Mouse Model Of Gastric Cancer Research Articles

Pyrolyzed deketene curcumin controls regulatory T cell generation and gastric cancer metabolism cooperate with 2-deoxy-d-glucose.

Pyrolyzed deketene curcumin GO-Y022 prevents carcinogenesis in a gastric cancer mouse model. However, it is still less clear if GO-Y022 affects tumor-induced immune suppression. In this study, we found that GO-Y022 inhibited Treg generation in the presence of transforming growth factor beta 1 (TGF-β). However, GO-Y022 showed less impact on Foxp3+ Tregs in the gastric tumor microenvironment. Gastric tumor cells produce a large amount of L-lactate in the presence of GO-Y022 and diminish the inhibitory role of GO-Y022 against Treg generation in response to TGF-β. Therefore, naïve CD4+ T cells co-cultured with GO-Y022 treated gastric tumor cells increased Treg generation. GO-Y022-induced tumor cell death was further enhanced by 2-deoxy-d-glucose (2DG), a glycolysis inhibitor. Combination treatment of GO-Y022 and 2DG results in reduced L-lactate production and Treg generation in gastric tumor cells. Overall, GO-Y022-treatment with restricted glucose metabolism inhibits gastric tumor cell survival and promotes anti-tumor immunity.

SOX9 Modulates the Transformation of Gastric Stem Cells Through Biased Symmetric Cell Division

Transformation of stem/progenitor cells has been associated with tumorigenesis in multiple tissues, but stem cells in the stomach have been hard to localize. We therefore aimed to use a combination of several markers to better target oncogenes to gastric stem cells and understand their behavior in the initial stages of gastric tumorigenesis. Mouse models of gastric metaplasia and cancer by targeting stem/progenitor cells were generated and analyzed with techniques including reanalysis of single-cell RNA sequencing and immunostaining. Gastric cancer cell organoids were genetically manipulated with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) for functional studies. Cell division was determined by bromodeoxyuridine-chasing assay and the assessment of the orientation of the mitotic spindles. Gastric tissues from patients were examined by histopathology and immunostaining. Oncogenic insults lead to expansion of SOX9+ progenitor cells in the mouse stomach. Genetic lineage tracing and organoid culture studies show that SOX9+ gastric epithelial cells overlap with SOX2+ progenitors and include stem cells that can self-renew and differentiate to generate all gastric epithelial cells. Moreover, oncogenic targeting of SOX9+SOX2+ cells leads to invasive gastric cancer in our novel mouse model (Sox2-CreERT;Sox9-loxp(66)-rtTA-T2A-Flpo-IRES-loxp(71);Kras(Frt-STOP-Frt-G12D);P53R172H), which combines Cre-loxp and Flippase-Frt genetic recombination systems. Sox9 deletion impedes the expansion of gastric progenitor cells and blocks neoplasia after Kras activation. Although Sox9 is not required for maintaining tissue homeostasis where asymmetric division predominates, loss of Sox9 in the setting of Kras activation leads to reduced symmetric cell division and effectively attenuates the Kras-dependent expansion of stem/progenitor cells. Similarly, Sox9 deletion in gastric cancer organoids reduces symmetric cell division, organoid number, and organoid size. In patients with gastric cancer, high levels of SOX9 are associated with recurrence and poor prognosis. SOX9 marks gastric stem cells and modulates biased symmetric cell division, which appears to be required for the malignant transformation of gastric stem cells.

Polyphyllin B Suppresses Gastric Tumor Growth by Modulating Iron Metabolism and Inducing Ferroptosis.

Gastric cancer (GC) is one of the most common malignant tumors in the world. GPx4, as the core regulator of ferroptosis, has become a potential molecular target for developing anticancer agents. In the present study, we found that GPx4 was overexpressed and negatively correlated with poor prognosis in GC, while it was associated with the GC development. Molecular docking and structure-based virtual screening assays were used to screen potential GPx4 inhibitors, and we identified a novel GPx4 inhibitor, polyphyllin B (PB), which can induce ferroptosis by down-regulating GPx4 expression in GC cells. It has also been shown to inhibit cell proliferation, suppress invasion and migration, induce apoptosis, and block the cell cycle progression in GC cells in vitro. Then, immunofluorescence and western blotting assay confirmed that PB can regulate the expression of LC3B, TFR1, NOCA4 and FTH1 in vitro, which suggested that suggest that PB may increase the level of Fe2+ by transporting Fe3+ into the cell by TFR1 and promoting NCOA4-dependent iron autophagy. In addition, PB can also suppresses tumor growth in an orthotopic mouse model of GC via regulating the expression of GPx4, TFR1, NOCA4 and FTH1 in vivo. In summary, we confirmed that GPx4 may be a potential target for GC treatment, PB may be a novel and promising drug for the treatment of GC, which shows good antitumor efficacy without causing significant host toxicity via inducing ferroptosis in both gastric cancer cells and mouse models.

Investigating the effect of ARHGEF10L gene on tumor growth in gastric cancer in a nude mouse model using quantitative MRI parameters.

The quantitative magnetic resonance imaging (MRI) parameters were initially used in the study of central nervous system diseases and has since been widely used in the diagnosis of breast, liver, rectum, and prostate diseases. In our study, we aimed to evaluate the effect of ARHGEF10L gene on tumor growth in gastric cancer in nude mice using quantitative MRI parameters. A nude mice model of gastric cancer was established, and the mice were divided into a control group and an shARHGEF10L group (N = 10). T2-fs and intravoxel incoherent motions (IVIM) imaging were performed in the mice coil with a 3.0 T MR system. The differences in quantitative parameters (apparent diffusion coefficient [ADC], D, D *, f values) were compared between both groups, and the effect of ARHGEF10L expression on tumor growth in tumor-bearing mice was investigated. The data were analyzed using Statistical Package for the Social Sciences (SPSS) 17.0 software package. The ADC and D values of tumor imaging in the shARHGEF10L group were higher than those in the control group, and the differences were statistically significant. There was no significant difference in the D* or F values between both groups. The ADC and D values of the quantitative IVIM imaging parameters can be used to effectively assess the growth of gastric cancer in nude mice, suggesting that ARHGEF10L may promote the growth of tumor cells.

Calponin 1 increases cancer-associated fibroblasts-mediated matrix stiffness to promote chemoresistance in gastric cancer

The mechanical microenvironment regulated by cancer-associated fibroblasts (CAFs) influence tumor progression. Chemotherapeutic interventions including 5-Fluorouracil (5-Fu) are commonly used for primary treatment of patients with advanced gastric cancer (GC), and the development of acquired resistance to 5-Fu limits the clinical efficacy of these chemotherapies. However, if and how the interplay between CAFs and the mechanical microenvironment regulates GC response to 5-Fu is poorly understood. In this study, we demonstrate that high-level expression of calponin 1(CNN1) in gastric CAFs predicts poor clinical outcomes of GC patients, especially for those treated with 5-Fu. CNN1 knockdown in CAFs improves the effectiveness of 5-Fu in reducing tumor growth in a mouse GC model and confers increased sensitivity to 5-Fu in a 3D culture system. Furthermore, CNN1 knockdown impairs CAF contraction and reduces matrix stiffness without affecting the expression of matrix proteins. Mechanistically, CNN1 interacts with PDZ and LIM Domain 7 (PDLIM7) and prevents its degradation by the E3 ubiquitin ligase NEDD4-1, which leads to activation of the ROCK1/MLC pathway. The increased matrix stiffness, in turn, contributes to 5-Fu resistance in GC cells by activating YAP. Taken together, our data reveal a critical role of the mechanical microenvironment in 5-Fu resistance, which is modulated by CNN1hi CAFs-mediated matrix stiffening, indicating that targeting CAFs may provide a novel option for overcoming drug resistance in GC.

Targeting nanoparticle-conjugated microbubbles combined with ultrasound-mediated microbubble destruction for enhanced tumor therapy

The stress of the abnormal stromal matrix of solid tumors is a major limiting factor that prevents drug penetration. Controlled, accurate, and efficient delivery of theranostic agents into tumor cells is crucial. Combining ultrasound with nanocarrierbased drug delivery systems have become a promising approach for targeted drug delivery in preclinical cancer therapy. In this study, to ensure effective tumor barrier penetration, access to the tumor microenvironment, and local drug release, we designed targeted nanoparticle (NP)-conjugated microbubbles (MBs); ultrasound could then help deliver acoustic energy to release the NPs from the MBs. The ultrasound-targeted MB destruction (UTMD) system of negatively charged NPs was conjugated with positively charged MBs using an ionic gelation method. We demonstrated the transfer of targeted NPs and their entry into gastric cancer cells through ligand-specific recognition, followed by enhanced cell growth inhibition owing to drug delivery-induced apoptosis. Moreover, the UTMD system combining therapeutic and ultrasound image properties can effectively target gastric cancer, thus significantly enhancing antitumor activity, as evident by tumor localization in an orthotopic mouse model of gastric cancer. The combination of ultrasound and NP-based drug delivery systems has become a promising approach for targeted drug delivery in preclinical cancer therapy.

DKK1 Promotes Tumor Immune Evasion and Impedes Anti-PD-1 Treatment by Inducing Immunosuppressive Macrophages in Gastric Cancer.

Tumor-associated macrophages (TAM) have key functions in promoting a suppressive tumor immune microenvironment (TIME) and immune evasion, which largely limit treatment effects of immune-checkpoint inhibitors (ICI) in different cancers, including gastric cancer. Dickkopf-1 (DKK1) is associated with tumor progression and has been shown to negatively regulate antitumor immunity, but the impact of DKK1 on the TIME remains incompletely understood. Here, we found that tumoral DKK1 expression is closely associated with worse survival and a suppressive TIME in gastric cancer patients. Results from in vitro coculture assays suggested that DKK1 induces macrophages to become immunosuppressive, thereby inhibiting antitumor responses of CD8+ T cells and natural killer (NK) cells. In vivo DKK1 blockade in syngeneic gastric cancer mouse models reprogramed TAMs to restore the immune activity in the TIME and triggered significant tumor regression. DKK1 blockade also directly reduced the growth of human gastric cancer tumors with high DKK1 expression in a xenograft model. Mechanistically, DKK1 interacted with cytoskeleton-associated protein 4 (CKAP4) on the macrophage surface and activated downstream PI3K-AKT signaling, which contributed to immune suppression. TAM reprogramming by DKK1 blockade also augmented the efficacy of programmed cell death protein-1 (PD-1) blockade in gastric cancer models. Therefore, our study provides novel insights into the role of DKK1 on tumor-intrinsic, innate, and adaptive antitumor immunity modulation and suggests that DKK1 is a promising immunotherapeutic target for enhanced PD-1 blockade therapy in gastric cancer.

1230P Immunotherapies for gastric cancer with CLDN18-ARHGAP fusion gene

Since gastric cancer (GC) is highly heterogeneous in histology and genomics, current treatments harvest efficacies only in limited patients. Sequencing data of previous studies have documented that CLDN18-ARHGAP fusion gene recurrently occurred in GC. However, therapies targeting the CLDN18-ARHGAP fusion gene have not been reported. Next-generation sequencing (NGS) was conducted to identify CLDN18-ARHGAP fusion gene in GC samples. Next, we screened immunogenic neoantigens derived from CLDN18-ARHGAP by CBA, ELISPOT and flow cytometry. Cytotoxicity of neoantigen reactive T (NRT) cells were assessed by killing assays and tumor suppression experiments. Also, we analyzed the tumor immune microenvironment (TIME) of GC with CLDN18-ARHGAP via bioinformatic analysis and in vivo immune analysis. The mechanism behind was explored by western blotting (WB) and fatty acid detection. We detected CLDN18-ARHGAP fusion genes in 9% (8/87) GC patients. To develop immunotherapies targeting these patients, we derived specific neoantigens, and found NRT cells induced by these neoantigens secreted IFN-γ 60 times that of the control group. They also showed stronger antitumor ability to GC cells with CLDN18-ARHGAP in the killing assay and tumor suppression experiments. Additionally, we found increased infiltration of regulatory T cells (Tregs) in GCs with CLDN18-ARHGAP by bioinformatic analysis of public databases and flow cytometry in subcutaneous GC mouse models. Mechanistically, CLDN18-ARHGAP could promote the secretion of fatty acids via activation of PI3K/AKT-mTOR-FAS pathway, thereby enhancing the metabolism and proliferation of Tregs. In mouse models, PI3K inhibitor (PI3Ki) could inverse the suppressive TIME induced by CLDN18-ARHGAP fusion genes and inhibit tumor growth. Our study was the first to identify the immunogenic neoantigens derived from CLDN18-ARHGAP fusion gene and verified the antitumor ability of NRT cells. Also, we revealed the role of CLDN18-ARHGAP fusion gene in promoting a suppressive TIME by facilitating the metabolism and proliferation of Tregs, which could be reversed by PI3Ki. Collectively, we demonstrated that CLDN18-ARHGAP is a potential target for immunotherapies in gastric cancer.

LncRNA TPTEP1 inhibits the migration and invasion of gastric cancer cells through miR-548d-3p/KLF9/PER1 axis

BackgroundDespite the development of many methods and new therapeutic agents, the survival and prognosis of patients with gastric cancer are still poor. The role of TPTEP1 in gastric cancer has not been reported. MethodsWound healing assay and transwell assay analysis TPTEP1/miR-548d-3p/KLF9/PER1 effect on migration and invasiveness of gastric cells. Western blot and RT-qPCR certificate TPTEP1/miR-548d-3p/KLF9/PER1transcription and expression of migration and invasion related genes. Luciferase assay was used to determine the adsorption of miR-548d-3p by TPTEP1 sponge, the targeting of miR-548d-3p to KLF9, and the binding of KLF9 to the promoter of PER1. immunohistochemical assay and H&E staining prove the function of TPTEP1 and miR-548d-3p in nude mice model of gastric cancer. ResultsTPTEP1 inhibited its expression by sponge adsorption of miR-548d-3p. miR-548d-3p targets KLF9 3′UTR to inhibit its expression, and KLF9 binds to the PER1 promoter to promote its expression.TPTEP1/KLF9/PER1 inhibits gastric cancer cell migration and invasion, and miR-548d-3p does the opposite. ConclusionsOur data suggest that TPTEP1 affects gastric cancer progression by regulating the miR-548d-3p/KLF9/PER1 axis. Targeting this pathway may provide new therapeutic opportunities for gastric cancer.

Gastric Microbiome Alterations Are Associated with Decreased CD8+ Tissue-Resident Memory T Cells in the Tumor Microenvironment of Gastric Cancer.

The host microbiota is closely associated with tumor initiation and progression in multiple solid tumors including gastric cancer. The aim of this study was to investigate in patients with gastric cancer whether there are alterations in gastric microbiota and any potential association these may have with immune dysregulation. 16S rRNA gene sequencing was used to analyze tumor microbiota of 53 patients with gastric cancer and gastric mucosal tissue microbiota of 30 patients with chronic gastritis. The effect of microbiota on the tumor microenvironment (TME) was studied by single-cell sequencing, immunohistochemistry, multiplex immunofluorescence staining, and flow cytometry, as well as in a mouse model of primary gastric cancer. The gastric cancer microbiota was characterized by reduced microbial diversity and enrichment of the Oceanobacter, Methylobacterium, and Syntrophomonas genera. Intratumoral Methylobacterium was significantly associated with poor prognoses in patients with gastric cancer. It also was inversely correlated with the frequency of CD8+ tissue-resident memory T (TRM) cells in the TME. TGFβ was significantly reduced in gastric cancer samples with higher abundance of Methylobacterium. Finally, we verified that Methylobacterium can decrease TGFβ expression and CD8+ TRM cells in the tumor by establishing a mouse model of primary gastric cancer. The results suggest that tumor microbiota and exhausted CD8+ TRM cells in the TME of gastric cancer are significantly correlated, and that Methylobacterium may play a role in gastric carcinogenesis.

Circ_0008287 promotes immune escape of gastric cancer cells through impairing microRNA-548c-3p-dependent inhibition of CLIC1

BackgroundAnalyses in silico suggested the upregulation of a circular RNA (circRNA), circ_0008287, in gastric cancer and possible interactions among microRNA (miR)-548c-3p, circ_0008287, and intracellular chloride channel protein 1 (CLIC1). This study aims to testify whether circ_0008287 can affect the immune escape of gastric cancer cells by regulating miR-548c-3p and CLIC1. MethodsRT-qPCR was performed to determine the expression pattern of circ_0008287 in gastric cancer cells. Gain- and loss-of function assays were then performed to assess the effects of circ_0008287 on malignant phenotypes of cancer cells. Interactions among circ_0008287, miR-548c-3p and CLIC1 were verified by dual luciferase reporter gene, RIP and FISH assays. Effects of CLIC1 on IFN-γ secretion and apoptosis in CD8 + T cells were evaluated by flow cytometry following co-culture of CD8 + T cells with cancer cells overexpressing/silencing CLIC1. A gastric cancer mouse model was further developed for in vivo investigation on effects of circ_0008287 on tumorigenesis and tumor metastasis. Resultscirc_0008287, an upregulated circRNA in gastric cancer cells, augmented the viability as well as invasive and migratory potentials of gastric cancer cells. By competitively binding to miR-548c-3, circ_0008287 increased the expression of CLIC1, which impaired the function of CD8 + T cells and promoted their apoptosis. After downregulation of circ_0008287, in vivo tumorigenesis and metastasis were suppressed. ConclusionHence, this study suggests the promotive role of circ_0008287 in gastric cancer progression and immune escape and further elucidates the underlying circ_0008287/miR-548c-3p/CLIC1 regulatory axis.

NCX1 coupled with TRPC1 to promote gastric cancer via Ca2+/AKT/β-catenin pathway

Plasma membrane Na+/Ca2+ exchanger 1 (NCX1) is a bidirectional ion transporter to operate in Ca2+ entry or exit modes, and TRPC1 is Ca2+-permeable channel. Both NCX1 and TRPC1 play critical roles in maintaining cytosolic free Ca2+ ([Ca2+]cyt) homeostasis in mammalian cells. Although either TRPC1 channel or Ca2+ entry mode of NCX1 is implicated in some tumorigenesis, it has not been explored if a coordination of NCX1 and TRPC1 involves in the pathogenesis of H. pylori-associated human gastric cancer (GC). Here we found the protein expression of NCX1 was significantly enhanced in human GC specimens, which correlated with tumor progression and poor survival in GC patients. TRPC1 and NCX1 were parallelly enhanced, co-localized and bound in human GC cells. By a functional coupling, TRPC1 drives NCX1 to the Ca2+ entry mode, raising [Ca2+]cyt in GC cells. Moreover, CaCl2, H. pylori and their virulence factors all enhanced expressions and activities of NCX1 and TRPC1, and evoked aberrant Ca2+ entry to promote proliferation, migration, and invasion of GC cells through AKT/β-catenin pathway. Tumor growth and metastasis also depended on the enhanced expression of NCX1 in subcutaneously xenografted GC mouse model. Overall, our findings indicate that TRPC1/NCX1 coupling may promote H. pylori-associated GC through the Ca2+/AKT/β-catenin pathway. Since the Ca2+ exit mode and the Ca2+ entry mode of NCX1 play different roles under mostly physiological and pathological conditions respectively, targeting TRPC1/NCX1 coupling could be a novel strategy for selectively blocking Ca2+ entry mode to potentially treat digestive cancer with less side effect.

Research on the Mechanism and Prevention of Hypertension Caused by Apatinib Through the RhoA/ROCK Signaling Pathway in a Mouse Model of Gastric Cancer.

Hypertension is one of the main adverse effects of antiangiogenic tumor drugs and thus limits their application. The mechanism of hypertension caused by tyrosine kinase inhibitors (TKIs) targeting vascular endothelial growth factors is mainly related to inhibition of the nitric oxide (NO) pathway and activation of the endothelin pathway, as well as vascular rarefaction and increased salt sensitivity; consequently, prevention and treatment differ for this type of hypertension compared with primary hypertension. Apatinib is a highly selective TKI approved in China for the treatment of advanced or metastatic gastric cancer. The RhoA/ROCK pathway is involved in the pathogenesis of hypertension and mediates smooth muscle contraction, eNOS inhibition, endothelial dysfunction and vascular remodeling. In this study, in vivo experiments were performed to explore whether the RhoA/ROCK signaling pathway is part of a possible mechanism of apatinib in the treatment of gastric cancer-induced hypertension and the impairment of vascular remodeling and left ventricular function. Y27632, a selective small inhibitor of both ROCK1 and ROCK2, was combined with apatinib, and its efficacy was evaluated, wherein it can reduce hypertension induced by apatinib treatment in gastric cancer mice and weaken the activation of the RhoA/ROCK pathway by apatinib and a high-salt diet (HSD). Furthermore, Y-27632 improved aortic remodeling, fibrosis, endothelial dysfunction, superior mesenteric artery endothelial injury, left ventricular dysfunction and cardiac fibrosis in mice by weakening the activation of the RhoA/ROCK pathway. The expression of RhoA/ROCK pathway-related proteins and relative mRNA levels in mice after apatinib intervention were analyzed by various methods, and blood pressure and cardiac function indexes were compared. Endothelial and cardiac function and collagen levels in the aorta were also measured to assess vascular and cardiac fibrosis and to provide a basis for the prevention and treatment of this type of hypertension.

Chitosan-Gelatin-EGCG Nanoparticle-Meditated LncRNA TMEM44-AS1 Silencing to Activate the P53 Signaling Pathway for the Synergistic Reversal of 5-FU Resistance in Gastric Cancer.

Chemoresistance is one of the leading causes of therapeutic failure in gastric cancer (GC) treatment. Recent studies have shown lncRNAs play pivotal roles in regulating GC chemoresistance. Nanocarriers delivery of small interfering RNAs (siRNAs) to silence cancer‐related genes has become a novel approach to cancer treatment research. However, finding target genes and developing nanosystems capable of selectively delivering siRNAs for disease treatment remains a challenge. In this study, a novel lncRNA TMEM44‐AS1 that is related to 5‐FU resistance is identified. TMEM44‐AS1 has the ability to bind to and sponge miR‐2355‐5p, resulting in the upregulated PPP1R13L expression and P53 pathway inhibition. Next, a new nanocarrier called chitosan‐gelatin‐EGCG (CGE) is developed, which has a higher gene silencing efficiency than lipo2000, to aid in the delivery of a si‐TMEM44‐AS1 can efficiently silence TMEM44‐AS1 expression to synergistically reverse 5‐FU resistance in GC, leading to a markedly enhanced 5‐FU therapeutic effect in a xenograft mouse model of GC. These findings indicate that TMEM44‐AS1 may estimate 5‐FU therapy outcome among GC cases, and that systemic si‐TMEM44‐AS1 delivery combined with 5‐FU therapy is significant in the treatment of patients with recurrent GC.

Abstract 5568: Revitalization of affinity-tuned CAR T cells via antigen-dependent release of interleukin-12

Abstract Introduction: The application of chimeric antigen receptor (CAR) T cells in solid tumors has met many challenges, arising from the paucity of effective yet safe targets and T cell dysfunction within the immunosuppressive milieu. Clinical trials over the years have reported severe on-target off-tumor toxicities associated with CAR T cell therapies targeting tumor-associated antigens (TAAs). Here, we propose a strategy to target TAAs effectively and safely: 1) improve tumor-targeting specificity by affinity-tuning; 2) armor T cells with antigen-dependent expression of interleukin-12 (IL-12) to compensate loss of CAR T cell activity caused by affinity-tuning. In this system, IL-12 expression is tightly regulated by the nuclear factor of the activated T cells promoter (NFAT) to limit IL-12 to the environ of activated affinity-tuned CAR T cells within the tumor. We demonstrate here the feasibility of this strategy using epithelial cell adhesion molecule (EpCAM) as a model TAA. Methods: Affinity variants were generated by alanine scanning mutagenesis in the complementary-determining region 3 (CDR3) of an EpCAM antibody (UBS54). The affinity of CAR variants was determined by flow cytometry-based saturation binding assay. On-target off-tumor cytotoxicity of CAR T cells was examined against human primary normal epithelial cells. In vivo anti-tumor efficacy was evaluated in mouse models of gastric cancer with cell line-derived (SNU-638 and MKN-45) and patient-derived xenografts. Results: Substitution of alanine into the UBS54 CDR3 led to identification of a tyrosine (6th residue in heavy chain CDR3) as a hot spot for affinity tuning. Among several more subtle amino acid substitutions for Tyr-6, we found that CAR molecule with valine substituent (Y6V) possesses 10 µM affinity toward EpCAM, rendering CAR T cells to be selective to EpCAM-high tumors while being not reactive to primary normal epithelial cells in vitro. In gastric cancer mouse models, compared to UBS54 CAR T cells that mediated rapid tumor remission, Y6V CAR T cells produced mainly partial responses. To revitalize affinity-tuned CAR T cells, we further engineered CAR T cells to release IL-12 under NFAT. We found that both the activity of the NFAT promoter and the level of IL-12 release were tightly regulated by the antigen density of targets when inducible IL-12 is combined with 10 µM affinity Y6V CAR; however, such dependence is lost when 1 µM affinity UBS54 was examined. In mouse models of gastric cancer, inducible lL-12 armored Y6V CAR T cells produced enhanced anti-tumor responses without elevating systemic exposure to IL-12, evidenced by low levels of IL-12 in mouse sera (below 100 pg/ml). Conclusions: Combination of affinity-tuned CAR with inducible expression of IL-12 is a promising strategy to develop CAR T cell therapy against TAAs with the potential for reduced on-target off-tumor toxicity and tumor resistance. Citation Format: Yanping Yang, Huan Yang, Yago Alcaina, Jaclyn E. McCloskey, Janusz Puc, Alyssa Birt, Yogindra Vedvyas, Juan Gonzalez-Valdivieso, Irene M. Min, Eric von Hofe, Moonsoo M. Jin. Revitalization of affinity-tuned CAR T cells via antigen-dependent release of interleukin-12 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5568.

Abstract 5849: Activation of STAT3 is mediated by FGFR4 induction via SRC signaling after H. pylori infection

Abstract Background: Gastric cancer is considered the 5th most common form of cancer and 4th leading cause of cancer-related deaths worldwide. Due to the late stage of diagnosis, gastric cancer carries poor prognosis and poor overall survival. Infection with H. pylori is the strongest known risk factor. Fibroblast Growth Factor Receptor 4 (FGFR4) belongs to a family of highly conserved tyrosine kinases. This study investigated the molecular mechanism of FGFR4 regulation in response to H. pylori infection. Methods and Results: Among all the FGFRs, FGFR4 mRNA expression level was the most significantly elevated in human gastric cancer samples as compared to normal samples, using Real-time qPCR. These results were confirmed using the TCGA and Geo database analysis, confirming that FGFR4 is highly expressed in gastric cancer. Using western blot, we found that FGFR4 is significantly expressed in dysplasia and adenocarcinoma lesions in the TFF1-KO gastric cancer mouse model. Following infection with H. pylori in in-vitro (J166, 7.13) and in-vivo (PMSS1) models, we detected a significant increase in the expression of FGFR4 at the protein and mRNA levels. We also detected activation of STAT3 in human gastric cell lines and mice gastric tissues. Furthermore, analysis of FGFR4 promoter revealed several putative binding sites for STAT3. To confirm that FGFR4 expression is dependent upon STAT3 activation, we performed ChIP assay and confirmed direct functional binding of STAT3 on FGFR4 promoter. Using the FGF19, the ligand for FGFR4, we found that FGF19-FGFR4 axis played a vital role in activating STAT3 through an SRC-dependent mechanism and discovered that there is a feedforward activation loop between FGFR4 and STAT3. Functionally, we found that FGFR4 protected against H. pylori-induced DNA damage and cell death. Conclusion: Our study established a feedforward activation loop between FGFR4 and STAT3 in response to H. pylori infection and FGFR4 could be a possible druggable moiety for future therapies in gastric cancer. Citation Format: Nadeem S. Bhat, Mohammed Soutto, Xing Zhang, Zheng Chen, Shoumin Zhu, Heng Lu, Dunfa Peng, Zekuan Xu, Wael El-Rifai. Activation of STAT3 is mediated by FGFR4 induction via SRC signaling after H. pylori infection [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 5849.

Huaier Inhibits Gastric Cancer Growth and Hepatic Metastasis by Reducing Syntenin Expression and STAT3 Phosphorylation

Gastric cancer (GC) is a common malignant tumor worldwide and poses a serious threat to human health. As a traditional Chinese medicine, Huaier (Trametes robiniophila Murr.) has been used in the clinical treatment of GC. However, the mechanism underlying the anticancer effect of Huaier remains poorly understood. In this study, we used in vivo imaging technology to determine the anticancer effect of the Huaier n-butanol extract (HBE) on orthotopic and hepatic metastasis of GC mouse models. We found that HBE suppressed tumor growth and metastasis without causing apparent host toxicity. Proteomic analysis of GC cells before and after HBE intervention revealed syntenin to be one of the most significantly downregulated proteins after HBE intervention. We further demonstrated that HBE suppressed the growth and metastasis of GC by reducing the expression of syntenin and the phosphorylation of STAT3 at Y705 and reversing the epithelial-mesenchymal transition (EMT). In addition, we confirmed that syntenin was highly expressed in GC tissue and correlated with metastasis and poor prognosis. In conclusion, our results suggest that Huaier, a clinically used anticancer drug, may inhibit the growth and liver metastasis of GC by inhibiting the syntenin/STAT3 signaling pathway and reversing EMT.

IL-33 promotes gastric tumour growth in concert with activation and recruitment of inflammatory myeloid cells.

Interleukin-33 (IL-33) is an IL-1 family cytokine known to promote T-helper (Th) type 2 immune responses that are often deregulated in gastric cancer (GC). IL-33 is overexpressed in human gastric tumours suggesting a role in driving GC progression although a causal link has not been proven. Here, we investigated the impact of IL-33 genetic deficiency in the well-characterized gp130F/F mouse model of GC. Expression of IL-33 (and it’s cognate receptor, ST2) was increased in human and mouse GC progression. IL-33 deficient gp130F/F /Il33−/− mice had reduced gastric tumour growth and reduced recruitment of pro-tumorigenic myeloid cells including key mast cell subsets and type-2 (M2) macrophages. Cell sorting of gastric tumours revealed that IL-33 chiefly localized to gastric (tumour) epithelial cells and was absent from tumour-infiltrating immune cells (except modest IL-33 enrichment within CD11b+ CX3CR1+CD64+MHCII+ macrophages). By contrast, ST2 was absent from gastric epithelial cells and localized exclusively within the (non-macrophage) immune cell fraction together with mast cell markers, Mcpt1 and Mcpt2. Collectively, we show that IL-33 is required for gastric tumour growth and provide evidence of a likely mechanism by which gastric epithelial-derived IL-33 drives mobilization of tumour-promoting inflammatory myeloid cells.

In situ gelatinase-responsive and thermosensitive nanocomplex for local therapy of gastric cancer with peritoneal metastasis.

Intraperitoneal chemotherapy (IPC) has been considered as an effective therapy for advanced gastric cancer (GC) especially those with peritoneal metastasis, while limited effectiveness, complications caused by chemotherapeutics and repeated infusion procedures restrict the application of IPC. In this study, to enhance the efficacy and safety of IPC, we intended to establish a biocompatible and biodegradable nanocomplex composed of intelligent gelatinase-responsive nanoparticles (NPs) and thermosensitive gel, which were prepared from different compositions of poly (ethyleneglycol)–poly (3-caprolactone) (PEG–PCL). Cancer stem cells (CSCs) inhibitor Salinomycin (SAL) and non-CSC inhibitor Docetaxel (DOC) were co-loaded in the NPs and delivered by liquid PEG-PCL-PEG gel (PECE) at room temperature, which was able to target tumor and formed a gel in situ at body temperature. Compared with free SAL-DOC solution administered at the same dose, PECE NP group inhibited intraperitoneal disseminated gastric cancer growth more remarkably, some of which even achieved complete response (CR) and continued for more than 2 weeks. Cytometric analysis of cellular suspension from abdominal tumor tissues showed that the proportion of CSCs (CD44+CD133+) and the expression of PD-L1 on the tumor cells in the PECE NP group were the lowest. In the allograft mouse models of GC, PECE NP significantly improved the infiltration of M1 macrophages into the tumor bed in vivo. This design may provide biodegradable smart drug-delivery system for potential application in IPC.

Tumor-derived IL-8 facilitates lymph node metastasis of gastric cancer via PD-1 up-regulation in CD8+ T cells

BackgroundThe pretherapeutic serum interleukin-8 (sIL-8) levels have been revealed to be increased in about half of patients with locally advanced gastric cancer. However, the roles of IL-8 in lymph node metastasis (LNM) and the underlying mechanisms remain unclear.Methods146 patients with primary gastric carcinoma were enrolled in this study. ELISA was used to measure IL-8 levels. The CD4/CD8 ratio and programmed cell death-1 (PD-1) expression of T cells in primary tumor tissues, tumor-draining lymph nodes (TDLNs) and non-draining lymph nodes (NDLNs) were assayed with flow cytometry. Protein expression of the molecules was determined with immunohistochemistry, western blotting or immunoprecipitation. The gastric cancer mouse tumor model with LNM was utilized to determine the role of IL-8 in regulation of tumor metastasis and progression.ResultsThe elevated sIL-8 levels were associated with LNM and poor prognosis in gastric cancer. Furthermore, sIL-8 was identified to be prominently produced by gastric cancer-associated fibroblasts (CAFs). Elevated IL-8 can up-regulate PD-1 expression in CD8+ T cells, resulting in immunosuppression in primary tumors and TDLNs, which enhances LNM of gastric cancer. Molecularly, IL-8 increases PD-1 expression through JAK2/STAT3 signaling activation, and inhibits its ubiquitination via Fbxo38 down-regulation. In addition, the in vivo studies in mouse gastric cancer model demonstrated that IL-8 promotes LNM via PD-1 up-regulation in CD8+ T cells.ConclusionThe present study elucidates the pro-metastatic role of elevated IL-8 in gastric cancer, and provides novel insights to enhance immune checkpoint blockade therapy for anti-PD-1 in gastric cancer.