Exploration of the regulatory mechanism of PD-L1 expression in pMMR type gastric cancer cell by cytotoxic drugs and anti angiogenic drugs

Exploration of the regulatory mechanism of PD-L1 expression in pMMR type gastric cancer cell by cytotoxic drugs and anti angiogenic drugs | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Exploration of the regulatory mechanism of PD-L1 expression in pMMR type gastric cancer cell by cytotoxic drugs and anti angiogenic drugs Jinming Li, Mengting Da, Xueying Zhou, Dengfeng Ren, Miaozhou Wang, and 6 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9102745/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract Background and Objectives: Advanced gastric cancer with proficient mismatch repair (pMMR) status responds poorly to immune checkpoint inhibitors (ICIs) and constitutes a significant proportion of cases, posing a major clinical challenge. Studies suggest that cytotoxic and anti-angiogenic drugs may influence ICI efficacy by modulating programmed death-ligand 1 (PD-L1) expression; however, their specific role and mechanisms in pMMR gastric cancer remain unclear. This study aimed to investigate the regulatory effects of cytotoxic drugs (5-fluorouracil, 5-FU; cisplatin) and an anti-angiogenic drug (apatinib) on PD-L1 expression in human pMMR-type AGS gastric cancer cells and explore the underlying signaling pathway mechanisms. Methods The impact of drug treatments at various concentrations on AGS cell proliferation was first assessed using the CCK-8 assay to select appropriate concentrations for subsequent experiments. Real-time quantitative PCR (qPCR) and Western blotting were employed to evaluate the effects of 72-hour drug treatment on PD-L1 mRNA and protein expression levels, respectively. Furthermore, Western blot analysis was used to examine drug-induced changes in the expression of key proteins (PI3K, Akt, MEK1, ERK) within the PI3K/Akt/mTOR and RAS/RAF/MEK/ERK signaling pathways, which are potentially involved in PD-L1 regulation. Results 1. Regulation of PD-L1 Expression: qPCR results showed that 5-FU (64 µM), cisplatin (16 µM), and apatinib (320 nM, 640 nM) significantly upregulated PD-L1 mRNA expression in AGS cells ( P < 0.05). Western blot analysis further confirmed that treatment with 5-FU (64 µM), cisplatin (16 µM), and apatinib (320 nM) significantly increased PD-L1 protein levels ( P < 0.05). 2. Alterations in Signaling Pathway Proteins: Western blot analysis indicated that treatments with 5-FU, apatinib, and cisplatin all upregulated PI3K protein expression ( P < 0.05). Regarding Akt protein, apatinib treatment increased its expression, whereas 5-FU and cisplatin decreased it ( P < 0.05). Within the MAPK pathway, 5-FU treatment upregulated the expression of MEK1 and ERK proteins, while apatinib and cisplatin treatments downregulated the expression of both proteins ( P < 0.05). Conclusion At specific concentrations, cytotoxic drugs (5-FU, cisplatin) and the anti-angiogenic drug (apatinib) can upregulate both mRNA and protein expression of PD-L1 in pMMR-type AGS gastric cancer cells and differentially modulate the expression of key proteins in the PI3K/Akt and MAPK signaling pathways. These findings provide preliminary in vitro experimental evidence and mechanistic insights supporting the potential of combining these drugs with ICIs to enhance therapeutic efficacy in pMMR gastric cancer. pMMR type gastric cancer PD-L1 cytotoxic drugs anti angiogenic drugs Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Background In patients with advanced gastric cancer, the survival benefit achieved with chemotherapy has long been difficult to surpass, while the introduction of immune checkpoint inhibitors(ICIs) has provided more therapeutic options. ICIs function by targeting the interaction between programmed death-1(PD-1) and programmed death-ligand 1(PD-L1). Numerous studies have demonstrated the efficacy of ICIs in treating advanced gastric cancer [ 1 – 8 ] . Despite their effectiveness, the issue of resistance to ICIs in advanced gastric cancer warrants attention [ 9 – 11 ] . Patients with proficient mismatch repairp(MMR) advanced gastric cancer exhibit resistance to ICI therapy, showing inferior responses compared to those with deficient mismatch repair(dMMR) tumors. This is because dMMR tumors, due to reduced mismatch repair function, have the potential to encode novel non-self antigens, subsequently attracting greater T-lymphocyte infiltration into the tumor and promoting dominant anti-tumor immunity. In contrast, pMMR gastric cancers lack this effect, resulting in weaker anti-tumor immune responses [ 12 – 22 ] , which may be one reason for ICI resistance in pMMR patients. In the KEYNOTE-059 study, the objective response rate (ORR) to pembrolizumab was 57% in patients with dMMR advanced gastric cancer, compared to only 9.0% in pMMR patients [ 23 ] . Overall survival (OS) follow-up data from the CheckMate 649 study showed a hazard ratio (HR) of 0.33 (95% CI 0.12–0.87, P < 0.05) for nivolumab plus chemotherapy versus chemotherapy alone in the dMMR subgroup, and an HR of 0.73 (95% CI 0.62–0.85, P < 0.05) in the pMMR subgroup. This indicates that while both dMMR and pMMR patients can benefit from ICI therapy, the survival benefit remains greater for dMMR patients [ 1 ] . A meta-analysis incorporating the KEYNOTE-062, CheckMate-649, JAVELIN Gastric 100, and KEYNOTE-061 studies further confirmed that dMMR patients derive greater OS benefit from anti-PD-1 therapy than pMMR patients [ 24 ] . Given that pMMR gastric cancers respond less effectively to ICIs and constitute the majority of cases (dMMR accounts for only about 6.2%) [ 25 ] , strategies to improve ICI efficacy in this large patient population require further exploration. Current research suggests that combining cytotoxic drugs (chemotherapy) or anti-angiogenic therapy with ICIs can enhance treatment outcomes in solid tumors. These studies indicate that cytotoxic and anti-angiogenic agents may influence ICI efficacy by modulating PD-L1 mRNA and protein expression levels [ 26 – 35 ] . Some studies also suggest that anti-angiogenic therapy and ICIs can directly inhibit tumor cell proliferation [ 36 , 37 ] . However, for patients with pMMR advanced gastric cancer, there is still a lack of research exploring the impact and underlying mechanisms of cytotoxic and anti-angiogenic drugs on ICI efficacy, both in vitro and in vivo. Chemotherapeutic agents can regulate cancer cell PD-L1 expression, thereby influencing the efficacy of ICIs [ 26 ] . Studies show that cisplatin upregulates PD-L1 mRNA and protein expression both in vitro and vivo. Its combination with anti-PD-L1 antibodies significantly inhibits tumor growth, a process potentially mediated by the PI3K/AKT signaling pathway, as AKT inhibitors can block this effect [ 27 ] . In head and neck squamous cell carcinoma, cisplatin similarly induces PD-L1 expression, suggesting synergistic potential with PD-1/PD-L1 inhibitors [ 28 ] . Other chemotherapeutic drugs exhibit similar effects. Paclitaxel and etoposide upregulate PD-L1 in breast cancer cells MCF-7 and MDA-MB-435, and fluorouracil has the same effect on cells like MDA-MB-468 [ 29 ] . In colon cancer SW480 and liver cancer HepG2 cells, paclitaxel-induced PD-L1 upregulation can be inhibited by MEK inhibitors, implicating the MAPK pathway in its regulation [ 30 ] . In summary, various chemotherapeutic drugs can upregulate PD-L1 by activating specific signaling pathways, providing a theoretical basis for combination strategies with immunotherapy. In light of the above, cytotoxic drugs such as cisplatin, paclitaxel, and fluorouracil may influence PD-L1 mRNA and protein expression in cancer cells through different mechanisms, potentially further affecting the therapeutic outcome of PD-L1/PD-1 inhibitors. However, research specifically investigating the effects of cisplatin, paclitaxel, fluorouracil, and other chemotherapeutic agents on PD-L1 expression in pMMR gastric cancer cells is limited and requires further exploration. Mechanisms by which anti-angiogenic drugs enhance ICI efficacy include modulating cancer cell PD-L1 expression and improving the tumor microenvironment (TME) [ 31 – 33 ] . Apatinib, a tyrosine kinase inhibitor (TKI) that selectively targets VEGFR2, exerts its anti-angiogenic effect by inhibiting VEGFR-induced endothelial cell proliferation and migration [ 34 , 35 ] . Research indicates that the impact of apatinib on PD-L1 expression is cancer-type specific: it downregulates PD-L1 in osteosarcoma by inhibiting STAT3 signaling [ 31 ] , whereas it upregulates PD-L1 in colon cancer CT26 cells and, in combination with anti-PD-1 antibodies, significantly inhibits tumor growth in mice [ 32 ] . However, the regulatory effect of apatinib on PD-L1 expression in gastric cancer remains unclear and warrants further investigation. Beyond modulating the immune microenvironment, apatinib can also directly inhibit gastric cancer cell proliferation and induce apoptosis, a mechanism potentially related to the inhibition of PI3K/Akt pathway phosphorylation [ 36 ] . Similarly, PD-1 inhibitors alone can directly inhibit gastric cancer cell proliferation by regulating the β-catenin, MAPK, and PI3K/Akt pathways [ 37 ] . These findings suggest that the combined application of apatinib and PD-1 inhibitors may synergistically enhance anti-tumor effects through multiple mechanisms. The expression of PD-L1 on cancer cells is regulated by multiple signaling pathways, with the RAS/RAF/MEK/MAPK-ERK and PI3K/PTEN/Akt/mTOR pathways being two key regulators. Their effects on PD-L1 expression vary across different cancer types such as melanoma, breast cancer, and non-small cell lung cancer [ 26 ] . Studies suggest that cytotoxic drugs and anti-angiogenic agents may regulate PD-L1 expression by influencing the PI3K/Akt and MAPK pathways, thereby affecting ICI efficacy. However, the specific pathways through which these drugs regulate PD-L1 expression in pMMR gastric cancer cells have not been elucidated and require further exploration. This study aims to explore the effects of cytotoxic drugs and anti-angiogenic agents on PD-L1 mRNA and protein expression in pMMR gastric cancer cells, along with the associated pathways. The goal is to investigate potential mechanisms for improving ICI efficacy in pMMR gastric cancer, thereby providing a basis for further research into methods to enhance the therapeutic outcomes of ICI treatment for this patient population. Materials and Methods Cell Lines and Culture Conditions Human gastric adenocarcinoma cell lines AGS were obtained from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. Cells were routinely cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin (100 U/mL penicillin, 100 µg/mL streptomycin). All cells were maintained in a humidified incubator at 37°C with 5% CO₂. The culture medium was refreshed every 2–3 days. Subculturing was performed using 0.25% trypsin-EDTA when cell confluence reached approximately 80–90%. Cell Viability Assay (CCK-8) Cell viability was assessed using the Cell Counting Kit-8 (CCK-8) assay. Cells were seeded into 96-well plates. The treatment groups and concentration gradients were as follows: 5-Fluorouracil (5-FU): 0, 2, 4, 8, 16, 32, 64, 128µM; cisplatin: 0, 2, 4, 8, 16, 32, 64, 128µM; apatinib: 0, 10, 20, 40, 80, 160, 320, 640nM. At each designated time point (0, 24, 48, 72, and 96 hours), the culture medium was carefully removed. Subsequently, 100 µL of fresh medium containing 10% CCK-8 reagent was added to each well. The plates were incubated for 2 hours at 37°C. The absorbance of each well was then measured at a wavelength of 450nm using a microplate reader. Each experimental condition was performed in triplicate. Protein Extraction Cells were washed twice with ice-cold phosphate-buffered saline (PBS). Total protein was extracted using RIPA lysis buffer freshly supplemented with 1× PhosSTOP phosphatase inhibitor cocktail and 1 mM phenylmethylsulfonyl fluoride (PMSF). Cells were scraped on ice and the lysates were transferred to pre-chilled microcentrifuge tubes. After incubation on dry ice for 30 minutes to facilitate complete lysis, the samples were centrifuged at 12,000 rpm for 15 minutes at 4°C. The supernatant containing the soluble proteins was carefully collected, aliquoted, and stored at -80°C until further use. Western Blotting Protein concentration was determined using a bicinchoninic acid (BCA) assay. Equal amounts of protein (typically 20–40 µg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electrophoretically transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% non-fat milk or bovine serum albumin (BSA) in Tris-buffered saline with Tween 20 (TBST) for 1 hour at room temperature. Subsequently, they were incubated overnight at 4°C with specific primary antibodies (e.g., anti-PD-L1). After extensive washing, membranes were incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL) detection system and imaged. RNA Extraction Total RNA was extracted from cultured cells using RNAiso Plus reagent according to the manufacturer's instructions. Briefly, cells were lysed directly in the culture dish by adding the reagent. Chloroform was added for phase separation, and the aqueous phase containing RNA was collected. RNA was precipitated with isopropanol, washed with 75% ethanol, and finally dissolved in RNase-free water. RNA concentration and purity were assessed by measuring the absorbance at 260 nm and 280 nm using a spectrophotometer. RNA integrity was confirmed by agarose gel electrophoresis. RNA samples were stored at -80°C. qPCR First, total RNA is extracted from samples, treated with DNase I, and its quality is verified. This RNA is then reverse transcribed into cDNA using reverse transcriptase with appropriate primers. The qPCR reaction is assembled by mixing the cDNA template, gene-specific primers, a detection system (SYBR Green dye or TaqMan probes), and a PCR master mix in a plate. The plate is run in a real-time PCR instrument under a thermal cycling program: initial denaturation (e.g., 95°C for 2–5 min), followed by 40–45 cycles of denaturation, annealing, and extension. Fluorescence is measured each cycle. A melt curve analysis is performed for SYBR Green assays to check specificity. Finally, the cycle threshold (Ct) values are used to calculate relative gene expression (e.g., via the 2^(-ΔΔCt) method), normalized to reference genes and a control. Statistical Analysis All quantitative data are presented as the mean±standard deviation (SD) from at least three independent experiments. Statistical comparisons between multiple groups were performed using one-way analysis of variance (ANOVA) followed by appropriate post-hoc tests. All statistical analyses were conducted using SPSS software (version 22.0). A p-value of less than 0.05 ( P < 0.05) was considered statistically significant. Result Detection of MMR status in AGS gastric cancer cells Prior to commencing the experiments, it was necessary to confirm that the AGS gastric cancer cell used in this study were of the pMMR (proficient mismatch repair) status. This was achieved by assessing the expression of four key MMR proteins (MSH6, MSH2, MLH1, and PMS2) in both cell lines via Western Blot analysis. The Western blot results showed that MSH6, MSH2, MLH1, and PMS2 proteins were expressed in AGS cancer cells (Figure S1 ) , indicating that the AGS gastric cancer cells used in this study were pMMR type. Baseline PD-L1 Expression Assay To evaluate the effect of drugs on PD-L1 expression, the basal expression level of PD-L1 mRNA in AGS gastric cancer cells was first examined. qPCR results demonstrated that PD-L1 mRNA is constitutively expressed in AGS cells. Western blot analysis further confirmed the normal expression of PD-L1 protein in AGS gastric cancer cells. CCK8 verifies the effects of cytotoxic drugs and anti angiogenic drugs on the proliferation of gastric cancer cells Experimental grouping The intervention groups were 5-FU group, cisplatin group, and apatinib group, with specific drug concentration gradients. Fluorouracil and cisplatin were cytotoxic drugs, while apatinib was an anti vascular drug. We referred to relevant literature to determine the drug concentration gradient for CCK8 detection [ 27 – 32 ] . CCK-8 assay We conducted CCK8 testing on patients in the 5-FU group, cisplatin group, and apatinib group. The concentration gradient of the 5-FU group was 0 uM、2uM、4uM、8uM、16uM、32uM、64uM、128uM, the concentration gradient of cisplatin group is 0uM, 2uM, 4uM, 8uM, 16uM, 32uM, 64uM, 128uM, while the concentration gradient of apatinib group is 0 nM、10nM、20nM、40nM、80nM、160nM、320nM、640nM, Measure the absorbance at 0h, 24h, 48h, 72h, and 96h after cultivation, repeat 3 times for each group, and calculate the average survival rate. The results showed that in the 5-FU group, the overall survival rate of AGS gastric cancer cells showed a decreasing trend with the prolongation of culture time. After 72 hours and 96 hours, there were statistically significant differences ( P < 0.05) in the survival rate of AGS gastric cancer cells at different concentration gradients compared to the control group ( Fig. 1 , Table S1 ) . In the cisplatin group, overall, the survival rate of AGS gastric cancer cells decreased with prolonged culture time, and this downward trend in survival rate was observed in various concentration gradients. After 72 and 96 hours of observation, except for 2uM, there was a statistically significant difference ( P < 0.05) in the survival rate of AGS gastric cancer cells compared to the control group in other concentration gradients ( Fig. 2 , Table S2 ) . In the apatinib group, the survival rate of AGS gastric cancer cells showed inconsistent trends with the prolongation of culture time at different concentrations of apatinib. After 72 hours of observation, the survival rate of AGS gastric cancer cells treated with 320nM and 640nM concentrations was lower than that of the control group ( P < 0.05). After 96 hours of observation, the survival rate of AGS gastric cancer cells treated with 640nM concentration was lower than that of the control group ( P < 0.05) (Fig. 3 , Table S3 ). qPCR detection verification of the effects of cytotoxicity and anti vascular drugs on PD-L1 mRNA expression in pMMR type AGS gastric cancer cells To investigate the effects of cytotoxic and anti-angiogenic drugs on PD-L1 mRNA expression in AGS gastric cancer cells, drug concentrations that effectively inhibited cell proliferation covering a broad range were selected based on CCK-8 assay results. The cells were treated with 5-FU (4µM, 16µM, 64µM), cisplatin (16µM, 32µM, 64µM), or apatinib (320nM, 640nM). After 72 hours of treatment, mRNA was extracted and subjected to qPCR analysis. The results showed that in the 5-FU treatment group, the 64µM concentration significantly upregulated PD-L1 mRNA expression, whereas the 4µM and 16µM concentrations showed no significant effect. In the apatinib treatment group, both the 320nM and 640nM concentrations significantly upregulated PD-L1 mRNA expression. In the cisplatin treatment group, the 16µM concentration significantly upregulated PD-L1 mRNA expression, while the 32µM and 64µM concentrations had no significant effect. All other tested concentrations showed no statistically significant difference compared to the control group ( P > 0.05), indicating no apparent regulatory effect on PD-L1 mRNA expression ( Fig. 4 , Table S4 ) . Western Blot experiment verifies the effects of cytotoxicity and anti vascular drugs on PD-L1 protein expression in pMMR type AGS gastric cancer cells To investigate the effects of cytotoxic and anti-angiogenic drugs on PD-L1 protein expression in AGS gastric cancer cell, Western blot analysis was performed. Based on prior qPCR results indicating upregulation of PD-L1 mRNA, cells were treated with 5-FU (64µM), cisplatin (16µM), or apatinib (320nM). Following a 72-hour drug treatment, proteins were extracted, and the experiment was conducted with three replicates per group. The results demonstrated that treatment with 5-FU (64µM), apatinib (320nM), and cisplatin (16µM) significantly upregulated PD-L1 protein levels in AGS gastric cancer cell compared to the control group. The differences in Western blot band intensity were statistically significant ( P < 0.05) ( Figs. 5 , 6 , Table S5 ) . Investigating the Regulation of key proteins in pathways associated with PD-L1 expression by Cytotoxic and Anti-Angiogenic Drugs in Gastric Cancer Cells To investigate the mechanisms by which cytotoxic and anti-angiogenic agents regulate PD-L1 protein expression in AGS gastric cancer cells, this study, based on literature reports, selected the RAS/RAF/MEK/ERK and PI3K/Akt/mTOR signaling pathways for exploration, as these pathways have been clearly shown to regulate PD-L1 in various cancers [ 26 ] . We analyzed key proteins within these pathways, including PI3K, Akt, MEK1, and ERK. The experiment utilized drug concentrations that upregulate both PD-L1 mRNA and protein expression in AGS cells (5-FU 64µM, cisplatin 16µM, apatinib 320nM). Cells were treated for 72 hours, with three replicates per group. Western blot analysis revealed that treatment with 5-FU, apatinib, and cisplatin significantly upregulated the expression of PI3K protein in AGS cells compared to the control group ( P < 0.05). Regarding AKT protein expression, apatinib treatment demonstrated an upregulatory effect ( P < 0.05), whereas 5-FU and cisplatin significantly downregulated its expression ( P < 0.05) ( Fig. 7 , 8 ) . Western Blot results demonstrated that 5-FU treatment significantly upregulated the expression levels of both MEK1 and ERK proteins in AGS cells ( P < 0.05). Conversely, treatment with apatinib and cisplatin significantly downregulated the expression of these two proteins ( P < 0.05) (Figure. 9, 10) . Discussion ICIs combined with chemotherapy have brought good survival benefits to patients with advanced gastric cancer [ 24 ] . However, due to the lack of the ability to generate new antigens in pMMR type gastric cancer with complete mismatch repair function, it is difficult to recruit enough tumor infiltrating lymphocytes to initiate effective anti-tumor immune responses [ 12 – 22 ] , which may lead to poor efficacy of ICIs in pMMR type advanced gastric cancer patients. This may be one of the reasons why pMMR type gastric cancer is resistant to ICIs. Given that pMMR type gastric cancer accounts for a high proportion in advanced gastric cancer (dMMR type only accounts for about 6.2%) [ 25 ] , it is particularly necessary to find strategies to improve the efficacy of ICIs in this group of patients. The expression level of PD-L1 is a potential pathway to overcome ICIs resistance. Some studies show that dMMR type advanced gastric cancer patients benefit more from ICIs treatment than pMMR type patients [ 1 , 23 ] . Due to the relatively poor response and high proportion of pMMR type gastric cancer to ICIs [ 25 ] , exploring methods to improve its efficacy is of great significance. At present, many studies have shown that cytotoxic drugs and anti angiogenic drugs can regulate the anti-tumor effect of ICIs by affecting the mRNA and protein expression of PD-L1 [ 26 – 35 ] , but there is still a lack of research on how these two types of drugs affect the efficacy and mechanism of ICIs in pMMR type gastric cancer. In recent years, many studies have focused on how to upregulate PD-L1 expression in cancer cells in order to enhance the combined therapeutic effect of immune checkpoint inhibitors (ICIs). At present, drugs used in combination with ICIs mainly include two categories: cytotoxic drugs and anti angiogenic drugs. In terms of cytotoxic drugs, existing evidence suggests that they may enhance ICIs therapy by upregulating PD-L1 expression. For example, cisplatin has been shown to upregulate the mRNA and protein levels of PD-L1 in lung cancer cells [ 28 ] . In head and neck squamous cell carcinoma, cisplatin also showed upregulation of PD-L1, and its combination with ICIs demonstrated stronger anti-tumor activity [ 28 ] . In addition, paclitaxel, fluorouracil and other drugs have also been reported to up regulate the expression of PD-L1 in breast cancer, colon cancer and liver cancer cell lines [ 30 ] . These findings collectively suggest that upregulating PD-L1 expression is a potential key mechanism for cytotoxic drugs to enhance the efficacy of ICIs and overcome drug resistance. However, it is not clear whether the above regulatory effects are applicable in pMMR gastric cancer cells with complete mismatch repair function. Therefore, this study explored this issue. Our results showed that qPCR detection indicated that 5-fluorouracil (5-FU) and cisplatin could upregulate the mRNA expression of PD-L1 in pMMR type AGS gastric cancer cells. Furthermore, Western Blot further confirmed that these two drugs can also upregulate the expression of PD-L1 protein at specific concentrations. Although differences in results were observed at individual drug concentrations, overall, specific cytotoxic drugs and their concentrations showed a clear upregulation trend on PD-L1 expression. Further experiments are needed to validate and refine this conclusion. The combination of anti angiogenic drugs and immune checkpoint inhibitors (ICIs) can enhance therapeutic efficacy, and its mechanisms are diverse, among which upregulation of PD-L1 expression in tumor cells is an important aspect. Research has shown that apatinib can upregulate PD-L1 in mouse colon cancer CT26 cells, and its combination with ICIs exhibits better anti-tumor effects [ 31 , 32 ] . In this study, qPCR experiments showed that apatinib upregulated PD-L1 mRNA in pMMR type AGS gastric cancer cells, and Western blot results also indicated its upregulation of PD-L1 protein expression. However, it is worth noting that other studies have reported that apatinib can inhibit the expression of PD-L1 protein in osteosarcoma KHOS and U2OS cells [ 31 ] . This suggests that the regulatory effect of anti angiogenic drugs on PD-L1 may have cancer specificity, and its specific mechanism still needs to be further explored. At present, it is believed that cytotoxic drugs and anti angiogenic drugs regulate PD-L1 expression, mainly involving key signaling pathways such as MAPK and PI3K/Akt. Numerous studies have confirmed that such drugs can affect the expression level of PD-L1 by regulating key proteins in the aforementioned pathways [ 30 , 36 ] . Therefore, analyzing the effects of these drugs on key proteins in related pathways is of great significance for elucidating their mechanisms of regulating PD-L1 expression in pMMR type gastric cancer cells, and providing theoretical basis for improving the efficacy of ICIs. Based on this, this study first analyzed the PI3K and Akt proteins in the PI3K/Akt/mTOR pathway. The results showed that treatment with 5-FU, apatinib, and cisplatin may upregulate the expression of PI3K protein in AGS gastric cancer cells. However, at the Akt protein level, apatinib showed an upregulation trend, while 5-FU and cisplatin may downregulate its expression. This indicates that even within the same signaling pathway, there may be differences in the response of different nodal proteins to the same drug. Due to the lack of functional validation using specific inhibitors of PI3K or Akt in this study, it cannot be determined whether these drugs affect PD-L1 expression by regulating this pathway. This inference requires further research to confirm. Secondly, we analyzed the MEK1 and ERK proteins in the MAPK pathway. The results showed that 5-FU may upregulate the expression of MEK1 and ERK proteins, while apatinib and cisplatin may downregulate the expression of these two proteins. It can be seen that in this pathway, there is a certain consistency in the response trend of MEK1 and ERK to specific drugs. Similarly, due to the lack of validation of MEK1 or ERK inhibitors, it is currently uncertain whether this pathway mediates the regulation of PD-L1 expression by drugs, and further experimental exploration is needed. In summary, this study investigated the regulatory effects of cytotoxic drugs (5-FU, cisplatin) and anti angiogenic drugs (apatinib) on PD-L1 in pMMR type AGS gastric cancer cells at the mRNA and protein levels, and preliminarily analyzed their potential signaling pathway mechanisms. The results showed that the above-mentioned drugs can upregulate the expression of PD-L1 and have a certain regulatory effect on key proteins in the PD-L1 regulatory pathway. This provides a partial mechanistic basis for the potential enhancement of the efficacy of pMMR type advanced gastric cancer by the combination of cytotoxic/anti angiogenic drugs and immune checkpoint inhibitors (ICIs) at the in vitro experimental level, and also lays a theoretical foundation for further exploration of reversing ICIs resistance in this type of gastric cancer. Declarations Author contributions All authors have made significant contributions to the work of this study, including the proposal of concepts, the design and implementation of the research, the acquisition, analysis, and statistical analysis of data, as well as the drafting and revision of the article. Disclosure The authors declare no potential conflicts of interest in this work. Declaration This research has not received any fund support. This study has been approved by the Ethics Committee of the Affiliated Hospital of Qinghai University, with the ethical approval number of SL-2023205. Funding Funding This work was supported by grants from the Youth Research Fund of Qinghai University in 2023 (No.2023-QYY-3) and Kunlun Talent High end Innovation and Entrepreneurship Talent project in Qinghai Province in 2023 (QingRenCaiZi 2023 No.9). References Janjigian YY, Shitara K, Moehler M, Garrido M, Salman P, Shen L, Wyrwicz L, Yamaguchi K, Skoczylas T, Campos Bragagnoli A, Liu T, Schenker M, Yanez P, Tehfe M, Kowalyszyn R, Karamouzis MV, Bruges R, Zander T, Pazo-Cid R, Hitre E, Feeney K, Cleary JM, Poulart V, Cullen D, Lei M, Xiao H, Kondo K, Li M, Ajani JA. First-line nivolumab plus chemotherapy versus chemotherapy alone for advanced gastric, gastro-oesophageal junction, and oesophageal adenocarcinoma (CheckMate 649): a randomised, open-label, phase 3 trial. Lancet 2021 07 03;398(10294) . DOI: 10.1016/S0140-6736(21)00797-2. J. Xu, H. Jiang, Y. Pan, K. Gu,S. Cang, L. Han,Y. Shu, J. Li, J. Zhao, H. Pan, S. Luo, Y. Qin, Q. Guo, Y. Bai, Y. Ling, Y. Guo, Z. Li, Y. Liu, Y. Wang, H. Zhou. LBA53 Sintilimab plus chemotherapy (chemo) versus chemo as first-line treatment for advanced gastric or gastroesophageal junction (G/GEJ) adenocarcinoma (ORIENT-16): First results of a randomized, double-blind, phase III study. ABSTRACT ONLY VOLUME 32, SUPPLEMENT 5, S1331, SEPTEMBER 01, 2021. DOI: https:// doi.org/ 10.1016/ j.annonc.2021.08.2133. Muto M, Nakata H, Ishigaki K, Tachibana S, Yoshida M, Muto M, Yanagawa N, Okumura T. Successful Treatment of Advanced Gastric Cancer with Brain Metastases through an Abscopal Effect by Radiation and Immune Checkpoint Inhibitor Therapy. J Gastric Cancer 2021 Sep;21(3) . DOI: 10.5230/jgc.2021.21.e24. Chen Y, Zhang C, Peng Z, Qi C, Gong J, Zhang X, Li J, Shen L. Association of Lymphocyte-to-Monocyte Ratio With Survival in Advanced Gastric Cancer Patients Treated With Immune Checkpoint Inhibitor. Front Oncol 2021;11. DOI：10.3389/fonc.2021.589022. Yang G, Zheng RY, Tan Q, Dong CJ, Jin ZS. Clinical characteristics and responses to chemotherapy and immune checkpoint inhibitor treatment for microsatellite instability gastric cancer. Am J Cancer Res 2020;10(12) . Song X, Qi W, Guo J, Sun L, Ding A, Zhao G, Li H, Qiu W, Lv J. Immune checkpoint inhibitor combination therapy for gastric cancer: Research progress. Oncol Lett 2020 Oct;20(4) . DOI：10.3892/ol.2020.11905 . Xie T, Zhang Z, Zhang X, Qi C, Shen L, Peng Z. Appropriate PD-L1 Cutoff Value for Gastric Cancer Immunotherapy: A Systematic Review and Meta-Analysis. Front Oncol 2021;11. DOI: 10.3389/fonc.2021.646355. Pietrantonio F, Randon G, Di Bartolomeo M, Luciani A, Chao J, Smyth EC, Petrelli F. Predictive role of microsatellite instability for PD-1 blockade in patients with advanced gastric cancer: a meta-analysis of randomized clinical trials. ESMO Open 2021 Feb;6(1). DOI: 10.1016/j.esmoop.2020.100036. Binnewies M, Roberts EW, Kersten K, Chan V, Fearon DF, Merad M, et al. Understanding the tumor immune microenvironment (TIME) for efective therapy. Nat Med. 2018;24(5):541–50. Giraldo NA, Sanchez-Salas R, Peske JD, Vano Y, Becht E, Petitprez F, et al. The clinical role of the TME in solid cancer. Br J Cancer. 2019;120(1):45–53. Saeed A, Park R, Sun W. The integration of immune checkpoint inhibitors with VEGF targeted agents in advanced gastric and gastroesophageal adenocarcinoma: a review on the rationale and results of early phase trials. J Hematol Oncol 2021 01 12;14(1) . DOI: 10.1186/s13045-021-01034-0. Eso Y, Shimizu T, Takeda H, Takai A, Marusawa H. Microsatellite instability and immune checkpoint inhibitors: toward precision medicine against gastrointestinal and hepatobiliary cancers. J Gastroenterol 2020 Jan;55(1) . DOI:10.1007/s00535-019-01620-7. Inderberg E M, Walchli S, Myhre M R, et al. T cell therapy targeting a public neoantigen in microsatellite instable colon cancer reduces in vivo tumor growth[J]. Oncoimmunology, 2017, 6(4): e1302631. Pietrantonio F, Lonardi S, Corti F, Infante G, Elez ME, Fakih M, Jayachandran P, Shah AT, Salati M. Nomogram to predict the outcomes of patients with microsatellite instability-high metastatic colorectal cancer receiving immune checkpoint inhibitors. J Immunother Cancer 2021 Aug;9(8) . DOI: 10.1136/jitc-2021-003370. Lengyel CG. Microsatellite Instability as a Predictor of Outcomes in Colorectal Cancer in the Era of Immune-Checkpoint Inhibitors. Curr Drug Targets 2021;22(9). DOI: 10.2174/1389450122666210325121322. Fucà G, Corti F, Ambrosini M, Intini R, Salati M, Fenocchio E, Manca P, Manai C, Daniel F. Prognostic impact of early tumor shrinkage and depth of response in patients with microsatellite instability-high metastatic colorectal cancer receiving immune checkpoint inhibitors. J Immunother Cancer 2021 Apr;9(4) . Cohen R, Colle R, Pudlarz T, Heran M, Duval A, Svrcek M, André T. Immune Checkpoint Inhibition in Metastatic Colorectal Cancer Harboring Microsatellite Instability or Mismatch Repair Deficiency. Cancers (Basel) 2021 Mar 08;13(5) . Ghidini M, Lampis A, Mirchev MB, Okuducu AF, Ratti M, Valeri N, Hahne JC. Immune-Based Therapies and the Role of Microsatellite Instability in Pancreatic Cancer. Genes (Basel) 2020 12 29;12(1) . DOI: 10.3390/genes12010033. Tian R, Hu J, Ma X, Liang L, Guo S. Immune-related gene signature predicts overall survival of gastric cancer patients with varying microsatellite instability status. Aging (Albany NY) 2020 12 09;13(2) . Wang Z, Zhao X, Gao C, Gong J, Wang X, Gao J, Li Z, Wang J, Yang B, Wang L, Zhang B. Plasma-based microsatellite instability detection strategy to guide immune checkpoint blockade treatment. J Immunother Cancer 2020 11;8(2) . Petrelli F, Ghidini M, Ghidini A, Tomasello G. Outcomes Following Immune Checkpoint Inhibitor Treatment of Patients With Microsatellite Instability-High Cancers: A Systematic Review and Meta-analysis. JAMA Oncol 2020 07 01;6(7). DOI: 10.1001/ jamaoncol.2020.1046. Jones NL, Xiu J, Rocconi RP, Herzog TJ, Winer IS. Immune checkpoint expression, microsatellite instability, and mutational burden: Identifying immune biomarker phenotypes in uterine cancer. Gynecol Oncol 2020 02;156(2) . DOI：10.1016/j.ygyno.2019.11.035. Fuchs C S, Doi T, Jang R W, et al. Safety and Efficacy of Pembrolizumab Monotherapy in Patients With Previously Treated Advanced Gastric and Gastroesophageal Junction Cancer: Phase 2 Clinical KEYNOTE-059 Trial[J]. JAMA Oncol,2018,4(5):e180013. Pietrantonio F, Randon G, Di Bartolomeo M, Luciani A, Chao J, Smyth EC, Petrelli F. Predictive role of microsatellite instability for PD-1 blockade in patients with advanced gastric cancer: a meta-analysis of randomized clinical trials. ESMO Open 2021 Feb;6(1). DOI: 10.1016/j.esmoop.2020.100036. Polonia A, Pinto R, Cameselle-Teijeiro J F, et al. Prognostic value of stromal tumour infiltrating lymphocytes and programmed cell death-ligand 1 expression in breast cancer[J]. J Clin Pathol,2017,70(10):860-867. Zerdes I, Matikas A, Bergh J, Rassidakis GZ, Foukakis T. Genetic, transcriptional and post-translational regulation of the programmed death protein ligand 1 in cancer: biology and clinical correlations. Oncogene 2018 08;37(34). DOI: 10.1038/s41388-018-0303-3. Fournel L, Wu Z, Stadler N, Damotte D, Lococo F, Boulle G, Ségal-Bendirdjian E, Bobbio A, Icard P, Trédaniel J, Alifano M, Forgez P. Cisplatin increases PD-L1 expression and optimizes immune check-point blockade in non-small cell lung cancer. Cancer Lett 2019 Nov 01;464. DOI: 10.1016/j.canlet.2019.08.005. Linda Tran, Clint T. Allen, Roy Xiao, Ellen Moore, Ruth Davis, So-Jin Park, Katie Spielbauer, Carter Van Waes, and Nicole C. Schmitt. Cisplatin Alters Antitumor Immunity and Synergizes with PD-1/PD-L1 Inhibition in Head and Neck Squamous Cell Carcinoma. American Association for Cancer Research.DOI: 10.1158/2326-6066.CIR-17-0235. Zhang P, Su DM, Liang M, Fu J. Chemopreventive agents induce programmed death-1-ligand 1 (PD-L1) surface expression in breast cancer cells and promote PD-L1-mediated T cell apoptosis. Mol Immunol. 2008;45:1470-6. Gong W, Song Q, Lu X, Gong W, Zhao J, Min P, et al. Paclitaxel induced B7-H1 expression in cancer cells via the MAPK pathway. J Chemother. 2011;23:295-9. Zheng B, Ren T, Huang Y, Guo W. Apatinib inhibits migration and invasion as well as PD-L1 expression in osteosarcoma by targeting STAT3. Biochem Biophys Res Commun. 2018;495(2):1695-701. ttps://doi.org/10.1016/j.bbrc.2017.12.032. Cai X, Wei B, Li L, Chen X, Liu W, Cui J, et al. Apatinib enhanced anti-PD-1 therapy for colon cancer in mice via promoting PD-L1 expression. Int Immunopharmacol. 2020;88:106858. https:// doi.org/ 10.1016/ j.intimp.2020.106858. Liang P, Ballou B, Lv X, Si W, Bruchez MP, Huang W, et al. Monotherapy and combination therapy using anti-angiogenic nanoagents to fight cancer. Adv Mater. 2021;33(15):e2005155. Zhang H. Apatinib for molecular targeted therapy in tumor. Drug Des Devel Ther. 2015;9:6075–81. Fathi Maroufi N, Rashidi MR, Vahedian V, Akbarzadeh M, Fattahi A, Nouri M. Therapeutic potentials of Apatinib in cancer treatment: possible mechanisms and clinical relevance. Life Sci. 2020;241:117106. https://doi. org/10.1016/j.lfs.2019.117106. Jia X, Wen Z, Sun Q, Zhao X, Yang H, Shi X, Xin T. Apatinib suppresses the Proliferation and Apoptosis of Gastric Cancer Cells via the PI3K/Akt Signaling Pathway. J BUON 2019 Sep-Oct;24(5) . Wang B, Qin L, Ren M, Sun H. Effects of Combination of Anti-CTLA-4 and Anti-PD-1 on Gastric Cancer Cells Proliferation, Apoptosis and Metastasis.Cell Physiol Biochem 2018;49(1) . DOI: 10.1159/000492876. Additional Declarations No competing interests reported. Supplementary Files TableS1.docx Table S1. Cell survival rates of AGS gastric cancer cells treated with 5-FU. TableS2.docx Table S2. Cell survival rate of AGS gastric cancer cells treated with cisplatin. TableS3.docx Table S3. Cell survival rate of AGS gastric cancer cells treated with apatinib. TableS4.docx Table S4. Effects of Cytotoxicity and Anti vascular Drugs on PD-L1 mRNA Expression in AGS Gastric Cancer Cells. TableS5.docx Table S5. Effects of Cytotoxicity and Anti vascular Drugs on PD-L1 Protein Expression in AGS Gastric Cancer Cells. FigureS1.jpg Figure S1. Detection of MMR protein in AGS gastric cancer cell. Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 17 Apr, 2026 Reviewers agreed at journal 24 Mar, 2026 Reviewers invited by journal 18 Mar, 2026 Editor assigned by journal 15 Mar, 2026 Submission checks completed at journal 13 Mar, 2026 First submitted to journal 12 Mar, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9102745","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":608359848,"identity":"3fe4c488-dc9a-41ac-aa4f-f44ae12ba831","order_by":0,"name":"Jinming Li","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jinming","middleName":"","lastName":"Li","suffix":""},{"id":608359849,"identity":"a065c4db-1109-4adf-ad22-3ea983010356","order_by":1,"name":"Mengting Da","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Mengting","middleName":"","lastName":"Da","suffix":""},{"id":608359850,"identity":"c44312b6-9ebc-4a73-a0d9-1bf43d63fd2e","order_by":2,"name":"Xueying Zhou","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Xueying","middleName":"","lastName":"Zhou","suffix":""},{"id":608359851,"identity":"9e0c7e9b-3762-425b-8472-04c88b050f7d","order_by":3,"name":"Dengfeng Ren","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Dengfeng","middleName":"","lastName":"Ren","suffix":""},{"id":608359852,"identity":"3b78b6ec-1aa7-4489-bad5-f4c932630d9b","order_by":4,"name":"Miaozhou Wang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Miaozhou","middleName":"","lastName":"Wang","suffix":""},{"id":608359853,"identity":"7f834334-8af0-4284-86d8-2b6491d1582b","order_by":5,"name":"Zhen Liu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Zhen","middleName":"","lastName":"Liu","suffix":""},{"id":608359854,"identity":"b5bde68d-f377-4d59-8671-8dc8f22d552c","order_by":6,"name":"Zhilin Liu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Zhilin","middleName":"","lastName":"Liu","suffix":""},{"id":608359855,"identity":"7dbc9a12-5eab-4af0-98e2-edace738d4bf","order_by":7,"name":"Zitao Li","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Zitao","middleName":"","lastName":"Li","suffix":""},{"id":608359856,"identity":"d3954ae8-121c-4740-a3d1-d669179f105d","order_by":8,"name":"Shifen Huang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Shifen","middleName":"","lastName":"Huang","suffix":""},{"id":608359857,"identity":"89c8657f-05d9-4f84-af58-a1225b626651","order_by":9,"name":"Yongzhi Chen","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Yongzhi","middleName":"","lastName":"Chen","suffix":""},{"id":608359858,"identity":"0d272e71-d552-4e60-b49a-318e318a09eb","order_by":10,"name":"Fuxing Zhao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA6UlEQVRIiWNgGAWjYBACNvb+j4///GCT42dvbHyQUFFDWAsfzwFjA94ePmPJnsPNBg/OHCOsRU4iwUyCh00uccOM9DbJhy3MRDiM50CahASPWeIGhsS2isQGNgb+9u4EAn5pOGxhYJFmvJ3hYNuNxB0yDBJnzm4gYMvBxhsJPMdkdzY2ArWcYWMwkMgloEUimUHiANt/xg2HGdsKEtuYidGSxiTZwMamuOEYYxsDcVp4zjAbM/awAQOZsVki4cwxHoJ+kW/vYXzMAIpK+ecPP/6oqJHjb+/FrwUD8JCmfBSMglEwCkYBVgAAa1dKc6Eg8yQAAAAASUVORK5CYII=","orcid":"","institution":"","correspondingAuthor":true,"prefix":"","firstName":"Fuxing","middleName":"","lastName":"Zhao","suffix":""}],"badges":[],"createdAt":"2026-03-12 09:10:49","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9102745/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9102745/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":105057546,"identity":"d4ede3cf-05a9-4540-b115-5133cac37c50","added_by":"auto","created_at":"2026-03-20 12:05:30","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":2204356,"visible":true,"origin":"","legend":"\u003cp\u003eCell survival rate analysis of AGS gastric cancer cells treated with different concentrations of 5-FU at different time points.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9102745/v1/e719a67e7ea843ba0bde6458.jpg"},{"id":105562875,"identity":"e94b2124-eb75-4be7-a94d-8ccffe7f93fe","added_by":"auto","created_at":"2026-03-27 12:45:05","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":2039755,"visible":true,"origin":"","legend":"\u003cp\u003eSurvival rate analysis of AGS gastric cancer cells treated with different concentrations of cisplatin at different time points.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9102745/v1/a833361f264765a5b793524a.jpg"},{"id":105057566,"identity":"26d1a24f-99e1-4e4b-8fe4-1b3d57c51a0c","added_by":"auto","created_at":"2026-03-20 12:05:35","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2040759,"visible":true,"origin":"","legend":"\u003cp\u003eSurvival rate analysis of AGS gastric cancer cells treated with different concentrations of apatinib at different time points.\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9102745/v1/0b32bda4bab657d2803d8077.jpg"},{"id":105057561,"identity":"ce14ca8b-9885-41e1-baf8-13d1e16b364e","added_by":"auto","created_at":"2026-03-20 12:05:31","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":200452,"visible":true,"origin":"","legend":"\u003cp\u003eqPCR detection verification of the effects of cytotoxicity and anti vascular drugs on PD-L1 mRNA expression in AGS gastric cancer cells(A: control group, B: 5-FU 4uM, C: 5-FU 16uM, D: 5-FU 64uM, E: apatinib 320 uM, F: apatinib 640 uM, G: cisplatin 16uM, H: cisplatin 32uM, I: cisplatin 64uM).\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9102745/v1/cebb4dbca9b9917a6871e991.jpg"},{"id":105057564,"identity":"69647361-8eba-4420-8c9c-076ecf48c308","added_by":"auto","created_at":"2026-03-20 12:05:31","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":681122,"visible":true,"origin":"","legend":"\u003cp\u003eWestern Blot detection of the effects of cytotoxicity and anti angiogenic drugs on PD-L1 protein expression in AGS gastric cancer cells.\u003c/p\u003e","description":"","filename":"Figure5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9102745/v1/504b0d16de0ee7eef88fc69f.jpg"},{"id":105057557,"identity":"f3ec6980-bdd3-4e91-9548-d79dc2f46ce2","added_by":"auto","created_at":"2026-03-20 12:05:30","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":786010,"visible":true,"origin":"","legend":"\u003cp\u003eWestern Blot detection of the effects of cytotoxicity and anti angiogenic drugs on PD-L1 protein expression in AGS gastric cancer cells(1: Control group, 2:5-FU 64uM, 3: Apatinib 320 nM, 4: Cisplatin 16uM).\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9102745/v1/910e1f2308a1b354ce0e40b1.jpg"},{"id":105057551,"identity":"c5efa2f5-bda6-41fe-9c38-393943f47801","added_by":"auto","created_at":"2026-03-20 12:05:30","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":787416,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of PI3K and Akt proteins in AGS gastric cancer cells treated with cytotoxicity and anti angiogenic drugs.\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9102745/v1/9bc357cffc3efbf0d2bce502.jpg"},{"id":105057553,"identity":"0ad5df1f-b70e-4c63-915f-ac9095dceb41","added_by":"auto","created_at":"2026-03-20 12:05:30","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":933439,"visible":true,"origin":"","legend":"\u003cp\u003eHistogram of PI3K and Akt protein expression in AGS gastric cancer cells treated with cytotoxicity and anti angiogenic drugs(1: Control group, 2: Cells+5-FU, 3: Cells+Apatinib, 4: Cells+Cisplatin).\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9102745/v1/ed7c6ca8600b78b1ba81f44a.jpg"},{"id":105057550,"identity":"c739119c-186f-4cf5-9811-1fc6592e3f6a","added_by":"auto","created_at":"2026-03-20 12:05:30","extension":"jpg","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":732572,"visible":true,"origin":"","legend":"\u003cp\u003eExpression of ERK and MEK1 proteins in AGS gastric cancer cells treated with cytotoxicity and anti angiogenic drugs.\u003c/p\u003e","description":"","filename":"Figure9.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9102745/v1/42fc55f5815ec5c52611f416.jpg"},{"id":105057549,"identity":"14cbe4bc-c95a-4829-bb4b-fd2b278bb005","added_by":"auto","created_at":"2026-03-20 12:05:30","extension":"jpg","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":917904,"visible":true,"origin":"","legend":"\u003cp\u003eHistogram of ERK and MEK1 protein expression in AGS gastric cancer cells treated with cytotoxicity and anti angiogenic drugs(1: Control group, 2: Cells+5-FU, 3: Cells+Apatinib, 4: Cells+Cisplatin).\u003c/p\u003e","description":"","filename":"Figure10.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9102745/v1/b5024dc83ef89454538a060f.jpg"},{"id":105907144,"identity":"aeb1f044-33e3-47d1-b964-8f90a7c32616","added_by":"auto","created_at":"2026-04-01 10:28:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":12195042,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9102745/v1/65ca709d-b6d8-4463-ad6b-a93cf3849c4a.pdf"},{"id":105057544,"identity":"960a6fb7-beb3-49ca-9d58-7250bfe46eb3","added_by":"auto","created_at":"2026-03-20 12:05:30","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":14409,"visible":true,"origin":"","legend":"\u003cp\u003eTable S1. Cell survival rates of AGS gastric cancer cells treated with 5-FU.\u003c/p\u003e","description":"","filename":"TableS1.docx","url":"https://assets-eu.researchsquare.com/files/rs-9102745/v1/35cf513f46a8b166f6d43439.docx"},{"id":105057548,"identity":"fe9c95d1-9f47-415d-9898-2a8673d3a587","added_by":"auto","created_at":"2026-03-20 12:05:30","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":14439,"visible":true,"origin":"","legend":"\u003cp\u003eTable S2. Cell survival rate of AGS gastric cancer cells treated with cisplatin.\u003c/p\u003e","description":"","filename":"TableS2.docx","url":"https://assets-eu.researchsquare.com/files/rs-9102745/v1/bc79c9b87fd123961d1a89d9.docx"},{"id":105057559,"identity":"297be6d3-3e35-4b3d-a38b-2de4de71d5c5","added_by":"auto","created_at":"2026-03-20 12:05:31","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":14469,"visible":true,"origin":"","legend":"\u003cp\u003eTable S3. Cell survival rate of AGS gastric cancer cells treated with apatinib.\u003c/p\u003e","description":"","filename":"TableS3.docx","url":"https://assets-eu.researchsquare.com/files/rs-9102745/v1/b5d80dfbbbac01adc84233b7.docx"},{"id":105057562,"identity":"92b91ae0-1acb-4f01-945a-285840833312","added_by":"auto","created_at":"2026-03-20 12:05:31","extension":"docx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":17023,"visible":true,"origin":"","legend":"\u003cp\u003eTable S4. Effects of Cytotoxicity and Anti vascular Drugs on PD-L1 mRNA Expression in AGS Gastric Cancer Cells.\u003c/p\u003e","description":"","filename":"TableS4.docx","url":"https://assets-eu.researchsquare.com/files/rs-9102745/v1/fa4662ced28189d31906f30a.docx"},{"id":105057552,"identity":"684e9b84-2be5-41c1-9f30-695d74bbd344","added_by":"auto","created_at":"2026-03-20 12:05:30","extension":"docx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":13441,"visible":true,"origin":"","legend":"\u003cp\u003eTable S5. Effects of Cytotoxicity and Anti vascular Drugs on PD-L1 Protein Expression in AGS Gastric Cancer Cells.\u003c/p\u003e","description":"","filename":"TableS5.docx","url":"https://assets-eu.researchsquare.com/files/rs-9102745/v1/cedd1eb627d30d20e05e2fdc.docx"},{"id":105057555,"identity":"80600c2e-0ffa-4da8-99d8-9644a8e6f6e9","added_by":"auto","created_at":"2026-03-20 12:05:30","extension":"jpg","order_by":6,"title":"","display":"","copyAsset":false,"role":"supplement","size":802462,"visible":true,"origin":"","legend":"\u003cp\u003eFigure S1. Detection of MMR protein in AGS gastric cancer cell.\u003c/p\u003e","description":"","filename":"FigureS1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9102745/v1/62941499aa9a36ada778d3a4.jpg"}],"financialInterests":"No competing interests reported.","formattedTitle":"Exploration of the regulatory mechanism of PD-L1 expression in pMMR type gastric cancer cell by cytotoxic drugs and anti angiogenic drugs","fulltext":[{"header":"Background","content":"\u003cp\u003eIn patients with advanced gastric cancer, the survival benefit achieved with chemotherapy has long been difficult to surpass, while the introduction of immune checkpoint inhibitors(ICIs) has provided more therapeutic options. ICIs function by targeting the interaction between programmed death-1(PD-1) and programmed death-ligand 1(PD-L1). Numerous studies have demonstrated the efficacy of ICIs in treating advanced gastric cancer\u003csup\u003e[\u003cspan additionalcitationids=\"CR2 CR3 CR4 CR5 CR6 CR7\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eDespite their effectiveness, the issue of resistance to ICIs in advanced gastric cancer warrants attention\u003csup\u003e[\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]\u003c/sup\u003e. Patients with proficient mismatch repairp(MMR) advanced gastric cancer exhibit resistance to ICI therapy, showing inferior responses compared to those with deficient mismatch repair(dMMR) tumors. This is because dMMR tumors, due to reduced mismatch repair function, have the potential to encode novel non-self antigens, subsequently attracting greater T-lymphocyte infiltration into the tumor and promoting dominant anti-tumor immunity. In contrast, pMMR gastric cancers lack this effect, resulting in weaker anti-tumor immune responses\u003csup\u003e[\u003cspan additionalcitationids=\"CR13 CR14 CR15 CR16 CR17 CR18 CR19 CR20 CR21\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e, which may be one reason for ICI resistance in pMMR patients.\u003c/p\u003e \u003cp\u003eIn the KEYNOTE-059 study, the objective response rate (ORR) to pembrolizumab was 57% in patients with dMMR advanced gastric cancer, compared to only 9.0% in pMMR patients\u003csup\u003e[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. Overall survival (OS) follow-up data from the CheckMate 649 study showed a hazard ratio (HR) of 0.33 (95% CI 0.12\u0026ndash;0.87, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) for nivolumab plus chemotherapy versus chemotherapy alone in the dMMR subgroup, and an HR of 0.73 (95% CI 0.62\u0026ndash;0.85, \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the pMMR subgroup. This indicates that while both dMMR and pMMR patients can benefit from ICI therapy, the survival benefit remains greater for dMMR patients\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]\u003c/sup\u003e. A meta-analysis incorporating the KEYNOTE-062, CheckMate-649, JAVELIN Gastric 100, and KEYNOTE-061 studies further confirmed that dMMR patients derive greater OS benefit from anti-PD-1 therapy than pMMR patients\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eGiven that pMMR gastric cancers respond less effectively to ICIs and constitute the majority of cases (dMMR accounts for only about 6.2%)\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e, strategies to improve ICI efficacy in this large patient population require further exploration. Current research suggests that combining cytotoxic drugs (chemotherapy) or anti-angiogenic therapy with ICIs can enhance treatment outcomes in solid tumors. These studies indicate that cytotoxic and anti-angiogenic agents may influence ICI efficacy by modulating PD-L1 mRNA and protein expression levels\u003csup\u003e[\u003cspan additionalcitationids=\"CR27 CR28 CR29 CR30 CR31 CR32 CR33 CR34\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. Some studies also suggest that anti-angiogenic therapy and ICIs can directly inhibit tumor cell proliferation\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. However, for patients with pMMR advanced gastric cancer, there is still a lack of research exploring the impact and underlying mechanisms of cytotoxic and anti-angiogenic drugs on ICI efficacy, both in vitro and in vivo.\u003c/p\u003e \u003cp\u003eChemotherapeutic agents can regulate cancer cell PD-L1 expression, thereby influencing the efficacy of ICIs\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Studies show that cisplatin upregulates PD-L1 mRNA and protein expression both in vitro and vivo. Its combination with anti-PD-L1 antibodies significantly inhibits tumor growth, a process potentially mediated by the PI3K/AKT signaling pathway, as AKT inhibitors can block this effect\u003csup\u003e[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]\u003c/sup\u003e. In head and neck squamous cell carcinoma, cisplatin similarly induces PD-L1 expression, suggesting synergistic potential with PD-1/PD-L1 inhibitors\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. Other chemotherapeutic drugs exhibit similar effects. Paclitaxel and etoposide upregulate PD-L1 in breast cancer cells MCF-7 and MDA-MB-435, and fluorouracil has the same effect on cells like MDA-MB-468\u003csup\u003e[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]\u003c/sup\u003e. In colon cancer SW480 and liver cancer HepG2 cells, paclitaxel-induced PD-L1 upregulation can be inhibited by MEK inhibitors, implicating the MAPK pathway in its regulation\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. In summary, various chemotherapeutic drugs can upregulate PD-L1 by activating specific signaling pathways, providing a theoretical basis for combination strategies with immunotherapy.\u003c/p\u003e \u003cp\u003eIn light of the above, cytotoxic drugs such as cisplatin, paclitaxel, and fluorouracil may influence PD-L1 mRNA and protein expression in cancer cells through different mechanisms, potentially further affecting the therapeutic outcome of PD-L1/PD-1 inhibitors. However, research specifically investigating the effects of cisplatin, paclitaxel, fluorouracil, and other chemotherapeutic agents on PD-L1 expression in pMMR gastric cancer cells is limited and requires further exploration.\u003c/p\u003e \u003cp\u003eMechanisms by which anti-angiogenic drugs enhance ICI efficacy include modulating cancer cell PD-L1 expression and improving the tumor microenvironment (TME)\u003csup\u003e[\u003cspan additionalcitationids=\"CR32\" citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]\u003c/sup\u003e. Apatinib, a tyrosine kinase inhibitor (TKI) that selectively targets VEGFR2, exerts its anti-angiogenic effect by inhibiting VEGFR-induced endothelial cell proliferation and migration\u003csup\u003e[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e. Research indicates that the impact of apatinib on PD-L1 expression is cancer-type specific: it downregulates PD-L1 in osteosarcoma by inhibiting STAT3 signaling\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e, whereas it upregulates PD-L1 in colon cancer CT26 cells and, in combination with anti-PD-1 antibodies, significantly inhibits tumor growth in mice\u003csup\u003e[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. However, the regulatory effect of apatinib on PD-L1 expression in gastric cancer remains unclear and warrants further investigation. Beyond modulating the immune microenvironment, apatinib can also directly inhibit gastric cancer cell proliferation and induce apoptosis, a mechanism potentially related to the inhibition of PI3K/Akt pathway phosphorylation\u003csup\u003e[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. Similarly, PD-1 inhibitors alone can directly inhibit gastric cancer cell proliferation by regulating the β-catenin, MAPK, and PI3K/Akt pathways\u003csup\u003e[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]\u003c/sup\u003e. These findings suggest that the combined application of apatinib and PD-1 inhibitors may synergistically enhance anti-tumor effects through multiple mechanisms.\u003c/p\u003e \u003cp\u003eThe expression of PD-L1 on cancer cells is regulated by multiple signaling pathways, with the RAS/RAF/MEK/MAPK-ERK and PI3K/PTEN/Akt/mTOR pathways being two key regulators. Their effects on PD-L1 expression vary across different cancer types such as melanoma, breast cancer, and non-small cell lung cancer\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. Studies suggest that cytotoxic drugs and anti-angiogenic agents may regulate PD-L1 expression by influencing the PI3K/Akt and MAPK pathways, thereby affecting ICI efficacy. However, the specific pathways through which these drugs regulate PD-L1 expression in pMMR gastric cancer cells have not been elucidated and require further exploration.\u003c/p\u003e \u003cp\u003eThis study aims to explore the effects of cytotoxic drugs and anti-angiogenic agents on PD-L1 mRNA and protein expression in pMMR gastric cancer cells, along with the associated pathways. The goal is to investigate potential mechanisms for improving ICI efficacy in pMMR gastric cancer, thereby providing a basis for further research into methods to enhance the therapeutic outcomes of ICI treatment for this patient population.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eCell Lines and Culture Conditions\u003c/h2\u003e \u003cp\u003eHuman gastric adenocarcinoma cell lines AGS were obtained from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. Cells were routinely cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% penicillin-streptomycin (100 U/mL penicillin, 100 \u0026micro;g/mL streptomycin). All cells were maintained in a humidified incubator at 37\u0026deg;C with 5% CO₂. The culture medium was refreshed every 2\u0026ndash;3 days. Subculturing was performed using 0.25% trypsin-EDTA when cell confluence reached approximately 80\u0026ndash;90%.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eCell Viability Assay (CCK-8)\u003c/h3\u003e\n\u003cp\u003eCell viability was assessed using the Cell Counting Kit-8 (CCK-8) assay. Cells were seeded into 96-well plates. The treatment groups and concentration gradients were as follows: 5-Fluorouracil (5-FU): 0, 2, 4, 8, 16, 32, 64, 128\u0026micro;M; cisplatin: 0, 2, 4, 8, 16, 32, 64, 128\u0026micro;M; apatinib: 0, 10, 20, 40, 80, 160, 320, 640nM. At each designated time point (0, 24, 48, 72, and 96 hours), the culture medium was carefully removed. Subsequently, 100 \u0026micro;L of fresh medium containing 10% CCK-8 reagent was added to each well. The plates were incubated for 2 hours at 37\u0026deg;C. The absorbance of each well was then measured at a wavelength of 450nm using a microplate reader. Each experimental condition was performed in triplicate.\u003c/p\u003e\n\u003ch3\u003eProtein Extraction\u003c/h3\u003e\n\u003cp\u003eCells were washed twice with ice-cold phosphate-buffered saline (PBS). Total protein was extracted using RIPA lysis buffer freshly supplemented with 1\u0026times; PhosSTOP phosphatase inhibitor cocktail and 1 mM phenylmethylsulfonyl fluoride (PMSF). Cells were scraped on ice and the lysates were transferred to pre-chilled microcentrifuge tubes. After incubation on dry ice for 30 minutes to facilitate complete lysis, the samples were centrifuged at 12,000 rpm for 15 minutes at 4\u0026deg;C. The supernatant containing the soluble proteins was carefully collected, aliquoted, and stored at -80\u0026deg;C until further use.\u003c/p\u003e\n\u003ch3\u003eWestern Blotting\u003c/h3\u003e\n\u003cp\u003eProtein concentration was determined using a bicinchoninic acid (BCA) assay. Equal amounts of protein (typically 20\u0026ndash;40 \u0026micro;g) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electrophoretically transferred onto polyvinylidene difluoride (PVDF) membranes. The membranes were blocked with 5% non-fat milk or bovine serum albumin (BSA) in Tris-buffered saline with Tween 20 (TBST) for 1 hour at room temperature. Subsequently, they were incubated overnight at 4\u0026deg;C with specific primary antibodies (e.g., anti-PD-L1). After extensive washing, membranes were incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour at room temperature. Protein bands were visualized using an enhanced chemiluminescence (ECL) detection system and imaged.\u003c/p\u003e\n\u003ch3\u003eRNA Extraction\u003c/h3\u003e\n\u003cp\u003eTotal RNA was extracted from cultured cells using RNAiso Plus reagent according to the manufacturer's instructions. Briefly, cells were lysed directly in the culture dish by adding the reagent. Chloroform was added for phase separation, and the aqueous phase containing RNA was collected. RNA was precipitated with isopropanol, washed with 75% ethanol, and finally dissolved in RNase-free water. RNA concentration and purity were assessed by measuring the absorbance at 260 nm and 280 nm using a spectrophotometer. RNA integrity was confirmed by agarose gel electrophoresis. RNA samples were stored at -80\u0026deg;C.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eqPCR\u003c/h2\u003e \u003cp\u003eFirst, total RNA is extracted from samples, treated with DNase I, and its quality is verified. This RNA is then reverse transcribed into cDNA using reverse transcriptase with appropriate primers. The qPCR reaction is assembled by mixing the cDNA template, gene-specific primers, a detection system (SYBR Green dye or TaqMan probes), and a PCR master mix in a plate. The plate is run in a real-time PCR instrument under a thermal cycling program: initial denaturation (e.g., 95\u0026deg;C for 2\u0026ndash;5 min), followed by 40\u0026ndash;45 cycles of denaturation, annealing, and extension. Fluorescence is measured each cycle. A melt curve analysis is performed for SYBR Green assays to check specificity. Finally, the cycle threshold (Ct) values are used to calculate relative gene expression (e.g., via the 2^(-ΔΔCt) method), normalized to reference genes and a control.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll quantitative data are presented as the mean\u0026plusmn;standard deviation (SD) from at least three independent experiments. Statistical comparisons between multiple groups were performed using one-way analysis of variance (ANOVA) followed by appropriate post-hoc tests. All statistical analyses were conducted using SPSS software (version 22.0). A p-value of less than 0.05 (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Result","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDetection of MMR status in AGS gastric cancer cells\u003c/h2\u003e \u003cp\u003ePrior to commencing the experiments, it was necessary to confirm that the AGS gastric cancer cell used in this study were of the pMMR (proficient mismatch repair) status. This was achieved by assessing the expression of four key MMR proteins (MSH6, MSH2, MLH1, and PMS2) in both cell lines via Western Blot analysis. The Western blot results showed that MSH6, MSH2, MLH1, and PMS2 proteins were expressed in AGS cancer cells\u003cb\u003e(Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e)\u003c/b\u003e, indicating that the AGS gastric cancer cells used in this study were pMMR type.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eBaseline PD-L1 Expression Assay\u003c/h2\u003e \u003cp\u003eTo evaluate the effect of drugs on PD-L1 expression, the basal expression level of PD-L1 mRNA in AGS gastric cancer cells was first examined. qPCR results demonstrated that PD-L1 mRNA is constitutively expressed in AGS cells. Western blot analysis further confirmed the normal expression of PD-L1 protein in AGS gastric cancer cells.\u003c/p\u003e \u003cp\u003e \u003cb\u003eCCK8 verifies the effects of cytotoxic drugs and anti angiogenic drugs on the proliferation of gastric cancer cells\u003c/b\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eExperimental grouping\u003c/h2\u003e \u003cp\u003eThe intervention groups were 5-FU group, cisplatin group, and apatinib group, with specific drug concentration gradients. Fluorouracil and cisplatin were cytotoxic drugs, while apatinib was an anti vascular drug. We referred to relevant literature to determine the drug concentration gradient for CCK8 detection\u003csup\u003e[\u003cspan additionalcitationids=\"CR28 CR29 CR30 CR31\" citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCCK-8 assay\u003c/h2\u003e \u003cp\u003eWe conducted CCK8 testing on patients in the 5-FU group, cisplatin group, and apatinib group. The concentration gradient of the 5-FU group was 0 uM、2uM、4uM、8uM、16uM、32uM、64uM、128uM, the concentration gradient of cisplatin group is 0uM, 2uM, 4uM, 8uM, 16uM, 32uM, 64uM, 128uM, while the concentration gradient of apatinib group is 0 nM、10nM、20nM、40nM、80nM、160nM、320nM、640nM, Measure the absorbance at 0h, 24h, 48h, 72h, and 96h after cultivation, repeat 3 times for each group, and calculate the average survival rate.\u003c/p\u003e \u003cp\u003eThe results showed that in the 5-FU group, the overall survival rate of AGS gastric cancer cells showed a decreasing trend with the prolongation of culture time. After 72 hours and 96 hours, there were statistically significant differences (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the survival rate of AGS gastric cancer cells at different concentration gradients compared to the control group \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e1\u003c/span\u003e, \u003cb\u003eTable \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the cisplatin group, overall, the survival rate of AGS gastric cancer cells decreased with prolonged culture time, and this downward trend in survival rate was observed in various concentration gradients. After 72 and 96 hours of observation, except for 2uM, there was a statistically significant difference (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) in the survival rate of AGS gastric cancer cells compared to the control group in other concentration gradients \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e2\u003c/span\u003e, \u003cb\u003eTable \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the apatinib group, the survival rate of AGS gastric cancer cells showed inconsistent trends with the prolongation of culture time at different concentrations of apatinib. After 72 hours of observation, the survival rate of AGS gastric cancer cells treated with 320nM and 640nM concentrations was lower than that of the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). After 96 hours of observation, the survival rate of AGS gastric cancer cells treated with 640nM concentration was lower than that of the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e3\u003c/span\u003e, Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eqPCR detection verification of the effects of cytotoxicity and anti vascular drugs on PD-L1 mRNA expression in pMMR type AGS gastric cancer cells\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo investigate the effects of cytotoxic and anti-angiogenic drugs on PD-L1 mRNA expression in AGS gastric cancer cells, drug concentrations that effectively inhibited cell proliferation covering a broad range were selected based on CCK-8 assay results. The cells were treated with 5-FU (4\u0026micro;M, 16\u0026micro;M, 64\u0026micro;M), cisplatin (16\u0026micro;M, 32\u0026micro;M, 64\u0026micro;M), or apatinib (320nM, 640nM). After 72 hours of treatment, mRNA was extracted and subjected to qPCR analysis.\u003c/p\u003e \u003cp\u003eThe results showed that in the 5-FU treatment group, the 64\u0026micro;M concentration significantly upregulated PD-L1 mRNA expression, whereas the 4\u0026micro;M and 16\u0026micro;M concentrations showed no significant effect. In the apatinib treatment group, both the 320nM and 640nM concentrations significantly upregulated PD-L1 mRNA expression. In the cisplatin treatment group, the 16\u0026micro;M concentration significantly upregulated PD-L1 mRNA expression, while the 32\u0026micro;M and 64\u0026micro;M concentrations had no significant effect. All other tested concentrations showed no statistically significant difference compared to the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05), indicating no apparent regulatory effect on PD-L1 mRNA expression \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e4\u003c/span\u003e, \u003cb\u003eTable \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eWestern Blot experiment verifies the effects of cytotoxicity and anti vascular drugs on PD-L1 protein expression in pMMR type AGS gastric cancer cells\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo investigate the effects of cytotoxic and anti-angiogenic drugs on PD-L1 protein expression in AGS gastric cancer cell, Western blot analysis was performed. Based on prior qPCR results indicating upregulation of PD-L1 mRNA, cells were treated with 5-FU (64\u0026micro;M), cisplatin (16\u0026micro;M), or apatinib (320nM). Following a 72-hour drug treatment, proteins were extracted, and the experiment was conducted with three replicates per group. The results demonstrated that treatment with 5-FU (64\u0026micro;M), apatinib (320nM), and cisplatin (16\u0026micro;M) significantly upregulated PD-L1 protein levels in AGS gastric cancer cell compared to the control group. The differences in Western blot band intensity were statistically significant (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) \u003cb\u003e(\u003c/b\u003e Figs.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e5\u003c/span\u003e, \u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e6\u003c/span\u003e, \u003cb\u003eTable \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cb\u003eInvestigating the Regulation of key proteins in pathways associated with PD-L1 expression by Cytotoxic and Anti-Angiogenic Drugs in Gastric Cancer Cells\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTo investigate the mechanisms by which cytotoxic and anti-angiogenic agents regulate PD-L1 protein expression in AGS gastric cancer cells, this study, based on literature reports, selected the RAS/RAF/MEK/ERK and PI3K/Akt/mTOR signaling pathways for exploration, as these pathways have been clearly shown to regulate PD-L1 in various cancers\u003csup\u003e[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]\u003c/sup\u003e. We analyzed key proteins within these pathways, including PI3K, Akt, MEK1, and ERK. The experiment utilized drug concentrations that upregulate both PD-L1 mRNA and protein expression in AGS cells (5-FU 64\u0026micro;M, cisplatin 16\u0026micro;M, apatinib 320nM). Cells were treated for 72 hours, with three replicates per group.\u003c/p\u003e \u003cp\u003eWestern blot analysis revealed that treatment with 5-FU, apatinib, and cisplatin significantly upregulated the expression of PI3K protein in AGS cells compared to the control group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Regarding AKT protein expression, apatinib treatment demonstrated an upregulatory effect (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05), whereas 5-FU and cisplatin significantly downregulated its expression (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) \u003cb\u003e(\u003c/b\u003eFig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e7\u003c/span\u003e, \u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e8\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWestern Blot results demonstrated that 5-FU treatment significantly upregulated the expression levels of both MEK1 and ERK proteins in AGS cells (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Conversely, treatment with apatinib and cisplatin significantly downregulated the expression of these two proteins (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) \u003cb\u003e(Figure. 9, 10)\u003c/b\u003e.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eICIs combined with chemotherapy have brought good survival benefits to patients with advanced gastric cancer\u003csup\u003e[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]\u003c/sup\u003e. However, due to the lack of the ability to generate new antigens in pMMR type gastric cancer with complete mismatch repair function, it is difficult to recruit enough tumor infiltrating lymphocytes to initiate effective anti-tumor immune responses\u003csup\u003e[\u003cspan additionalcitationids=\"CR13 CR14 CR15 CR16 CR17 CR18 CR19 CR20 CR21\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/sup\u003e, which may lead to poor efficacy of ICIs in pMMR type advanced gastric cancer patients. This may be one of the reasons why pMMR type gastric cancer is resistant to ICIs. Given that pMMR type gastric cancer accounts for a high proportion in advanced gastric cancer (dMMR type only accounts for about 6.2%)\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e, it is particularly necessary to find strategies to improve the efficacy of ICIs in this group of patients.\u003c/p\u003e \u003cp\u003eThe expression level of PD-L1 is a potential pathway to overcome ICIs resistance. Some studies show that dMMR type advanced gastric cancer patients benefit more from ICIs treatment than pMMR type patients\u003csup\u003e[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]\u003c/sup\u003e. Due to the relatively poor response and high proportion of pMMR type gastric cancer to ICIs\u003csup\u003e[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]\u003c/sup\u003e, exploring methods to improve its efficacy is of great significance. At present, many studies have shown that cytotoxic drugs and anti angiogenic drugs can regulate the anti-tumor effect of ICIs by affecting the mRNA and protein expression of PD-L1\u003csup\u003e[\u003cspan additionalcitationids=\"CR27 CR28 CR29 CR30 CR31 CR32 CR33 CR34\" citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]\u003c/sup\u003e, but there is still a lack of research on how these two types of drugs affect the efficacy and mechanism of ICIs in pMMR type gastric cancer.\u003c/p\u003e \u003cp\u003eIn recent years, many studies have focused on how to upregulate PD-L1 expression in cancer cells in order to enhance the combined therapeutic effect of immune checkpoint inhibitors (ICIs). At present, drugs used in combination with ICIs mainly include two categories: cytotoxic drugs and anti angiogenic drugs. In terms of cytotoxic drugs, existing evidence suggests that they may enhance ICIs therapy by upregulating PD-L1 expression. For example, cisplatin has been shown to upregulate the mRNA and protein levels of PD-L1 in lung cancer cells\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. In head and neck squamous cell carcinoma, cisplatin also showed upregulation of PD-L1, and its combination with ICIs demonstrated stronger anti-tumor activity\u003csup\u003e[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]\u003c/sup\u003e. In addition, paclitaxel, fluorouracil and other drugs have also been reported to up regulate the expression of PD-L1 in breast cancer, colon cancer and liver cancer cell lines\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]\u003c/sup\u003e. These findings collectively suggest that upregulating PD-L1 expression is a potential key mechanism for cytotoxic drugs to enhance the efficacy of ICIs and overcome drug resistance. However, it is not clear whether the above regulatory effects are applicable in pMMR gastric cancer cells with complete mismatch repair function. Therefore, this study explored this issue. Our results showed that qPCR detection indicated that 5-fluorouracil (5-FU) and cisplatin could upregulate the mRNA expression of PD-L1 in pMMR type AGS gastric cancer cells. Furthermore, Western Blot further confirmed that these two drugs can also upregulate the expression of PD-L1 protein at specific concentrations. Although differences in results were observed at individual drug concentrations, overall, specific cytotoxic drugs and their concentrations showed a clear upregulation trend on PD-L1 expression. Further experiments are needed to validate and refine this conclusion.\u003c/p\u003e \u003cp\u003eThe combination of anti angiogenic drugs and immune checkpoint inhibitors (ICIs) can enhance therapeutic efficacy, and its mechanisms are diverse, among which upregulation of PD-L1 expression in tumor cells is an important aspect. Research has shown that apatinib can upregulate PD-L1 in mouse colon cancer CT26 cells, and its combination with ICIs exhibits better anti-tumor effects\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]\u003c/sup\u003e. In this study, qPCR experiments showed that apatinib upregulated PD-L1 mRNA in pMMR type AGS gastric cancer cells, and Western blot results also indicated its upregulation of PD-L1 protein expression. However, it is worth noting that other studies have reported that apatinib can inhibit the expression of PD-L1 protein in osteosarcoma KHOS and U2OS cells\u003csup\u003e[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]\u003c/sup\u003e. This suggests that the regulatory effect of anti angiogenic drugs on PD-L1 may have cancer specificity, and its specific mechanism still needs to be further explored.\u003c/p\u003e \u003cp\u003eAt present, it is believed that cytotoxic drugs and anti angiogenic drugs regulate PD-L1 expression, mainly involving key signaling pathways such as MAPK and PI3K/Akt. Numerous studies have confirmed that such drugs can affect the expression level of PD-L1 by regulating key proteins in the aforementioned pathways\u003csup\u003e[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]\u003c/sup\u003e. Therefore, analyzing the effects of these drugs on key proteins in related pathways is of great significance for elucidating their mechanisms of regulating PD-L1 expression in pMMR type gastric cancer cells, and providing theoretical basis for improving the efficacy of ICIs. Based on this, this study first analyzed the PI3K and Akt proteins in the PI3K/Akt/mTOR pathway. The results showed that treatment with 5-FU, apatinib, and cisplatin may upregulate the expression of PI3K protein in AGS gastric cancer cells. However, at the Akt protein level, apatinib showed an upregulation trend, while 5-FU and cisplatin may downregulate its expression. This indicates that even within the same signaling pathway, there may be differences in the response of different nodal proteins to the same drug. Due to the lack of functional validation using specific inhibitors of PI3K or Akt in this study, it cannot be determined whether these drugs affect PD-L1 expression by regulating this pathway. This inference requires further research to confirm. Secondly, we analyzed the MEK1 and ERK proteins in the MAPK pathway. The results showed that 5-FU may upregulate the expression of MEK1 and ERK proteins, while apatinib and cisplatin may downregulate the expression of these two proteins. It can be seen that in this pathway, there is a certain consistency in the response trend of MEK1 and ERK to specific drugs. Similarly, due to the lack of validation of MEK1 or ERK inhibitors, it is currently uncertain whether this pathway mediates the regulation of PD-L1 expression by drugs, and further experimental exploration is needed.\u003c/p\u003e \u003cp\u003eIn summary, this study investigated the regulatory effects of cytotoxic drugs (5-FU, cisplatin) and anti angiogenic drugs (apatinib) on PD-L1 in pMMR type AGS gastric cancer cells at the mRNA and protein levels, and preliminarily analyzed their potential signaling pathway mechanisms. The results showed that the above-mentioned drugs can upregulate the expression of PD-L1 and have a certain regulatory effect on key proteins in the PD-L1 regulatory pathway. This provides a partial mechanistic basis for the potential enhancement of the efficacy of pMMR type advanced gastric cancer by the combination of cytotoxic/anti angiogenic drugs and immune checkpoint inhibitors (ICIs) at the in vitro experimental level, and also lays a theoretical foundation for further exploration of reversing ICIs resistance in this type of gastric cancer.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors have made significant contributions to the work of this study, including the proposal of concepts, the design and implementation of the research, the acquisition, analysis, and statistical analysis of data, as well as the drafting and revision of the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDisclosure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no potential conflicts of interest in this work.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research has not received any fund support. This study has been approved by the Ethics Committee of the Affiliated Hospital of Qinghai University, with the ethical approval number of SL-2023205.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunding This work was supported by grants from the Youth Research Fund of Qinghai University in 2023 (No.2023-QYY-3) and Kunlun Talent High end Innovation and Entrepreneurship Talent project in Qinghai Province in 2023 (QingRenCaiZi 2023 No.9).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eJanjigian YY, Shitara K, Moehler M, Garrido M, Salman P, Shen L, Wyrwicz L, Yamaguchi K, Skoczylas T, Campos Bragagnoli A, Liu T, Schenker M, Yanez P, Tehfe M, Kowalyszyn R, Karamouzis MV, Bruges R, Zander T, Pazo-Cid R, Hitre E, Feeney K, Cleary JM, Poulart V, Cullen D, Lei M, Xiao H, Kondo K, Li M, Ajani JA. First-line nivolumab plus chemotherapy versus chemotherapy alone for advanced gastric, gastro-oesophageal junction, and oesophageal adenocarcinoma (CheckMate 649): a randomised, open-label, phase 3 trial. Lancet 2021 07 03;398(10294) . DOI: 10.1016/S0140-6736(21)00797-2. \u003c/li\u003e\n\u003cli\u003eJ. Xu, H. Jiang, Y. Pan, K. Gu,S. Cang, L. Han,Y. Shu, J. Li, J. Zhao, H. Pan, S. Luo, Y. Qin, Q. Guo, Y. Bai, Y. Ling, Y. Guo, Z. Li, Y. Liu, Y. Wang, H. Zhou. LBA53 Sintilimab plus chemotherapy (chemo) versus chemo as first-line treatment for advanced gastric or gastroesophageal junction (G/GEJ) adenocarcinoma (ORIENT-16): First results of a randomized, double-blind, phase III study. ABSTRACT ONLY VOLUME 32, SUPPLEMENT 5, S1331, SEPTEMBER 01, 2021. DOI: https:// doi.org/ 10.1016/ j.annonc.2021.08.2133.\u003c/li\u003e\n\u003cli\u003eMuto M, Nakata H, Ishigaki K, Tachibana S, Yoshida M, Muto M, Yanagawa N, Okumura T. Successful Treatment of Advanced Gastric Cancer with Brain Metastases through an Abscopal Effect by Radiation and Immune Checkpoint Inhibitor Therapy. J Gastric Cancer 2021 Sep;21(3) . DOI: 10.5230/jgc.2021.21.e24. \u003c/li\u003e\n\u003cli\u003eChen Y, Zhang C, Peng Z, Qi C, Gong J, Zhang X, Li J, Shen L. Association of Lymphocyte-to-Monocyte Ratio With Survival in Advanced Gastric Cancer Patients Treated With Immune Checkpoint Inhibitor. Front Oncol 2021;11. DOI：10.3389/fonc.2021.589022. \u003c/li\u003e\n\u003cli\u003eYang G, Zheng RY, Tan Q, Dong CJ, Jin ZS. Clinical characteristics and responses to chemotherapy and immune checkpoint inhibitor treatment for microsatellite instability gastric cancer. Am J Cancer Res 2020;10(12) .\u003c/li\u003e\n\u003cli\u003eSong X, Qi W, Guo J, Sun L, Ding A, Zhao G, Li H, Qiu W, Lv J. Immune checkpoint inhibitor combination therapy for gastric cancer: Research progress. Oncol Lett 2020 Oct;20(4) . DOI：10.3892/ol.2020.11905 .\u003c/li\u003e\n\u003cli\u003eXie T, Zhang Z, Zhang X, Qi C, Shen L, Peng Z. Appropriate PD-L1 Cutoff Value for Gastric Cancer Immunotherapy: A Systematic Review and Meta-Analysis. Front Oncol 2021;11. DOI: 10.3389/fonc.2021.646355.\u003c/li\u003e\n\u003cli\u003ePietrantonio F, Randon G, Di Bartolomeo M, Luciani A, Chao J, Smyth EC, Petrelli F. Predictive role of microsatellite instability for PD-1 blockade in patients with advanced gastric cancer: a meta-analysis of randomized clinical trials. ESMO Open 2021 Feb;6(1). DOI: 10.1016/j.esmoop.2020.100036. \u003c/li\u003e\n\u003cli\u003eBinnewies M, Roberts EW, Kersten K, Chan V, Fearon DF, Merad M, et al. Understanding the tumor immune microenvironment (TIME) for efective therapy. Nat Med. 2018;24(5):541\u0026ndash;50.\u003c/li\u003e\n\u003cli\u003eGiraldo NA, Sanchez-Salas R, Peske JD, Vano Y, Becht E, Petitprez F, et al. The clinical role of the TME in solid cancer. Br J Cancer. 2019;120(1):45\u0026ndash;53.\u003c/li\u003e\n\u003cli\u003eSaeed A, Park R, Sun W. The integration of immune checkpoint inhibitors with VEGF targeted agents in advanced gastric and gastroesophageal adenocarcinoma: a review on the rationale and results of early phase trials. J Hematol Oncol 2021 01 12;14(1) . DOI: 10.1186/s13045-021-01034-0.\u003c/li\u003e\n\u003cli\u003eEso Y, Shimizu T, Takeda H, Takai A, Marusawa H. Microsatellite instability and immune checkpoint inhibitors: toward precision medicine against gastrointestinal and hepatobiliary cancers. J Gastroenterol 2020 Jan;55(1) . DOI:10.1007/s00535-019-01620-7. \u003c/li\u003e\n\u003cli\u003eInderberg E M, Walchli S, Myhre M R, et al. T cell therapy targeting a public neoantigen in microsatellite instable colon cancer reduces in vivo tumor growth[J]. Oncoimmunology, 2017, 6(4): e1302631.\u003c/li\u003e\n\u003cli\u003ePietrantonio F, Lonardi S, Corti F, Infante G, Elez ME, Fakih M, Jayachandran P, Shah AT, Salati M. Nomogram to predict the outcomes of patients with microsatellite instability-high metastatic colorectal cancer receiving immune checkpoint inhibitors. J Immunother Cancer 2021 Aug;9(8) . DOI: 10.1136/jitc-2021-003370. \u003c/li\u003e\n\u003cli\u003eLengyel CG. Microsatellite Instability as a Predictor of Outcomes in Colorectal Cancer in the Era of Immune-Checkpoint Inhibitors. Curr Drug Targets 2021;22(9). DOI: 10.2174/1389450122666210325121322. \u003c/li\u003e\n\u003cli\u003eFuc\u0026agrave; G, Corti F, Ambrosini M, Intini R, Salati M, Fenocchio E, Manca P, Manai C, Daniel F. Prognostic impact of early tumor shrinkage and depth of response in patients with microsatellite instability-high metastatic colorectal cancer receiving immune checkpoint inhibitors. J Immunother Cancer 2021 Apr;9(4) .\u003c/li\u003e\n\u003cli\u003eCohen R, Colle R, Pudlarz T, Heran M, Duval A, Svrcek M, Andr\u0026eacute; T. Immune Checkpoint Inhibition in Metastatic Colorectal Cancer Harboring Microsatellite Instability or Mismatch Repair Deficiency. Cancers (Basel) 2021 Mar 08;13(5) .\u003c/li\u003e\n\u003cli\u003eGhidini M, Lampis A, Mirchev MB, Okuducu AF, Ratti M, Valeri N, Hahne JC. Immune-Based Therapies and the Role of Microsatellite Instability in Pancreatic Cancer. Genes (Basel) 2020 12 29;12(1) . DOI: 10.3390/genes12010033.\u003c/li\u003e\n\u003cli\u003eTian R, Hu J, Ma X, Liang L, Guo S. Immune-related gene signature predicts overall survival of gastric cancer patients with varying microsatellite instability status. Aging (Albany NY) 2020 12 09;13(2) . \u003c/li\u003e\n\u003cli\u003eWang Z, Zhao X, Gao C, Gong J, Wang X, Gao J, Li Z, Wang J, Yang B, Wang L, Zhang B. Plasma-based microsatellite instability detection strategy to guide immune checkpoint blockade treatment. J Immunother Cancer 2020 11;8(2) .\u003c/li\u003e\n\u003cli\u003ePetrelli F, Ghidini M, Ghidini A, Tomasello G. Outcomes Following Immune Checkpoint Inhibitor Treatment of Patients With Microsatellite Instability-High Cancers: A Systematic Review and Meta-analysis. JAMA Oncol 2020 07 01;6(7). DOI: 10.1001/ jamaoncol.2020.1046. \u003c/li\u003e\n\u003cli\u003eJones NL, Xiu J, Rocconi RP, Herzog TJ, Winer IS. Immune checkpoint expression, microsatellite instability, and mutational burden: Identifying immune biomarker phenotypes in uterine cancer. Gynecol Oncol 2020 02;156(2) . DOI：10.1016/j.ygyno.2019.11.035. \u003c/li\u003e\n\u003cli\u003eFuchs C S, Doi T, Jang R W, et al. Safety and Efficacy of Pembrolizumab Monotherapy in Patients With Previously Treated Advanced Gastric and Gastroesophageal Junction Cancer: Phase 2 Clinical KEYNOTE-059 Trial[J]. JAMA Oncol,2018,4(5):e180013.\u003c/li\u003e\n\u003cli\u003ePietrantonio F, Randon G, Di Bartolomeo M, Luciani A, Chao J, Smyth EC, Petrelli F. Predictive role of microsatellite instability for PD-1 blockade in patients with advanced gastric cancer: a meta-analysis of randomized clinical trials. ESMO Open 2021 Feb;6(1). DOI: 10.1016/j.esmoop.2020.100036.\u003c/li\u003e\n\u003cli\u003ePolonia A, Pinto R, Cameselle-Teijeiro J F, et al. Prognostic value of stromal tumour infiltrating lymphocytes and programmed cell death-ligand 1 expression in breast cancer[J]. J Clin Pathol,2017,70(10):860-867.\u003c/li\u003e\n\u003cli\u003eZerdes I, Matikas A, Bergh J, Rassidakis GZ, Foukakis T. Genetic, transcriptional and post-translational regulation of the programmed death protein ligand 1 in cancer: biology and clinical correlations. Oncogene 2018 08;37(34). DOI: 10.1038/s41388-018-0303-3. \u003c/li\u003e\n\u003cli\u003eFournel L, Wu Z, Stadler N, Damotte D, Lococo F, Boulle G, S\u0026eacute;gal-Bendirdjian E, Bobbio A, Icard P, Tr\u0026eacute;daniel J, Alifano M, Forgez P. Cisplatin increases PD-L1 expression and optimizes immune check-point blockade in non-small cell lung cancer. Cancer Lett 2019 Nov 01;464. DOI: 10.1016/j.canlet.2019.08.005. \u003c/li\u003e\n\u003cli\u003eLinda Tran, Clint T. Allen, Roy Xiao, Ellen Moore, Ruth Davis, So-Jin Park, Katie Spielbauer, Carter Van Waes, and Nicole C. Schmitt. Cisplatin Alters Antitumor Immunity and Synergizes with PD-1/PD-L1 Inhibition in Head and Neck Squamous Cell Carcinoma. American Association for Cancer Research.DOI: 10.1158/2326-6066.CIR-17-0235.\u003c/li\u003e\n\u003cli\u003eZhang P, Su DM, Liang M, Fu J. Chemopreventive agents induce programmed death-1-ligand 1 (PD-L1) surface expression in breast cancer cells and promote PD-L1-mediated T cell apoptosis. Mol Immunol. 2008;45:1470-6.\u003c/li\u003e\n\u003cli\u003eGong W, Song Q, Lu X, Gong W, Zhao J, Min P, et al. Paclitaxel induced B7-H1 expression in cancer cells via the MAPK pathway. J Chemother. 2011;23:295-9.\u003c/li\u003e\n\u003cli\u003eZheng B, Ren T, Huang Y, Guo W. Apatinib inhibits migration and invasion as well as PD-L1 expression in osteosarcoma by targeting STAT3. Biochem Biophys Res Commun. 2018;495(2):1695-701. ttps://doi.org/10.1016/j.bbrc.2017.12.032.\u003c/li\u003e\n\u003cli\u003eCai X, Wei B, Li L, Chen X, Liu W, Cui J, et al. Apatinib enhanced anti-PD-1 therapy for colon cancer in mice via promoting PD-L1 expression. Int Immunopharmacol. 2020;88:106858. https:// doi.org/ 10.1016/ j.intimp.2020.106858.\u003c/li\u003e\n\u003cli\u003eLiang P, Ballou B, Lv X, Si W, Bruchez MP, Huang W, et al. Monotherapy and combination therapy using anti-angiogenic nanoagents to fight cancer. Adv Mater. 2021;33(15):e2005155.\u003c/li\u003e\n\u003cli\u003eZhang H. Apatinib for molecular targeted therapy in tumor. Drug Des Devel Ther. 2015;9:6075\u0026ndash;81.\u003c/li\u003e\n\u003cli\u003eFathi Maroufi N, Rashidi MR, Vahedian V, Akbarzadeh M, Fattahi A, Nouri M. Therapeutic potentials of Apatinib in cancer treatment: possible mechanisms and clinical relevance. Life Sci. 2020;241:117106. https://doi. org/10.1016/j.lfs.2019.117106.\u003c/li\u003e\n\u003cli\u003eJia X, Wen Z, Sun Q, Zhao X, Yang H, Shi X, Xin T. Apatinib suppresses the Proliferation and Apoptosis of Gastric Cancer Cells via the PI3K/Akt Signaling Pathway. J BUON 2019 Sep-Oct;24(5) .\u003c/li\u003e\n\u003cli\u003eWang B, Qin L, Ren M, Sun H. Effects of Combination of Anti-CTLA-4 and Anti-PD-1 on Gastric Cancer Cells Proliferation, Apoptosis and Metastasis.Cell Physiol Biochem 2018;49(1) . DOI: 10.1159/000492876.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"medical-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"medo","sideBox":"Learn more about [Medical Oncology](https://www.springer.com/journal/12032)","snPcode":"12032","submissionUrl":"https://submission.nature.com/new-submission/12032/3","title":"Medical Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"pMMR type gastric cancer, PD-L1, cytotoxic drugs, anti angiogenic drugs","lastPublishedDoi":"10.21203/rs.3.rs-9102745/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9102745/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground and Objectives:\u003c/h2\u003e \u003cp\u003eAdvanced gastric cancer with proficient mismatch repair (pMMR) status responds poorly to immune checkpoint inhibitors (ICIs) and constitutes a significant proportion of cases, posing a major clinical challenge. Studies suggest that cytotoxic and anti-angiogenic drugs may influence ICI efficacy by modulating programmed death-ligand 1 (PD-L1) expression; however, their specific role and mechanisms in pMMR gastric cancer remain unclear. This study aimed to investigate the regulatory effects of cytotoxic drugs (5-fluorouracil, 5-FU; cisplatin) and an anti-angiogenic drug (apatinib) on PD-L1 expression in human pMMR-type AGS gastric cancer cells and explore the underlying signaling pathway mechanisms.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eThe impact of drug treatments at various concentrations on AGS cell proliferation was first assessed using the CCK-8 assay to select appropriate concentrations for subsequent experiments. Real-time quantitative PCR (qPCR) and Western blotting were employed to evaluate the effects of 72-hour drug treatment on PD-L1 mRNA and protein expression levels, respectively. Furthermore, Western blot analysis was used to examine drug-induced changes in the expression of key proteins (PI3K, Akt, MEK1, ERK) within the PI3K/Akt/mTOR and RAS/RAF/MEK/ERK signaling pathways, which are potentially involved in PD-L1 regulation.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003e1. Regulation of PD-L1 Expression: qPCR results showed that 5-FU (64 \u0026micro;M), cisplatin (16 \u0026micro;M), and apatinib (320 nM, 640 nM) significantly upregulated PD-L1 mRNA expression in AGS cells (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Western blot analysis further confirmed that treatment with 5-FU (64 \u0026micro;M), cisplatin (16 \u0026micro;M), and apatinib (320 nM) significantly increased PD-L1 protein levels (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). 2. Alterations in Signaling Pathway Proteins: Western blot analysis indicated that treatments with 5-FU, apatinib, and cisplatin all upregulated PI3K protein expression (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Regarding Akt protein, apatinib treatment increased its expression, whereas 5-FU and cisplatin decreased it (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Within the MAPK pathway, 5-FU treatment upregulated the expression of MEK1 and ERK proteins, while apatinib and cisplatin treatments downregulated the expression of both proteins (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eAt specific concentrations, cytotoxic drugs (5-FU, cisplatin) and the anti-angiogenic drug (apatinib) can upregulate both mRNA and protein expression of PD-L1 in pMMR-type AGS gastric cancer cells and differentially modulate the expression of key proteins in the PI3K/Akt and MAPK signaling pathways. These findings provide preliminary in vitro experimental evidence and mechanistic insights supporting the potential of combining these drugs with ICIs to enhance therapeutic efficacy in pMMR gastric cancer.\u003c/p\u003e","manuscriptTitle":"Exploration of the regulatory mechanism of PD-L1 expression in pMMR type gastric cancer cell by cytotoxic drugs and anti angiogenic drugs","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-20 12:05:24","doi":"10.21203/rs.3.rs-9102745/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-04-18T01:11:02+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"201521469956170063297305869477193203859","date":"2026-03-24T19:45:00+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-03-18T04:15:38+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-03-16T02:55:28+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-03-13T09:59:09+00:00","index":"","fulltext":""},{"type":"submitted","content":"Medical Oncology","date":"2026-03-12T09:04:41+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"medical-oncology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"medo","sideBox":"Learn more about [Medical Oncology](https://www.springer.com/journal/12032)","snPcode":"12032","submissionUrl":"https://submission.nature.com/new-submission/12032/3","title":"Medical Oncology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"5cf1ed34-1aa4-4d9e-9007-2299a16df2d0","owner":[],"postedDate":"March 20th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-03-20T12:05:25+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-20 12:05:24","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9102745","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9102745","identity":"rs-9102745","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}