Identification And Annotation Of Molecular Signature From Metastatic Cancer Stem Cells Of Colorectal Cancer

Background&ObjectiveColorectal cancer (CRC) is a leading cause of morbidity and mortality worldwide. Metastasis is the main cause compromising patients with colorectal cancer. In the course of the disease, many patients will present metastatic lesion in liver and lung. Before tumor cells from a primary tumor mass invade surrounding tissues and vessels, cells must acquire the ability to migrate. In fact, only a small fraction of cells released from a primary tumor can successfully form distant lesions. Without systematic study, researches on metastasis so far have mainly focused on genetic changes of single or few metastasis-related molecules or proteins. Little is known about the key factors triggering tumorigenic cells to initiate further invasion and metastasis. To clarify the molecular mechanism of metastasis in colorectal cancer, proteomic strategies with high reproducibility, including two-dimensional electrophoresis (2-DE) separation and mass spectrometry (MS) identification with advantage of high resolution, were employed to separate and identify differentially expressed proteins between highly and lowly metastatic subpopulations and pivotal factors associated with colorectal cancer metastasis. Once the key factors in metastasis were confirmed, effectual biotherapeutic targets and reliable diagnosis markers could be developed.It was first extensively documented in leukaemia and multiple myeloma that only a small subset of cancer cells is capable of consecutive proliferation. Cancer stem cells (CSCs), a subpopulation of cells within the cancer bulk, can self-renew, differentiate into multiple lineages, and drive tumor growth. As the whole cancer population is composed of heterogeneous cells, CSCs also exhibit a hierarchical differentiation process which produces progenitor cells, transit-amplifying cells and terminated matured cancer cells. Independent subclonal populations within the tumor are endowed with different functional properties, but only selected clones have the potential to metastasize to distant organs."Migrating cancer stem (MCS)-cells" was coined to name CSCs in possession of following capability:CSCs experience EMT to disseminate; disseminating CSCs retain stemness features to form metastatic colonies. Since CSCs is the tumor initiating cells, MCSC therefore is the source of tumor metastasis. Functional proteins and signal-processing mechanism of MCSC which are different from those of CSC maybe play a paramount role to illuminate the mechanism of metastasis.Available evidences has already revealed that cancer cells undergoing metastasis are driven by a group of related genes and their products, Molecular Signatures that composed of a group of related genes can reflect the process of metastasis much better. Molecular signature is a serial genes conferring particular biological characteristic, which systemically describes a cascade homeostasis more accurately than single gene pattern. Comparative proteomics is a powerful method to obtain molecular signature. At present, by the analyses of comparative proteomics, new candidate molecules were identified from prostate cancer, breast cancer, colon cancer, ovarian cancer and esophageal cancer, and some of them may become new molecular marker for diagnosis and treatment in the future. Nevertheless, how to isolate MCSC, which molecule can be identified for MCSC marker, why MCSC conceive metastatic capacity, all the researches works about that are very useful to interpret the mechanism of metastasis and to establish an effective anti-metastasis therapy.In this study,12colorectal cancer stem cell clone spheres were isolated from SW480cell lines by limiting dilution and serum-free culture. We have made an attempt to identify proteins associated with colorectal cancer metastasis by comparing the metastatic potentials among different clone spheres and their paternal cell line SW480through two-dimensional electrophoresis (2-DE) separation and mass spectrometry (MS). We fortunately have found out a series of protein cluster involved in metastatic process and addressed the question whether they are new proteins for metastasis. Moreover, we also set up the experiments to annotate the possible roles mediated by these new candidate proteins.Methods1. Isolation, cultivation and identification of colorectal cancer stem cell.Using the limited dilution and serum-free method, we cultivated single-cell-derived monoclonal cell clone spheres from the colorectal cancer cell line SW480. Western blotting, immunofluorescence and flow cytometry were conducted to detect the expression of CD133, CD44and CK7in the clone spheres. Self-renewness and differentiation of CSCs were confirmed with soft agar experiments and nude mice subcutaneous tumor forming assay.2. Metastatic potential comparison of CSCs clone spheres1×106single cell derived clone sphere and SW480which were in a logarithmic phase were injected into a subcutaneous nude mouse. When the tumor was up to about8mm in diameter, it was removed in aseptic conditions and then put into ice-cold serum-free RPMI-1640medium. One tumor mass of lmm3was sutured with7/0suture needle to cecal wall in every one of five anesthetized6-week-old BALB/c-nu nude mice with1%pentobarbital (0.1ml/kg). Then, put the cecum back into the abdominal cavity and sutured the abdominal incision.6-8weeks later, all the mice were executed, specimens of various organs from all the mice were observed under gross examination and light microscope.Nude mice subcutaneous tumor forming assay was performed to detect tumorigenicity of high and no metastasis clone sphere and SW480cell line; trans-well chamber assay were used to examine their in vitro mobility; expression of fibronectin, vimentin and E-cadherin were detected by western blot and immunofluorescence.3. Protein expression profiles of cancer stem clone spheres with different metastatic potential and their parent SW480were determined using2-DE analysis and MS identification.To better understand the mechanism underlying the colorectal cancer metastasis and to search potential markers for MCSC, differential proteome analysis on high and no metastasis clone sphere were conducted using various proteomics approaches. Western blot and semi-quantitative RT-PCR analysis were used to further verify candidate proteins in order to ensure the reliability of previous proteomic results.4. Expressional and functional verification of candidate proteins from colorectal MCSC.In our previous experiments, we screened a series of MCSC-associated proteins. Further confirmation and verification were needed to clarify their precise biological and clinical significance. Among the candidate protein, we selected interesting MCSC-associated protein CLIC4, ERp29, Smac and ISG15to investigate their function in colorectal cancer development and metastasis.Results1. Isolation, cultivation and identification of colorectal cancer stem cell.Cells from SW480were plated on1096-well plates at a concentration of a single cell per well with serum-free media. Wells containing either none or more than one cell were excluded from further analysis.12cancer stem cell clone spheres were collected. The colony-forming efficiency is1.2%(12/1000). The gold standard assay of cancer stem cell—serial transplantation in animal models in this study revealed that500cells from clone sphere can form tumor using nude mice subcutaneous tumor forming assay, but500cells from SW480failed to form tumor.2. Compare the invasion and metastasis potential of colorectal CSC clone spheres and SW480Surgical orthotopic implantation (SOI) nude mice model of colorectal cancer was established to compare metastatic potentials of12colorectal cancer stem cell spheroids and their parent SW480. Different metastatic potentials were found in12colorectal cancer stem cell clone spheres in SOI nude mice model of colorectal cancer. Among the clone spheres studied, one clone spheres with high metastatic potential isolated as L7, one clone sphere with no metastatic potential isolated as L4, were found in SOI nude mice model of colorectal cancer.Trans-well chamber assay showed highly metastatic cancer stem cell clone sphere L7have more enhanced mobility than no metastatic cancer stem cell clone sphere L4and that the difference of mobility between them was significant (F=117.857,P<0.001). Subcutaneous tumor formation experiment revealed highly metastatic cell sublines possessed higher metastatic potentials than no metastatic cell sublines (F=53.229, P=0.000).3. Compare EMT properties of colorectal MCSC and no-MCSC clone spheres Epithelial mesenchymal transformation (EMT) refers to the transfer of epithelial cells to fibroblasts or mesenchymal cells morphologically and the acquirement of metastatic ability, which is a basic physiological and pathological phenomena and playing an important role in embryonic development. We found that MCSC presented high expression of vimentin and fibronectin and low level of E-cadherin. MCSC develop systemic secondary cancerous mass may result from a stronger EMT capacity than that of nMCSC.4. Isolation of colorectal cancer metastasis cancer stem cell-associated proteins27CRC metastasis cancer stem cell-associated proteins were screened and identified using proteomic strategy based on2-DE analysis.16high expressed protein spots and11low expressed protein spots were detected in MCSC. Among these proteins, four proteins including CLIC4、Sma、ERp29、ISG15were found to be associated with metastasis. However, the roles of the four proteins were still unkown to be related with metastasis and CSC. The mRNA and protein expressions of CLIC4、Smac、ERp29、ISG15in MCSC、nMCSC and SW480cells detected by real-time PCR,western blot and immunofluorescence were consistent with2-DE analysis.5. Function annotation of molecular signature from metastatic cancer stem cells of colorectal cancerCLIC4, Smac, ERp29and ISG15were analyzed by immunohistochemistry in421cases of primary colorectal cancer with a follow-up period of10years. Results showed that CLIC4, Smac, ERp29and ISG15were closely correlated with metastasis of patients (P<0.05). Biological characteristic changes were verified by gene over-expression method with ervery one of the four proteins transfected into nMCSC of colorectal cancer. nMCSC-CLIC4, nMCSC-ERp29, nMCSC-Smac cells showed a significant proliferation compared with nMCSC in vitro plate colony assay. In addition, MCSC-ISG15cells had a significant increased ability to form colonies in plate as compared with MCSC cells. The result of in vitro and in vivo migration assay showed that MCSC-ISG15cells had significantly reduced invasiveness as compared with MCSC cells, nMCSC-CLIC4, nMCSC-ERp29, nMCSC-Smac cells showed significant increased invasiveness compared with nMCSC.Conclution1.12colonospheres from SW480were isolated and stably clutured by serum-free media and limited dilution method. It also indicated that limited dilution should be a successful method to separate cancer stem cells spheres.2. Different cancer stem cell spheroids from SW480exhibit heterogeneity in metastatic potential and tumorgenicity in in vitro assay, in vivo tumor growth and metastasis assays.3. Proteomic profile differences may give rise to the heterogeneity of metastasis.16over-expressed proteins and11under-expressed proteins in MCSC cells were screened and identified from MCSC cells proteins, W480cells proteins and nMCSC cells proteins using proteomic strategy based on2-DE analysis.4. CLIC4, ERp29, Smac and ISG15were closely correlated with metastasis of colorectal cancer. The four proteins may paly roles on maintaining sternness, migration and invasion of colorectal cancer MCSC. Moleular signature identified from MCSC predicts an unfavorable prognosis and metastasis of colorectal cancer patients.New Findings1. Successfully comfirmed molecular tags from MCSC of colorectal cancer.2. Molecular signature of MCSC provides a new target for diagnostic makers and treatment of metastasis after explicitation of relationship between cancer tissue expression and metastases. 3. Molecular signature of MCSC expressed in primary colorectal cancer tissues is related to poor survival and reveals that the molecular basis for metastatic prognosis, diagnosis and targeted therapy.4. MCSC is the culprit for metastatic colorectal cancer, which provides a new theoretical basis for anti-metastatic experiments and clinical treatment.