A Study On Metastasis Of Colorectal Cancer And Fibroblast To Myofibroblast Transformation Induced By CLIC4

BackgroudColorectal cancer (CRC) is a prevalent GI tumor with a worldwide occurrence of1,020,000and a annual mortality of53,000. CRC as the fourth most common cancer holds an occurrence of17,000in China and90%of mortality of CRC patiens are contributed by metastasis. It is critical for researchs to identify effective methods to achieve early diagnosis, metastasis prevention and better survival.Cancer cells must go through cell structure remodeling, adhesive dysfunction and movement enhancement to obtain metastasis, which is a multistep process managed by various elements inside and out of cells. Cancer stem cells is a subpopulation of cells within the cancer bulk, can self-renew, differentiate into multiple lineages, and drive tumor growth. CSCs as a promising tactics has receives growing attention from scientific community. However CSCs alone are not capable of sowing tumor cells to distant organ, metastasis requires suitable niche both in the local lesion and distant organic. Tumor Niche is composed by endocellular cells, fibroblast, inflammatary cells, smooth muscle cells and others ECM members as well as their excreted factor The visionary ’soil and seed’ hypothesis adjust by Fidler in2003has clearly demonstrate that the potential of a tumour cell to metastasize depends on its interactions with the homeostatic factors that promote tumour-cell growth, survival, angiogenesis, invasion and metastasis.In other words, the outcome of metastasis depends on multiple interactions (’cross-talk’) of metastasizing cells with homeostatic mechanisms, which the tumour cells can usurp. Therapy of metastases,therefore, should be targeted not only against the cancer cells themselves, but also against the homeostatic factors that promote tumour-cell growth, survival, angiogenesis, invasion and metastasis.This study is part of a whole research protocol designed to gain a closer look at colorectal cancer stem cells and microenvironment. Previous srtudy has isolated CSC colones from22colorectal tumor specimens and divided them into MCSC(metastatic cancer stem cells, MCSC) and non-MCSC(non-metastatic cancer stem cells) according to their metastatic potential in orthotopic xenograft mouse model. Two-dimensional electrophoresis (2-DEE) separation and mass spectrometry (MS) were employed to compare the protomics of the two panels of CSCs. We fortunately have identified nine proteins involved in metastatic process, including CLIC4(chlorideintracellular channel protein4). In survival analysis of413cases of colorectal cancer with10-year follow-up, CLIC4was found highly correlated with postoperative metastasis and patiens with a low CLIC4express stand a better chance of long-term survival. Though there are growing evidences revealing that CLIC4is related to fibroblast to myofibroblast in various malignant tumors, no pertaining study has appeared on colorectal cancer. This stusy was aimed to explore the role CLIC4playing in colorectal cancer associated fibroblast to myofibroblast differentiation and the effect CAF have on colorectal cells with different CLIC4expression. Methods1. Isolation, cultivation and identification of colorectal cancer assciated fibrobalst.Colorectal tumor tissue obtained from surgical specimen were cut into small cube of lmm3, followed by cultivation in20%FBS DMEM with Penicillin-Streptomycin (1μg/ml) for a week. After5or6Media changes, cells of polarized, elongate shape with narrow protrusions and wide lamellas can be seen microscopically. Potential fibroblast cells were subjected to flow cytometry to detect the expression of Keratin, Desmin and FSP..2. Construction of CLIC4-overexpressed colorectal cell linesC17and C40cells maintained in DMEM supplemented with10%FBS without antibiotics, on10cm culture plates to60%confluency. Sixteen hours later transfection of cells took place. As a transfection reagent Lipofectamine was used according to the manufacture’s protocol.3μg of the envelop plasmid pLVTHM,3μg of the packaging plasmid pLVTHM-CLIC4,2.5μg of the Rev-expression plasmid pRSV/Rev and10μg of the shRNA lentiviral construct were added in1mL of DMEM medium, serum and antibiotics free.40μL of lipofectamine was diluted in1mL of DMEM without serum and antibiotics and incubated for5min. DNA and lipofecta分é'Ÿe parts were mixed and incubated for20min before added to the cells.24hours later, the medium was discarded and replaced with5mL of DMEM with10%FCS, without antibiotics. Virus was stored at-80. cells were plated in6well plates and16hours later were viral transduced. This was performed using1mL of lentiviral supernatant, diluted in1mL of DMEM complete medium and4μL of protein sulphate. Cells were centrifuged at2,500rpm for90min at RT. The medium was replaced with fresh complete DMEM and changed again24hours later3. Effect of various CLIC4expression on fibroblast differentiation. Colorectal cancer cells with different CLIC4expression were cocultured with cancer associated fibroblast and fibrblast cell lines for1week. MACS protocol was emplayed to separate cocultured cells for futher Western Blot and Transwell study. To explore effects each cell has on their cocultured counterpart, Western Blot test for a SMA expression of cancer cells before and after cocultivation and a SMA expression of fibroblast cells after seperation were performed. Immunofluorescence of mixture cells were conduct to confirm the result of Western Blot. Colorectal cancer cells before and after cocultivation were subjuct to Transwell test to examine the effctts that fibroblast have on cancer invasion.To better understand the in vivo effect of coculture system on its individual members, subcutaneous xenograft mouse model were constructed to compar the tumogenesis of different cell mixtures.Results1. Isolation, cultivation and identification of colorectal cancer assciated fibrobalst.Colorectal cancer associated cells were sucessfully obtained from surgical spicimen. Under microscopic examinaton, fibroblasts like cells were seen. flow cytometry reveal that Desmin, Keratin and FSP were deteced in0.7%,0.8%and99.5%of cells respectively, which confirm the cells as fibroblasts..2. Construction of CLIC4-overexpressed colorectal cell linesAll steps of lentivirus overexpression system were conducted following proper instruction, RT-PCR and Western Blot test prove the efficiency of lentiviral transfection. C40-CLIC4obtain a significantly higher CLIC4expression than other cells, while C40has the lowest expression of all5cells.3. Effect of various CLIC4expression on fibroblast differentiation. fibroblast to myofibroblast differentiation is found in both CCAF and MRC-5 cells after cocultivation with colorectal cancers, however the increase of a-SMA is consistent with the trend of CLIC4expression of colorectal cells. CCAF and MRC-5also achieve higher a-SMA expression after treatment with recombinant huamn CLIC4, and the increse is consistent with the doses of the CLIC4, which highly suggest that CLIC4paly a critical role in fibroblast to myofibroblast in colorectal cancerl. In vivo xenograft model show C40-CLIC4with highest CLIC4expression can chieve largest mean tumor bulk and fastest tumor growth in coculture mixture after30days’ observation.the tumor volume and growth pattern of other4combination also confirm the in vitro result.Conclution1. primary culture of colorectal cancer associated fibroblast can be sucessfully established with10%FBS DMEM.2. CLIC4is highly involved in invasion and metastasis of colorectal cancer.3. CLIC4-overexpressed colorectal cancer cells can significantly increase the fibroblast to myofibroblast differentiation.4. colorectal cancer associated fibroblast improve migration ability of cancer cells and the increase is correlated with CLIC4expression.5. colorectal cancer with CLIC4overexpression show more impotent tumorgenesis with the co-stimulation of CCAF.