Cancer-associated fibroblasts induced by colorectal cancer cells inhibit γδT cells killing function through the ERK/COX-2/PGE2 pathways

doi:10.21203/rs.3.rs-3874261/v1

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Cancer-associated fibroblasts induced by colorectal cancer cells inhibit γδT cells killing function through the ERK/COX-2/PGE2 pathways

https://doi.org/10.21203/rs.3.rs-3874261/v1

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The tumor microenvironment (TME) is a highly ordered ecosystem in which several non-malignant cell types, including immune cells, cancer-associated fibroblasts (CAFs), and other cell types, are encircled by cancer cells. Colorectal cancer (CRC) is determined by the reciprocal contact between cancer cells and host cells. γδT cells represent a significant subset of the natural immune system and are vital in the battle against CRC. Yet, the ability of γδT cells to kill is limited in the CRC microenvironment. The purpose of this work is to investigate how CRC cells-induced CAFs affect γδT cells, which offers fresh perspectives on how to broaden CRC treatment approaches. The findings demonstrated that fibroblast activation, proliferation, and clone formation were induced by CRC cell culture supernatants via the ERK signaling pathway. On the basis of analyzing the correlation between γδT cells and CAFs, we further demonstrated that the generated CAFs can inhibit the killing function of γδT cells to CRC cells. In CAFs co-cultured with γδT cells, ERK inhibitors can decrease the expression levels of COX-2 and PGE2, and partially reverse the inhibition of CAFs on γδT cell killing function. According to our research, CAFs generated by CRC cells in the TME may decrease the ability of γδT cells to destroy themselves and facilitate the development of an immunosuppressive microenvironment by activating the ERK/COX-2/PGE2 pathway. They establish the groundwork for assessing how ERK inhibitors alleviate γδT cell immune suppression in the CRC microenvironment, investigating novel targets for CRC therapy, and formulating clinical CRC treatment plans.

Cancer-related fibroblasts

γδT cells

COX-2

PGE2

ERK

Colorectal cancer (CRC), one of the most common malignant tumors of the gastrointestinal tract, typically develops around the rectum-colon junction. Early identification of CRC is now extremely difficult, and the vast majority of patients are diagnosed with numerous tumor metastases [1, 2]. Cancer cell metastasis involves contact with the host immune system, which occurs mostly in the tumor microenvironment (TME). Despite the fact that numerous studies are currently focusing on the genetic and epigenetic factors behind CRC growth, we still have a limited understanding of the complex TME.

TME is composed of several cell types such as tumor cells, fibroblasts, and immune cells, and it contains important regulatory and driving factors that are involved in cancer cell metastasis, invasion, and drug resistance creation. It has been associated to tumor occurrence and progression [3]. Fibroblasts are found throughout the lamina propria of neighboring colorectal mucosal epithelium in normal human colorectal stromal cells. Their primary role is to respond to tissue damage by releasing growth factors that promote regeneration and repair while preserving distinct tissue structures and functions [4]. Cancer-associated fibroblasts (CAFs) have long been thought to be carcinogenic components of CRC due to their role in angiogenesis, immunological suppression, and production of molecules that promote cancer cell proliferation and migration [5, 6]. The majority of CAFs are identified as a unique subset of activated fibroblasts in tumor tissue and are created by the differentiation of intrinsic fibroblasts. In order to support cancer cell proliferation, invasion, and migration, activated fibroblasts acquire additional secretory phenotype, a distinct extracellular matrix (ECM) remodeling ability, potent autocrine activation, and a dynamic immunological regulatory signaling role [7]. Considering the significance of CAFs in the development and occurrence of tumors, a thorough investigation into CAF-related data will yield innovative concepts for tumor-causing variables and therapeutic intervention tactics.

About 5–10% of human peripheral blood lymphocytes are γδT cells. γδT cells, which are the primary components of intraepithelial lymphocytes, are more prevalent in colon mucosa [8, 9]. In several types of cancer, γδT cells have been found and identified as infiltrating lymphocytes in solid tumor tissues, such as CRC [10–11]. Nonetheless, it has been documented that TME is usually an immunosuppressive milieu [12]. The highly complex interactions among different cell subsets within the immunosuppressive TME usually diverted or impaired the cytotoxic functions of γδT cells [13]. Therefore, overcoming the obstacles set by immunosuppressive CRC TME and recovering the cytotoxic function of γδT cells is critical for the future development of immunotherapy. Our previous research has shown that conditioned media (CM) from CRC cells can trigger the production of colorectal CAFs [14]. However, the effect of CAFs to γδT cells has yet to be identified.

The effect of CRC-induced CAFs on peripheral blood γδT cells from CRC patients was investigated in this study. According to our research, CAFs generated by CRC cells may decrease the ability of γδT cells to destroy themselves and facilitate the development of an immunosuppressive microenvironment by activating the ERK/COX-2/PGE2 pathway. They establish the groundwork for assessing how ERK inhibitors alleviate γδT cell immune suppression in the CRC microenvironment, investigating novel targets for CRC therapy, and formulating clinical CRC treatment plans.

Subjects. Between May 2022 and October 2023, twenty CRC patients were selected from Renmin Hospital and Taihe Hospital in Shiyan. Patients with colorectal cancer (CRC) are all recently diagnosed and untreated. Hubei University of Medicine's Clinical Ethics Committee gave its clearance for this work (2023-TH-048). Each subject provided informed consent before taking part in the research.

Preparation of CM for Human CRC Cells. In a 10 cm cell plate, 8×10⁶ CRC cells (HCT116 and Caco−2 cells were used in this investigation) in the logarithmic growth phase were grown. The old culture media was discarded and replaced with new, serum-free culture medium once the cells had grown to 70–80% confluence. The cell supernatant was extracted after 48 hours and centrifuged for 10 minutes at 3000 rpm/min. At−80°C, the supernatant was gathered, filtered, and frozen. When in use, the cells were blended with fresh and full culture media at a ratio of 1:1 to ensure an adequate supply for the cells.

Colony formation assay. 500 CCD-18Co (A cell line exhibiting fibroblast morphology that was isolated from the normal colon tissue) cells under logarithmic growth phase were added to one well of the 6-well plate. And then control CM, HCT116-CM, Caco-2-CM, HCT116-CM + iERK (ERK inhibitor: SCH772984), Caco-2-CM + iERK were added corresponding well respectively. Each group has three repeated wells. The culture medium was changed every two days within 14 days. After removing the culture media, the cells were fixed in 4% paraformaldehyde for 15 minutes, stained with crystal violet, and the number of colonies was counted in order to see how the colonies were forming.

Real-time unlabeled dynamic cell analysis technology (RTCA). CCD-18Co cells in logarithmic growth phase with good growth density and good condition were digested by trypsin and made into a 4×10⁴/mL cell suspension. An E-plate was filled with 50 µL of mixed CM and set on a DP detection platform to measure the baseline. Each well was then filled with 50 µL of the aforesaid cell suspension and 50 µL of CM. The cells were maintained in an incubator at 37°C and 5% CO₂ to ensure optimal growth conditions. The cell index was assessed every 15 minutes. The corresponding cell proliferation curve was obtained by dynamically measuring the cell proliferation index of the experimental group and the control group.

Isolation of Peripheral blood mononuclear cells (PBMCs). PBMCs from peripheral blood samples of the participants in this study were isolated through density-gradient centrifugation using Ficoll-Paque (Sigma, 17144002). Isolated PBMCs were cultivated in the presence of RPMI 1640 medium (supplemented with 10% fetal bovine serum) at 37°C in an incubator with 5% CO₂.

MACS magnetic bead sorting. Using an anti-TCR γ/δ MicroBead Kit from the Miltenyi business (Miltenyi, 130-050-701), γδT cells were separated from PBMCs in accordance with the manufacturer's instructions. The PBMCs were labeled fluorescently and magnetically with anti-Hapten MicroBeads-FITC after being first treated with anti-TCR γ/δ Hapten-Antibodies. After that, the cell suspension was put onto a MACSR Column and it was positioned inside a MACS Separator's magnetic field. The unlabeled cells were passing through the column while the magnetically labeled γδT cells were kept inside. These cells that were passing through were gathered as non-γδT cells. The positively selected γδT cells can be eluted from the magnetically held column once the column has been removed from the magnetic field. Using flow cytometry, the purity of γδT cells was shown to be greater than 95%.

Real-time PCR (RT-PCR). RT-PCR was used to assess the target gene mRNA expression levels in CCD-18Co and γδT cells. After being extracted, the total RNA of these cell types was reverse transcribed into cDNA. The expression levels of α-SMA, FAP, FN, TGF-β, granzyme, perforin, COX-2 and PGE2 were then determined. GAPDH served as the standard control. The relative mRNA expression levels of these genes were then analyzed with Bio-Rad CFX manager 3.1 software.

Western blotting. After being washed with PBS, CCD-18Co cells were lysed using RIPA buffer that contained protease inhibitors. The total protein extract was resolved by SDS-polyacrylamide gel electrophoresis. Following the transfer of proteins onto a PVDF membrane, the relevant monoclonal antibody (α-Tubulin, α-SMA, FAP, FN, TGF-β, ERK, and p-ERK) was incubated with the membrane. After the membrane was incubated with the corresponding secondary antibodies, the protein bands were detected by visualization with an ECL kit.

Co-culture of CAFs and γδT cells. The indirect cell-cell interaction/contact was done using Transwell plates (6.5 mm insert, 5.0 µm pore size polycarbonate membranes, Corning, 3421) as well as cell-cell co-culture system. Briefly, CAFs were placed in the lower chamber and grown to confluence in complete medium. γδT cells (2×10⁵) were loaded in the upper chamber. Transwell plates were then incubated for 6 hours at 37°C in a 5% CO₂ humidified atmosphere.

Flow cytometry. γδT cells were treated with 200µL of BD Cytofix/Cytoperm fixation and permeabilization solution (Becton Dickinson, 554714) for 20 minutes at 4°C prior to intracellular labeling. To determine the expression levels, γδT cells were then washed and stained with anti-granzyme (Biolegend, 372204) and anti-perforin (Becton Dickinson, 556437) antibodies. Using a Beckman MoFlo XDP flow cytometer, stained cells were examined.

Cytotoxicity assays. CytoTox 96® non-radioactive cytotoxicity assay (Promega 1780) was used for the detection of the killing of the colon cancer cells (HCT116) by γδT cells, according to the manufacturer’s protocol. The target cells (HCT116) were seeded onto 96-well plates at a density of 5 × 10³ cells per well. The γδT cells isolated from the peripheral blood of the CRC patients, which were used as effector cells, were added to each well at a effector cells to target cells ratios of 1:1, 2.5:1, and 5:1 and incubated at 37°C in a 95% humidified atmosphere with 5% CO₂ for 4 h. The cytotoxic activity was measured based on the LDH released from the supernatant, which was obtained and analyzed using the CytoTox 96 assay. HCT116 cell-death was calculated using the following equation: % cytotoxicity = (Experimental – Effector spontaneous – target spontaneous)/(target maximum – target spontaneous) × 100, according to the manufacturer’s protocol. Maximum and spontaneous both refer to LDH release.

TISID database analysis. The Tumor and Immune System Interaction Data Bank (TISIDB) is an online resource that combines various disparate data formats. Using the lymphocyte module, we investigated the connection between COAD's CAF-related genes and γδT cells.

Statistical analysis. Software such as GraphPad 8.0 or SPSS 19.0 were used to examine all of the data. Independent sample t tests and paired sample t tests were used to generate and statistically analyze the data. A p-value of less than 0.05 was deemed statistically significant.

CRC-CM can induce the formation of CAFs

Serum-free CM was collected from monolayer grown HCT116 and Caco-2 cells for 48 hours, and added to fibroblast CCD-18Co cells for another 72h. In CCD-18Co cells, it was found that HCT116-CM and Caco-2-CM significantly increased the mRNA levels of CAF-related molecular markers like α-SMA, FAP, FN, and TGF-β (Fig. 1A). In the meantime, western blotting reveals that these CAF-related molecular markers have increased in response to both HCT116-CM and Caco-2-CM (Fig. 1B). Subsequently, we observed the increse in the capacity to form clones and multiply CCD-18Co cells cultured with HCT116-CM and Caco-2-CM, respectively (Figs. 1C and 1D). The findings demonstrated that CRC-CM might improve fibroblasts' capacity to proliferate and generate clones, suggesting that CRC-CM could trigger the development of CAFs.

CRC cells induce fibroblast activation via the ERK signaling pathway

We observed the activation of associated signaling pathways in CCD-18Co cells following treatment with CRC-CM in order to explore the precise method by which CRC cells affect fibroblasts. It was discovered that the ERK signaling pathway can be activated by HCT116-CM and Caco-2-CM. Next, we used ERK inhibitor (SCH772984, iERK) to stop CCD-18Co cells from expressing ERK. Before adding conditioned or control medium, the fibroblasts were pretreated with iERK at this dose (300 nM) for 4 hours. The phosphorylation level of the ERK protein in CCD-18Co cells was found following an additional 72-hour treatment. The results showed that iERK significantly inhibited the expression of α-SMA in CRC-CM induced CCD-18Co cells (Fig. 2A). Subsequently, we introduced iERK into the CCD-18Co treated by HCT116-CM and Caco-2-CM, and identified them by RTCA assays and clone creation. We discovered that, in contrast to the group lacking iERK, iERK dramatically reduced the ability of CCD-18Co cells to proliferate and generate clones when stimulated by CRC-CM (Figs. 2B and 2C). The results showed that CRC cells induce fibroblast activation through the ERK signaling pathway.

CAFs generated by CRC-CM have the inhibitroy effect to γδT cells from CRC patients.

Next, we're looking into whether CAFs brought on by CRC-CM affect γδT cells function in patients with CRC. First, we examined the relationship between γδT cells and CAF-related biomarkers using the TISID database. It was proposed that there is a substantial correlation between γδT cells and α-SMA (ACTG2), FAP, FN, and TGF-β (Fig. 3A). To examine the effects of CAFs on the cytotoxic function of peripheral γδT cells, 2×10⁵ isolated γδT cells were co-cultured with CAFs induced by CRC-CM. Following a 24-hours co-cultivation period, the expression level of granzyme and perforin in γδT cells was examined. Real-time PCR and FACS data shown that CAFs may drastically reduce the expression levels of granzyme and perforin in peripheral γδT cells from CRC patients. (Figs. 3B, 3C). Moreover, it was also suggested that CAFs generated by CRC-CM might inhibit the cytotoxic function of CRC patients’ peripheral γδT cells to HCT-116 cells (Fig. 3D).

ERK inhibitors can reverse the inhibitory effect of CAFs on γδT cells.

Granzyme and perforin expression in CRC patients' γδT cells was examined using SCH772984 in order to assess the significance of ERK signaling pathway activation by CAFs on the cytotoxic function of these cells. For this reason, γδT cells were extracted from blood samples of six patients with CRC, and they were co-cultured with CCD-18Co cell, CAFs generated by CRC-CM or the sequential combination of iERK and CAFs. Our results further supported the findings in Fig. 3 by demonstrating that CAFs addition could decrease the production of both granzyme (Figs. 4A and 4C) and perforin (Figs. 4B and 4C). However, in the γδT cells of CRC patients, CAF-induced cytotoxic function attenuation could be partially reversed upon the addition of iERK (Fig. 4A-C). This finding suggested that the effects of CAFs on the γδT cells of CRC patients are significantly influenced by the activation of the ERK signaling pathway.

CAFs generated by CRC cells inhibit γδT cells killing function through the ERK / COX-2 /PGE2 pathways

It was discovered that cyclooxygenase-2 (COX-2) and other pro-inflammatory factors are highly expressed by CAFs in human malignancies [15, 16]. One of COX-2's downstream enzymatic products is prostaglandin E2 (PGE2). It has been documented that PGE2 and COX-2 synthesis is aided by the stimulation of the ERK signaling pathway [17, 18]. Thus, we investigated whether the ERK signaling pathway's activation in CAFs could also have an impact on the expression of COX-2 and PGE-2. γδT cells were isolated from 6 CRC patients by MACS and then were co-cultured with CCD-18Co cells, CAFs generated by CRC-CM or the sequential combination of iERK and CAFs. Our results showed that CAFs have the increase in the expression of COX-2 (Fig. 5A) and PGE-2 (Fig. 5B). After iERK was added together with CAFs in CRC patient’s γδT cells, it could be observed that the increased expression of COX-2 and PGE-2 could be partially restored to the levels comparable to the control group (Figs. 5A and 5B). These results demonstrated that CAFs induced by CRC cells inhibit γδT cells killing function through the ERK/COX-2/PGE2 pathways.

Low diagnostic efficiency, vulnerability to medication resistance, and distant metastases are the primary reasons of poor outcomes for CRC. The TME, which is composed of numerous types of cells and cytokines that govern and drive cancer progression, is the internal and exterior environment of CRC occurrence, growth, and metastasis [1, 3]. This study focuses on relationship in fibroblasts, cancer cells and γδT cells in the CRC’s TME.

Fibroblasts are responsible for the synthesis and secretion of matrix elements like collagen, which are necessary for the maintenance of proper tissue structure and pleiotropic function. Fibroblasts are uniformly dispersed in the lamina propria of neighboring colorectal mucosal epithelium in normal colorectal tissue; in CRC tissue, this cohesive structure is destroyed, as evidenced by stromal and tumor cell aggregation and widespread angiogenesis. According to current research, the interaction and relationship between tumor cells and fibroblasts in the TME is one of the factors that leads to the production of CAFs [6]. CAFs are the most numerous cellular components in the TME, and they play a diverse and critical function in tumor growth. On the other hand, tumor cells can activate fibroblasts and induce the development of CAFs when cocultured with fibroblasts via signal transduction mediated by specific cytokines [19]. In this investigation, we discovered that CRC secretion factors can trigger the development of typical CAFs in activated CCD-18Co cells, as evidenced by an increase in associated molecular markers including α-SMA, FAP, FN, and TGF-β (Fig. 1). Studies have shown that α-SMA is highly expressed in tumors with higher levels of invasiveness and malignancy, and it plays a significant role in the transdifferentiation of fibroblasts into CAFs [20]. TGF-β is abundant in the CRC microenvironment, which can induce the expression of α-SMA in fibroblasts to some extent. Additionally, it has been observed that FAP expression is lower in normal tissues than in malignant tissues. Furthermore, there is a strong correlation between the levels of α-SMA and FAP expression and tumor invasion intensity, distant metastasis, clinical advanced stage, and poor prognosis [21]. Activated fibroblasts have the ability to produce a significant amount of FN and promote the migration of cancer cells in comparison to normal fibroblasts [22]. In order to comprehend the specific mechanism of the interaction between CRC cells and fibroblasts, we investigated the activation of their associated pathways. It was discovered that CRC cells use the ERK signaling pathway to activate fibroblasts (Fig. 2).

γδT cells are important lymphocytes in antitumor immunosurveillance because of their MHC-unrestricted cytotoxic activity [23]. γδT cells are considered to be one of the main components of intraepithelial lymphocytes in colon mucosal tissue, playing an important role in the process of combating CRC [24]. But in the TME of CRC, the killing effect of γδT cells is inhibited [25–27]. Therefore, a better knowledge of CAF's mechanism and potential impact on γδT cell killing function will be helpful in the treatment of CRC. Studies have demonstrated that CAFs can control αβT cell activity, activate Treg cells in pancreatic cancer [28], encourage CD8⁺T cell exhaustion in lung cancer [29], and prevent CD8⁺T cell invasion in breast cancer [30]. However, its impact on γδT cells has not yet been documented.

The results of this study indicate that γδT cells and CAF-related molecular markers, including as α-SMA, FAP, FN, and TGF-β, exhibit a strong association. Upon co-culturing CAFs and γδT cells, it was also observed that CAFs might reduce the production of granzyme and perforin, two molecules necessary for cytotoxic action in γδT cells. We first found that HCT116-CM and Caco-2-CM may activate the ERK signaling pathway, which is necessary for the detection of machnisem. Furthermore, iERK significantly reduced the expression of α-SMA in CRC-CM-induced CCD-18Co cells. Meanwhile, when stimulated by CRC-CM, iERK significantly decreased the capacity of CCD-18Co cells to multiply and produce clones. The findings demonstrated that the ERK signaling pathway is used by CRC cells to activate fibroblasts (Fig. 2). Granzyme and perforin expression levels in γδT cells that were initially repressed have increased when ERK inhibitors were added to the co-culturation system of CAFs and γδT cells (Fig. 4). These findings imply that CAFs' ability to suppress γδT cells is mostly dependent on the ERK signaling pathway.

It was discovered that a subset of fibroblasts that expressed COX-2 changed the immunological milieu of the lungs by inhibiting monocytes and driving dysfunctional dendritic cells (DCs) [31]. The upregulation of COX-2 is crucial for the interaction between tumor cells and γδT cell subsets, which affects the cytotoxicity or proliferation of γδT cells and limits the effectiveness of γδT cell-based immunotherapy [32]. Our earlier findings also showed that leukemia inhibitory factor (LIF) inhibits the COX-2/PGE2 pathway, which in turn prevents γδT cells from being cytotoxic [33]. In the investigation, we discovered that when co-culturing with γδT cells, CAFs may enhance the synthesis of pro-tumor chemicals COX-2 and PGE2. Following the introduction of ERK inhibitors into the co-cultivation system between CAFs and γδT cells, we observed that the initially elevated expression levels of PGE2 and COX-2 in CAFs had been suppressed (Fig. 5). Thus, the above results indicated that CAFs inhibit γδT cells killing function through the ERK/COX-2/PGE2 pathways.

In conclusion, our study revealed that CAFs generated by CRC cells inhibit γδT cells killing function through the ERK/COX-2/PGE2 pathways (Fig. 5C). Thus, more research is required to determine how to counteract CAFs' impact on γδT cells in CRC patients.

Author Contributions: Conceived and designed the experiments: Xueyan Xi, Boyu Du. Performed the experiments: Ying Chen, Ting Deng, Jinyi Zhu, Ke Qu, Yilei Ke. Analyzed the data: Yan Zhao, Shuaitong Liu, Fangzhou Li, Qingqing Yu. Contributed reagents/materials/analysis tools: Gang Li, Yunhe Zhu. Wrote the paper: Xueyan Xi, Boyu Du.

Funding declaration: This work is supported by grants from the National Natural Science Foundation of China (81772649). The Scientific and Technological Project of Shiyan City of Hubei Province (2021K62). Open Project of Hubei Key Laboratory of Wudang Local Chinese Medicien Research (Hubei Unviersity of Medicine) (WDCM2023024). National Training Program of Innovation and Entrepreneurship for Undergraduates (S202313249001, S202313249003). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data availability statement: Data availability is not applicable to this article as no new data were created or analyzed in this study.

Conflict of interest: The authors declare no conflicts of interest.

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No competing interests reported.

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Cancer-associated fibroblasts induced by colorectal cancer cells inhibit γδT cells killing function through the ERK/COX-2/PGE2 pathways

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CRC-CM can induce the formation of CAFs

CRC cells induce fibroblast activation via the ERK signaling pathway

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