Modification of the Hi-C Technology for Molecular Genetic Analysis of Formalin-Fixed Paraffin-Embedded Sections of Tumor Tissues.

Molecular genetic analysis of tumor tissues is the most important step towards understanding the mechanisms of cancer development; it is also necessary for the choice of targeted therapy. The Hi-C (high-throughput chromatin conformation capture) technology can be used to detect various types of genomic variants, including balanced chromosomal rearrangements, such as inversions and translocations. We propose a modification of the Hi-C method for the analysis of chromatin contacts in formalin-fixed paraffin-embedded (FFPE) sections of tumor tissues. The developed protocol allows to generate high-quality Hi-C data and detect all types of chromosomal rearrangements. We have analyzed various databases to compile a comprehensive list of translocations that hold clinical importance for the targeted therapy selection. The practical value of molecular genetic testing is its ability to influence the treatment strategies and to provide prognostic insights. Detecting specific chromosomal rearrangements can guide the choice of the targeted therapies, which is a critical aspect of personalized medicine in oncology.

Complex Analysis of Single-Cell RNA Sequencing Data.

Single-cell RNA sequencing (scRNA-seq) is a revolutionary tool for studying the physiology of normal and pathologically altered tissues. This approach provides information about molecular features (gene expression, mutations, chromatin accessibility, etc.) of cells, opens up the possibility to analyze the trajectories/phylogeny of cell differentiation and cell-cell interactions, and helps in discovery of new cell types and previously unexplored processes. From a clinical point of view, scRNA-seq facilitates deeper and more detailed analysis of molecular mechanisms of diseases and serves as a basis for the development of new preventive, diagnostic, and therapeutic strategies. The review describes different approaches to the analysis of scRNA-seq data, discusses the advantages and disadvantages of bioinformatics tools, provides recommendations and examples of their successful use, and suggests potential directions for improvement. We also emphasize the need for creating new protocols, including multiomics ones, for the preparation of DNA/RNA libraries of single cells with the purpose of more complete understanding of individual cells.

The trimeric coiled-coil HSBP1 protein promotes WASH complex assembly at centrosomes.

The Arp2/3 complex generates branched actin networks that exert pushing forces onto different cellular membranes. WASH complexes activate Arp2/3 complexes at the surface of endosomes and thereby fission transport intermediates containing endocytosed receptors, such as α5ß1 integrins. How WASH complexes are assembled in the cell is unknown. Here, we identify the small coiled-coil protein HSBP1 as a factor that specifically promotes the assembly of a ternary complex composed of CCDC53, WASH, and FAM21 by dissociating the CCDC53 homotrimeric precursor. HSBP1 operates at the centrosome, which concentrates the building blocks. HSBP1 depletion in human cancer cell lines and in Dictyostelium amoebae phenocopies WASH depletion, suggesting a critical role of the ternary WASH complex for WASH functions. HSBP1 is required for the development of focal adhesions and of cell polarity. These defects impair the migration and invasion of tumor cells. Overexpression of HSBP1 in breast tumors is associated with increased levels of WASH complexes and with poor prognosis for patients.

Technologies for Viable Circulating Tumor Cell Isolation.

The spread of tumor cells throughout the body by traveling through the bloodstream is a critical step in metastasis, which continues to be the main cause of cancer-related death. The detection and analysis of circulating tumor cells (CTCs) is important for understanding the biology of metastasis and the development of antimetastatic therapy. However, the isolation of CTCs is challenging due to their high heterogeneity and low representation in the bloodstream. Different isolation methods have been suggested, but most of them lead to CTC damage. However, viable CTCs are an effective source for developing preclinical models to perform drug screening and model the metastatic cascade. In this review, we summarize the available literature on methods for isolating viable CTCs based on different properties of cells. Particular attention is paid to the importance of in vitro and in vivo models obtained from CTCs. Finally, we emphasize the current limitations in CTC isolation and suggest potential solutions to overcome them.

Inactivation of PTEN and ZFHX3 in Mammary Epithelial Cells Alters Patterns of Collective Cell Migration.

Whole exome sequencing of invasive mammary carcinomas revealed the association of mutations in PTEN and ZFHX3 tumor suppressor genes (TSGs). We generated single and combined PTEN and ZFHX3 knock-outs (KOs) in the immortalized mammary epithelial cell line MCF10A to study the role of these genes and their potential synergy in migration regulation. Inactivation of PTEN, but not ZFHX3, induced the formation of large colonies in soft agar. ZFHX3 inactivation in PTEN KO, however, increased colony numbers and normalized their size. Cell migration was affected in different ways upon PTEN and ZFHX3 KO. Inactivation of PTEN enhanced coordinated cell motility and thus, the collective migration of epithelial islets and wound healing. In contrast, ZFHX3 knockout resulted in the acquisition of uncoordinated cell movement associated with the appearance of immature adhesive junctions (AJs) and the increased expression of the mesenchymal marker vimentin. Inactivation of the two TSGs thus induces different stages of partial epithelial-to-mesenchymal transitions (EMT). Upon double KO (DKO), cells displayed still another motile state, characterized by a decreased coordination in collective migration and high levels of vimentin but a restoration of mature linear AJs. This study illustrates the plasticity of migration modes of mammary cells transformed by a combination of cancer-associated genes.

Mutational drivers of cancer cell migration and invasion.

Genomic instability and mutations underlie the hallmarks of cancer-genetic alterations determine cancer cell fate by affecting cell proliferation, apoptosis and immune response, and increasing data show that mutations are involved in metastasis, a crucial event in cancer progression and a life-threatening problem in cancer patients. Invasion is the first step in the metastatic cascade, when tumour cells acquire the ability to move, penetrate into the surrounding tissue and enter lymphatic and blood vessels in order to disseminate. A role for genetic alterations in invasion is not universally accepted, with sceptics arguing that cellular motility is related only to external factors such as hypoxia, chemoattractants and the rigidity of the extracellular matrix. However, increasing evidence shows that mutations might trigger and accelerate the migration and invasion of different types of cancer cells. In this review, we summarise data from published literature on the effect of chromosomal instability and genetic mutations on cancer cell migration and invasion.

P-cadherin-induced decorin secretion is required for collagen fiber alignment and directional collective cell migration.

Directional collective cell migration (DCCM) is crucial for morphogenesis and cancer metastasis. P-cadherin (also known as CDH3), which is a cell-cell adhesion protein expressed in carcinoma and aggressive sarcoma cells and associated with poor prognosis, is a major DCCM regulator. However, it is unclear how P-cadherin-mediated mechanical coupling between migrating cells influences force transmission to the extracellular matrix (ECM). Here, we found that decorin, a small proteoglycan that binds to and organizes collagen fibers, is specifically expressed and secreted upon P-cadherin, but not E- and R-cadherin (also known as CDH1 and CDH4, respectively) expression. Through cell biological and biophysical approaches, we demonstrated that decorin is required for P-cadherin-mediated DCCM and collagen fiber orientation in the migration direction in 2D and 3D matrices. Moreover, P-cadherin, through decorin-mediated collagen fiber reorientation, promotes the activation of ß1 integrin and of the ß-Pix (ARHGEF7)/CDC42 axis, which increases traction forces, allowing DCCM. Our results identify a novel P-cadherin-mediated mechanism to promote DCCM through ECM remodeling and ECM-guided cell migration.

Prognosis of Different Types of Non-Small Cell Lung Cancer Progression: Current State and Perspectives.

Despite advances in diagnostics and therapy of non-small cell lung cancer (NSCLC), the problem of prognosis and prevention of tumor progression is still highly important. Even if NSCLC is diagnosed in the early stages, almost a quarter of patients develop relapse; most of them die from recurrent disease. A large number of different markers have been proposed to predict the risk of NSCLC progression; however, none of them are used in clinical practice. It is obvious that this situation is related to the economic and methodological complexity of the proposed markers and/or their insufficient efficiency due to a lack of effective study models and tumor heterogeneity. Another reason may be that potential markers are developed for NSCLC progression in general, which is represented by at least four pathogenetically-distinct processes: synchronous lymph node metastasis, local, regional, and distant recurrence. In this review, we summarize data from published literature on clinicopathological, genetic, and molecular factors associated with different types of NSCLC progression and emphasize challenges and approaches to developing prognostic factors. In conclusion, we highlight the importance of further studies to reveal molecular mechanisms of NSCLC progression and the need for differential analysis of markers of local, regional, and distant recurrences for disease prognosis.

Heterogeneous Manifestations of Epithelial-Mesenchymal Plasticity of Circulating Tumor Cells in Breast Cancer Patients.

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).

MT1-MMP targeting to endolysosomes is mediated by upregulation of flotillins.

Tumor cell invasion and metastasis formation are the major cause of death in cancer patients. These processes rely on extracellular matrix (ECM) degradation mediated by organelles termed invadopodia, to which the transmembrane matrix metalloproteinase MT1-MMP (also known as MMP14) is delivered from its reservoir, the RAB7-containing endolysosomes. How MT1-MMP is targeted to endolysosomes remains to be elucidated. Flotillin-1 and -2 are upregulated in many invasive cancers. Here, we show that flotillin upregulation triggers a general mechanism, common to carcinoma and sarcoma, which promotes RAB5-dependent MT1-MMP endocytosis and its delivery to RAB7-positive endolysosomal reservoirs. Conversely, flotillin knockdown in invasive cancer cells greatly reduces MT1-MMP accumulation in endolysosomes, its subsequent exocytosis at invadopodia, ECM degradation and cell invasion. Our results demonstrate that flotillin upregulation is necessary and sufficient to promote epithelial and mesenchymal cancer cell invasion and ECM degradation by controlling MT1-MMP endocytosis and delivery to the endolysosomal recycling compartment.

Heterogeneity of Circulating Tumor Cells in Breast Cancer: Identifying Metastatic Seeds.

Metastasis being the main cause of breast cancer (BC) mortality represents the complex and multistage process. The entrance of tumor cells into the blood vessels and the appearance of circulating tumor cells (CTCs) seeding and colonizing distant tissues and organs are one of the key stages in the metastatic cascade. Like the primary tumor, CTCs are extremely heterogeneous and presented by clusters and individual cells which consist of phenotypically and genetically distinct subpopulations. However, among this diversity, only a small number of CTCs is able to survive in the bloodstream and to form metastases. The identification of the metastasis-initiating CTCs is believed to be a critical issue in developing therapeutic strategies against metastatic disease. In this review, we summarize the available literature addressing morphological, phenotypic and genetic heterogeneity of CTCs and the molecular makeup of specific subpopulations associated with BC metastasis. Special attention is paid to the need for in vitro and in vivo studies to confirm the tumorigenic and metastatic potential of metastasis-associating CTCs. Finally, we consider treatment approaches that could be effective to eradicate metastatic CTCs and to prevent metastasis.

Heterogeneity of Circulating Tumor Cells in Neoadjuvant Chemotherapy of Breast Cancer.

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.

Recurrence of squamous cell lung carcinoma is associated with the co-presence of reactive lesions in tumor-adjacent bronchial epithelium.

Recurrences occur in 30 % of lung cancer patients after radical therapy; however, known prognostic factors are not always effective. In this study, we investigated whether the frequency of squamous non-small cell lung cancer (NSCLC) recurrence depends on the presence of reactive lesions in tumor-adjacent bronchial epithelium. Specimens of adjacent lung tissue from 104 patients with squamous NSCLC were used for the determination of basal cell hyperplasia (BCH) and squamous metaplasia (SM) and for the analysis of the expression of Ki-67, p53, Bcl-2, and CD138. We found that recurrence was observed in 36.7 % of patients with BCH combined with SM (BCH + SM+) in the same bronchus, compared with 1.8 % in patients with isolated BCH (BCH + SM-; odds ratio (OR) 31.26, 95 % confidence interval (CI) 3.77-258.60; p = 0.00002). The percentage of Ki-67-positive cells was significantly higher in BCH + SM+ than in BCH + SM- (34.9 vs. 18.3 %; effect size 2.86, 95 % CI 2.23-3.47; p = 0.003). P53 expression was also more significant in BCH + SM+ than in BCH + SM- (14.4 vs. 9.6 %; effect size 1.22, 95 % CI 0.69-1.76; p = 0.0008). In contrast, CD138 expression was lower in BCH + SM+ than in BCH + SM- (21.8 vs. 38.5 %; effect size -6.26, 95 % CI -7.31 to -5.22; p = 0.003). Based on our results, we concluded that the co-presence of reactive bronchial lesions is associated with the development of recurrent squamous NSCLC and may be a negative prognostic indicator. In addition, significant differences in Ki-67, p53, and CD138 expression exist between isolated BCH and BCH combined with SM that probably reflect part of biological differences, which could relate to the mechanism of lung cancer recurrence.

Integrin-associated transcriptional characteristics of circulating tumor cells in breast cancer patients.

Background: Integrins enable cell communication with the basal membrane and extracellular matrix, activating signaling pathways and facilitating intracellular changes. Integrins in circulating tumor cells (CTCs) play a significant role in apoptosis evasion and anchor-independent survival. However, the link between CTCs expressing different integrin subunits, their transcriptional profile and, therefore, their functional activity with respect to metastatic potential remains unclear. Methods: Single-cell RNA sequencing of CD45-negative cell fraction of breast cancer patients was performed. All CTCs were divided into nine groups according to their integrin profile. Results: СTCs without the gene expression of integrins or with the expression of non-complementary α and ß subunits that cannot form heterodimers prevailed. Only about 15% of CTCs expressed integrin subunits which can form heterodimers. The transcriptional profile of CTCs appeared to be associated with the spectrum of expressed integrins. The lowest potential activity was observed in CTCs without integrin expression, while the highest frequency of expression of tumor progression-related genes, namely genes of stemness, epithelial-mesenchymal transition (EMT), invasion, proinflammatory chemokines and cytokines as well as laminin subunits, were observed in CTCs co-expressing ITGA6 and ITGB4. Validation on the protein level revealed that the median of integrin ß4+ CTCs was higher in patients with more aggressive molecular subtypes as well as in metastatic breast cancer patients. One can expect that CTCs with ITGA6 and ITGB4 expression will have pronounced metastatic potencies manifesting in expression of EMT and stemness-related genes, as well as potential ability to produce chemokine/proinflammatory cytokines and laminins.

Metastasis prevention: How to catch metastatic seeds.

Despite considerable advances in the evolution of anticancer therapies, metastasis still remains the main cause of cancer mortality. Therefore, current strategies for cancer cure should be redirected towards prevention of metastasis. Targeting metastatic pathways represents a promising therapeutic opportunity aimed at obstructing tumor cell dissemination and metastatic colonization. In this review, we focus on preclinical studies and clinical trials over the last five years that showed high efficacy in suppressing metastasis through targeting lymph node dissemination, tumor cell extravasation, reactive oxygen species, pre-metastatic niche, exosome machinery, and dormancy.

Heterogeneity of Circulating Epithelial Cells in Breast Cancer at Single-Cell Resolution: Identifying Tumor and Hybrid Cells.

Circulating tumor cells and hybrid cells formed by the fusion of tumor cells with normal cells are leading players in metastasis and have prognostic relevance. This study applies single-cell RNA sequencing to profile CD45-negative and CD45-positive circulating epithelial cells (CECs) in nonmetastatic breast cancer patients. CECs are represented by transcriptionally-distinct populations that include both aneuploid and diploid cells. CD45- CECs are predominantly aneuploid, but one population contained more diploid than aneuploid cells. CD45+ CECs mostly diploid: only two populations have aneuploid cells. Diploid CD45+ CECs annotated as different immune cells, surprisingly harbored many copy number aberrations, and positively correlated to tumor grade. It is noteworthy that cancer-associated signaling pathways areabundant only in one aneuploid CD45- CEC population, which may represent an aggressive subset of circulating tumor cells. Thus, CD45- and CD45+ CECs are highly heterogeneous in breast cancer patients and include aneuploid cells, which are most likely circulating tumor and hybrid cells, respectively, and diploid cells. DNA ploidy analysis can be an effective instrument for identifying tumor and hybrid cells among CECs. Further follow-up study is needed to determine which subsets of circulating tumor and hybrid cells contribute to breast cancer metastasis.

Tumor Hybrid Cells: Nature and Biological Significance.

Metastasis is the leading cause of cancer death and can be realized through the phenomenon of tumor cell fusion. The fusion of tumor cells with other tumor or normal cells leads to the appearance of tumor hybrid cells (THCs) exhibiting novel properties such as increased proliferation and migration, drug resistance, decreased apoptosis rate, and avoiding immune surveillance. Experimental studies showed the association of THCs with a high frequency of cancer metastasis; however, the underlying mechanisms remain unclear. Many other questions also remain to be answered: the role of genetic alterations in tumor cell fusion, the molecular landscape of cells after fusion, the lifetime and fate of different THCs, and the specific markers of THCs, and their correlation with various cancers and clinicopathological parameters. In this review, we discuss the factors and potential mechanisms involved in the occurrence of THCs, the types of THCs, and their role in cancer drug resistance and metastasis, as well as potential therapeutic approaches for the prevention, and targeting of tumor cell fusion. In conclusion, we emphasize the current knowledge gaps in the biology of THCs that should be addressed to develop highly effective therapeutics and strategies for metastasis suppression.

Metastasis prevention: targeting causes and roots.

The spread of tumor cells from the primary focus, metastasis, is the main cause of cancer mortality. Therefore, anticancer therapy should be focused on the prevention of metastatic disease. Key targets can be conditions in the primary tumor that are favorable for the appearance of metastatic cells and the first steps of the metastatic cascade. Here, we discuss different approaches for targeting metastasis causes (hypoxia, metabolism changes, and tumor microenvironment) and roots (angiogenesis, epithelial-mesenchymal transition, migration, and invasion). Also, we emphasize the challenges of the existing approaches for metastasis prevention and suggest opportunities to overcome them. In conclusion, we highlight the importance of clinical evaluation of the agents showing antimetastatic effects in vivo, especially in patients with early-stage cancers, the identification of metastatic seeds, and the development of therapeutics for their eradication.

Metastasis Prevention: Focus on Metastatic Circulating Tumor Cells.

Metastasis is the main cause of cancer death. Metastatic foci are derived from tumor cells that detach from the primary tumor and then enter the circulation. Circulating tumor cells (CTCs) are generally associated with a high probability of distant metastasis and a negative prognosis. Most CTCs die in the bloodstream, and only a few cells form metastases. Such metastatic CTCs have a stem-like and hybrid epithelial-mesenchymal phenotype, can avoid immune surveillance, and show increased therapy resistance. Targeting metastatic CTCs and their progenitors in primary tumors and their descendants, particularly disseminated tumor cells, represents an attractive strategy for metastasis prevention. However, current therapeutic strategies mainly target the primary tumor and only indirectly affect metastasis-initiating cells. Here, we consider potential methods for preventing metastasis based on targeting molecular and cellular features of metastatic CTCs, including CTC clusters. Also, we emphasize current knowledge gaps in CTC biology that should be addressed to develop highly effective therapeutics and strategies for metastasis suppression.