Metastatic Potential Of Tumor Cells Research Articles

Identification of Matrine as a Novel Regulator of the CXCR4 Signaling Axis in Tumor Cells.

Matrine, a quinolizidine alkaloid, is commonly employed for treating various viral and inflammatory disorders. Here, we have evaluated matrine for its activity on C-X-C chemokine receptor type 4 (CXCR4) and matrix metalloproteinases (MMP-9/2) expression, and its potential to affect tumor metastasis and invasion. The effects of matrine on CXCR4, MMP-9/2, and nuclear factor κB (NF-κB) activation in lung (A549), prostate (DU145), and pancreas (MIA PaCa-2) cells were investigated by diverse techniques. The expression level of CXCR4 and MMP-9/2 was analyzed by western blot analysis and reverse transcription polymerase chain reaction. NF-κB activation was also evaluated by western blot analysis, electrophoretic mobility shift assay as well as immunocytochemical experiments. Furthermore, we monitored cell invasion and metastasis activities by wound healing and Boyden chamber assays. We noted that matrine induced a down-regulation of CXCR4 and MMP-9/2 at both protein and mRNA levels. In addition, matrine negatively regulated human epidermal growth factor receptor 2 (HER2) and C-X-C Motif Chemokine Ligand 12 (CXCL12)-induced CXCR4 expression. Moreover, NF-κB suppression by matrine led to inhibition of metastatic potential of tumor cells. Our results suggest that matrine can block the cancer metastasis through the negative regulation of CXCR4 and MMP-9/2 and consequently it can be considered as a potential candidate for cancer therapy.

Comparison of oncologic outcomes for robotic-assisted radical cystectomy (RARC) versus open radical cystectomy (ORC) among locally advanced and node-positive patients: An analysis of the National Cancer Database (NCDB).

e17002 Background: Despite recent concerns of atypical recurrence following RARC, utilization of the modality is increasing. The presumed mechanisms are especially relevant in more aggressive disease, where the metastatic potential of tumor cells may be greater. This study aims to compare the oncologic efficacy of RARC to ORC among patients with stage pT3-4 or node-positive bladder cancer. Methods: A retrospective cohort analysis of pT3-4N0-3 and pT(any)-4N1-3 patients who underwent RARC or ORC from 2010-2016 was performed using the NCDB. Appropriate univariate and multivariable analysis were performed between treatment cohorts. Results: RARC was significantly associated with superior unadjusted survival compared to ORC (median survival relative to ORC; 23.6 mo. vs. 21.6 mo.; P= 0.001). Additionally, RARC was associated with lower proportions of unadjusted 30- and 90-day mortality, positive margin status, and shorter surgical inpatient stay (all respective P< 0.05). However, after adjusting for confounding covariates, multivariable analysis revealed no difference in mortality hazard or odds of secondary outcomes with the exception of inpatient stay (Table). RARC was also significantly associated with higher lymph node yield (increased incidence of > 14 lymph nodes examined relative to ORC; 55% vs. 40%; P< 0.01). Conclusions: RARC is no less safe than ORC for patients with locally advanced or node-positive bladder cancer on the basis of overall, 30- and 90-day survival outcomes. Unadjusted mortality and surgical outcomes in this population demonstrate advantages to the robotic modality. Perioperative benefits may favor RARC, but further randomized control studies are necessary to better elucidate differences between surgical approaches. [Table: see text]

NLRP3 inflammasome in immune cells regulates metastatic potential of cancer

Abstract One of the main features of tumor microenvironment is inflammation accompanied with the activation of immune cells and the production of pro-inflammatory cytokines. IL-1b, one of the critical cytokines released by immune cells to promote tumor growth and metastasis by acting on its receptor, IL-1R, and to culminate in the secondary activation of immune cells and the expression of immune cytokines. Inflammasome plays a critical role in production of mature IL-1b through activation of capspase-1. Therefore, we investigated whether regulation of NLRP3 inflammasome in macrophages affected the metastatic potential of tumor cells. Activation of NLRP3 inflammasome was induced by ATP in bone marrow-derived primary mouse macrophages. The metastatic potential of tumor cells was determined by transwell migration and invasion assays. Wild-type macrophages stimulated with ATP increased the migration and invasion of melanoma cells (B16F10). However, NLRP3- or caspase-1-knockout macrophages showed greatly decreased ability to promote the migration and invasion of melanoma cells. Treatment with celastrol, an inhibitor of NLRP3 inflammasome, blocked the ability of macrophages to stimulate migration and invasion of melanoma cells. The results show that inhibition of the NLRP3 inflammasome in macrophages by genetic deficiency or a pharmacological inhibitor leads to decrease of the metastatic potential of tumor cells. The results would provide a novel anti-cancer immuntherapy to regulate the activity of NLRP3 inflammasome in tumor microenvironment. [Supported by grants from BK21 PLUS program, NRF-2019R1A2C2085739, and NRF-2017R1A4A1015036].

Hsp90 facilitates acquired drug resistance of tumor cells through cholesterol modulation however independent of tumor progression

Acquired multidrug resistance of cancer cells challenges the chemotherapeutic interventions. To understand the role of molecular chaperone, Hsp90 in drug adapted tumor cells, we have used in vitro drug adapted epidermoid tumor cells as a model system. We found that chemotherapeutic drug adaptation of tumor cells is mediated by induced activities of both Hsp90 and P-glycoprotein (P-gp). Although the high-affinity conformation of Hsp90 has correlated with the enhanced drug efflux activity, we did not observe a direct interaction between P-gp and Hsp90. The enrichment of P-gp and Hsp90 at the cholesterol-rich membrane microdomains is found obligatory for enhanced drug efflux activity. Since inhibition of cholesterol biosynthesis is not interfering with the drug efflux activity, it is presumed that the net cholesterol redistribution mediated by Hsp90 regulates the enhanced drug efflux activity. Our in vitro cholesterol and Hsp90 interaction studies have furthered our presumption that Hsp90 facilitates cholesterol redistribution. The drug adapted cells though exhibited anti-proliferative and anti-tumor effects in response to 17AAG treatment, drug treatment has also enhanced the drug efflux activity. Our findings suggest that drug efflux activity and metastatic potential of tumor cells are independently regulated by Hsp90 by distinct mechanisms. We expose the limitations imposed by Hsp90 inhibitors against multidrug resistant tumor cells.

Galectin-3 Regulates the Expression of Tumor Glycosaminoglycans and Increases the Metastatic Potential of Breast Cancer.

Galectin-3 (Gal-3) is a multifunctional β-galactoside-binding lectin that once synthesized is expressed in the nucleus, cytoplasm, cell surface, and extracellular environment. Gal-3 plays an important role in breast cancer tumors due to its ability to promote interactions between cell-cell and cell-extracellular matrix (ECM) elements, increasing tumor survival and metastatic dissemination. Still, the mechanism by which Gal-3 interferes with tumor cell migration and metastasis formation is complex and not fully understood. Here, we showed that Gal-3 knockdown increased the migration ability of 4T1 murine breast cancer cells in vitro. Using the 4T1 orthotopic breast cancer spontaneous metastasis mouse model, we demonstrated that 4T1-derived tumors were significantly larger in the presence of Gal-3 (scramble) in comparison with Gal-3 knockdown 4T1-derived tumors. Nevertheless, Gal-3 knockdown 4T1 cells were outnumbered in the bone marrow in comparison with scramble 4T1 cells. Finally, we reported here a decrease in the content of cell-surface syndecan-1 and an increase in the levels of chondroitin sulfate proteoglycans such as versican in Gal-3 knockdown 4T1 cells both in vitro and in vivo. Overall, our findings establish that Gal-3 downregulation during breast cancer progression regulates cell-associated and tumor microenvironment glycosaminoglycans (GAGs)/proteoglycans (PG), thus enhancing the metastatic potential of tumor cells.

SNHG12: An LncRNA as a Potential Therapeutic Target and Biomarker for Human Cancer.

Limitations in current diagnostic procedures warrant identification of new methodologies to improve diagnoses of cancer patients. In this context, long non-coding RNAs (lncRNAs) have emerged as stable biomarkers which are expressed abundantly in tumors. Importantly, these can be detected at all stages of tumor development, and thus may provide potential biomarkers and/or therapeutic targets. Recently, we suggested that aberrant levels of lncRNAs can be used to determine the invasive and metastatic potential of tumor cells. Further, direct correlations of lncRNAs with cancer-derived inflammation, metastasis, epithelial-to-mesenchymal transition, and other hallmarks of cancer indicate their potential as biomarkers and targets for cancer. Thus, in this review we have discussed the importance of small nucleolar RNA host gene 12 (SNHG12), a lncRNA, as a potential biomarker for a variety of cancers. A meta-analysis of a large cohort of cancer patients revealed that SNHG12 may also serve as a potential target for cancer-directed interventions due to its involvement in unfolded protein responses, which many tumor cells exploit to both evade immune-mediated attack and enhance the polarization of effector immune cells (e.g., macrophages and T cells). Thus, we propose that SNHG12 may serve as both a biomarker and a druggable therapeutic target with promising clinical potential.

Inhibition of NLRP3 inflammasome in tumor microenvironment leads to suppression of metastatic potential of cancer cells

Tumor microenvironment favors tumor cells to promote their growth and metastasis such as migration, invasion, and angiogenesis. IL-1β, one of the inflammatory cytokines released from myeloid cells in tumor microenvironment, plays an important role in development and progress of tumor. The activation of inflammasome is a critical step to secrete mature IL-1β through stepwise reactions to activate capspase-1. Therefore, we investigated whether the inhibition of NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome in macrophages regulated the metastatic potential of tumor cells. NLRP3 inflammasome was activated by ATP in bone marrow-derived primary mouse macrophages. The metastatic potential of mouse melanoma cell line (B16F10) was determined by migration and invasion assays with transwell system. ATP-treated wild-type macrophages increased the migration and invasion of melanoma cells. However, NLRP3- or caspase-1-knockout macrophages exhibited greatly diminished ability to promote the migration and invasion of melanoma cells. In addition, treatment with celastrol, an inhibitor of NLRP3 inflammasome, reduced the potency of macrophages to stimulate migration and invasion of melanoma cells. The results demonstrate that inhibition of the NLRP3 inflammasome in macrophages by genetic deficiency or a pharmacological inhibitor is linked to suppression of the metastatic potential of tumor cells. The results would provide a novel anti-cancer strategy to modulate tumor microenvironment by suppressing NLRP3 inflammasome and consequently reducing IL-1β production.

Photoacoustic-imaging-guided therapy of functionalized melanin nanoparticles: combination of photothermal ablation and gene therapy against laryngeal squamous cell carcinoma.

Multimodality therapy under imaging-guidance is significant to improve the accuracy of cancer treatment. In this study, a photoacoustic imaging (PAI)-guided anticancer strategy based on poly-l-lysine functionalized melanin nanoparticles (MNP-PLL) was developed to treat laryngeal squamous cell carcinoma (LSCC). As a promising alternative to traditional therapies for LSCC, MNP-PLL/miRNA nanoparticles were combined with photothermal ablation against primary tumors and miR-145-5p mediated gene therapy for depleting the metastatic potential of tumor cells. Furthermore, taking advantage of the photoacoustic properties of melanin, PAI guided therapy could optimize the time point of NIR irradiation to maximize the efficacy of photothermal therapy (PTT). The in vitro and in vivo results proved that the combined treatments displayed the most significant tumor suppression compared with monotherapy. By integrating thermo-gene therapies into a theranostic nanoplatform, the MNP-PLL/miR-145-5p nanoparticles significantly suppressed the LSCC progression, indicating their great potential use for cancer therapy.

Hypoxia and the Tumor Secretome.

Metastasis remains the leading cause of cancer-related deaths. To date, there are no specific treatments targeting disseminated disease. New therapeutic options will become available only if we enhance our understanding of mechanisms underlying metastatic spread. A large body of literature shows that the metastatic potential of tumor cells is strongly influenced by microenvironmental cues such as low oxygen (hypoxia). Clinically, hypoxia is a hallmark of most solid tumors and is associated with increased metastasis and poor survival in a variety of cancer types. Mechanistically, hypoxia influences multiple steps within the metastatic cascade and particularly impacts the interactions between tumor cells and host stroma at both primary and secondary sites. Here we review current evidence for a hypoxia-induced tumor secretome and its impact on metastatic progression. These studies have identified potential biomarkers and therapeutic targets that could be integrated into strategies for preventing and treating metastatic disease.

Single-Cell Imaging of Metastatic Potential of Cancer Cells

SummaryMolecular imaging of metastatic “potential” is an unvanquished challenge. To engineer biosensors that can detect and measure the metastatic “potential” of single living cancer cells, we carried out a comprehensive analysis of the pan-cancer phosphoproteome to search for actin remodelers required for cell migration, which are enriched in cancers but excluded in normal cells. Only one phosphoprotein emerged, tyr-phosphorylated CCDC88A (GIV/Girdin), a bona fide metastasis-related protein across a variety of solid tumors. We designed multi-modular biosensors that are partly derived from GIV, and because GIV integrates prometastatic signaling by multiple oncogenic receptors, we named them “‘integrators of metastatic potential (IMP).” IMPs captured the heterogeneity of metastatic potential within primary lung and breast tumors at steady state, detected those few cells that have acquired the highest metastatic potential, and tracked their enrichment during metastasis. These findings provide proof of concept that IMPs can measure the diversity and plasticity of metastatic potential of tumor cells in a sensitive and unbiased way.

Relationship between pretreatment concentration of plasma Epstein-Barr virus DNA and tumor burden in nasopharyngeal carcinoma: An updated interpretation.

BackgroundPretreatment plasma Epstein‐Barr virus (EBV) DNA is an important tumor marker and prognostic factor in nasopharyngeal carcinoma (NPC). This study aimed to clarify the relationship between plasma EBV DNA level and tumor burden.Materials and MethodsPretreatment tumor burden was measured by radiologically delineated volumes, including nasopharynx tumor volume (GTVnx) and malignant nodes volume (GTVnd); pretreatment level of plasma EBV DNA was quantified by quantitative polymerase chain reaction. The relationship between natural logarithm of EBV DNA (ln‐DNA) and square root of tumor volume (sq‐GTV) was analyzed by Pearson correlation coefficient and partial correlation coefficient. Correlations in subgroups of tumor and nodal stages were also analyzed. A linear regression model was constructed to evaluate the contribution of tumor volumes to plasma EBV DNA. The prognostic effects of EBV DNA independent of tumor burden were evaluated.ResultsTwo thousand two hundred and forty nine nonmetastatic NPC patients with detectable plasma EBV DNA were included in correlation analyses. Ln‐DNA showed significant correlation with sq‐GTVnx (r = 0.171) and sq‐GTVnd (r = 0.339) separately. Together, sq‐GTVnx and sq‐GTVnd could only explain 12.9% of the ln‐DNA. Tumor and nodal stages of disease could clearly influence the strength of relationship in subgroup analysis. After excluding confounding volume information, EBV DNA still can predict death and distant metastasis, but not locoregional relapse.ConclusionThis study suggests that plasma EBV DNA is not only an index of tumor burden, but may also reflect other tumor features, such as accessibility to circulation, angiogenesis, tumor cell kinetics, metabolic activity, and metastatic potential, among others.

The expression profile of integrin receptors and osteopontin in thyroid malignancies varies depending on the tumor progression rate and presence of BRAF V600E mutation

Thyroid cancer (TC) is one of the most common malignancy of the human endocrine system. BRAF V600E mutation is the most frequent genetic alteration of papillary carcinoma, the most frequent TC, which effects RAS-RAF-MEK intracellular signaling pathway. These alterations in RAS-RAF-MEK pathway lead to changes in expression levels of cell membrane integrin receptors and their ligand - extracellular matrix protein osteopontin, which in turn increases the metastatic potential of tumor cells. Thus, integrins and their ligand osteopontin can be considered as potential biomarkers of tumor progression and aggressive tumor phenotypes.The aim of the study was to evaluate the expression levels of integrin receptors ITGA2, ITGA3, ITGAV, ITGA6, ITGA9, ITGB1, ITGB3 and their ligands OPNa, OPNb in the thyroid cancer with different BRAF V600E mutation status. MethodsThyroid tumor samples of 70 patients obtained during surgical treatment were analyzed. Expression levels of the investigated genes were evaluated by real time RT-PCR. Fluorescent immunohistochemistry (IHC) was used to confirm the PCR results and to estimate the amount of protein levels. For IHC frozen sections were used. BRAF V600E mutation was determined using allele-specific amplification. Nonparametric criteria (Kruskal Wallis, Wilcoxon and Mann-Whitney tests) were used to evaluate group differences. P values of less than 0.05 were considered as statistically significant. ResultsA higher gene expression level of ITGA2 (1.9-fold, p = 0.037), ITGA3 (21.1-fold, p = 0.041) and ITGA5 (2.08-fold, p = 0.048) was observed in papillary thyroid cancer (PTC) tissue in comparison with median expression level in control samples (conventionally normal tissue of thyroid gland). These changes were confirmed by IHC (significant changes for α2 integrin). ITGAV expression level was statistically significantly higher in follicular thyroid cancer (FTC) (2.0-fold, p = 0.040). Next, high gene expression levels in tissue samples of lymph node metastases were observed for ITGA5 (2.92-fold, p = 0.015), OPNb (4.36-fold, p = 0.037). For genes ITGA3 (37.48-fold, p = 0.017790), ITGA6 (18.76-fold, p = 0.028921) and ITGA9 (12.52-fold, p = 0.026710) higher expression level was detected in T3-4 tumors (TNM) compared to tumors classified as T1-2. Presence of BRAF V600E mutation was identified in 20 samples of PTC of 40 (50%). A significant increase of the expression level only of ITGA3 (3.1-fold, p = 0. 0422) was observed in BRAF V600E positive samples. Further, changes in expression levels of integrins and osteopontin were assessed in benign and malignant neoplasms. In PTC samples higher expression of ITGA2 (2.8-fold, p = 0.005), ITGA6 (2.11, p = 0.03) and ITGB1 (2.32-fold, p = 0.02) was detected. In FTC expression level of ITGA6 (2.67, p = 0.007) was higher than in benign thyroid nodules. ConclusionIdentified changes in expression levels of the studied genes indicate that they could play an important role in tumor progression, and their expression could be affected by the product of mutant BRAF gene. Integrins and their ligand osteopontin might be considered as potential markers in determining prognosis and treatment of TC.

Therapeutic Targeting of mTOR in T-Cell Acute Lymphoblastic Leukemia: An Update.

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive blood malignancy that arises from the clonal expansion of transformed T-cell precursors. Although T-ALL prognosis has significantly improved due to the development of intensive chemotherapeutic protocols, primary drug-resistant and relapsed patients still display a dismal outcome. In addition, lifelong irreversible late effects from conventional therapy are a growing problem for leukemia survivors. Therefore, novel targeted therapies are required to improve the prognosis of high-risk patients. The mechanistic target of rapamycin (mTOR) is the kinase subunit of two structurally and functionally distinct multiprotein complexes, which are referred to as mTOR complex 1 (mTORC1) and mTORC2. These two complexes regulate a variety of physiological cellular processes including protein, lipid, and nucleotide synthesis, as well as autophagy in response to external cues. However, mTOR activity is frequently deregulated in cancer, where it plays a key oncogenetic role driving tumor cell proliferation, survival, metabolic transformation, and metastatic potential. Promising preclinical studies using mTOR inhibitors have demonstrated efficacy in many human cancer types, including T-ALL. Here, we highlight our current knowledge of mTOR signaling and inhibitors in T-ALL, with an emphasis on emerging evidence of the superior efficacy of combinations consisting of mTOR inhibitors and either traditional or targeted therapeutics.

The versatile nature of miR-9/9* in human cancer.

miR-9 and miR-9* (miR-9/9*) were first shown to be expressed in the nervous system and to function as versatile regulators of neurogenesis. The variable expression levels of miR-9/9* in human cancer prompted researchers to investigate whether these small RNAs may also have an important role in the deregulation of physiological and biochemical networks in human disease. In this review, we present a comprehensive overview of the involvement of miR-9/9* in various human malignancies focusing on their opposing roles in supporting or suppressing tumor development and metastasis. Importantly, it is shown that the capacity of miR-9/9* to impact tumor formation is independent from their influence on the metastatic potential of tumor cells. Moreover, data suggest that miR-9/9* may increase malignancy of one cancer cell population at the expense of another. The functional versatility of miR-9/9* emphasizes the complexity of studying miRNA function and the importance to perform functional studies of both miRNA strands in a relevant cellular context. The possible application of miR-9/9* as targets for miRNA-based therapies is discussed, emphasizing the need to obtain a better understanding of the functional properties of these miRNAs and to develop safe delivery methods to target specific cell populations.

The coagulome and the oncomir: impact of cancer-associated haemostatic dysregulation on the risk of metastasis.

Patients with cancer are at high risk of both thromboembolic and haemorrhagic events during the course of their disease. The pathogenesis of haemostatic dysfunction in cancer is complex and involves the interplay of multiple factors. There is growing evidence that interactions between malignancies and the coagulation system are not random but can represent coordinated and clinically-significant adaptations that enhance tumour cell survival, proliferation and metastatic potential. A detailed understanding of the interactions between the haemostatic systems and the pathophysiology of metastasis may not only provide insight into strategies that could potentially reduce the incidence of thrombohaemorrhagic events and complications, but could also help design strategies that are capable of modifying tumour biology, progression and metastatic potential in ways that could enhance anticancer therapies and thereby improve overall survival.

Artemin regulates CXCR4 expression to induce migration and invasion in pancreatic cancer cells through activation of NF-κB signaling

Pancreatic ductal adenocarcinoma (PDAC) is the most lethal human malignant tumor because of the early onset of local invasion and distant metastasis. Perineural invasion is a prominent characteristic of pancreatic adenocarcinoma, which is a multifactorial process that involves various signaling molecules from different signaling pathways. The glial cell line-derived neurotrophic factor family of ligands was reported to be involved in perineural invasion in pancreatic cancer. Artemin is one member of the glial cell line-derived neurotrophic factor family of ligands. Although Artemin has previously been demonstrated to promote invasiveness of pancreatic cancer, the mechanisms remain poorly understood. In this study, we performed an analysis to determine the effects of Artemin on modulating tumor cell metastatic potential and invasion activity and explored its mechanisms in pancreatic cancer. We indicated that Artemin and CXCR4 were overexpressed in cancer tissues and widely expressed in pancreatic cancer cell lines. We observed that activation of ERK1/2 and Akt in Artemin-treated cells led to enhanced nuclear accumulation of NF-κB, which then induced CXCR4 expression. Through regulation of the expression of CXCR4, Artemin functionally promoted the migration and invasion in pancreatic cancer cells. The present study indicated that Artemin induced CXCR4 expression by activating Akt and ERK 1/2/NF-κB signaling, thereby modulating tumor cell metastatic potential and invasion activity in pancreatic cancer by regulating SDF-1α/CXCR4 axis. Artemin might be an effective and potent therapeutic target for pancreatic cancer metastasis, especially in perineural invasion.

GLMEEMSAL epitope common in different isoforms of hMena elicits in vitro activation of cytotoxic T cells and stimulates specific antitumor immunity in BALB/c mice

BackgroundAlternative expression of human ortholog of murine Mena (hMena) hMena/hMena11a and hMena/hMenaΔv6 isoforms regulate the invasiveness and metastatic potential of tumor cells. It is then important to identify epitopes of these proteins that can elicit antitumor immune response to contribute to the elimination of cells with metastatic potential. MethodsWe assayed the capacity of the peptide GLMEEMSAL, common in hMena/hMena11a and hMena/hMenaΔv6 isoforms, to generate an antitumor immune response through an in vitro vaccination system with mature dendritic cells (MDC) loaded with this peptide and in vivo immunization using a tumor model with the mammary adenocarcinoma JC cell line to induce tumors in BALBc mice. ResultsMDC loaded with the peptide GLMEEMSAL elicited strong proliferation and activation of CD8+ T lymphocytes. The CTLs generated with this system were capable to lyse specifically BrCa and CeCa cell lines expressing either hMena/hMena11a or hMena/hMenaΔv6. Immunization with GLMEEMSAL provided protective and therapeutic antitumor activity as well as increased survival in BALB/c mice. ConclusionThese results are highly relevant for the use of common peptides among the different isoforms of hMena to develop immunotherapy protocols to counteract the growth and metastatic potential of tumors with over-expression of hMena.

Blood Shear Stress Selects Metastasis-Initiating Cells with Metastatic Advantages

Metastasis is the most lethal and malignant state of cancer, which accounts for over 90% of cancer-related deaths. Tumor cells from the primary lesion metastasize to distant organs mainly through hematogenous dissemination. The presence and frequency of circulating tumor cells in blood are correlated with poor prognosis and overall survival. It has been proposed that circulating tumor cells contain a rare subset of metastasis-initiating cells (MICs) with the ability to generate clinically significant metastatic tumors in distant organs. These cells must hold survival advantages in the metastatic cascade and cancer stem cell properties that are essential in metastasis formation. Although the influence of blood circulation has been extensively studied, the roles of blood shear flow in metastatic potential of tumor cells are unclear. In particular, it remains largely elusive whether and how hematogenous shear stress selects MICs with metastatic advantages from bulk tumor cells. This study developed a circulatory microfluidic system, in which single breast cancer cell suspensions with differential metastatic potential were treated under various conditions to eradicate the majority of cells and optimize the levels of shear stress and circulating time. Tumor cells with high metastatic potential survived better in fluid shear flow in a force-magnitude and circulating-time dependent manner. Importantly, the surviving cells exhibited metastasis-initiating abilities, including high migratory and invasive potential, self-renewal and tumorigenicity, survival advantages, and chemoresistance. Mechanistically, fluid shear stress significantly affected epithelial-mesenchymal transition (EMT) plasticity and YAP/TAZ activity that influenced tumor cell survival in blood circulation. Silencing YAP/TAZ or critical transcription factors in EMT attenuated MIC properties of the surviving tumor cells selected by blood shear stress. Taken together, these results suggest that blood shear stress selects MICs with stemness and metastatic advantages that depend on EMT and YAP/TAZ.

Treatment with Src inhibitor Dasatinib results in elevated metastatic potential in the 4T1 murine mammary carcinoma model.

The src inhibitor Dasatinib has been widely studied as an anti-metastatic agent. The aims of this study were to examine the effect of Src inhibition on the metastatic potential of the 4T1 murine mammary carcinoma. Src is a non-receptor tyrosine kinase well-known to contribute to the metastatic potential of tumour cells. It does so through alteration of signalling pathways important to metastasis. Elevated levels of Src are common in many cancer types, and have been correlated with tumour progression and poor patient prognosis. This study examined whether disruption of the Src signalling pathway could inhibit metastases formation. The Src inhibitor Dasatinib was evaluated in vitro and in vivo using the highly metastatic 4T1 murine mammary adenocarcinoma cell line. In vitro assays included growth curve, western blot, migration, and invasion assays. In vivo assays included intradermal and tail vein injection models. In vitro data were analysed using one-way ANOVA with Dunnett's multiple comparisons in GraphPad Prism 6.0. In vivo data were analysed using GraphPad Prism 6.0, using the Wilcoxon matched pairs test. Dasatinib is effective at inhibiting in vitro phosphorylation of Src, migration and invasion in the 4T1 cell line, as well as angiogenesis in vivo. In vitro treatment with Dasatinib impaired the metastatic ability of tumour cells as assessed by a tail vein injection model. However, both the syngeneic BALB/c and the athymic nu/nu mice receiving oral doses of the drug developed significantly higher numbers of 4T1 lung metastases. This effect was not seen in a different breast carcinoma cell line, the MDA-MB-231-4175-LM2, nor was this effect seen in the murine fibrosarcoma KHT cell line. The 4T1 cell line is not an appropriate model to study Src inhibition.

Quantitative assesment of coexpression of cytokeratins and mesenchimal cells marker vimentin in serous ovarian cancer

Background. Epithelial-mesenchymal transition (EMT) is a factor related to metastatic potential of tumor cells. The most distinguishing feature of this transformation is expression of protein vimentin, which is not common for epithelial cells. Data of EMT level and clinical significance of the marker is ambiguous in prognosis of tumor different localization including ovarian cancer. Objective is characterization of EMT in ovarian cancer tissue based on quantitative analysis of co-expression level of epithelial cytokeratins and mesenchymal cells marker vimentin. Materials and methods. A quantitative assessment of co-expression level of cytokeratins and vimentin was performed by double immunofluorescence staining method (58 surgery specimens of ovarian cancer stage III), associated with flow cytometry. Results. Double immunofluorescence staining method, developed and used in the current study, was used for quantitative assessment of EMT level based on value of cytokeratin and vimentin co-expression in epithelial cells. Epithelial cells co-expressed these markers, were detected in 100 % tumor investigated with individual value differences from 10 to 86 %. Average co-expression level of cytokeratins and vimentin in subgroups with high and low level value related to median 42 % significantly varied and were 28,5 ± 7,5 and 56,3 ± 11,8 % (p = 0,02) respectively. Conclusions Serous ovarian cancer is molecular heterogeneous group with principal differences in level of EMT tumor phenotype between various patients. In half of the cases the disease is characterized by high metastatic potential of tumor cells. Co-expression of cyto-keratins and vimentin in all the tumors investigated is evidenced clinical significance of this molecular characteristic in ovarian cancer pathogenesis.