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.

The biological properties of circulating tumor cells (CTCs), and their dynamics during neoadjuvant chemotherapy are important, both for disease progression prediction and therapeutic target determination, with the aim of preventing disease progression. The aim of our study was to estimate of different CTC subsets in breast cancer during the NACT (neoadjuvant chemotherapy). The prospective study includes 27 patients with invasive breast cancer, T2-4N0-3M0, aged 32 to 60 years. Venous heparinized blood samples, taken before and after biopsy, after each courses of chemotherapy (on days 3–7), and before surgical intervention, served as the material for this study. Different subsets of circulating tumor cells were determined on the basis of the expression of EpCAM, CD45, CD44, CD24, and N-Cadherin using flow cytometry. As the result of this study, it has been observed that significant changes in the quantity of the different subsets of circulating tumor cells in patients’ blood were observed after carrying out the 3rd course of NACT. NACT causes significant changes in the quantity of six CTC subsets, with various combinations of stemness and epithelial-mesenchymal transition (EMT) properties.

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).

Inflammatory infiltration of tumor stroma is an integral reflection of reactions that develop in response to any damage to tumor cells including immune responses to antigens or necrosis caused by vascular disorders. In this review, we use the term “immune-inflammatory response” (IIR) that allows us to give an integral assessment of the cellular composition of the tumor microenvironment. Two main types of IIRs are discussed: type 1 and 2 T-helper reactions (Th1 and Th2), as well as their inducers: immunosuppressive responses and reactions mediated by Th22 and Th17 lymphocytes and capable of modifying the main types of IIRs. Cellular and molecular manifestations of each IIR type are analyzed and their general characteristics and roles in tissue regeneration and tumor growth are presented. Since inflammatory responses in a tumor can also be initiated by innate immunity mechanisms, special attention is given to inflammation based on them. We emphasize that processes accompanying tissue regeneration are prototypes of processes underlying cancer progression, and these processes have the same cellular and molecular substrates. We focus on evidence that tumor progression is mainly contributed by processes specific for the second phase of “wound healing” that are based on the Th2-type IIR. We emphasize that the effect of various types of immune and stroma cells on tumor progression is determined by the ability of the cells and their cytokines to promote or prevent the development of Th1- or Th2-type of IIR. Finally, we supposed that the nonspecific influence on the tumor caused by the cytokine context of the Th1- or Th2-type microenvironment should play a decisive role for suppression or stimulation of tumor growth and metastasis.

Most studies on CTCs have focused on isolating cells that express EpCAM. In this study, we emphasize the presence of EpCAM-negative and EpCAM^low CTCs, in addition to EpCAM^high CTCs, in early BC. We evaluated stem cell markers (CD44/CD24 and CD133) and EMT markers (N-cadherin) in each subpopulation. Our findings indicate that all stemness variants were present in both EpCAM^high and EpCAM-negative CTCs, whereas only one variant of stemness (nonCD44+CD24−/CD133+) was observed among EpCAM^low CTCs. Nearly all EpCAM^high CTCs were represented by CD133+ stem cells. Notably, the hybrid EMT phenotype was more prevalent among EpCAM-negative CTCs. scRNA-seq of isolated CTCs and primary tumor partially confirmed this pattern. Therefore, further investigation is imperative to elucidate the prognostic significance of EpCAM-negative and EpCAM^low CTCs.