characterization of stemlike features of CTCs, we suggest the simultaneous use of the three stem markers.

To date, there is indisputable evidence of significant CTC heterogeneity in carcinomas, in particular breast cancer. The heterogeneity of CTCs is manifested in the key characteristics of tumor cells related to metastatic progression – stemness and epithelial–mesenchymal (EMT) plasticity. It is still not clear what markers can characterize the phenomenon of EMT plasticity in the range from epithelial to mesenchymal phenotypes. In this article we examine the manifestations of EMT plasticity in the CTCs in breast cancer. The prospective study included 39 patients with invasive carcinoma of no special type. CTC phenotypes were determined by flow cytometry before any type of treatment. EMT features of CTC were assessed using antibodies against CD45, CD326 (EpCam), CD325 (N-cadherin), CK7, Snail, and Vimentin. Circulating tumor cells in breast cancer are characterized by pronounced heterogeneity of EMT manifestations. The results of the study indicate that the majority of heterogeneous CTC phenotypes (22 out of 24 detectable) exhibit epithelial–mesenchymal plasticity. The variability of EMT manifestations does not prevent intravasation. Co-expression of EpCAM and CK7, regardless of the variant of co-expression of Snail, N-cadherin, and Vimentin, are associated with a low number of CTCs. Intrapersonal heterogeneity is manifested by the detection of several CTC phenotypes in each patient. Interpersonal heterogeneity is manifested by various combinations of CTC phenotypes in patients (from 1 to 17 phenotypes).

In recent years, the new direction such as identification of informative circulating markers reflecting molecular genetic changes in the DNA of tumor cells was actively developed. Smoking-related DNA adducts are very promising research area, since they indicate high pathogenetic importance in the lung carcinogenesis and can be identified in biological samples with high accuracy and reliability using highly sensitive mass spectrometry methods (TOF/TOF, TOF/MS, MS/MS). The appearance of DNA adducts in blood or tissues is the result of the interaction of carcinogenic factors, such as tobacco constituents, and the body reaction which is determined by individual characteristics of metabolic and repair systems. So, DNA adducts may be considered as a cumulative mirror of heterogeneous response of different individuals to smoking carcinogens, which finally could determine the risk for lung cancer. This review is devoted to analysis of the role of DNA adducts in lung carcinogenesis in order to demonstrate their usefulness as cancer associated markers. Currently, there are some serious limitations impeding the widespread use of DNA adducts as cancer biomarkers, due to failure of standardization of mass spectrometry analysis in order to correctly measure the adduct level in each individual. However, it is known that all DNA adducts are immunogenic, their accumulation over some threshold concentration leads to the appearance of long-living autoantibodies. Thus, detection of an informative pattern of autoantibodies against DNA adducts using innovative multiplex ELISA immunoassay may be a promising approach to find lung cancer at an early stage in high-risk groups (smokers, manufacturing workers, urban dwellers).

Following the discovery of circulating tumor cells (CTCs) in the peripheral blood of cancer patients, CTCs were initially postulated to hold promise as a valuable prognostic tool through liquid biopsy. However, a decade and a half of accumulated data have revealed significant complexities in the investigation of CTCs. A challenging aspect lies in the reduced expression or complete loss of key epithelial markers during the epithelial-mesenchymal transition (EMT). This likely hampers the identification of a pathogenetically significant subset of CTCs. Nevertheless, there is a growing body of evidence regarding the prognostic value of such molecules as CD24 expressing in the primary breast tumor. Herewith, the exact relevance of CD24 expression on CTCs remains unclear. We used two epithelial markers (EpCAM and cytokeratin 7/8) to assess the count of CTCs in 57 breast cancer patients, both with (M0^mts) and without metastasis (M0) during the follow-up period, as well as in M1 breast cancer patients. However, the investigation of these epithelial markers proved ineffective in identifying cell population expressing different combinations of EpCAM and cytokeratin 7/8 with prognostic significance for breast cancer metastases. Surprisingly, we found CD24+ circulating cells (CCs) in peripheral blood of breast cancer patients which have no epithelial markers (EpCAM and cytokeratin 7/8) but was strongly associated with distant metastasis. Namely, the count of CD45-EpCAM-CK7/8-CD24+ N-cadherin—CCs was elevated in both groups of patients, those with existing metastasis and those who developed metastases during the follow-up period. Simultaneously, an elevation in these cell counts beyond the established threshold of 218.3 cells per 1 mL of blood in patients prior to any treatment predicted a 12-fold risk of metastases, along with a threefold decrease in distant metastasis-free survival over a 90-month follow-up period. The origin of CD45-EpCAM-CK7/8-CD24+ N-cadherin—CCs remains unclear. In our opinion their existence can be explained by two most probable hypotheses. These cells could exhibit a terminal EMT phenotype, or it might be immature cells originating from the bone marrow. Nonetheless, if this hypothesis holds true, it's worth noting that the mentioned CCs do not align with any of the recognized stages of monocyte or neutrophil maturation, primarily due to the presence of CD45 expression in the myeloid cells. The results suggest the presence in the peripheral blood of patients with metastasis (both during the follow-up period and prior to inclusion in the study) of a cell population with a currently unspecified origin, possibly arising from both myeloid and tumor sources, as confirmed by the presence of aneuploidy.