Ying Hu, Yi Liu, Cheng-Jiang Wei, Ya-Zhen Zhu, Hao Lai, Yan Feng, Yuan Lin?

1. The Second Ward of Gastrointestinal Surgery,Guangxi Medical University Cancer Hospital

2. Colorectal Cancer Clinical Diagnosis and Treatment Center

3. Department of Research,Guangxi Medical University Cancer Hospital;Nanning 530021,China

Keywords:Tumor microenvironment E p i t h e l i a l-m e s e n c h y m a l transformation Malignant tumor Mesenchymal stem cells

ABSTRACT Objective: To establish a co-culture model of colon cancer cells or mesenchymal stem cells with normal colon epithelial cells in vitro for simulating the tumor microenvironment in vitro,and to investigate the effect of colon cancer cells or mesenchymal stem cells on the migration and epithelial-mesenchymal transformation (EMT) of normal colon epithelial cells in the coculture environment. Method: Co-culture model of colon cancer cell line SW480 or human umbilical cord mesenchymal stem cells (hUC-MSCs) with human normal colon epithelial cells (NCM460) was established. Morphological changes of NCM460 after co-culture were evaluated by microscopic observation, and migration ability of NCM460 was measured by wound healing and Transwell assay. Meanwhile, Western blot was used to detect the EMT markers of NCM460. Results: There was no significant change in cell morphology of NCM460. Transwell assay results showed that when hUC-MSC or SW480 were co-cultured with NCM460, there is a trend of enhancement of the migration ability of NCM460, and the expression of Vimentin was up-regulated to some extent (P<0.05), E-cadherin and wound healing ability were not significantly changed (P>0.05).When NCM460 were cocultured with MSCs and SW480 at the same time, NCM460's wound healing ability and migration ability were significantly enhanced, meanwhile Vimentin protein expression was significantly upregulated and E-cadherin protein was significantly down-regulated significantly (P<0.05).Conclusion: Colon cancer cells can increase the migration ability of normal colon epithelial cells and upregulate the expression of EMT markers. Mesenchymal stem cells may play a role in this process.

1. Introduction

The composition of the tumor microenvironment (TME) varies in different tumors, and its hallmark members include immune cells, stromal cells, blood vessels and extracellular matrix[1]. As an important role in tumor occurrence and development, TME is an active promoter of tumor progression. At the same time,there is a dynamic interaction between tumor cells and TME components. Under the influence of tumor cells, other cells in the TME undergo changes, which forming a microenvironment that contribute to tumor growth further more. For example, the acidic microenvironment contributed by tumor cells[2], the polarization of macrophages[3], and the state of effector T cells[4] all play a role in promoting the growth of tumor cells. Most of the researches focus more on immune cells, mesenchymal cells, etc., and there are few studies concentrate on the role of normal epithelial cells. A research paper recently published in Nature showed that normal lung epithelial cells located around lung metastases of breast cancer can be induced to dedifferentiate, and express epithelial cell adhesion molecule (EpCAM), even exhibited stronger proliferation ability and stem cell activity which promoted the growth and survival of tumor cells[5]. The study showed that normal cells in the TME also undergone changes in the microenvironment. Whether similar changes occur in the colon epithelial cells surrounding the cancer cells and its clinical significance have not been studied.

Mesenchymal stem cells are important part of the tumor stroma, and their tendency of homing to tumor site has been observed in various cancer[6], and can be induced to become tumor-associated fibroblasts(CAFs) under the influence of the tumor microenvironment which play a role in promoting tumor growth, invasion and metastasis[7,8].The effect of mesenchymal stem cells on normal cells has also not attracted attention. In this study, the co-culture model was used to simulate the microenvironment to explore the induction potential of colorectal cancer cells to normal epithelial cells, and the effect of mesenchymal stem cells in the process.

2. Data and Methods

2.1 Cell culture

Colon cancer cell line SW480 was purchased from Guangzhou Cellcook Biotech company, cultured in L-15 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin. The human umbilical cord mesenchymal stem cells (hUC-MSCs) were acquired from Beijing Cyagen Biosciences, cultured with specific basal medium within P10 generation. Human normal colorectal epithelial cell line NCM460 was purchased from GuangZhou Jennio Biotech company, cultured in 1640 medium containing 10% fetal bovine serum FBS and 1% penicillin-streptomycin. All cells were cultured in a 37°C cell incubator with 5% CO2.

2.2 Wound healing assay

Plate NCM460 evenly in a 6-well plate at a density of 2.5 × 105cells per well. When the density of NCM460 cells reaches about 80%, manually make three vertical scratches in the 6-well plate with the tip of 200μL-pipette. Take pictures and record with a Zeiss microscope at the time point of 0h, 6h, and12h after scratching respectively, then calculate the distance of the blank space.

Experimental grouping: NCM460 cells were all placed in the lower chamber, and corresponding cells were placed in the upper chamber according to the experimental groups: Control group (no cells were placed in the upper chamber, and the ratio of the number of cells in the upper and lower chambers was 0:1), hUC-MSCs Group (hUCMSCs were placed in the upper chamber, hUC-MSCs:NCM460 1:1), SW480 group (SW480 were placed in the upper chamber,SW480:NCM460 1:1),and SW480+hUC-MSCs group (hUCMSCs and SW480 were placed in the upper chamber hUC-MSCs:SW480:NCM460 1:1:1).

2.3 Transwell migration assay

Take an 8 μm chamber (the upper chamber) of a 24-well plate,plate NCM460 cells at a density of 2.5×104/well in the chamber,supplement the volume to 500 μl, and plate hUC-MSCs(2.5×104/well), SW480(2.5×104/well), SW480+hUC-MSCs (2.5×104/well+2.5×104/well) in the lower chamber respectively. Blank group(medium only) was set. After co-culture for 24 hours, the cells were fixed with 4% paraformaldehyde for 30 minutes, and the cells were stained with 0.1% crystal violet for 20 minutes. The cells that passed through were counted according to the stained nucleus using the microscope, 3-5 fields were randomly selected from each well.

2.4 Western blot

After the cells were co-cultured in a 6-well plate for 24 hours in different groups, the proteins of NCM460 in the lower chamber were extracted by adding RIPA Lysis Buffer with PMSF (100:1),and the concentration was assessed by BCA BCA Protein Assay Kit(BeyonTime Biotechnology, Shanghai, China).

2.5 Statistical methods

All statistical analyses and plots were performed in GraphPad Prism 8.0; cell counts were performed using Image J; Wound healing distances were expressed as mean (Mean) ± standard deviation (SD).Unpaired Student's t test was used for comparison between two groups, one-way analysis of variance (One-Way ANOVA) was used for comparison between multiple groups, and L-SD was used for multiple comparison between two samples.

3. Result

3.1 No obvious change in cell morphology after co-culture

Changes in cell morphology were observed at 0h and 24h. After 24h of culture, no significant changes in cell morphology were found between groups after 24h of co-culture, as shown in Figure 1.

Figure1 Morphological observation of NCM460 cells in each group after 24h co-culture through inverted microscope

3.1 Enhanced wound healing ability of NCM460 cells in coexistence of hUC-MSCs and SW480

Compared with the control group, the wound healing distance of hUC-MSCs+SW480 group increased at 6h and 12h, (6h:(131.33±23.12) μm vs (60.33±6.06) μm, F=7.167, P<0.05; 24h:(602.00±89.21) μm vs (423.00±19.92) μm, F=7.012, P<0.05), the difference was statistically significant, and there was no statistical significance when compare the control group with other groups(Table 1, Figure 2B).

Figure2 The wound healing ability of NCM460 was enhanced by hUCMSCs+SW480 group. (A) The wound healing rate of NCM460 cells in each group at 0, 6 and 12h was observed in the bright field. (B)Histogram of healing rate based on three repeated experiments. (P value of each group compared with the control group: *P< 0.05 **P< 0.01)

Table1 Wound healing rate of NCM460 at 6 h and 12 h(x±s)

3.2 hUC-MSCs+SW480 enhanced the migration ability of NCM460

The results showed that compared with the control group, the migration ability of NCM460 in the lower chamber of each experimental group had enhanced at different levels, but the hUCMSCs+SW480 group significantly enhanced the migration ability of NCM460 (120.00±3.00 vs 1.67±1.53), the difference was statistically significant (P<0.001), as shown in Table 2 and Figure 3.

Figure3 hUC-MSCs+SW480 enhanced the migration ability of NCM460(A)Migrated NCM460 stained by crystal violet(×200) (B)Histogram of the migrated cells (*P< 0.05, **P< 0.01, ***P<0.001)

Table2 Results of NCM460 cell migration experiment( x±s)

3.3 hUC-MSCs+SW480 induces changes in markers of EMT in NCM460

Compared with the control group, the expression of Vimentin increased in each experimental group, among them the relative expression level of hUC-MSCs+SW480 group was the highest(0.84±0.51 vs 0.12±0.13), and the difference was statistically significant (P<0.001); Compared with the control group, there was a downregulated trend of the expression of E-Cadherin in NCM460 cells in hUC-MSC group and SW480 group after indirect coculture, but only in the hUC-MSC+SW480 group, the difference was statistically significant ((0.74±0.06 vs 0.96±0.06), P<0.05), as shown in Table 3 and Figure 4.

Figure4 hUC-MSCs+SW480 upregulated Vimentin and downregulated the E-Cadherin of NCM460. (A) E-cadherin and vimentin in NCM460 cells,GADPH as the internal reference. (B) Semi-quantitative analysis of Vimentin and E-cadherin expression according to Western blot results.

Table3 Semi-quantitative analysis of Vimentin and E-cadherin(x±s)

4. Discussion

Tumor cells can "engineer" a variety of cellular components in the tumor microenvironment, enabling them to develop and transform into a state that supports tumor cell growth, thereby building the tumor microenvironment into a state that is more conducive to its own development. At present, most studies focus on immune cells and CAFs in the tumor microenvironment. Changes that occur in normal cells around tumors have only been sporadically mentioned in a few studies[5,9,10]. This study confirmed that tumor cells can induce behavioral and molecular expression changes in surrounding normal intestinal epithelial cells in vitro through a co-culture model,and found that the presence of mesenchymal stem cells can enhance this effect.

The induction of tumor cells to various components of the TME is ubiquitous. For example, tumor cells can induce normal fibroblasts or their precursor cells to express markers of CAF by secreting a variety of growth factors or other mechanisms[11]. At the same time,tumor cells can also regulate the function and status of immune cells, such as inhibiting the activation of CD8+T cells[12], regulating the polarization of macrophage and neutrophil toward a tumorpromoting phenotype[13,14], and the effect on regulatory T cells of tumor cell-specific site mutations[15] and so on. However, since most of the epithelial cells are highly differentiated cells, and no changes in histopathological morphology are often observed, most studies ignore that normal epithelium may be affected by tumor cells and the surrounding tumor microenvironment. In a study published in Nature, OMBRATO et al. performed tSNE analysis on the RNAseq expression profiling data of fluorescently labeled normal lung epithelial cells around breast cancer lung metastases and found that they were induced to differentiate into two clusters, E-Cadherinnegative and positive epithelial cells, and E-cadherin-negative epithelial cells specifically expressed Ly6a and Tm4sm1 lung progenitor cell markers. At the same time, the qPCR results also showed that the expression of alveolar lineage markers of the labeled lung epithelial cells was generally reduced, and the E-cadherin was significantly reduced. On the contrary, the expressions of Vimentin,Snail and Twist proteins increased to a certain extent, indicating that breast cancer lung metastases can induce surrounding normal lung Epithelial cells undergo dedifferentiation and exhibit enhanced proliferation and stem cell activity, and are involved in promoting tumor cell growth and survival[5]. Similarly, this study found that colorectal cancer cells had similar induction effects on normal colonic epithelial cells. In the absence of significant changes in cell morphology (Figure 1), the migration ability of normal epithelial cells can be significantly enhanced (Figure 3), and similar changes in the expression of epithelial-mesenchymal transition markers (Figure 4), The difference in wound healing assay was not significant (Figure 2), which may be related to the short co-culture time in this study.

At present, there have been lots of researches on the complex interaction mechanism between mesenchymal stem cells and colorectal cancer cells. For example, in colorectal cancer,mesenchymal stem cells can induce the EMT of colorectal cancer cells through their conditioned medium or direct cell-to-cell contact,promoting tumor cell migration and proliferation[16,17]. MSCs can regulate the cell cycle of tumor cells and inhibit cell apoptosis by activating the AMPK/mTOR signaling pathway[18]. The literature suggests that the interaction between them are based on direct cellto-cell contact or through cytokine paracrine effects[19-21]. For example, studies have found that MSCs can regulate the transcription of colorectal cancer cells through microRNAs carried by their extracellular vesicles, thereby mediating the invasion and immune escape of tumor cells through the AKT pathway[22]. The promotion of MSCs to tumor cells is in turn related to the induction of tumor cells into tumor-associated fibroblasts (CAFs)[23-25]. This study found that MSCs could enhance the migration ability of NCM460 and upregulate the expression of EMT marker——Vimentin in normal epithelial cells, and the co-existence of MSCs and SW480 significantly enhanced this induction (Figure 3, Figure 4). Given the complex regulatory relationship between mesenchymal stem cells and colorectal cancer cells, we speculated that the enhanced induction of normal intestinal epithelial cells by tumor cells in the presence of mesenchymal stem cells in this study would also There is an inducing effect of tumor cells on mesenchymal stem cells, which leads to changes in the secretion profile of mesenchymal stem cells,and further enhances the changes in the biological behavior and gene expression of normal intestinal epithelial cells. It is speculated that further experiments are needed in the future. verify.This study explores the possibility and the potential mechanism that normal intestinal epithelial cells can be affected by tumor cells, and the results suggested that components of the microenvironment such as mesenchymal stem cells, can enhance this process. A simplified coculture model was applied in this research for exploring the effect of the microenvironment on normal epithelial cells. In this research system important influencing factors such as immune cells and cell matrix components in the TME were not included. On the other hand, it remains unclear that whether the behavioral changes and EMT of normal intestinal epithelial cells exist in vivo. In addition,the specific clinical significance for tumor growth, development,recurrence, drug resistance and other processes needs to be confirmed and discussed in more in-depth studies.