| Background/ObjectiveColorectal cancer (CRC) is one of the serious harm to human health and the most common malignant tumors. The disease incidence of CRC is the second malignant tumors in the western developing countries. With the change of dietary pattern and elevation of living standard in our country, the disease incidence and mortality of colorectal cancer showed a clear upward trend. However, CRC lost operation opportunity due to rapid progress, difficulties in early diagnosis of CRC and often the patient received treatment in the late stages. Some patients who received chemotherapy remain in relapsing period at the early stage, which directly lead to poor prognosis and high mortality. Thus, studying the pathogenesis of colorectal cancer and detecting therapeutic targets are very important for the treatment of CRC. The occurrence and development of tumor is a complicated process that involved many factors such as alteration of multiple genes and development of multiple steps. A recent study by Fang DD et al. discovered that CD133+colorectal cancer cells possessed stem cell characteristics, and evidence attested CD133as a marker of colorectal cancer stem cells (CCSCs). Dalerba P and His colleagues demonstrated CD44which was regarded as a CCSCs marker. CD133, CD44, ESA, ALDH1, etc, are common CSCs markers and among those, CD133is accepted by most scholars. CD133+cancer stem cells are effective method to enrich CCSCs, and had been used for many tumors. Colo205cells were cultured in SSM and SFM, and the expression of CD133and CD44was detected in our initial research, highlighted to be the foundation for studying CCSCs for this experiment.Representing a small subpopulation of the tumor mass, cancer stem cells (CSCs) are believed to have a capacity of self-renewal, proliferation, differentiation and strong tumorigenicity. Furthermore, they seem to be closely related with tumorigenesis, development, recurrence, metastasis, and drug resistance. CSCs are the main reason of tumor growth, tumor recurrence and metastasis. Therefore, CSCs can be a hot spot in tumor research. Tumor immune editorial theory considers that, there are interactions between the immune system and tumor cells. More over, tumor cell antigenicity is remodeled by the immune system and tumor cells affect the function of the immune system in the process of development of tumor. When the cells were detected malignant transformation, the interaction between the immune system and tumor cells is divided into three stages:clearance, equilibrium and escape. The immune system identifies the tumor cells and plays the killing effect before the occurrence of the tumor. However, CSCs might evade the immune system to suppress immune system or promote immune tolerance in tumor. Thus, there is a close relationship between tumor immune and CCSCs which is an important cause of tumor development.Cancer stem cell theory suggests that pathogenesis, metastases, recurrence of colon cancer is associated with CCSCs. However, the identification of colorectal cancer stem cells by immune system, the effect and mechanism of colorectal cancer stem cells in the induction of tumor immunoregulation and tolerance still remains unsolved. Furthermore, immunotherapy targeting colorectal cancer stem cells has not been used widely. Thus, the research is to study the relationship between colorectal cancer and tumor immunity, which would provide a new insight into tumor research.CD200have a function of immune regulatory. CD200is a type I membrane glycoprotein and highly conserved member of the immunoglobulin superfamily and is commonly expressed in cells of the myeloid lineage such as macrophages, dendritic cells, neutrophils, mast cells,eosinophils and also found on B cells, activated T cells, etc. Moreaux J et al. showed that CD200mRNA was strongly expressed in colon cancer, kidney cancer, head and neck cancer, etc. Besides, high expression in patients had worse prognosis. Kawasaki BT et al. found that CD200co-expression with stem cell markers such as CD44and CD133found on prostate, breast, brain, and colon cancers, and proposed CD200is a CSCs surface marker for them. Recent studies had demonstrated that CD200had an important role in organ transplantation, autoimmune disease and tumor immunity, which was related to the occurrence and development of the disease. Currently, the researches of CD200in CCSCs is at the beginning, this experiment is to study the relationship between CD200and colorectal cancer stem cells marker CD133and xenograft transplantation assay, which lay a foundation for CD200-mediated colorectal tumor immunity research.Material and Methods1. Cells culture Colo205colon cancer cell lines were purchased from the Type Culture Collection of the Chinese Academy of Sciences, Shanghai, China. The serum-supplimented medium (SSM) consisted of RPMI-1640supplemented with10%fetal calf serum. The serum-free medium(SFM) was prepared from1:1(v/v) Dulbecco’s modified Eagle’s medium and Ham’s F-12nutrient mixture (DMEM/F12; HyClone, USA), B27supplement (1:50; Gibco, USA),20ng/mL epidermal growth factor (EGF; PeproTech, USA),10ng/mL basic fibroblast growth factor (bFGF; PeproTech, USA),10ng/mL leukemia inhibitory factor (LIF;Prospec, USA). Colo205cells were cultured in SSM. Cells at the exponential growth phase were washed with phosphate buffer saline (PBS) and digested with trypsin, followed by resuspension in SFM.2. Flow cytometry Cells were collected separately from SSM and SFM group by trypsin digestion, followed by washing and resuspending in PBS at a concentration of1×106/mL. Cells were incubated with FCR blocking reagent, allophycocyanin (APC)-conjugated anti-CD200and phycoerythrin (PE)-conjugated anti-CD133monoclonal antibodies(Miltenyibiotec, Germany) for30min at4℃. As a control, some cells were also incubated with FCR blocking reagent, APC-conjugated mouse IgG1and PE-conjugated mouse IgG2a(BD, USA).The fluorescence intensity was measured by flow cytometry(BD, USA).3. cells Immunofluorescence Colon cancer cells in SFM group were collected by centrifugation, the SSM group as a control, then resuspended in PBS and smear. They were followed by fixation in4%paraformaldehyde for30minutes at room temperature. Cells were then incubated for5minutes with0.2%Triton X-100and washed again in PBS. Blocking was performed for30minutes with10%normal goat serum. Cells were then incubated with primary antibodies (both anti-CD133,1:100, Miltenyibiotec, Germany and anti-ALDH1,1:200, Abcam, USA) or (both anti-CD200,1:100, Abeam, USA and anti-ALDHl,1:200), respectively, for1hour at room temperature. After washing with PBS, incubation was performed with a secondary antibody (anti mouse-594for CD133and CD200,1:200; anti-rabbit488for ALDH1,1:200) for1hour at room temperature and washed again. Nuclei were stained with DAPI before anti-flourescent quencher sealing. Fluorescence images were captured by microscope equipped with a digital camera.4. Fluorescence activated cell sorter Colon cancer cells in SFM group were collected by centrifugation, and then resuspending in PBS at a concentration of1×108/mL. Cells were incubated with FCR blocking reagent, anti-CD200-APC and anti-CD133-PE monoclonal antibodies for30min at4℃. Cells with marker CD133+CD200-,CD133"CD200+,CD133+CD200+and CD133’CD200-were obtained respectively from the SFM group using flow cytometry. CD133+CD200" cells, CD133’CD200+cells, CD133+CD200+cells and CD133"CD200-cells were cultured in SFM and analyzed by flow cytometry.5. Cells morphology and growth observed with an inverted microscope Colo205colon cancer cell lines were cultured in SSM or SFM, and the cells growth was observed by inverted microscope. CD133+CD200-, CD133-CD200+, CD133+CD200+and CD133-CD200-cells were cultured in SFM and observed using the same method.6. Subcutaneous xenograft assay in NOD/SCID mice(1) All animal experiments were approved by the Animal Ethics Committee of Southern Medical University and the protocol of animal treatment was approved by the Institutional Animal Care and Use Committee. Six to eight week old female NOD/SCID mice were used in these studies. To study the tumorigenic potential of CD133+CD200" cells,CD133"CD200+cells,CD133+CD200+cells and CD133’CD200-cells isolated from colon cancer spheres in SFM, four groups of cells were mechanically and enzymatically dissociated into single-cell suspension in PBS at concentrations of1×103,1×104and1×105. CD133+CD200’cells,CD133"CD200+cells and CD133+CD200+cells were mixed1:1(v/v) with Matrigel(BD, USA) and injected subcutaneously into upper left of the back of NOD/SCID mice (Beijing Hua Fu Kang Bio-Technology company,China),respectively. CD133-CD200-cells were injected into lower right of the back as a control. We observed tumor growth and record the changes, Tumor length and width were measured using a venire caliper three times per week. Tumor volumes were calculated as following formula:V=(π×L×W2)/6(V, Volume; L, Length; W, Width). Finally, we plotted a tumor growth curve. Thirty-two days after the injection, mice were sacrificed, tumor nodules were immediately removed, fixed in4%paraformaldehyde, and embedded in paraffin. Four micrometer-thick sections were cut, mounted on poly-L-lysine-coated slides, dried overnight at37℃, dewaxed in xylene, rehydrated according to standard histopathological procedure, and stained with H&E.(2)To determine ALDH1immunoreactivity, the antigen was retrieved with ethylenediamine tetraacetic acid repair fluid, via microwave heating (high power for5min, followed by low power for8min). Sections were incubated overnight at4℃with anti-human ALDH1antibody (1:200; Abcam). Immunohistochemistry was performed using a chemmateTM dako envisionTM detection kit as described by the manufacturer (DAKO).7. Statistical analyzes Analysis was completed with a statistical software package for desktop computer (SPSS16.0). Data are reported as means±SD. Comparison of the mean in two groups were preformed with Independent-Samples T Test and2Independent Samples Tests. Multiple groups comparison analyses by One-Way ANOVQ and K Independent Samples Tests. P values of less than0.05(two tailed) were considered to be significant.Results1.Detection of the proportion of CD133+cells and CD200+cells The percentage of CD133+CD200-cells,CD133-CD200+cells and CD133+CD200+cells were (1.68±0.94)%,(0.94±0.67)%,(0.68±0.22)%in SSM group and were(19.72±2.36) %,(9.56±1.00)%,(3.02±0.93)%in SFM group. The proportion of CD133+CD200-,CD133’CD200+,CD133+CD200+cells were significantly higher in SFM groups than in SSM groups (P<0.05). Flow cytometry results showed that CD133+CD200-cells, CD133-CD200+cells, CD133+CD200+cells and CD133-CD200-cells were cultured in SFM and analyzed by flow cytometry, which could reach over90%in purity.2. Cells morphology and growth Colo205cells were beginning to form irregular cancer spheres in SFM after seven days, and cells were closely connected, new spheres were generated next to present spheres. However, cells cloud not form cancer spheres in SSM. Cells marked with CD133+CD200-,CD133-CD200+, CD133+CD200+and CD133-CD200-were obtained respectively from the SFM group using flow cytometry. Only CD133+CD200-cells and CD133+CD200+cells showed the ability to form cancer spheres in SFM, while CD133-CD200+cells and CD133-CD200-cells failed.3. Cells immunofluorescence The expression of CD133, CD200and ALDH1in colon cancer cells in SFM group and regular Colo205cells in SSM group was detected by immune fluorescence. We found that high expression of CD133, CD200and ALDH1in colon cancer spheres. More over, CD133and ALDH1or CD200and ALDH1were found to be co-expressed, but showed low or negative expression in regular Colo205cells.4. The xenograft transplantation assay To study the tumorigenicity of CD133+CD200-, CD133-CD200+, CD133+CD200+and CD133-CD200-cells, we injected the cells into NOD/SCID mice. The study showed that103CD133+CD200-cells and CD133+CD200+cells were found to be sufficient to induce tumorigenesis, while the same number of CD133-CD200+cells and CD133-CD200-cells failed to induce visible tumors32days after injection. More over,104CD133+CD200+cells were fastest to form tumor in four groups, while CD133’CD200-cells groups failed. The volume of tumors generated by104CD133+CD200+group was significantly greater than CD133+CD200-or CD133’CD200+groups after32days.105tumor cells of four groups could form tumor in our study. In vivo, CD133+CD200+cells group was the most tumorigenicity in four groups. The mean weight of tumors in104CD133+CD200+, CD133+CD200-and CD133-CD200+cells groups were (0.46±0.16) g,(0.27±0.05) g and (0.07±0.02) g, respectively. CD133+CD200+cells tumors were significantly bigger than another two groups. CD133+CD200"cells group was significantly heavier than CD133-CD200+cells group(P<0.05).5. H&E analysis and the expression of ALDH1of tumor in NOD/SCID mice. The histochemical assay showed that105CD133+CD200-cells,CD133-CD200+cells and CD133+CD200+cells groups positively expressed ALDH1, but CD133-CD200-cells group was weakly positive. In addition, tumor tissues were showed poorly differentiated by H&E staining in four groups.Conclusion1. CD200co-expressed with CD133+Colo205colorectal cancer cells This study found that the proportion of CD133+cells and CD200+cells were significantly higher in SFM group than in SSM group by flow cytometry, which suggested CD133+cells and CD200+cells could show the ability of self-renewal and proliferation in SFM. Moreover, we found that CD200co-expressed with CD133+Colo205colorectal cancer cells.2. High expression of CD133, CD200and ALDHl in colorectal cancer spheres Colo205cells had the ability to form colorectal cancer spheres which strongly expressed CD133, CD200and ALDH1,more over, CD133co-expressed with ALDH1, and CD200co-expressed with ALDH1. CD133is a marker of undifferentiated, it is suggested that colorectal cancer spheres might enrich undifferentiated cells. CD200strongly expressed in colorectal cancer spheres and co-expressed with colorectal cancer stem cells marker ALDH1, which inferred there was a close relationship between CD200and CCSCs.3. CD133+CD200+cells had a capacity of forming cancer spheres and showed the stronger tumorigenicity CD133+CD200+cells had a capacity of forming cancer spheres and could show a characteristic of self-renewal and proliferation in SFM. Xenograft transplantation assay showed that CD133+CD200+cells had stronger tumorigenicity.4. CD133+CD200+cells may be one of the subpopulation of CCSCs and CD200may be one of the candidate marker of CCSCs CD133+CD200+cells were few number of the tumor cells which accorded with one of definition of cancer stem cells. Our date suggests CD133+CD200+cells had stronger ability of self-renewal, proliferation and tumorigenicity, in addition, it could form cancer spheres in SFM and CD200co-expressed with colorectal cancer stem cells markers CD133and ALDH1. CD200was closely related to the mechanism of immune escape of tumor immunity and tumor metastases. Thus, we inferred CD133+CD200+cells might be one of the subpopulation of CCSCs and CD200might be one of the candidate marker of CCSCs.5. ALDH1played an important role in CCSCs and the development of colorectal cancer High expression of ALDH1in cancer spheres and the xenograft transplantation assay showed the expression of ALDH1in the tumor tissues in NOD/SCID mice. Therefore, the detection of the expression of ALDH1in clinic provides a new insight into CRC diagnosis and prognosis evaluation. |
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