Estimation of cancer cell migration in biomimetic random/oriented collagen fiber microenvironments

Increasing data indicate that cancer cell migration is regulated by extracellular matrixes and their surrounding biochemical microenvironment,playing a crucial role in pathological processes such as tumor invasion and metastasis.However,conventional two-dimensional cell culture and animal models have limitations in studying the influence of tumor microenvironment on cancer cell migration.Fortunately,the further development of microfluidic technology has provided solutions for the study of such questions.We utilize microfluidic chip to build a random collagen fiber microenvironment(RFM)model and an oriented collagen fiber microenvironment(OFM)model that resemble early stage and late stage breast cancer microenvironments,respectively.By combining cell culture,biochemical concentration gradient construction,and microscopic imaging techniques,we investigate the impact of different collagen fiber biochemical microenvironments on the migration of breast cancer MDA-MB-231-RFP cells.The results show that MDA-MB-231-RFP cells migrate further in the OFM model compared to the RFM model,with significant differences observed.Furthermore,we establish concentration gradients of the anticancer drug paclitaxel in both the RFM and OFM models and find that paclitaxel significantly inhibits the migration of MDA-MB-231-RFP cells in the RFM model,with stronger inhibition on the high concentration side compared to the low concentration side.However,the inhibitory effect of paclitaxel on the migration of MDA-MB-231-RFP cells in the OFM model is weak.These findings suggest that the oriented collagen fiber microenvironment resembling the late-stage tumor microenvironment is more favorable for cancer cell migration and that the effectiveness of anticancer drugs is diminished.The RFM and OFM models constructed in this study not only provide a platform for studying the mechanism of cancer development,but also serve as a tool for the initial measurement of drug screening.

27-Hydroxycholesterol-induced EndMT acts via STAT3 signaling to promote breast cancer cell migration by altering the tumor microenvironment

Objective:The endothelial to mesenchymal transition(EndMT)plays a major role in cancer metastasis by regulating the complexity of the tumor microenvironment(TME).Here,we investigated whether 27-hydroxycholesterol(27 HC)induces EndMT in endothelial cells(ECs).Methods:EndMT markers in the human microvascular endothelial cell-1(HMEC-1)cell line and human umbilical vein endothelial cells(HUVECs)stimulated with 27 HC were evaluated with Western blot.Epithelial to mesenchymal transition(EMT)markers in breast cancer(BC)cells cultured in conditioned medium were investigated with quantitative real time polymerase chain reaction(qRT-PCR).The MMP-2 and MMP-9 mRNA expression and activity were detected with qRT-PCR and gelatin zymography assays,respectively.The effect of activated STAT3 on 27 HC-induced EndMT was validated by Western blot,immunofluorescence staining,and cell transfection assays.The migration ability of BC cells was evaluated with Transwell assays.Results:We found that 27 HC induced EndMT in HMEC-1 and HUVECs,and 27 HC-induced EndMT facilitated EMT and BC cell migration.The 27 HC-induced EMT of BC cells also promoted EndMT and HUVEC migration.Investigation of the underlying molecular mechanisms revealed that STAT3 knockdown repressed EndMT in HUVECs as well as migration in BC cells induced with 27 HC.In addition,C646 and resveratrol,inhibitors of STAT3 acetylation,repressed the expression of Ac-STAT3,p-STAT3,and EndMT markers in HUVECs exposed to 27 HC;these HUVECs in turn attenuated the migration ability of BC cells in 27 HC-induced EndMT.Conclusions:Cross-talk between 27 HC-induced EndMT and EMT was observed in the TME.Moreover,activation of STAT3 signaling was found to be involved in 27 HC-induced EndMT.

SLC38A1 Promotes Proliferation and Migration of Human Colorectal Cancer Cells

Current studies have demonstrated that SLC38A1 proteins play a causal role in neoplastic cell transformation. The twofold aim of this study was to provide insight into whether a variance in the expression of SLC38A1 exists between human colorectal cancer and healthy human tissues and to determine how silencing or overexpressing the SLC38A1 gene could affect the proliferation, viability and migration of colorectal cancer cells. Immunohistochemical staining was used to analyze the expression of SLC38A1 in colorectal cancer tissues and adjacent normal mucosa in 77 patients who underwent surgical resection. The expression of SLC38A1 in colorectal cancer tissues and cell lines was detected using RT-PCR and Western blotting. Two colorectal cancer cell lines SW480 and HCT116 were used to examine whether silencing SLC38A1 with si RNA and overexpressing SLC38A1 with sh RNA could affect cell viability and migration. As a result, the SLC38A1 protein was very low or undetectable in the normal colon mucosa. In contrast, strong staining of SLC38A1 protein was found in the cytoplasm in 79.2% colorectal cancer samples. More pronounced SLC38A1 expression in colorectal cancer tissues was significantly associated with tumor node metastasis（TNM） stage. Inhibition of SLC38A1 reduced tumour growth and suppressed proliferation and migration of SW480 cells. In contrast, overexpression of SLC38A1 had the opposite effects on HCT116 cells. SLC38A1 is overexpressed in colorectal cancer, which suggests that it is associated with tumour progression. These results encourage the exploration of SLC38A1 as a target for intervention in colorectal cancer.

Inhibitory activities of plumbagin on cell migration and invasion and inducing activity on cholangiocarcinoma cell apoptosis

Objective: To investigate the cytotoxic, apoptotic and inhibitory activities on cell migration and invasion of plumbagin in the human cholangiocarcinoma(CCA) cell line(CL-6) in comparison with human embryonic fibroblast cell line(OUMS). Methods: Cytotoxicity activity was evaluated using MTT assay. Inhibitory effect on cell migration and invasion were investigated using label-free real-time cell analysis and QCM ECMatrix cell invasion chamber, respectively. Apoptotic activity was evaluated using flow cytometry and Cell Event? Caspase 3/7 assay. Results: Based on results of the cytotoxicity test in CL-6 cells, 50% inhibitory concentration(IC_(50), Mean±SD) values of plumbagin and the standard drug 5-fluorouracil were(24.00±3.33) and(1 036.00±137.77) μmol/L, respectively. The corresponding values for OUMS cells were(57.00±5.23) and(2 147.00±209.98) μmol/L, respectively. The selectivity index was 2.28. The inhibitory activities of plumbagin on cell migration and invasion were potent and concentration-dependent with IC_(50) of 25.0 μmol/L and complete inhibition at 25.0 μmol/L. Flow cytometry analysis showed that plumbagin at 12.5 μmol/L(half IC_(50)) induced CL-6 cell apoptosis(43.24% of control) through stimulation of caspase 3/7 activities. Complete cell apoptosis was observed at 12.5 μmol/L. Conclusions: The cytotoxic activity and inhibition of migration and invasion including apoptosis induction in the human CCA cell line(CL-6) suggest that plumbagin could be a promising candidate for CCA chemotherapeutics. However, its relatively low selective cytotoxic effect on CCA cells is a major concern.

Effects of Exogenous VEGF_(165)b on Invasion and Migration of Human Lung Adenocarcinoma A549 Cells

Vascular endothelial growth factor 165 （VEGF165）-mediated autocrine stimulation of tumor cells enhances the progression to a malignant phenotype. VEGF165b competes with VEGF165 and binds to vascular endothelial growth factor receptor （VEGFR）, resulting in inhibition of downstream signal transduction pathways. This study was designed to investigate the role of VEGF165b in the migration and invasion of human lung adenocarcinoma A549 cells. The full-length of VEGF165b was constructed and cloned into an expression plasmid （pVEGF165b）, and then transfected into A549 cells. Dimethylthiazolyl-2, 5-diphenyltetrazolium bromide （MTT） assay was used to detect the effect of VEGF165b on proliferation of transfected cells. Reverse transcription polymerase chain reaction （RT-PCR） was employed to examine the effect of VEGF165b on the expression of VEGF165 in transfected cells. Wound-healing assays were used to investigate the effect of VEGF165b on migration of transfected cells. Matrix metalloproteinase （MMPs） activity assay and in vitro invasion assay were used to determine the role of VEGF165b in invasion of transfected cells. There was no significant change in proliferation of A549 cells after transfection of pVEGF165b, but the expression of VEGF165, migration and invasion in A549 cells were inhibited. Furthermore, exogenous VEGF165b inhibited the activity of MMP9 in the supernatant of A549 cells and the subsequent invasion capacity of those cells. We therefore conclude that exogenous VEGF165b can inhibit the expression of VEGF165, as well as the migration and invasion of A549 cells, but has no effect on the proliferation of A549 cells.

Knockdown of GRHL3 Inhibits Activities and Induces Cell Cycle Arrest and Apoptosis of Human Colorectal Cancer Cells

The Grainyhead-like 3（GRHL3） is involved in epidermal barrier formation, neural tube closure and wound repair. Previous studies have suggested that GRHL3 has been linked to many different types of cancers. However, to date, its effects on human colorectal cancer（CRC） has not been clarified yet. Our microarray analysis has indicated predominant GRHL3 expression in CRC. The purpose of this study was to investigate the expression and significance of GRHL3 in CRC tumorigenesis using CRC tissues and paired paracancerous tissues, as well as using distinct CRC cell lines（HT29 and DLD1）. We observed increased GRHL3 expression at both m RNA and protein levels in CRC tissues and CRC cell lines using quantitative real-time polymerase chain reaction（q RT-PCR） and Western blotting. Moreover, silencing GRHL3 with si RNA could suppress CRC cell proliferation, viability and migration in vitro. We also found that knockdown of GRHL3 could promote cell cycle arrest at G0/G1 phase in HT29 cells and DLD1 cells, and induce cell apoptosis in HT29 cells. Together, our study revealed the down-regulation of GRHL3 in vitro could inhibit CRC cell activity and trigger cell cycle arrest at G0/G1 phase and apoptosis.

Effects of bisphenol compounds on the growth and epithelial mesenchymal transition of MCF-7 CV human breast cancer cells

Bisphenol-A（BPA） has been considered as an endocrine disrupting chemical（EDC） because it can exert estrogenic properties.For bisphenol-S（BPS） and bisphenol-F（BPF） that are BPA analogs and substitutes,their risk to estrogendependent cancer has been reported rarely compared with the numerous cases of BPA.In this study,we examined whether BPA,BPS,and BPF can lead to the proliferation,migration,and epithelial mesenchymal transition（EMT） of MCF-7 clonal variant（MCF-7 CV） breast cancer cells expressing estrogen receptors（ERs）.In a cell viability assay,BPA,BPS,and BPF significantly increased proliferation of MCF-7 CV cells compared to control（DMSO） as did17β-estradiol（E2）.In Western blotting assay,BPA,BPS,and BPF enhanced the protein expression of cell cycle progression genes such as cyclin D1 and E1.In addition,MCF-7 CV cells lost cell to cell contacts and acquired fibroblast-like morphology by the treatment of BPA,BPS,or BPF for 24 hours.In cell migration assay,BPA,BPS,and BPF accelerated the migration capability of MCF-7 CV cells as did E2.In relation with the EMT process,BPA,BPS,and BPF increased the protein expression of N-cadherin,while they decreased the protein expression of Ecadherin.When BPA,BPS,and BPF were co-treated with ICI 182,780,an ER antagonist,proliferation effects were reversed,the expression of cyclin D1 and cyclin E1 was downregulated,and the altered cell migration and expression of N-cadherin and E-cadherin by BPA,BPS,and BPF were restored to the control level.Thus,these results imply that BPS and BPF also have the risk of breast cancer progression as much as BPA in the induction of proliferation and migration of MCF-7 CV cells by regulating the protein expression of cell cycle-related genes and EMT markers via the ER-dependent pathway.

The role of YAP in the control of the metastatic potential of oral cancer

The Yes-associated protein(YAP)is a downstream effector of the Hippo pathway and acts as a key transcription co-factor to regulate cell migration,proliferation,and survival.The Hippo pathway is evolutionarily conserved and controls tissue growth and organ size.Dysregulation and heterogeneity of this pathway are found in cancers,including oral squamous cell carcinoma(OSCC),leading to overexpression of YAP and its regulated proliferation machinery.The activity of YAP is associated with its nuclear expression and is negatively regulated by the Hippo kinase-mediated phosphorylation resulting in an induction of its cytoplasmic translocation.This review focuses on the role of YAP in OSCC in the context of cancer metastatic potential and highlights the latestfindings about the heterogeneity of YAP expression and its nuclear transcription activity in oral cancer cell lines.The review also discusses the potential target of YAP in oral cancer therapy and the recentfinding of the unprecedented role of the desmosomal cadherin desmoglein-3(DSG3)in regulating Hippo-YAP signaling.

Mechanical transmission enables EMT cancer cells to drive epithelial cancer cell migration to guide tumor spheroid disaggregation

Cell phenotype heterogeneity within tumor tissue,especially which due to the emergence of epithelial-mesenchymal transition(EMT) in cancer cells,is associated with cancer invasion and metastasis.However,our understanding of the cellular mechanism(s)underlying the cooperation between EMTcell and epithelial cancer cell migration remains incomplete.Herein,heterotypic tumor spheroids containing both epithelial and EMT cancer cells were generated in vitro.We observed that EMT cells dominated the peripheral region of the self-organized heterotypic tumor spheroid.Furthermore,our results demonstrated that EMT cells could serve as leader cells to improve the collective migration efficiency of epithelial cancer cells and promote dispersion and invasion of the tumor spheroids,which was regulated by the force transition between EMT cells and epithelial cancer cells.Mechanistically,our data further suggest that force transmission is mediated by heterophilic N-cadherin/E-cadherin adhesion complexes between EMT and epithelial cancer cells.Impairment of N-cadherin/E-cadherin adhesion complex formation abrogated the ability of EMT cells to guide epithelial cancer cell migration and blocked the dispersion of tumor spheroids.Together,our data provide new insight into the mechanical interaction between epithelial and EMT cancer cells through heterophilic cadherin adhesion,which enables cooperative tumor cell migration,highlighting the role of EMT cells in tumor invasion.

Effects of human microRNA-181a-5p on proliferation and migration of gastric cancer cells

Objective To preliminarily explore the effects of human microRNA-181a on migration of gastric cancer cells and its mechanism.Methods The expression of miRNA-181a-5p in gastric cancer cell line GC9811 and peritoneal high metastasis gastric cancer cell line GC9811-P were tested by quantitative real-time polymerase chain reaction(qRT-PCR).GC9811 cell line was

A review of microfluidic approaches for investigating cancer extravasation during metastasis

Metastases,or migration of cancers,are common and severe cancer complications.Although the 5-year survival rates of primary tumors have greatly improved,those of metastasis remain below 30%,highlighting the importance of investigating specific mechanisms and therapeutic approaches for metastasis.Microfluidic devices have emerged as a powerful platform for drug target identification and drug response screening and allow incorporation of complex interactions in the metastatic microenvironment as well as manipulation of individual factors.In this work,we review microfluidic devices that have been developed to study cancer cell migration and extravasation in response to mechanical(section‘Microfluidic investigation of mechanical factors in cancer cell migration’),biochemical(section‘Microfluidic investigation of biochemical signals in cancer cell invasion’),and cellular(section‘Microfluidic metastasis-on-a-chip models for investigation of cancer extravasation’)signals.We highlight the device characteristics,discuss the discoveries enabled by these devices,and offer perspectives on future directions for microfluidic investigations of cancer metastasis,with the ultimate aim of identifying the essential factors for a‘metastasis-on-a-chip’platform to pursue more efficacious treatment approaches for cancer metastasis.