Tumor-associated fibroblasts promote the migration and invasion of Hepatocellular carcinoma cells by regulating THY-1

doi:10.21203/rs.3.rs-1531403/v1

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Tumor-associated fibroblasts promote the migration and invasion of Hepatocellular carcinoma cells by regulating THY-1

https://doi.org/10.21203/rs.3.rs-1531403/v1

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We investigate the role of Cancer-associated fibroblasts (CAFs) in regulating the epithelial-mesenchymal transition (EMT) and migration of Hepatocellular carcinoma cells. Culture Hepatocellular carcinoma cell lines (HepG2 and Huh7) and primary fibroblasts, isolate and identify primary fibroblasts; Western blot and real-time quantitative PCR techniques to detect the expression of thymocyte differentiation antigen (Thy-1) in the cells; And establish a co-culture system of Hepatocellular carcinoma cells and CAFs, divide the cells into Hepatocellular carcinoma cell group, co-culture system group (blank control group), co-culture system + isotype IgG antibody group (negative control group), co-culture system + anti-Thy-1 Antibody group. Transwell experiment detects the effect of Thy-1 on the migration ability of Hepatocellular carcinoma cells in the co-culture system; Western blot technology detects the effect of Thy-1 on the epithelial (E-cad) and stroma (N-cad and ZEB2) of Hepatocellular carcinoma cell lines in the co-culture system The effect of phenotypic marker protein expression. The CAFs were successfully isolated, and there was no significant difference in the morphology of normal fibroblasts. Both showed a spindle morphology. It was found that the expression of α-smooth muscle actin (α-SMA) in CAFs was significantly higher than that of matched NFs cells, suggesting that The isolated CAFs can be used as follow-up related experimental research; Western blot and real-time quantitative PCR technology detection indicates that Thy-1 mRNA and protein in CAFs are highly expressed, which has obvious statistical significance compared with paired NFs, and Thy- 1 Low expression in Hepatocellular carcinoma cell lines; after CAFs are co-cultured with Hepatocellular carcinoma cells, the migration ability of Hepatocellular carcinoma cell lines is significantly enhanced, and the expression of E-cad is significantly reduced, and the expression of N-cad and ZEB2 is obvious However, after pre-incubation with Thy-1 antibody in CAFs in the co-culture system, it was found that compared with the negative control group, the migration ability and EMT effect of Hepatocellular carcinoma cell lines were significantly weakened. Thy-l over-expressed in CAFs cells enhances the migration ability and EMT effect of HCC cells in the in vitro co-culture system.

Tumor-associated fibroblasts

thymocyte differentiation antigen-1

epithelial-mesenchymal transition

Hepatocellular carcinoma

The incidence of Hepatocellular carcinoma is high, the five-year survival rate is low, and the early diagnosis rate is only maintained at about 10–20%. At present, surgical resection is the only possible cure, but most of them still have the risk of tumor recurrence or metastasis^[1–3]. Therefore, understanding the molecular mechanism in the development process has great clinical value for the prevention and treatment of Hepatocellular carcinoma. Tumor tissues often achieve self-regulation through extracellular matrix (ECM) remodeling to gain invasion ability and metastasize to other tissues ^{[4, 5]}. In this process, CAFs cell subsets play important biological functions^[6]. CAFs can enhance tumor growth and invasion ability and reduce the sensitivity to chemotherapeutics; can enhance blood vessel formation, immune cell infiltration and tumor cell cloning ability ; can also induce tumor cells to develop EMT, which is an important biological process in epithelial cells to acquire migration and invasion capabilities^[7–9]. Epithelial phenotype transforms to mesenchyme, loses cell polarity, loses connection with basement membrane, and gains higher migration and invasion ability. Thymocyte differentiation antigen-l (Thy-l) is a type of glycolipid acyl alcohol, which is mainly anchored on the cell surface, and its genes are highly conserved^[10]. As a molecular marker on the surface of stem cells, it mainly plays an important role in axon maturation. However, it has been reported in the literature that it plays an important role in tumor cell apoptosis and metastasis, and can mediate the connection between cells.

In this study, the isolated and identified CAFs/NFs were co-cultured with Hepatocellular carcinoma cell lines HepG2 and Huh7 to detect the metastatic ability of Hepatocellular carcinoma cells and the expression of EMT molecular indicators to determine whether CAFs affect tumor cell migration and EMT; and verify it; Whether CAFs are overexpressed through Thy-1 to induce cell migration and EMT.

Material

Clinical data

Hepatocellular carcinoma tissue specimens were from 5 patients who underwent radical resection of Hepatocellular carcinoma in the Third Xiangya Hospital of Central South University. All patients did not receive chemotherapy and radiotherapy before surgery. Primary Hepatocellular carcinoma must be diagnosed by histological examination, and the normal tissues adjacent to the cancer were not infiltrated by cancer cells.

Cell lines

HepG2 and Huh7 Hepatocellular carcinoma cell lines were purchased from the Cell Resource Center, Shanghai Academy of Sciences, Chinese Academy of Sciences.

Main reagents

Fetal bovine serum were purchased from Gibco, the United States; trypsin, RPMI1640 medium, and phosphate buffered saline (PBS) were purchased from HyClone, the United States; mouse anti-human E-cadherin (E-cadherin, E -cad), N-cadherin (N-cadherin, N-cad), zinc finger protein 2 (ZEB2), Thy-l and GAPDH antibodies were purchased from Sigma-Aldrich, USA; HRP-labeled goat anti-mouse secondary antibody was purchased From Zymed, USA; goat anti-mouse FITC-labeled fluorescent secondary antibody was purchased from Earthox, USA; gene primers were purchased from Shanghai Shenggong Company; total RNA extraction kit was purchased from Beijing Soleibao Biotechnology Co., Ltd.; cDNA reversal kit and SYBR Real-time quantitative reagents were purchased from TaKaRa, Japan; Transwell microplates were purchased from Corning, USA.

Method

Primary separation of CAFs by tissue block method

Five fresh Hepatocellular carcinoma tissue specimens were taken during the operation, soaked in PBS, washed with ophthalmological scissors, and covered with serum in 28 cm2 petri dishes, placed in ^5% CO2, 37 Incubate in an incubator _at °C for 2 hours. After it is firmly attached, add about 2 ml of RPMI1640 culture medium containing 20% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. Change the medium every 2 to 3 days until After the cells crawl out of the tissue block, remove the tissue block. After the cells are spread over the entire culture dish, trypsinization is used to purify the cells. The method of culturing normal gastric tissue NFs is the same.

Establish an in vitro co-cultivation system

To adjust the cell density of CAFs in logarithmic growth phase to about 1×10⁵ cells/ml, place them in a 6-well plate, about 2 ml/well, culture in an incubator for 24 hours; adjust the logarithm Growth stage Hepatocellular carcinoma cells are ^about 1×106 cells/ml, and they are seeded on the upper layer of 0.4µm Transwell cell, about 1 ml/well. Transfer the Transwell cell seeded with Hepatocellular carcinoma cell lines to a 6-well plate containing fibroblasts Incubate in an incubator for 48 h.

Indirect immunofluorescence observation of the expression of α-SMA protein in fibroblasts

After collecting the cells of each group, fix them with 4% paraformaldehyde and wash them with PBS for 5 min for 3 times. After 0.5% Trition X-100 breaks the membrane, wash with PBS 5 min 3 times, 5% BSA-PBS at room temperature for 30 min, add α-SMA antibody, incubate overnight at 4°C, wash with PBS for 5 min 3 times, add fluorescent secondary antibody, incubate for 2 h at room temperature in the dark, PBS Wash 3 times for 5 min, and finally add DAPI to stain the nucleus for 5 min. After rinsing with PBS, the slides are mounted with glycerol and observed under a fluorescence microscope.

Transwell cell migration experiment to detect cell migration ability

Put an 8µm Transwell cell into a 24-well culture plate inoculated with CAFs, add 10% FBS RPMI1640 medium 600µl/JL, and add serum-free medium to the cell. The tumor cell suspension (1×106 cells/ml), 200µl/well. Repeat 3 samples in each group and place them in an incubator for 24 hours; take out the Transwell chamber, remove non-penetrating cells, fix with methanol for 30 minutes, wash with PBS and stain with 1% crystal violet for 20 minutes. Count the penetrating cells under the microscope.

Western blot

To detect the expression of the target protein in each group of cells. After the BCA protein concentration kit determines the protein concentration, take the same amount of protein electrophoresis and transfer it to the nitrocellulose membrane; after the 3% skimmed milk powder is blocked and eluted, the primary antibody 4 Incubate overnight at °C, add secondary antibody after washing the membrane, and incubate for 2 h at room temperature; after washing the membrane again, perform enhanced chemiluminescence (ECL) development in a dark room, and analyze the optical density value of each band with the image analysis software Quantity One.

Real-time quantitative PCR to detect the expression of α-SMA and Thy-1 Collect each group of cells, TRIZOL extracts the total RNA of the cells, and measure its concentration and purity, then use the reverse transcription kit to obtain cDNA, and use the ABI7500 Fast System as the template. PCR amplification.And set GAPDH as an internal reference gene. The sequence of each primer is as follows: α-SMA: upstream 5'-CAGGGCTGTTTTCCCATCCAT-3', downstream 5'-CCATGTTCTATCGGGTACTTC-3'; Thy-1: upstream 5'-CGGTCCAGTTGCCTTCTCCC-3', downstream 5'-GAGTGGCTGTCTGTGTGGGG-3'; GAPDH: upstream 5'- CCT-CAACGACCACTGTCA-3', downstream 5'-TTACTCCTTGGAGGCCATGT-3'.The PCR reaction conditions were as follows: denaturation at 95°C for 30 s; 95°C, 5 s, 60°C, 34 s, and 40 cycles.Set 3 replicate holes for each sample. And use ^2−△△CT to analyze the expression of the target gene.

Statistical analysis

Data is expressed, \(\bar {x} \pm s\)using SPSS 19.0 software for statistical analysis, after normality and homogeneity of variance tests, the difference between multiple groups of data is compared by single-factor analysis of variance, and the comparison between the two groups is by LSD- t test. Set α = 0.05 as the inspection level.

Morphological observation and identification of primary cells CAFs/NFs isolated from patients with Hepatocellular carcinoma From the morphological point of view, the isolated CAFs are spindle-shaped, long spindle-shaped or star-shaped, and there is no significant difference compared with NFs. CAFs can also be triangular or polygonal. After passage, the division cycle of CAFs becomes shorter and pseudopods and cell processes appear. When the cell density is low, the cells can present a network structure; and when the cell density is high, the cells The contact inhibition or density inhibition is weakened and fuse into a fiber bundle structure. In terms of molecular expression, α-SMA can be used as one of the marker molecules to identify these two types of fibroblasts because of the obvious difference in expression between CAFs and NFs.Therefore, when identifying the isolated fibroblasts in this experiment, real-time quantitative PCR, Western blot and immunofluorescence techniques were used to detect the gene and protein expression of α-SMA.The results showed that the expression levels of α-SMA genes and proteins in the isolated CAFs cells were significantly higher than the molecular expression levels in NFs cells (t = 3.89, P < 0.01; t = 4.01, P < 0.01). In addition, CAFs can be seen The fluorescence intensity of α-SMA in the cytoplasm is obviously matched with NFs cells, as shown in Table 1, Figs. 1 and 2.

Detection of Thy-1 expression in each group of cells Real-time quantitative PCR and Western blot techniques were used to analyze Thy-1 mRNA and protein expression in mesenchymal fibroblasts and Hepatocellular carcinoma cell lines. The results showed that there was no obvious expression of Thy-1 in hepatocellular carcinoma cell lines, while interstitial fibroblasts CAFs/NFs highly expressed Thy-1; and compared with neighboring NFs cells, Thy-1 in CAFs of patients The expression levels of were significantly increased, and the difference was statistically significant (P < 0.05). See Table 2 and Fig. 3.

Related research on Thy-1 regulation of hepatocellular carcinoma cell epithelial-mesenchymal transition and metastasis in CAFs Western blot technology was used to detect the expression changes of epithelial and mesenchymal marker molecules in hepatocellular carcinoma cell lines in the co-culture system to analyze Thy-1 in mesenchymal transition Biological role. The results showed that, compared with the Hepatocellular carcinoma cell group, the expression of hepatocarcinoma epithelial cell marker molecules in the co-culture system was significantly reduced (P < 0.01), while the expression of mesenchymal cell marker molecules was significantly increased (p < 0.01); Compared with the negative control group, the Thy-1 Ab group can significantly attenuate the biological effects of down-regulation of epithelial cell marker molecules and up-regulation of mesenchymal marker molecules in this co-culture system, and the difference is statistically significant (P < 0. 01). See Fig. 4.

Transwell chamber was used to detect the migration ability of cells in the co-culture system to analyze the influence of CAFs on tumor cell migration. The results showed that compared with Hepatocellular carcinoma cells, the co-culture system significantly increased the migration and mobility of cells (P < 0.01); and compared with the negative control group, Thy-1 Ab group also significantly antagonized cell migration in the co-culture system The difference in exercise ability was statistically significant (P < 0.01). See Fig. 5 for details.

Table 1

Relative expression of α-SMA in primary cells NFs/CFs\(\left( {\bar {x} \pm s} \right)\)
CAFs	6.02 ± 1.71¹⁾	3.40 ± 2.02¹⁾
NFs	3.52 ± 1.03	1.13 ± 3.19
Note: Compared to the NFs group,1) P < 0. 01.

Table 2

Expression of Thy-1 from individual group\(\left( {\bar {x} \pm s} \right)\)
Groups	Expression of protein	Expression of mRNA
HepG2	0. 81 ± 1.43	0.95 ± 4.19
Huh7	0. 63 ± 2.94	1.11 ± 2.14
CAFs-1	2. 46 ± 2.10¹⁾	2.85 ± 3.79¹⁾
NFs-1	1.29 ± 3.12	1.21 ± 2.64
CAFs-2	3.25 ± 1.26¹⁾	4.51 ± 3.84¹⁾
NFs-2	2.46 ± 2.91	2.39 ± 3.51
CAFs-3	3. 83 ± 2.41¹⁾	3. 95 ± 1.29²⁾
NFs-3	1.53 ± 4.01	2.51 ± 3.03
Note: Compared to the NFs group, 1) P < 0.01, 2) P < 0.01.

Fibroblasts are the main mesenchymal cells that exist in the tumor microenvironment, but their specific biological role in the tumorigenesis and development process is still not completely clear^[11]. After CAFs are activated, they may promote the invasion and metastasis of tumor cells through paracrine methods. It has been confirmed that CAFs are co-cultured with corresponding tumor cells, leading to the activation of CAFs in breast cancer, colon cancer and squamous cell carcinoma, and enhancing the invasion ability of tumor cells ^{[12, 13]}. The prerequisite for the study of CAFs-related molecular biological functions is the successful isolation of primary cells. CAFs and NFs have different biological functions and phenotypic characteristics. In the primary cell culture and isolation and identification, this study referred to foreign scholars' isolation and purification methods of prostate cancer, breast cancer, Hepatocellular carcinoma and other tumor-related CAFs ^{[14, 15]}.

In this study, the detection of α-SMA protein and mRNA expression on the isolated fibroblasts found that the isolated primary cells have a typical CAFs phenotype, which can be used as a follow-up study of related invasion experiments and EMT experiments.

Thy-1 is a phosphatidylinositol glycoprotein with a relative molecular mass of 25 000 to 37 000 Da ^, which regulates a variety of biological functions in almost all types of fibroblasts that have been studied so far^[16]. It not only supports the structure of fibroblasts, but also balances cell proliferation, migration and regulates the synthesis of immune inflammatory mediators. Western blot and real-time quantitative PCR technology were used to detect Thy-1 protein and mRNA expression in different groups of cells. Thy-1 is weakly expressed in simple tumor cells, mainly in the CAFs of the co-culture system; it is found that the expression of Thy-1 is significantly higher than that of paired NFs in the CAFs of the patient's tissues. 1 It may be overexpressed in CAFs to regulate a series of biological functions, but the specific mechanism needs to be further studied.

Previous scholars have found that the invasiveness of simple rat colon cancer cells is weak when cultured in vitro, but when it is co-cultured with CAFs, the invasive ability is significantly increased, and the number of CAFs is proportional to the tumor cell's effect on lymphatic vessels and lymph nodes. Invasiveness is positively correlated ^[17]. Although many studies have found that CAFs have an effect on the migration ability of tumor cells, there are few reports on the effect of CAFs on Hepatocellular carcinoma cells. In this study, successfully isolated CAFs were co-cultured with Hepatocellular carcinoma cell lines, and paired NFs were used as controls to analyze the effect of CAFs on the migration ability of Hepatocellular carcinoma cells. The results confirmed that, similar to colon cancer cells, simple hepatocellular carcinoma cell lines have weaker invasion ability, but CAFs in the co-culture system can significantly enhance the migration and invasion ability of Hepatocellular carcinoma cells. This biological effect has been modified by anti-Thy-1 monoclonal Antibodies are significantly inhibited. In addition, this study also involves the biological role of Thy-1 in regulating EMT in tumor cells. The main reason for tumor cell infiltration and migration is the acquisition of certain phenotypic characteristics, which leads to the loss of polarity of epithelial cells, and the weakening of the adhesion between cells leads to epithelial cells with mesenchymal cell morphology. Among them, the reduction or loss of E-cad is the most important landmark change formed by EMT. EMT-related protein molecules E-cad, N-cad and ZEB2 in the culture system all have significant changes, all suggesting that under the action of CAFs, tumor cells may enhance tumor cell migration through EMT. Previous studies have found that there is overexpression of Thy-1 in CAFs. Using this as a starting point, blocking the expression of Thy-1 can prevent the weakening or lack of E-cad in epithelial cells and the overexpression of mesenchymal cell phenotype to a certain extent. .

In short, CAFs can induce EMT in tumor cells, which makes the cells lose the epithelial cell polarity and the connection with the basement membrane and invade surrounding tissues. The overexpression of Thy-1 may be involved in the EMT and invasion effects of Hepatocellular carcinoma cells. However, how Thy-1 in CAFs cells participates in regulating this series of biological functions and which downstream molecules are mobilized and activated. These need to be further explored.

CAFs can induce EMT in tumor cells, which makes the cells lose the epithelial cell polarity and the connection with the basement membrane and invade surrounding tissues. The overexpression of Thy-1 may be involved in the EMT and invasion effects of Hepatocellular carcinoma cells.

Acknowledgements

None

Authors’ contributions

Conceptualization and methodology: Xin-Ming Pan.

Formal analysis and data curation: Xin-Ming Pan.

Validation and investigation:Zheng Wang.

Writing—original draft preparation and writing—review and editing:Zheng Wang and Xin-Ming Pan.

Approval of final manuscript: all authors

Funding

No funding was received.

Availability of data and materials

The analyzed data sets generated during the present study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate

All specimens were collected and used with the informed consent of patients and their families, in line with the ethical standards established by the Human Testing Committee and approved by the Institutional Medical Ethics Committee of Third Xiangya Hospital of Central South University（No:2019CSU-018）.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

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No competing interests reported.

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Tumor-associated fibroblasts promote the migration and invasion of Hepatocellular carcinoma cells by regulating THY-1

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