Mouse Model Of Gastric Cancer Research Articles

Metastatic gastric cancer mouse models.

Metastatic gastric cancer mouse models.

Somatic mouse models of gastric cancer reveal genotype-specific features of metastatic disease.

Metastatic gastric carcinoma is a highly lethal cancer that responds poorly to conventional and molecularly targeted therapies. Despite its clinical relevance, the mechanisms underlying the behavior and therapeutic response of this disease are poorly understood owing, in part, to a paucity of tractable models. Here we developed methods to somatically introduce different oncogenic lesions directly into the murine gastric epithelium. Genotypic configurations observed in patients produced metastatic gastric cancers that recapitulated the histological, molecular and clinical features of all nonviral molecular subtypes of the human disease. Applying this platform to both wild-type and immunodeficient mice revealed previously unappreciated links between the genotype, organotropism and immune surveillance of metastatic cells, which produced distinct patterns of metastasis that were mirrored in patients. Our results establish a highly portable platform for generating autochthonous cancer models with flexible genotypes and host backgrounds, which can unravel mechanisms of gastric tumorigenesis or test new therapeutic concepts.

Babao dan inhibits gastric cancer progression in vivo Through multiple signaling pathways

Objective: The aim of this study was to explore the effects of Babao dan (BBD), a traditional Chinese medicine, on gastric cancer (GC) progression in vivo. Materials and Methods: A subcutaneous xenograft mouse model of GC was established using MGC80-3 cells. The terminal deoxynucleotidyl transferase-mediated dUTP: 2'-deoxyuridine 5'-triphosphate -biotin nick-end labeling method was adopted to detect cell apoptosis in vivo. The expression levels of proteins associated with proliferation, apoptosis, and angiogenesis were measured by immunohistochemical staining or western blotting (WB). The activation and protein levels of p-c-Jun N-terminal kinase (JNK), p-p38, p-extracellular-regulated kinase 1/2, p-nuclear factor-κB (NF-κB), and p-STAT3 were examined by Bio-plex and WB. Results: BBD significantly inhibited tumor growth in GC mouse models with no adverse effect on body weight or organ function. It was also found that BBD significantly suppressed the proliferation of GC tumor cells, induced the apoptosis of tumor cells, and inhibited angiogenesis through inactivating with mitogen-activated protein kinase, NF-κB, and STAT3 pathways. Conclusions: BBD exerts suppressive effects on GC tumor growth by regulating multiple pathways in vivo, which may provide a novel treatment option for GC therapy.

Mucoadhesive Carrier-Mediated Oral Co-delivery of Bcl2 Inhibitors Improves Gastric Cancer Treatment.

Gastric cancer treatment is challenging due to the lack of early-stage diagnostic technology and targeted delivery systems. Currently, the available treatments for gastric cancer are surgery, chemotherapy, immunotherapy, and radiation. These strategies are either invasive or require systemic delivery, exerting toxicities within healthy tissues. By creation of a targeted delivery system to the stomach, gastric cancer can be treated in the early stages. Such an approach reduces the negative effects on the rest of the body by minimizing systemic absorbance and random localization. With this in mind, we developed a mucoadhesive vehicle composed of β-Glucan And Docosahexaenoic Acid (GADA) for controlled drug/gene delivery. In the current study, we investigated the therapeutic effect of codelivery Bcl2 inhibitors navitoclax (NAVI) and siRNA (Bcl2) via oral using GADA. The therapeutic efficacy of the GADA-mediated oral NAVI/siRNA was investigated in a gastric cancer mouse model. Higher Bcl2 inhibition efficacy was observed in Western blotting and TUNEL assay in mice treated with GADA/NAVI/siRNA compared to free NAVI, siRNA, and NAVI/siRNA. Histology (H&E) and immunohistochemistry (Ki67, TUNEL, and BCl2) analyses confirmed a significant reduction of the tumor region. Interaction between GADA and mucus resulted in retention for over 6 h and thereby sustained local payload release. The developed oral carrier GADA is an emerging vehicle that has promising potential in oral delivery of both small and large molecules, and their mucoadhesive property results in improved therapeutic efficacy with minimal side effects compared to conventional treatment. This study opens a new window for the effective delivery of oral medicine for the treatment of gastric cancer and other gastrointestinal diseases.

177Lu]Lu-labeled anti-claudin-18.2 antibody demonstrated radioimmunotherapy potential in gastric cancer mouse xenograft models.

Gastric cancer (GC), one of the most prevalent and deadliest tumors worldwide, is often diagnosed at an advanced stage with limited treatment options and poor prognosis. The development of a CLDN18.2-targeted radioimmunotherapy probe is a potential treatment option for GC. The CLDN18.2 antibody TST001 (provided by Transcenta) was conjugated with DOTA and radiolabeled with the radioactive nuclide 177Lu. The specificity and targeting ability were evaluated by cell uptake, imaging and biodistribution experiments. In BGC823CLDN18.2/AGSCLDN18.2 mouse models, the efficacy of [177Lu]Lu-TST001 against CLDN18.2-expressing tumors was demonstrated, and toxicity was evaluated by H&E staining and blood sample testing. [177Lu]Lu-TST001 was labeled with an 99.17%±0.32 radiochemical purity, an 18.50 ± 1.27 MBq/nmol specific activity and a stability of ≥ 94% after 7 days. It exhibited specific and high tumor uptake in CLDN18.2-positive xenografts of GC mouse models. Survival studies in BGC823CLDN18.2 and AGSCLDN18.2 tumor-bearing mouse models indicated that a low dose of 5.55 MBq and a high dose of 11.10 MBq [177Lu]Lu-TST001 significantly inhibited tumor growth compared to the saline control group, with the 11.1 MBq group showing better therapeutic efficacy. Histological staining with hematoxylin and eosin (H&E) and Ki67 immunohistochemistry of residual tissues confirmed tumor tissue destruction and reduced tumor cell proliferation following treatment. H&E showed that there was no significant short-term toxicity observed in the heart, spleen, stomach or other important organs when treated with a high dose of [177Lu]Lu-TST001, and no apparent hematotoxicity or liver toxicity was observed. In preclinical studies, [177Lu]Lu-TST001 demonstrated significant antitumor efficacy with acceptable toxicity. It exhibits strong potential for clinical translation, providing a new promising treatment option for CLDN18.2-overexpressing tumors, including GC.

Anti-tumor effect of Fasudil, a ROCK inhibitor, on gastric cancer in mice

Gastric cancer remains one of the most common malignant tumors and a leading cause of death. However, there are few reports about the anti-tumor effect of Fasudil (Fas) on the stomach. Establishment of nude mice model of gastric cancer to evaluate the anti-tumor effect of Fas. The mice in each group were weighed every week. Serum and tumor tissue cytokines are detected by commercial kits. Serum and tumor tissue superoxide dismutase and malondialdehyde were detected by commercial kits. Histopathological changes of the tumor were detected by HE staining. Expression of ROS/ TXNIP/NLRP3/Caspase-1 pathway in tumor tissues was detected by Western blot. Our results showed that Fas increased and decreased the body weight of tumor mice, increased serum and tumor tissue cytokines contents of tumor mice, increased serum and tumor tissue oxidative stress of tumor mice, and increased Bax, Caspase-3, Caspase-9 protein expression, reduced Bcl-2 protein expression, also regulated ROS/TXNIP/NLRP3/Caspase-1 pathway expression. Our experiments show that Fas has a significant anti-gastric cancer effect, and its mechanism is related to the regulation of oxidative stress, inflammation and apoptosis in vivo.

Xiaotan Sanjie recipe, a compound Chinese herbal medicine, inhibits gastric cancer metastasis by regulating GnT-V-mediated E-cadherin glycosylation

ObjectiveXiaotan Sanjie recipe (XTSJ), a Chinese herbal compound medicine, exerts a significant inhibitory effect on gastric cancer (GC) metastasis. This work investigated the mechanism underlying the XTSJ-mediated inhibition of GC metastasis. MethodsThe effect of XTSJ on GC metastasis and the associated mechanism were investigated in vitro, using GC cell lines, and in vivo, using a GC mouse model, by focusing on the expression of Glc-N-Ac-transferase V (GnT-V; encoded by MGAT5). ResultsThe migration and invasion ability of GC cells decreased significantly after XTSJ administration, which confirmed the efficacy of XTSJ in treating GC in vitro. XTSJ increased the accumulation of E-cadherin at junctions between GC cells, which was reversed by MGAT5 overexpression. XTSJ administration and MGAT5 knockdown alleviated the structural abnormality of the cell–cell junctions, while MGAT5 overexpression had the opposite effect. MGAT5 knockdown and XTSJ treatment also significantly increased the accumulation of proteins associated with the E-cadherin-mediated adherens junction complex. Furthermore, the expression of MGAT5 was significantly lower in the lungs of BGC-823-MGAT5 + XTSJ mice than in those of BGC-823-MGAT5 + solvent mice, indicating that the ability of gastric tumors to metastasize to the lung was decreased in vivo following XTSJ treatment. ConclusionXTSJ prevented GC metastasis by inhibiting the GnT-V-mediated E-cadherin glycosylation and promoting the E-cadherin accumulation at cell–cell junctions.

Combined inhibition of Bcl-2 family members and YAP induces synthetic lethality in metastatic gastric cancer with RASA1 and NF2 deficiency

BackgroundTargetable molecular drivers of gastric cancer (GC) metastasis remain largely unidentified, leading to limited targeted therapy options for advanced GC. We aimed to identify molecular drivers for metastasis and devise corresponding therapeutic strategies.MethodsWe performed an unbiased in vivo genome-wide CRISPR/Cas9 knockout (KO) screening in peritoneal dissemination using genetically engineered GC mouse models. Candidate genes were validated through in vivo transplantation assays using KO cells. We analyzed target expression patterns in GC clinical samples using immunohistochemistry. The functional contributions of target genes were studied through knockdown, KO, and overexpression approaches in tumorsphere and organoid assays. Small chemical inhibitors against Bcl-2 members and YAP were tested in vitro and in vivo.ResultsWe identified Nf2 and Rasa1 as metastasis-suppressing genes through the screening. Clinically, RASA1 mutations along with low NF2 expression define a distinct molecular subtype of metastatic GC exhibiting aggressive traits. NF2 and RASA1 deficiency increased in vivo metastasis and in vitro tumorsphere formation by synergistically amplifying Wnt and YAP signaling in cancer stem cells (CSCs). NF2 deficiency enhanced Bcl-2-mediated Wnt signaling, conferring resistance to YAP inhibition in CSCs. This resistance was counteracted via synthetic lethality achieved by simultaneous inhibition of YAP and Bcl-2. RASA1 deficiency amplified the Wnt pathway via Bcl-xL, contributing to cancer stemness. RASA1 mutation created vulnerability to Bcl-xL inhibition, but the additional NF2 deletion conferred resistance to Bcl-xL inhibition due to YAP activation. The combined inhibition of Bcl-xL and YAP synergistically suppressed cancer stemness and in vivo metastasis in RASA1 and NF2 co-deficiency.ConclusionOur research unveils the intricate interplay between YAP and Bcl-2 family members, which can lead to synthetic lethality, offering a potential strategy to overcome drug resistance. Importantly, our findings support a personalized medicine approach where combined therapy targeting YAP and Bcl-2, tailored to NF2 and RASA1 status, could effectively manage metastatic GC.

Tumor Site-Specific Cleavage Improves the Antitumor Efficacy of Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs) play important roles in tumor therapy. However, traditional ADCs are limited by the extremely large molecular weight of the antibody molecules, which results in low permeability into solid tumors. The use of small ADCs may be expected to alleviate this problem, but this switch brings the new limitation of a greatly shortened blood circulation half-life. Here, we propose a new cleavable ADC design with excellent tumor tissue permeability and a long circulation half-life by fusing the small ADC ZHER2-MMAE with the Fc domain of the antibody for circulation half-life extension, and inserting a digestion sequence between them to release the small ADC inside tumors for better tumor penetration. The experimental results showed that the designed molecule Fc-U-ZHER2-MMAE has a significantly increased blood circulation half-life (7.1 h, 59-fold longer) compared to the small ADC ZHER2-MMAE, and significantly improved drug accumulation ability at tumor sites compared to the conventional full-length antibody-coupled ADC Herceptin-MMAE. These combined effects led to Fc-U-ZHER2-MMAE having significantly enhanced tumor treatment ability, as shown in mouse models of NCI-N87 gastric cancer and SK-OV-3 ovarian cancer, where Fc-U-ZHER2-MMAE treatment achieved complete regression of tumors in all or a portion of animals with no obvious side effects and an MTD exceeding 90 mg/kg. These data demonstrate the therapeutic advantages of this cleavable ADC strategy, which could provide a new approach for ADC design.

HOX Antisense Intergenic RNA Inhibitor Loaded on Magnetic Nanoparticles Inhibits Gastric Cancer Progression Through miR-20a-5p

Magnetic nanoparticles are easy to operate and highly efficient and acts as ideal molecular carriers. At the same time, research has found that inhibiting Homeotic Complex Gene transcript antisense RNA (HOTAIR) can inhibit tumor development. Therefore, this study intends to use magnetic nanoparticles to carry HOTAIR inhibitors to explore its role in gastric cancer progression. Magnetic nanoparticles loaded with HOTAIR inhibitor and mouse model of gastric cancer were constructed. Serum HOTAIR level was detected by ELISA, tumor volume and mass changes in mice were observed, and histopathology was assessed by HE staining. The effect of HOTAIR inhibitor loaded magnetic nanoparticles on miR-20a-5p expression and downstream apoptosis genes/proteins was also evaluated. (1) si-HOTAIR is the silent gene of HOTAIR. The obtained si-HOTAIR magnetic nanoparticles were used in various research groups. It was found that HOTAIR was highly expressed in gastric cancer mice. (2) HOTAIR inhibitor intervention reduced tumor mass and volume, and changed gastric histopathological conditions. (3) HOTAIR inhibitors inhibited the activities of gastric cancer MFC cells, promoted apoptosis, and downregulated miR-20a-5p. (4) Compared with other groups, miR-20a-5p in the cells of si-HOTAIR-loaded magnetic nanoparticles group was reduced and cancer progression was significantly inhibited. Magnetic nanoparticles loaded with HOTAIR inhibitors have a strong anti-tumor effect and this effect is achieved by inhibiting miR-20a-5p. At the same time, this process is related to Bcl-2.

β-Glucan-Mediated Oral Codelivery of 5FU and Bcl2 siRNA Attenuates Stomach Cancer.

Based on cancer-related deaths, stomach cancer is ranked fifth, and first among Hispanics. Lack of technologies for early diagnosis and unavailability of target-specific therapeutics are largely the causes of the poor therapeutic outcomes from existing chemotherapeutics. Currently available therapeutic modalities are invasive and require systemic delivery, although the cancer is localized in the stomach at its early stage. Therefore, we hypothesize that an oral local delivery approach can extend the retention duration of the therapeutics modalities within the stomach and thereby enhance therapeutic efficacy. To accomplish this, we have developed a ß-glucan (BG)-based oral delivery vehicle that can adhere to the mucus lining of the stomach for an extended period while controlling the release of Bcl2 siRNA and 5-fluorouracil (5FU) payload for over 6 h. We found that Bcl2 siRNA selectively knocked down the Bcl2 gene in a C57BL/6 stomach cancer mouse model followed by upregulation of apoptosis and remission of cancer. BG was found to be very effective in maintaining the stability of siRNA for at least 6 h, when submerged in simulated gastric juice tested in vitro. To investigate the potential therapeutic effects in vivo, we used a stomach cancer mouse model, where C57BL/6 mice were treated with 5FU, BG/5FU, siRNA, BG/siRNA, and BG/5FU/siRNA. Higher inhibition of Bcl2 and therapeutic efficacy were observed in mice treated with BG/5FU/siRNA confirmed with Western blotting and a TUNEL assay. Significant reduction in the tumor region was observed with histology (H&E) and immunohistochemistry (Ki67, TUNEL, and Bcl2) analyses. Overall, the oral formulation shows improved efficacy with nonsignificant side effects compared to the conventional treatment tested in the gastric cancer mouse model.

Effect of MDSC-targeted TFF2-MSA with PD-1 blockade therapy in advanced gastric cancer models.

e16037 Background: Recent studies revealed chemotherapy increases anti-PD1 response of gastric cancer (GC) by reducing myeloid-derived suppressor cells (MDSCs). However, a more specific MDSC-targeted treatment is needed to further improve anti-PD1 efficacy in advanced GC. Trefoil factor family 2 (TFF2), a partial agonist of CXCR4 and a secreted anti-inflammatory peptide, can suppress MDSC expansion. Here, we developed a novel MDSC-targeted peptide TFF2-MSA and investigated whether it can synergize with anti-PD1 in syngeneic GC mouse models. Methods: Murine serum albumin (MSA) was fused to murine TFF2 to generate TFF2-MSA peptide with an extended serum half-life. ACKP (Atp4b-Cre; Cdh1-/-; LSL-KrasG12D; Trp53-/-) GC cells developed from a highly malignant diffuse GC mouse model were grafted subcutaneously into HDC-GFP transgenic mice in which the immunosuppressive histidine decarboxylase (HDC)-expressing myeloid cells were traced with GFP. These recipient mice then received either TFF2-MSA or anti-PD-1 antibody or both when tumors reached 150 mm3. At the endpoint, flow cytometry analysis was performed to examine immune profiles. In parallel, a group of the s.c. tumor bearing mice were followed up for survival differences in response to the treatments. In the orthotopic model, ACKP-luc cells were implanted to stomach submucosa, and recipient mice received the same treatments started after 1 week. In the metastasis model, subcutaneous flank tumors were resected in ACKP-grafted mice once tumors reached 500 mm3 to permit spontaneous lung metastasis, and mice then received the same treatments. Results: While either TFF2-MSA or PD-1 antibody showed little benefit as a single agent (TGI 15% and 25% respectively, p>0.05), their combination dramatically suppressed ACKP s.c. tumor growth (TGI 78%, p<0.0001) and markedly prolonged median survival (64 days vs. 32.5 days in control) in a synergistic manner. The combination therapy efficiently reduced tumor HDC-GFP+ MDSCs by 67%, and profoundly increased tumor-infiltrating cytotoxic CD8+ T cells by 18 fold. The myeloid/lymphoid progenitor and output ratio in the bone marrow were decreased by the combination, to a similar level of tumor-free mice. While standard chemotherapy (5-fluorouracil and oxaliplatin) showed no efficacy, adding chemotherapy to the combination further improved treatment efficacy (TGI 91%) and extended median survival to 73 days. In addition, in the orthotopic model, the TFF2-MSA/PD-1 antibody combination eradicated GC in 80% mice without recurrence compared to 0% with either monotherapy. Finally, the combination of TFF2-MSA/PD-1 antibody significantly reduced 80% lung metastasis (vs. control, p<0.0001), compared to minimal inhibition with either monotherapy (p>0.05). Conclusions: Targeting MDSCs using TFF2-MSA synergizes with PD-1 blockade therapy in advanced and metastatic syngeneic mouse models of gastric cancer.

RUNX2 promotes gastric cancer progression through the transcriptional activation of MGAT5 and MMP13.

RUNX2 is overexpressed in gastric cancer but the mechanism(s) through which it promotes tumor progression remain undefined. Here, we investigated the role of RUNX2 on gastric cancer pathogenesis at the molecular level. The qRT-PCR and western bolt were utilized to examine the mRNA and protein levels. CCK-8, Transwell and wound healing assays were used to measure cell proliferation, invasion and migration. CHIP-PCR gel electrophoresis was used to verify RUNX2 as a transcription factor for MMP13 and MGAT5. The in vivo assay was utilized to assess tumor growth. In vivo assay was used to evaluate tumor growth, aberrant expression of RUNX2 and lung metastasis of gastric cancer. RUNX2 is overexpressed in MKN-45 and AGS cells. Genetic RUNX2 silencing reduced the proliferation, invasion and migration of MKN-45 and AGS cells. Analysis of the gastric cancer samples from the database revealed a significant positive correlation between MGAT5, MMP13, and RUNX2 expression. JASPAR analysis revealed that there was a potential binding site of RUNX2 in the promoter regions of MGAT5 and MMP13, and the experimental results confirmed that RUNX2 could regulate the expression of MGAT5 and MMP13 respectively. In vivo assays confirmed the aberrant expression of RUNX2 in mouse models of gastric cancer and reduced growth and lung metastasis in RUNX2 silenced xenograft tumors assessed. Collectively, these data reveal that RUNX2 enhances MGAT5 and MMP13 expression in gastric cancer cells and represents a biomarker and potential therapeutic target for gastric cancer therapy.

Tumor-infiltrating mast cells stimulate ICOS+ regulatory T cells through an IL-33 and IL-2 axis to promote gastric cancer progression

IntroductionIn solid tumors, regulatory T cell (Treg) and mast cell perform different roles depending on the microenvironment. Nevertheless, mast cell and Treg-mediated interactions in gastric cancer (GC) are unclear, as are their regulation, function, and clinical significance. ObjectiveThe present study demonstrated the mechanism of tumor-infiltrating mast cells stimulating ICOS+ regulatory T cells via the IL-33/IL-2 axis to promote the growth of gastric cancer. MethodsAnalyses of 98 patients with GC were conducted to examine mast cell counts, ICOS+ Tregs, and the levels of IL-33 or IL-2. Isolated ICOS+ Treg and CD8+ T cell were stimulated, cultured and tested for their functional abilities in vitro and in vivo. ResultsGC patients exhibited a significantly more production of IL-33 in tumors. Mast cell stimulated by tumor-derived IL-33 exhibited a prolonged lifespan through IL-33 mediated inhibition of apoptosis. Moreover, mast cells stimulated by tumor-derived IL-33 secreted IL-2, which induced Treg expansion. These inducible Tregs displayed an activated immunosuppressive phenotype with positive expression for the inducible T cell co-stimulator (ICOS). In vitro, IL-2 from IL to 33-stimulated mast cells induced increased numbers of ICOS+ Tregs with increased immunosuppressive activity against proliferation and effector function of CD8+ T cell. In vivo, ICOS+ Tregs were treated with anti-IL-2 neutralizing antibody followed by co-injection with CD8+ T cells in GC mouse model, which showed an increased CD8+ T cell infiltration and effector molecules production, meanwhile tumor growth and progression were inhibited. Besides, reduction in GC patient survival was associated with tumor-derived ICOS+ Tregs. ConclusionOur results highlight a crosstalk between GC-infiltrating mast cells and ICOS+ Tregs and provide a novel mechanism describing ICOS+ Treg expansion and induction by an IL-33/mast cell/IL-2 signaling axis in GC, and also provide functional evidence that the modulation of this immunosuppressive pathway can attenuate GC-mediated immune tolerance.

Abstract 1193: Sensory innervation directly promotes gastric cancer and modulates the tumor microenvironment

Abstract Tumor innervation contributes directly to cancer progression, but there appears to be organ-specific variation in the type of innervation. While our previous studies showed cholinergic signaling promoted gastric cancer and vagal denervation inhibited gastric cancer, it is noteworthy that sensory nerves comprise 80% of the vagal axons. To investigate the role of sensory innervation in gastric carcinogenesis, we performed retrograde neural tracing and identified CGRP+ peptidergic neurons in nodose ganglia and dorsal root ganglia as primary gastric sensory neurons. While VAChT+ parasympathetic nerves are the main nerves innervating the normal stomach, CGRP+ sensory nerves show the greatest degree of expansion in mouse gastric tumors. Indeed, mouse gastric tumors show upregulation of NGF, a growth factor essential for sensory nerve survival, and gastric NGF overexpression in an NGF knock-in mouse also resulted in increased sensory innervation. Inhibition of NGF/Trk signaling by Entrectinib markedly attenuates NGF-dependent sensory nerve expansion. In addition to overall increased axonogenesis of sensory nerves, mouse models of gastric cancer showed upregulation of αCGRP peptide in gastric tumors and Calca mRNA in gastric sensory neurons, indicating increased activation of sensory neurons during gastric carcinogenesis. To study the effect of such activation, sensory neurons were then directly activated by treatment of Trpv1-Cre;hM3Dq mice with clozapine-N-oxide (CNO), which led to marked gastric hyperplasia with increased proliferation. Interestingly, in an acetic acid-induced ulcer model, activation of sensory neurons reduced the ulcer size and strongly promoted gland regeneration. Activation of sensory neurons, in a syngeneic, orthotopic model of gastric cancer in the Trpv1-Cre;hM3Dq mice, led to a significant increase in tumor volume, Ki-67+ cancer cells, CD31+ vessels and M2 macrophage polarization, as well as decreased CD8+ T cells. Analysis of available single-cell transcriptome data from mouse and human stomach showed that Calcrl/Ramp1 (CGRP receptors) are expressed in gastric epithelial cells and most stromal cells, while the expression levels of Tacr1 (receptor for Substance P) and Sstr1-5 (receptors for Somatostatin) were much lower. Importantly, some Calcrl/Ramp1+ cells are proliferative cells with strong Ki-67 expression. Our data show that Calcrl is also overexpressed in mouse gastric tumors, while TCGA data reveals significant upregulation of CALCRL in human gastric cancers and that high expression of CALCRL is correlated with poor overall survival. Finally, we established a co-culture model using DRGs and gastric cancer organoids, and found that sensory nerves can generate synaptic-like terminals on cancer organoids. In conclusion, our data indicate that sensory innervation directly promotes gastric cancer growth and also modulates the tumor microenvironment. Citation Format: Xiaofei Zhi, Feijing Wu, Jin Qian, Yosuke Ochiai, Guodong Lian, Ermanno Malagola, Timothy C. Wang. Sensory innervation directly promotes gastric cancer and modulates the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1193.

Abstract 2380: Fluorescence labeling of human gastric cancer using novel tumor specific near infrared labeled antibodies

Abstract Introduction: Gastric cancer has a global incidence of ~1 million cases per year. Currently, complete surgical resection is the only opportunity for cure for patients. Bright labeling of tumor margins with tumor specific markers could improve outcomes. This report demonstrates the advantages of targeting gastric cancer with humanized anti-CEA and TAG-72 antibodies in mouse models. Methods: A cell line of human gastric cancer, MKN45, and a patient-derived tumor, KG8, were used to create xenograft models. To establish orthotopic models for each, ~1mm tumor fragments were harvested from subcutaneous models of these lines and sutured to the lesser curvature of the stomach. Humanized versions of TAG-72 (huCC44) and CEA (M5A) were conjugated to the near infrared dye IRDye800CW. IgG was conjugated to IRDye800CW and used as a control. For CEA-IR800 and IgG-IR800, mice received 50 µg IV and were imaged after 72 hours. For Tag72-IR800, mice received 75 µg IV and were imaged after 96 hours. Fluorescence imaging was performed on the mice using the LI-COR Pearl Imaging System. A tumor to background ratio (TBR) was calculated by dividing mean fluorescence intensity of the tumor versus normal gastric tissue. Results: Humanized anti-CEA and TAG-72 brightly labeled multiple mouse models of human gastric cancer (Table 1). The TBR for TAG-72 labeling of KG8 was 2.777. CEA had a superior TBR of 3.528 compared to 0.606 for the control (IgG) in KG8 models. In MKN models labelled with CEA, an excellent average TBR of 7.407 were seen compared to 0.668 for the control. Conclusion: Humanized anti-CEA and TAG-72 antibodies conjugated to near-infrared fluorescent dyes provide specific labeling of gastric cancers in orthotopic models. These tumor-specific fluorescent antibodies are promising potential clinical tools for visualization of gastric cancer margins at the time of surgical resection and improved surgical outcomes. Orthotopic Model Antibody Tumor to Background Ratio KG8 anti-TAG-72 2.777 KG8 anti-CEA 3.528 KG8 Control 0.606 MKN anti-CEA 7.407 MKN Control 0.668 Citation Format: Kristin E. Cox, Michael A. Turner, Siamak Amirfakhri, Thinzar M. Lwin, Pradipta Ghosh, Marygorret Obonyo, Kaitlyn M. Kelly, Robert M. Hoffman, Paul J. Yazaki, Michael Bouvet. Fluorescence labeling of human gastric cancer using novel tumor specific near infrared labeled antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2380.

Abstract 2938: Preclinical development of a novel bispecific mAb-Trap fusion protein, IMM2902, targeting both HER2 and CD47 as cancer immunotherapy

Abstract The overexpression of human epidermal growth factor receptor 2 (HER2) has been identified in a variety of solid tumors including breast cancer (BC), gastric cancer and is associated with tumor recurrence, brain metastasis, and an overall poor prognosis. As such, HER2 has become a major target in the research and development of anticancer drugs. CD47, an integrin-associated protein, is expressed in many human cancer cells, especially upregulated preferentially in HER2-expressing cells. Co-expression of HER2 and CD47 is more frequently detected in recurrent BC patients with poor prognosis when comparing with the counterpart primary tumors. IMM2902 is a first-in-class, clinical-stage, novel recombinant bispecific mAb-Trap fusion protein targeting both CD47 and HER2 simultaneously. The binding activities of IMM2902, measured as EC50, to total CD47+ and HER2+ in BT474, NCI-N87 and SKOV-3 cancer cell lines are 16, 11 and 5 nM respectively. The affinity of IMM2902 to HER2 is about 15 times higher than that of CD47. IMM2902 not only prevents the engagement of CD47 with SIRPα and hence blocks “don’t eat me” signal of CD47/SIRPα, but also induces strong antibody-dependent cell-mediated cytotoxicity (ADCC, EC50 = 0.0045 - 0.0544 nM) and antibody-dependent cellular phagocytosis (ADCP, EC50 = 0.05 - 0.13 nM) activity with specifically engineered IgG1 Fc fragment. Most importantly, IMM2902 induces accelerated HER2 degradation in tumor tissues. Despite its high affinity to CD47, IMM2902 does not binds to CD47 on erythrocyte membrane, therefore, unlike conventional CD47 mAb, IMM2902 demonstrated a minimal impact on red blood cells (RBC). In preclinical research, IMM2902 has demonstrated a much stronger antitumor activity with excellent tolerability in a mouse model of human breast cancer (dose range 3.5 - 10 mg/kg) and a mouse model of human gastric cancer (dose range 1 -18 mg/kg) than that of trastuzumab (targeting HER2 only) alone, IMM01 (a SIRPα-Fc fusion protein) alone, and the combination of trastuzumab and IMM01. In HER2-low SUN-1 xenograft models, IMM2902 has showed a strong tumor growth inhibition at doses between 2 to 18 mg/kg. Intriguingly, IMM2902 also has exhibited potent tumor killing activity against breast cancer resistant to trastuzumab (HCC-1954 and patient-derived xenografts herceptin-resistant model) at doses between 3.5 to 10 mg/kg. Given the strong preclinical antitumor activity as well as the favorable safety profile, IMM2902 may serve as a potent immunotherapy for HER2-expressing cancers via dual blockade of CD47 and HER2. A Phase 1 clinical trial exploring safety, tolerability, and preliminary efficacy of IMM2902 in patients with HER2-expressing advanced solid tumors is currently ongoing in both China (CXSL2101035) and USA (NCT05076591). Citation Format: Binglei Zhang, Song Li, Dianze Chen, Dandan Liu, Huiqin Guo, Chunmei Yang, Li Zhang, Wei Zhang, Xiaoping Tu, Liang Peng, Gui Zhao, Ruliang Zhang, Frank X. Gan, Wenzhi Tian, Fan Zhang, Yongping Song. Preclinical development of a novel bispecific mAb-Trap fusion protein, IMM2902, targeting both HER2 and CD47 as cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2938.

Abstract 709: AST-301, a pDNA-based therapeutic cancer vaccine encoding HER2 ICD, improves the efficacy of checkpoint blockade therapy in CD34+ humanized gastric cancer mouse model

Abstract PD-1/PD-L1 play a crucial role in modulating immune response and promoting self-tolerance through activating apoptosis of antigen-specific T cells and suppressing apoptosis of regulatory T cells. Although immunotherapy to block this may provide a greater survival benefit for some patients, the therapeutic effect shown in clinical trials is limited by shortage of pre-existing CD8+ T cells infiltrating the tumor. AST-301 is a pDNA-based therapeutic cancer vaccine encoding HER2 ICD sequence. It was effective in eliciting HER2-specific T cells and antibody immunity in the majority of breast and ovarian cancer patients who completed the vaccine regimen and has proven safety as well as long-term immunological memory efficacy of T-cell immune by the phase 1 study (PN 109, NCT00436254). Our previous study has shown that AST-301 has anti-tumor effects by mediating NK cells in human HER2+ gastric cancer cell (NCI-N87) xenograft athymic mice. Here, we aimed to determine whether combined administration of AST-301 can enhance the anti-tumor effect of immune checkpoint inhibitor (ICI) using NCI-N87 xenograft humanized mouse. To confirm the tumor suppressive effect of pembrolizumab (anti-PD-1) treatment combined with AST-301 in the hu-CD34+ hematopoietic stem cell-engrafted NSG mice that formed tumors with NCI-N87, AST-301 (100 μg) and pembrolizumab (5 mg/kg) were administered into mice once a week for a total of 3 times and one booster doses of AST-301 was injected at week 4. Pembrolizumab low dose (5 mg/kg) test group administered with AST-301 had a relatively higher tumor growth inhibition (TGI) effect than the same concentration of Pembrolizumab alone group. TGI effect (%) of G2 (a combination of AST-301 and Pembrolizumab 5 mg/kg), G3 (Pembrolizumab 5 mg/kg), and G4 (Pembrolizumab 10 mg/kg) were 54%, 47%, and 78%, respectively. To evaluate the mechanism underlying the enhanced anti-tumor effect due to the combined administration of AST-301 and pembrolizumab, we measured the ratio of immune cells isolated from spleen and tumor in mice. in G2, the ratio of helper T cells and cytotoxic T cells in splenocytes higher than the vehicle-treated control group (G1), and the ratio of cytotoxic T cells infiltrated the tumor was also detected high in some subjects compared to those in all the other three groups. These results suggest that AST-301 can improve HER2+ gastric cancer treatment outcomes of immune checkpoint blockade therapy by inducing a robust cytotoxic CD8+ T cell response to tumor cells. Currently, we are ongoing study to prove the anti-tumor synergistic effects of the combination of AST-301 and various FDA-approved Antibody Drug Conjugates (ADCs). This effort is focused on narrowing the administration doses to low levels to resolve the safety issue of ADC or ICI, and it will strongly support the diverse applicability of the cancer therapeutic vaccine. Citation Format: Hyo-Hyun Park, Hye-Ran Kim, Jin Kyeong Choi, Jee Hyun Choi, JinHo Kang, Jinback Lim, Hyo Jung Lim, Jong Yeong Lee, Eunkyo Joung, Hun Jung. AST-301, a pDNA-based therapeutic cancer vaccine encoding HER2 ICD, improves the efficacy of checkpoint blockade therapy in CD34+ humanized gastric cancer mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 709.

Abstract 5088: MDSC-targeted TFF2-MSA synergizes with PD-1 blockade therapy in diffuse-type gastric cancer

Abstract Aims: Recent studies revealed myeloid-derived suppressor cell (MDSC) in the tumor microenvironment as an appealing therapeutic target to sensitize intestinal-type gastric cancer (GC) to PD-1 blockade therapy. However, whether MDSC inhibition can improve survival in the more aggressive diffuse-type GC is unknown. Trefoil factor family 2 (TFF2), a secreted anti-inflammatory peptide, suppresses MDSC expansion partly via CXCR4 receptor. Here, we developed a novel MDSC-targeted peptide TFF2-MSA and investigated whether it can synergize with anti-PD1 to prolong survival in syngeneic diffuse GC mouse models. Methods: Murine serum albumin (MSA) was appended to the murine TFF2 to generate TFF2-MSA peptide with an extended serum half-life. ACKP (Atp4b-Cre; Cdh1-/-; LSL-KrasG12D; Trp53-/-) GC cells developed from a highly malignant diffuse GC mouse model were grafted subcutaneously into HDC-GFP transgenic mice in which the more immunosuppressive histidine decarboxylase (HDC)-expressing myeloid cells were traced with GFP. These recipient mice subsequently received either TFF2-MSA or anti-PD-1 antibody or both. Further, to assess treatment efficacy in inhibiting spontaneous lung metastasis, subcutaneous flank tumors were resected in ACKP cell-grafted mice once tumors reached 500 mm3, and mice then received treatment with TFF2-MSA or anti-PD-1 or the combination. At the endpoint, flow cytometry and histopathological analyses were performed to examine immune profiles and tumor metastasis. In another model, ACKP-luc cells were implanted orthotopically to stomach submucosa, and recipient mice received the same treatments started from 1 week later to assess their efficacy. Results: While either TFF2-MSA or PD-1 antibody showed little benefit as a single agent (TGI 15% and 25% respectively, p>0.05), their combination dramatically suppressed ACKP s.c. tumor growth (TGI 78%, p<0.0001) and markedly prolonged median survival (64 days vs. 32.5 days in control) of host mice in a synergistic manner. The combination therapy efficiently reduced HDC-GFP+ MDSCs (by 67% vs. control, p<0.01), and profoundly increased tumor-infiltrating cytotoxic CD8+ T cells (by 18 fold, p<0.001). Further, while standard chemotherapy (5-fluorouracil and oxaliplatin) showed limited efficacy in this model, addition of chemotherapy to the TFF2-MSA/PD-1 antibody combination further improved treatment efficacy (TGI 91%) and extended median survival to 73 days. In addition, combination of TFF2-MSA/PD-1 antibody significantly reduced 80% lung metastasis (vs. control, p<0.0001), in contrast to minimal inhibition observed with either monotherapy (p>0.05). Finally, in the orthotopic model, the combo regimen eradicated GC in 50% mice compared to 0% in either monotherapy treatment. Conclusion: Targeting MDSCs using TFF2-MSA synergizes with PD-1 blockade therapy in advanced and metastatic syngeneic mouse models of diffuse-type GC. Citation Format: Jin Qian, Sandra Ryeom, Bruce Daugherty, Seth Lederman, Timothy C. Wang. MDSC-targeted TFF2-MSA synergizes with PD-1 blockade therapy in diffuse-type gastric cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5088.

LncRNA H19-rich extracellular vesicles derived from gastric cancer stem cells facilitate tumorigenicity and metastasis via mediating intratumor communication network

BackgroundExtracellular vesicles (EVs) transport biologically active molecules, and represent a recently identified way of intercellular communication. Recent evidence has also reported that EVs shed by cancer stem cells (CSCs) make a significant contribution to carcinogenesis and metastasis. Here, this study aims to explore the possible molecular mechanism of CSCs-EVs in gastric cancer (GC) by mediating intratumor communication network.MethodsCSCs and non-stem cancer cells (NSCCs) were sorted from GC cells, and EVs were isolated from CSCs. H19 was knocked down in CSCs, and CSCs-EVs or CSCs-EVs containing shRNA-H19 (CSCs-EVs-sh-H19) were co-cultured with NSCCs, followed by evaluation of the malignant behaviors and stemness of NSCCs. Mouse models of GC were established and injected with CSCs-EVs from sh-H19-treated NSCCs in vivo.ResultsCSCs had notable self-renewal and tumorigenic capacity compared with NSCCs. CSCs promoted the malignant behaviors of NSCCs and expression of stemness marker proteins through secretion of EVs. Inhibited secretion of CSCs-EVs curtailed the tumorigenicity and metastasis of NSCCs in vivo. H19 could be delivered by CSCs-EVs into NSCCs. H19 promoted the malignant behaviors of NSCCs and stemness marker protein expression in vitro along with tumorigenicity and liver metastasis in vivo, which was mechanistically associated with activation of the YAP/CDX2 signaling axis.ConclusionTaken together, the present study points to the importance of a novel regulatory axis H19/YAP/CDX2 in carcinogenic and metastatic potential of CSCs-EVs in GC, which may be potential targets for anticancer therapy.