Mice Liver Sinusoidal Endothelial Cells Promote Invasiveness And Metastases Of Colorectal Cancer Stem Cells

Colorectal cancer (CRC) is the third most common malignancy worldwide. Approximately one half of colorectal cancer patients develop hepatic metastases involving of causes two-thirds of deaths. Metastasis brings out therapeutic failure and a poor survival rate. Therefore, it seems that effective anti-metastatic therapies could fight against cancer in improving medical administration and patients’ survival resulting from understanding metastatic mechanism. Various factors play important roles in the multiple steps of metastatic procedure associated with many genes turning on or turning off. Cancer cells orchestrate microenvironmental elements elaborately adjusting secondary tumors products, in which target organ provides a suitable dwelling place is a key role for cancer cell residence. Cancer cells begin with detaching from primary site, invading lymphatic or blood vessels, surviving in the circulation, adhering and exudating target organ terminated vessels, immigrating into specific distant site, and ultimately establishing a secondary tumor. In this process, if any of the steps is blocked, metastasis could not happen. As a matter of fact, many evidences have been revealed that very few cancer cells are able to make a successful journey to produce metastasis. These small subsets of cancer cells are named cancer stem cells (CSC), which were first identified and isolated from leukaemia and multiple myeloma. CSC as a subpopulation of cancer cells has capacities of self-renewal, proliferation and differentiation and initiating new tumors. More and more evidences in the last decade have been demonstrated that CSC isthe culprits of cancer. Since a tumor bulk consist of heterogeneous cells subsets, CSC show a distinct different differentiation process which produces progenitor cells, transit-amplifying cells and terminated matured cancer cells. In2007, metastasic cancer stem cell (MCSC) was isolated and identified from pancreatic carcinoma by Hermann et al. A new concept is gradually accepted by pathologists and clinicians that MCSC may be the wanted crimer committed to metastases.CSC/MCSC is sufficient to develop metastases at distant sites but they should depend on microenvironment of primary and secondary sites. Microenvironments support CSC/MCSC detaching from primary sites and entering into circulation. Blood vessel endothelial cells preserve CSC/MCSC adhering to target organ and promoting invasiveness and implanting into the interstitial tissue. Additionally, CSC/MCSC might also adapt to the adoptive soil so that they could resident the place in which for self-renewal, amplification and differentiation. Now it is well-understood that the CSC/MCSC is the real seed of tumor metastasis. And the microenvironment is the soil of tumor growth. The malignant cells do not exist in isolation but are intimately connected to the non-tumor cells of their microenvironment; these cells include endothelial cells, leukocytes, macrophages, fibroblasts, bone marrow-derived cells and other tissue components, including adipocytes and neural elements and so on, all of them composite a supporting growth and stemness chamber for CSC which is defined as niche in recent years. Epithelial-mesenchymal transition (EMT) plays an important role in invasiveness and metastasis; conversely, CSC participates into EMT process not only maintaining self-renewal capacities but also promoting distant metastases of CSC. The living microenvironment elaborates spatial-temporal factors for regulating CSC growth, differentiation, migration and invasiveness. Endothelial cells of microcirculation might make a decision of metastatic target organ and select given CSCsto arrest blood vessel terminals. Tumor cells rely on EMT features to obtain metastatic potentials at first, then tumor cells depolarize and detach from extracellular matrix to leave from primary site and enhance their motility. It is a possible therapeutic method to block metastasis with interrupting balance between CSC/MCSC and niche, abrogating CSC/MCSC sticking to endothelia and rendering EMT to mesenchymal-epithelial transition (MET).In our previous studies, colorectal cancer stem cell clone spheres were isolated from SW480cell lines by limiting dilution and serum-free culture, and then CSC with different metastatic potentials were identified from xenografting into nude mice caecal wall for hepatic metastatic experiments. Therefore, we have obtained the metastasis cancer stem cells (MCSC) and non-metastasis cancer stem cells (nMCSC). Liver sinusoidal endothelial cells (LSEC) were disassociated from nude mice livers by magnetic active cell sorting method with anti-CD146microbead. LSEC samples were examined to make sure the cells homogeneity and expression of von-Willebrand factor (vWF) by immunocellular chemistry (ICC) according to LESC morphology and phenotype. In addition, apoptotic cells of LESC were labeled with Hochest33342under observation of fluorescence microscope. The colorectal cancer cells and LSEC were put into one Petri dish for culturing. In vitro, series experiments were compared the co-cultured cells adhesion abilities in parental tumors cells and LSEC or MCSC with different metastatic potentials and LSEC by Trans well chamber invasive assays and confocal laser-microscopic examinations. Then, surgical orthotopic implantation (SOI) nude mice models of colorectal cancer were applied to compare the metastatic potentials between SW480and SW480/LSEC, MCSC and MCSC/LSEC, nMCSC and nMCSC/LSEC. Finally, in vivo assays revealed that earlier and more diffuse secondary foci produced in mice livers could result from LSEC promoting EMT to control invasiveness and metastases of colorectal CSC.Methods1. Isolation, cultivation and identification of LSECs> Magnetic active cell sorting (MACS) was used to isolate LSEC from nude mice livers which were removed under sterile condition and cut into species, then the livers were digested with type IV collagenase in order to get single cells suspension at37℃. Single cells were subsequently captured by anti-CD146microbead linked to LSEC with MACS. These cells were identified the cells homogeneity and expression of von-Willebrand factor (vWF) by immunocellular chemistry (ICC). The purity of LSECs of isolation was examined with fluorescence-activated cell sorter (FACS). In addition, apoptotic cells of LESC were labeled with Hochest33342/PI double staining under observation of fluorescence microscope.2. Adhesive pattern of different metastatic potentials between colorectal cancer cells and LSEC106cells of SW480, MCSCs or nMCSCs were co-cultured with LSECs in petri dishes. Invasive abilities were assayed the co-cultured cells with transwell chamber to assess tumor cells chemotaxis of LSECs. The adhesive patterns and morphological changes were obtained from immunofluorescent observations of confocal laser-microscopic examinations.3. Metastatic potentials of SW480and SW480/LSECs, MCSC and MCSC/LSEC, nMCSC and nMCSC/LSEC1×106SW480, MCSC or nMCSC in a logarithmic phase were respectively injected into nude mice subcutaneously. The other three groups were injected in the same amount of cells SW480/LSECs. MCSC/LSEC. nMCSC/LSEC. Tumors volume was regularly observed at intervals to compare the growth rate of different cells. When the tumors were up to8～10mm in diameter, they were removed in aseptic conditions and then put into ice-cold serum-free RPMI-1640medium. Every tumor mass of lmm3was sutured with7/0suture needle to cecal wall in every one of six anesthetized6-week-old BALB/c-nu nude mice with1%pentobarbital (0.1ml/kg). Then, the cecum was put back into the abdominal cavity and sutured the abdominal incision. Thirty-eight days later, all the mice were executed, specimens of various organs from all the mice were observed under gross examination and light microscope to confirm metastatic foci.Results1. Isolation, cultivation and identification of LSECHomogeneous LSEC obtained from MACS of CD146microbeads and immunocytochemistrical assays. The isolated cells were typical cobblestone in morphology. Expression of von-Willebrand factor (vWF) in the LSEC was up to95.0±0.4%by ICC. The survival time of LSEC was three weeks in vitro. Under observation of fluorescence microscope, the apoptotic cells were not obviously along with the incubation time by Hochest33342.2. The adhesive pattern of different metastatic potential of colorectal cancer cells and LSECEvery field of stuck LSEC to tumor cells from SW480, MCSC and nMCSC was95.667±6.028,160.33±7.37and76.67±10.12respectively by two-dimension co-culture systems evaluation, and statistical data was demonstrated that MCSC were the most powerful cells to attract LSEC (F=89.712, P＜0.001). nMCSC also showed a weakly attractant capacity to LSEC. The difference of three groups are statistically significant(F=89.712, P＜0.001). When the upper chamber of Transwell invasive system was filled with SW480, MCSC and nMCSC respectively, and the lower chambers were laid up with LSEC, MCSC were the most number of tumor cells which penetrated into the lower chambers with cell counting and confocal laser-microscopic observation.3. Compare the invasion and metastasis potentials between SW480and SW480/LSECs, MCSC and MCSC/LSEC, nMCSC and nMCSC/LSECSubcutaneous tumor formation experiments revealed that the simple cancer cells and the mixed cells had no statistical significance (p>0.05). Surgical orthotopic implantation (SOI) nude mice models of colorectal cancer were established to compare metastatic potentials between SW480and SW480/LSECs, MCSC and MCSC/LSEC, nMCSC and nMCSC/LSEC. Different metastatic potentials were found in these six groups in SOI nude mice. Among the six groups, SW480/LSECs, MCSC/LSEC, nMCSC/LSEC had higher metastatic potential than the corresponding groups as SW480, MCSC and nMCSC.4. Spindle cells transition indicated EMT properties of MCSC attributing to an enhanced metastatic potential by LSECAll of the tumor cells demonstrated spindle shaped transitions during cecal wall invasion from SW480/LSEC, MCSC/LSEC and nMCSC/LSEC nude mice transplantation, but not shown from SW480, MCSC and nMCSC nude mice. This portrait indicated EMT transformation resulting from sarcoid changes derived from LSEC stimulation. MCSC/LSEC nude mice presented the most number of spindle cells, which suggested a possible reason of earlier metastases and more secondary nodules of liver governed from EMT at primary site.Conclusion1. MACS is an effective method to easily isolate homogeneous LSEC from nude mice livers. LSEC cultured in vitro displayed stable morphologic pattern and phenotype.2. Different metastatic potential tumor cells demonstrated different adhesive abilities sticking to LSEC. MCSC had a powerful adhesion ability to attract LSEC, which enhanced distant implantations.3. LSEC did not modify tumorigenic capacities of SW480parental cells or CSC of colorectal cancer after xenografting experiments, but they did strengthen the metastatic potentials of colorectal CSC. Earlier and more diffuse metastatic nodules formation might result from enhanced EMT features of CSC at primary site by LSEC.New Findings1. MACS is an easy and successful method to isolate liver sinusoidal endothelial cells from nude mice livers.2. Metastatic cancer stem cells of colorectal cancer are susceptible to adhere target organ endothelium and in favor of invasiveness and metastasis.3. Microcirculational endothelial cells of target organ promoted metastasis ability of colorectal cancer stem cells might come from an enhanced EMT feature.