Gastric Cancer Patient-derived Xenograft Research Articles

Dual targeting non-overlapping epitopes in HER2 domain IV substantially enhanced HER2/HER2 homodimers and HER2/EGFR heterodimers internalization leading to potent antitumor activity in HER2-positive human gastric cancer

BackgroundTrastuzumab and pertuzumab combination has been approved for the treatment of patients with HER2-positive metastatic breast cancer. However, trastuzumab and pertuzumab combination did not show improvement in overall survival in patients with HER2-positive metastatic gastric cancer.MethodsWe developed a new HER2-targeted monoclonal antibody, HLX22, targeting HER2 subdomain IV as trastuzumab but with non-overlapping epitopes. We examined the antitumor effects of this novel HER2-antibody in gastric cell lines and cell line-derived xenograft (CDX) and patient-derived xenograft (PDX) models.ResultsHLX22 in combination with HLX02 (trastuzumab biosimilar) induced enhancement of HER2/HER2 homodimers and HER2/EGFR heterodimers internalization, which ultimately led to the reduction in signal transductions involving STAT3, P70 S6, and AKT; gene expressions of FGF-FGFR-PI3K-MTOR, EGF-EGFR-RAS, TGF-β-SMAD, PLCG and cell cycle progression related pathways that favor tumor development, proliferation, progression, migration and survival in gastric cancer cell line NCI-N87 were also reduced. These differing but complementary actions contributed to the synergistic antitumor efficacy of the HLX22 and HLX02 combination in gastric cancer cell lines, CDX and PDX. In addition, HLX22 in combination with HLX02 demonstrated stronger antitumor efficacy than HLX02 and HLX11 (a potential pertuzumab biosimilar) combination treatment both in vitro and in vivo.ConclusionsThese results suggested that the application of non-competing antibodies HLX22 and HLX02 targeting HER2 subdomain IV together may be of substantial benefit to gastric cancer patients who currently respond suboptimal to trastuzumab therapy.

Abstract 2118: SGN-CEACAM5C/SAR445953, a novel topoisomerase I inhibitor antibody-drug conjugate targeting CEACAM5, has potent anti-tumor activity in CRC, PDAC, GC and lung cancer tumor models

Abstract Carcinoembryonic antigen cell adhesion molecule 5, CEACAM5, is a glycosylphosphatidylinositol-anchored glycoprotein expressed on the cell surface of most of the colorectal cancer (CRC) and pancreatic ductal adenocarcinoma (PDAC) and of more than 2/3 of gastric cancer (GC) and non-small cell lung cancer (NSCLC) while normal tissue expression is limited. The high prevalence of CEACAM5 expression prompted us to develop a novel CEACAM5 topoisomerase I inhibitor (Topo1i) antibody drug conjugate (ADC) with a DAR of 8, SGN-CEACAM5C/SAR445953. The anti-CEACAM5 antibody was chosen based on its high selectivity for CEACAM5 and its potential to direct cytotoxic payloads to tumor. The Topo1i payload was optimized for potency and enhanced bystander activity. SGN-CEACAM5C/SAR445953 is rapidly internalized and demonstrates in vitro cytotoxicity with EC50 values in the sub-nM range while it induces no toxicity on CEACAM5-negative cells. The potent anti-tumor activity is mediated by direct cytotoxicity on CEACAM5-expressing tumor cells and by a strong bystander effect due to the diffusion of the payload to the neighboring CEACAM5-negative tumor cells. Accordingly, the ADC incubated in a co-culture of CEACAM5-positive and -negative cells at a ratio of 1/1 induces a ~50% growth inhibition of CEACAM5-negative cells. Interestingly, with incubated ADC, only 1% of CEACAM5-positive cells in co-culture are sufficient to provide 11% growth inhibition of CEACAM5-negative cells. In vivo, SGN-CEACAM5C/SAR445953 is stable in circulation in SCID mice with a t1/2 close to 15 days. In vivo efficacy at 1, 3 and 10 mg/kg (single administration) was evaluated in panels of 4 CRC, 3 PDAC, 4 NSCLC and 3 GC patient-derived xenograft (PDX) models. At 10 mg/kg, the ADC elicits antitumor regression in 14/14 models. At 3 mg/kg, tumor regression occurs in 12/14 models. This potent, specific and dose dependent anti-tumor activity was further confirmed in Single Mouse Trials (SMT) of 20 CRC PDX models, 31 lung cancer PDX models and 19 gastric cancer PDX models. SMT consists in use of one animal per PDX model per treatment arm and for which the evaluation of efficacy is based on RECIST (Response Evaluation Criteria In Solid Tumors) criteria used in clinic. In CRC, gastric and lung cancer SMT, disease control rates are 95%, 84% and 87%, respectively with overall response rates of 55%, 68% and 71% including 15%, 10% and 26% of complete responses, respectively. The high anti-tumor activity across panels of PDX models of several CEACAM5 positive indications supports further evaluation of SGN-CEACAM5C/SAR445953 in patients with CRC, PDAC, GC and lung cancers (NCT06131840). Citation Format: Yves Baudat, Celine Nicolazzi, Johann Petur Sigurjonsson, Celine Amara, Astrid Clarke, Ryan lyski, Dave Meyer, Valeria Fantin, Marielle Chiron, Stephanie Decary. SGN-CEACAM5C/SAR445953, a novel topoisomerase I inhibitor antibody-drug conjugate targeting CEACAM5, has potent anti-tumor activity in CRC, PDAC, GC and lung cancer tumor models [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2118.

Biological and targeting differences between the rare KRAS A146T and canonical KRAS mutants in gastric cancer models.

Gastric cancer (GC) is the third leading cause of cancer-related death worldwide, with a poor prognosis for patients with advanced disease. Since the oncogenic role of KRAS mutants has been poorly investigated in GC, this study aims to biochemically and biologically characterize different KRAS-mutated models and unravel differences among KRAS mutants in response to therapy. Taking advantage of a proprietary, molecularly annotated platform of more than 200 GC PDXs (patient-derived xenografts), we identified KRAS-mutated PDXs, from which primary cell lines were established. The different mutants were challenged with KRAS downstream inhibitors in in vitro and in vivo experiments. Cells expressing the rare KRAS A146T mutant showed lower RAS-GTP levels compared to those bearing the canonical G12/13D mutations. Nevertheless, all the KRAS-mutated cells displayed KRAS addiction. Surprisingly, even if the GEF SOS1 is considered critical for the activation of KRAS A146T mutants, its abrogation did not significantly affect cell viability. From the pharmacologic point of view, Trametinib monotherapy was more effective in A146T than in G12D-mutated models, suggesting a vulnerability to MEK inhibition. However, in the presence of mutations in the PI3K pathway, more frequently co-occurrent in A146T models, the association of Trametinib and the AKT inhibitor MK-2206 was required to optimize the response. A deeper genomic and biological characterization of KRAS mutants might sustain the development of more efficient and long-lasting therapeutic options for patients harbouring KRAS-driven GC.

Interaction between the EPHB2 receptor and EFNB1 ligand drives gastric cancer invasion and metastasis via the Wnt/β-catenin/FAK pathway

The survival benefit for patients with gastric cancer (GC) is modest due to its high transfer potential. Targeted therapy for metastasis-related genes in GC may be a viable approach, however, inhibitors specifically targeting GC are limited. In this study, GC patient-derived xenografts (PDX) with metastatic burden were established via orthotopic transplantation. PCR-Array analysis of primary and metastatic tumors revealed EPH receptor B2 (EPHB2) as the most significantly upregulated gene. The interaction between the EPHB2 receptor and its cognate-specific EFNB1 ligands was high in GC and correlated with a poor prognosis. Fc-EFNB1 treatment increased the invasion and migration abilities of GC cells and induced a high EPHB2 expression. EPHB2 knockdown in GC cells completely abolished the ephrin ligand-induced effects on invasion and migration abilities. Signal transduction analysis revealed Wnt/β-catenin and FAK as downstream signaling mediators potentially inducing the EPHB2 phenotype. In conclusion, the observed deregulation of EPHB2/EFNB1 expression in GC enhances the invasive phenotype, suggesting a potential role of EPHB2/EFNB1 compound in local tumor cell invasion and the formation of metastasis.

First-in-Human Study with Preclinical Data of BCL-2/BCL-xL Inhibitor Pelcitoclax in Locally Advanced or Metastatic Solid Tumors.

B-cell lymphoma-extra-large (BCL-xL) regulates apoptosis and is an attractive anticancer therapeutic target. However, BCL-xL inhibition also kills mature platelets, hampering clinical development. Using an innovative prodrug strategy, we have developed pelcitoclax (APG-1252), a potent, dual BCL-2 and BCL-xL inhibitor. Aims of this study were to characterize the antitumor activity and safety of pelcitoclax and explore its underlying mechanisms of action (MOA). Cell line-derived xenograft and patient-derived xenograft (PDX) models were tested to evaluate antitumor activity and elucidate MOA. Subjects (N = 50) with metastatic small-cell lung cancer and other solid tumors received intravenous pelcitoclax once or twice weekly. Primary outcome measures were safety and tolerability; preliminary efficacy (responses every 2 cycles per RECIST version 1.1) represented a secondary endpoint. Pelcitoclax exhibited strong BAX/BAK‒dependent and caspase-mediated antiproliferative and apoptogenic activity in various cancer cell lines. Consistent with cell-based apoptogenic activity, pelcitoclax disrupted BCL-xL:BIM and BCL-xL:PUMA complexes in lung and gastric cancer PDX models. Levels of BCL-xL complexes correlated with tumor growth inhibition by pelcitoclax. Combined with taxanes, pelcitoclax enhanced antitumor activity by downregulating antiapoptotic protein myeloid cell leukemia-1 (MCL-1). Importantly, pelcitoclax was well tolerated and demonstrated preliminary therapeutic efficacy, with overall response and disease control rates of 6.5% and 30.4%, respectively. Most common treatment-related adverse events included transaminase elevations and reduced platelets that were less frequent with a once-weekly schedule. Our data demonstrate that pelcitoclax has antitumor activity and is well tolerated, supporting its further clinical development for human solid tumors, particularly combined with agents that downregulate MCL-1.

CLDN18.2 and 4-1BB bispecific antibody givastomig exerts antitumor activity through CLDN18.2-expressing tumor-directed T-cell activation

BackgroundClaudin18.2 (CLDN18.2) is a tight junction protein that has been identified as a clinically proven target in gastric cancer. Stimulation of 4-1BB with agonistic antibodies is also a promising strategy...

Comparison of renal clearance of [18F]AlF-RESCA-HER2-BCH and [18F]AlF-NOTA-HER2-BCH in mice and breast cancer patients.

A novel HER2 affibody-based molecular probe, [18F]AlF-RESCA-HER2-BCH, was developed for reducing renal uptake, evaluated, and compared with [18F]AlF-NOTA-HER2-BCH. In preclinical studies, micro-PET/CT was performed using HER2-positive gastric cancer patient-derived xenografts (PDX) model at 0.5-1 (dynamic), 2, 4, and 6h post-injection. For blocking experiment, 0.5mg cold affibody was co-injected with probes. Biodistribution were performed on HER2-positive PDX models at 2h post-injection. For clinical study, PET/CT images were acquired at 2h and 4h after injection of 231.29 ± 17.77MBq [18F]AlF-NOTA-HER2-BCH or [18F]AlF-RESCA-HER2-BCH in five breast cancer patients (4 HER2-positive and 1 HER2-low). Standardized uptake values (SUVs) were measured in tumors and source-organs for semi-quantitative analysis. The OLINDA/EXM software (version 1.2) was used to calculate the radiation doses. [18F]AlF-NOTA-HER2-BCH and [18F]AlF-RESCA-HER2-BCH were stably labeled with [18F]F, with high binding specificity and affinity to HER2. Micro-PET/CT of both tracers could clearly visualize HER2-positive PDX tumors with high uptake of 16.24 ± 1.74% ID/g and 14.39 ± 2.45% ID/g at 2h post-injection. The renal accumulation of [18F]AlF-RESCA-HER2-BCH was significantly lower than that of [18F]AlF-NOTA-HER2-BCH (5.16 ± 0.22% ID/g vs. 158.73 ± 5.44% ID/g at 2h, p < 0.0001). In the clinical study, both [18F]AlF-NOTA-HER2-BCH and [18F]AlF-RESCA-HER2-BCH demonstrated favorable tumor targeting and image contrast. [18F]AlF-RESCA-HER2-BCH showed a higher SUVmax in both primary tumor and metastases, and a significantly higher target-to-nontarget ratio in metastases than [18F]AlF-NOTA-HER2-BCH. Moreover, [18F]AlF-RESCA-HER2-BCH had lower renal accumulation (43.56 ± 7.88 vs. 79.81 ± 3.81 at 2h, p < 0.0001; 33.23 ± 6.89 vs. 78.63 ± 4.00 at 4h, p < 0.0001) as well as a significantly lower renal absorbed dose than [18F]AlF-NOTA-HER2-BCH (0.4450 ± 0.1117mGy/MBq vs. 0.8030 ± 0.1604mGy/MBq, p < 0.01). [18F]AlF-RESCA-HER2-BCH tended to provide better image contrast than [18F]AlF-NOTA-HER2-BCH with a higher target-to-nontarget ratio in detection of metastases. Notably, [18F]AlF-RESCA-HER2-BCH had lower renal accumulation than [18F]AlF-NOTA-HER2-BCH.

Development and comparison of 68Ga/18F/64Cu-labeled nanobody tracers probing Claudin18.2

Claudin 18.2 (CLDN18.2) is an emerging target for the treatment of gastric cancers. We aim to develop tracers to image the expression of CLDN18.2. A humanized nanobody targeting CLDN18.2 (clone hu19V3) was produced and labeled with 68Ga, 64Cu, and 18F. The tracers were investigated in subcutaneous and metastatic models established using two different mouse types (nude and Balb/c mice) and two different cell lines (CHO-CLDN18.2 and CT26-CLDN18.2). Gastric cancer patient-derived xenograft (PDX) models were further established for validation experiments. Three novel CLDN18.2-targeted tracers (i.e., [68Ga]Ga-NOTA-hu19V3, [64Cu]Cu-NOTA-hu19V3, and [18F]F-hu19V3) were developed with good radiochemical yields and excellent radiochemical purities. [68Ga]Ga-NOTA-hu19V3 immuno-positron emission tomography (immunoPET) rapidly delineated subcutaneous CHO-CLDN18.2 lesions and CT26-CLDN18.2 tumors, as well as showing excellent diagnostic value in PDX models naturally expressing CLDN18.2. While [68Ga]Ga-NOTA-hu19V3 had high kidney accumulation, [64Cu]Cu-NOTA-hu19V3 showed reduced kidney accumulation and improved image contrast at late time points. Moreover, [18F]F-hu19V3 was developed via click chemistry reaction under mild conditions and precisely disseminated CHO-CLDN18.2 lesions in the lungs. Furthermore, region of interest analysis, biodistribution study, and histopathological staining results correlated well with the invivo imaging results. Taken together, immunoPET imaging with the three tracers can reliably visualize CLDN18.2 expression.

Abstract 1143: ATG-022, an antibody-drug conjugate targeting Claudin 18.2, demonstrated potent in vivo efficacy in gastric cancer patient-derived xenografts

Abstract Background: Antibody-drug conjugates (ADCs) have become promising antitumor agents in recent years. ADCs are comprised of a monoclonal antibody, targeting antigens expressed at higher levels on tumor cells than on normal cells, cross-linked to small molecule payloads with cytotoxic activity. Human Claudin 18.2 (CLDN18.2) is overexpressed in a large proportion of gastric and pancreatic cancers, with restricted normal tissue expression. The monoclonal antibody targeting CLDN18.2 (IMAB362) has demonstrated promising clinical benefit in combination with chemotherapies for gastric cancer patients. However, the same antibody shows suboptimal efficacy in patients with low CLDN18.2 expression. Here we report the potent in vivo efficacy of ATG-022, a CLDN18.2 ADC, in multiple gastric cancer patient-derived xenograft (PDX) models, including those with low CLDN18.2 expression. Methods: The binding affinity of ATG-022 with CLDN18.2 was detected by SPR and FACs analysis. The in vitro 50% inhibition concentration (IC50) of ATG-022 was determined in CLDN18.2 positive cell lines using CellTiter-Glo luminescent cell viability assay. In vivo efficacy of ATG-022 was evaluated in a series of gastric cancer PDX models with different expression level of CLDN18.2 The expression level of CLDN18.2 was determined using IHC staining. ATG-022 was dosed twice at 1 mg/kg, 3 mg/kg or 10 mg/kg every two weeks. Results: ATG-022 binds to CLDN18.2 protein with sub-nM affinity. It induced potent in vitro cytotoxicity in CHOK1 cells overexpressing CLDN18.2, with an IC50 of 5-7nM. Bystander killing by ATG-022 was observed. ATG-022 demonstrated potent in vivo antitumor efficacy in a gastric cancer PDX model with high CLDN18.2 expression. Intravenous dosing of 1 mg/kg, 3 mg/kg, and 10 mg/kg ATG-022 induced 35.41%, 71.56% tumor growth inhibition (%TGI) and tumor regression respectively. In a PDX model with low CLDN18.2 expression, 3 mg/kg ATG-022 or 10 mg/kg clinical benchmark CLDN18.2-ADC did not inhibit tumor growth, while 10 mg/kg ATG-022 induced tumor regression. Conclusions: ATG-022 demonstrated potent in vitro and in vivo antitumor effects, with in vivo efficacy observed in CLDN18.2-low expression PDX models, suggesting a promising therapeutic strategy for gastric cancer patients with a broad range of CLDN18.2 expression levels. Citation Format: Peng Chen, Yun Liu, Min Deng, Linjie Tian, Kevin Lynch, Bo Shan, Jay Mei, Bing Hou. ATG-022, an antibody-drug conjugate targeting Claudin 18.2, demonstrated potent in vivo efficacy in gastric cancer patient-derived xenografts [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 1143.

Inhibition of STAT3-ferroptosis negative regulatory axis suppresses tumor growth and alleviates chemoresistance in gastric cancer

Chemotherapy is still one of the principal treatments for gastric cancer, but the clinical application of 5-FU is limited by drug resistance. Here, we demonstrate that ferroptosis triggered by STAT3 inhibition may provide a novel opportunity to explore a new effective therapeutic strategy for gastric cancer and chemotherapy resistance. We find that ferroptosis negative regulation (FNR) signatures are closely correlated with the progression and chemoresistance of gastric cancer. FNR associated genes (GPX4, SLC7A11, and FTH1) and STAT3 are upregulated in 5-FU resistant cells and xenografts. Further evidence demonstrates that STAT3 binds to consensus DNA response elements in the promoters of the FNR associated genes (GPX4, SLC7A11, and FTH1) and regulates their expression, thereby establishing a negative STAT3-ferroptosis regulatory axis in gastric cancer. Genetic inhibition of STAT3 activity triggers ferroptosis through lipid peroxidation and Fe2+ accumulation in gastric cancer cells. We further develop a potent and selective STAT3 inhibitor, W1131, which demonstrates significant anti-tumor effects in gastric cancer cell xenograft model, organoids model, and patient-derived xenografts (PDX) model partly by inducing ferroptosis, thus providing a new candidate compound for advanced gastric cancer. Moreover, targeting the STAT3-ferroptosis circuit promotes ferroptosis and restores sensitivity to chemotherapy. Our finding reveals that STAT3 acts as a key negative regulator of ferroptosis in gastric cancer through a multi-pronged mechanism and provides a new therapeutic strategy for advanced gastric cancer and chemotherapy resistance.

Lenvatinib inhibits the growth of gastric cancer patient-derived xenografts generated from a heterogeneous population

BackgroundLenvatinib is a multitargeted tyrosine kinase inhibitor that is being tested in combination with immune checkpoint inhibitors to treat advanced gastric cancer; however, little data exists regarding the efficacy of lenvatinib monotherapy. Patient-derived xenografts (PDX) are established by engrafting human tumors into immunodeficient mice. The generation of PDXs may be hampered by growth of lymphomas. In this study, we compared the use of mice with different degrees of immunodeficiency to establish PDXs from a diverse cohort of Western gastric cancer patients. We then tested the efficacy of lenvatinib in this system.MethodsPDXs were established by implanting gastric cancer tissue into NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) or Foxn1nu (nude) mice. Tumors from multiple passages from each PDX line were compared histologically and transcriptomically. PDX-bearing mice were randomized to receive the drug delivery vehicle or lenvatinib. After 21 days, the percent tumor volume change (%Δvtumor) was calculated.Results23 PDX models were established from Black, non-Hispanic White, Hispanic, and Asian gastric cancer patients. The engraftment rate was 17% (23/139). Tumors implanted into NSG (16%; 18/115) and nude (21%; 5/24) mice had a similar engraftment rate. The rate of lymphoma formation in nude mice (0%; 0/24) was lower than in NSG mice (20%; 23/115; p < 0.05). PDXs derived using both strains maintained histologic and gene expression profiles across passages. Lenvatinib treatment (mean %Δvtumor: -33%) significantly reduced tumor growth as compared to vehicle treatment (mean %Δvtumor: 190%; p < 0.0001).ConclusionsNude mice are a superior platform than NSG mice for generating PDXs from gastric cancer patients. Lenvatinib showed promising antitumor activity in PDXs established from a diverse Western patient population and warrants further investigation in gastric cancer.

Inhibition of Musashi-1 enhances chemotherapeutic sensitivity in gastric cancer patient-derived xenografts.

Musashi-1 (MSI1), a neural RNA-binding protein, is considered a gastric and intestinal stem cell marker. Although the function of MSI1 in gastric cancer has attracted increasing interest, it is not known whether MSI1 can be used as a biomarker to monitor gastric cancer development and response to treatment. Here, the role of MSI1 in the chemotherapeutic sensitivity of gastric cancer was investigated. Patients with high MSI1 levels had poor outcomes, implicating the gene in the development and progression of the disease. We overexpressed and silenced MSI1 in the human gastric cancer cell lines MKN45 and HGC27, finding that knockdown reduced proliferation, invasion, and migration, while promoting apoptosis. A patient-derived xenograft gastric cancer model was constructed in which mice received chemical drugs, si-MSI1, or a drug-si-MSI1 combination. It was found that blocking MSI1 expression reduced gastric cancer drug tolerance. The combination treatment with si-MSI1 was superior to 5F-dUMP and cisplatin, either separately or in combination, indicating that including si-MSI1 was better than drug therapy alone. Transcriptome sequencing analysis showed that MSI1 altered cell cycle regulation and growth signal transduction, including that of blood vessel epicardial substance (BVES). These results suggest that MSI1 reduces the tolerance of gastric cancer to chemical drugs through modulation of MSI1/BVES signaling.

GPR30 Activation Promotes the Progression of Gastric Cancer and Plays a Significant Role in the Anti-GC Effect of Huaier.

Gastric cancer (GC) is one of the most common types of cancer. The n-butanol extract of Huaier (NEH) is the alcohol-soluble part extracted by the systematic solvent method, which is effective against gastric cancer (GC). However, the mechanism of action of NEH remains unclear. In this study, we aim to evaluate the clinical relevance of GPR30 expression in GC patients and the role of the GPR30/PI3K/AKT signalling pathway in the anti-GC effect of NEH. The expression of GPR30 was examined using immunohistochemistry. Cell counting kit 8 (CCK-8) assay, wound healing, and transwell experiments were used to investigate the viability, migration, and invasion of gastric cancer cells. Western blotting was used to detect the expression of GPR30 and its downstream signalling molecules of the PI3K/AKT signalling pathway. Gastric cancer patient-derived xenografts (PDX) mouse model was used to evaluate the antitumor effect of NEH in vivo. In addition, the graded doses and the maximum tolerated dose of NEH were administered intraperitoneally into the mice for acute toxicity test. We demonstrate that GPR30 expression in GC tissues was significantly higher than that in corresponding adjacent noncancerous tissues and the expression of GPR30 was correlated with a poor prognosis in GC patients. Moreover, GPR30 expression was involved in the migration and invasion of GC cells in vitro. Additionally, we found that NEH can suppress the growth of GC in patient-derived xenograft tumors in vivo. Furthermore, NEH inhibited the proliferation, migration, and invasion in GC cells in a concentration-dependent manner through inhibiting the GPR30-mediated PI3K/AKT signalling pathway in vitro. Acute toxicity test showed that NEH caused no toxic reaction or death and the maximum tolerated dose of NEH in mice was greater than 1600 mg/kg. Our results demonstrate that the high expression of GPR30 is an independent factor of poor prognosis in patients with GC and NEH could be a new agent for the treatment of gastric cancer.

From AVATAR Mice to Patients: RC48-ADC Exerted Promising Efficacy in Advanced Gastric Cancer With HER2 Expression.

RC48-ADC is a novel humanized antibody specific for human epidermal growth factor receptor 2 (HER2)in conjugation with a microtubule inhibitor via a cleavable linker. This study was to evaluate the antitumor activity and mechanism of RC48-ADC in gastric cancer (GC) and explore the population that may benefit from RC48-ADC treatment. Four human GC cell lines and nine patient-derived xenograft (PDX) models were exploited to evaluate the antitumor effect of RC48-ADC or trastuzumab treatment in vitro and in vivo. The expression and phosphorylation of HER2 were assessed by immunohistochemistry (IHC) staining. Critical molecules of downstream PI3K/AKT and cell cycle and apoptosis signaling pathways were detected and quantified by immunoblotting. Combined with preliminary results of preclinical research, three patients with IHC3+, IHC2+/FISH+, and IHC2+/FISH- of HER2 were enrolled to verify the efficacy of RC48-ADC treatment in advanced GC. In vitro, RC48-ADC had superior antiproliferative effects in a dose-dependent manner on GC cells, especially on HER2-positive cells. In vivo, RC48-ADC exceeded trastuzumab in GC PDX models with HER2 expression, even in models with moderate to low expression of HER2. Further exploration of mechanism showed that RC48-ADC exerted the antitumor effect by inhibiting phosphorylation of HER2, inducing G2/M phase arrest and cell apoptosis in HER2-expressed PDX models. In clinical practice, RC48-ADC had satisfactory efficacy in HER2-positive and HER2 moderately expressed GC patients and demonstrated promising efficacy in HER2-positive patients who have progressed after anti-HER2 therapy. In conclusion, RC48-ADC exerted promising antitumor activity in HER2-positive as well as score of 2+ in IHC and ISH-negative AGC patients after progression of systematic treatment.

Multimodal Imaging Technology Effectively Monitors HER2 Expression in Tumors Using Trastuzumab-Coupled Organic Nanoparticles in Patient-Derived Xenograft Mice Models.

Trastuzumab is a monoclonal antibody targeting human epidermal growth factor 2 (HER2), which has been successfully used in the treatment of patients with breast cancer and gastric cancer; however, problems concerning its cardiotoxicity, drug resistance, and unpredictable efficacy still remain. Herein, we constructed novel organic dopamine–melanin nanoparticles (dMNs) as a carrier and then surface-loaded them with trastuzumab to construct a multifunctional nanoprobe named Her-PEG-dMNPs. We used micro-PET/CT and PET/MRI multimodality imaging to evaluate the retention effect of the nanoprobe in HER2 expression in gastric cancer patient-derived xenograft (PDX) mice models after labeling of the radionuclides 64Cu or 124I and MRI contrast agent Mn2+. The nanoprobes can specifically target the HER2-expressing SKOV-3 cells in vitro (3.61 ± 0.74 vs. 1.24 ± 0.43 for 2 h, P = 0.002). In vivo, micro-PET/CT and PET/MRI showed that the 124I-labeled nanoprobe had greater contrast and retention effect in PDX models than unloaded dMNPs as carrier (1.63 ± 0.07 vs. 0.90 ± 0.04 at 24 h, P = 0.002), a similarity found in 64Cu-labeled Her-PEG-dMNPs. Because 124I has a longer half-life and matches the pharmacokinetics of the nanoparticles, we focused on the further evaluation of 124I-Her-PEG-dMNPs. Furthermore, immunohistochemistry staining confirmed the overexpression of HER2 in the animal model. This study developed and validated novel HER2-specific multimodality imaging nanoprobes for quantifying HER2 expression in mice. Through the strong retention effect of the tumor site, it can be used for the promotion of monoclonal antibody treatment effect and process monitoring.

Lenvatinib Inhibits the Growth of Gastric Cancer Patient-Derived Xenografts Derived from a Heterogeneous Western Population

Karalis, John D. MD; Yoon, Lynn Y. BS; Hammer, Suntrea MD; Reznik, Scott I. MD, FACS; Mansour, John C. MD, FACS; Yopp, Adam C. MD, FACS; Polanco, Patricio M. MD, FACS; Zhu, Hao MD; Porembka, Matthew R. MD, FACS; Wang, Sam C. MD, FACS Author Information

LncRNA PINK1-AS promotes Gαi1-driven gastric cancer tumorigenesis by sponging microRNA-200a.

Gastric cancer (GC) is one of the leading causes of human mortality around the world. We have previously shown that Gαi1 (the inhibitory subunit 1 of the heterotrimeric guanine nucleotide-binding protein) recruitment to ligand-activated receptor tyrosine kinases (RTKs) is essential for signaling. Testing its role in GC cancer-promoting functions, we found that Gαi1 is upregulated in human GC, correlating with poor overall survival. In established and primary human GC cells, Gαi1 shRNA (small hairpin RNA) or knockout produced significant anti-GC cell activity, proliferation and migration was inhibited, and apoptosis was activated. Conversely, ectopic Gαi1 overexpression promoted proliferation and migration of GC cells in vitro. By examining the tumor-suppressive miRNA microRNA-200a (miR-200a), we found that miR-200a directly silenced Gαi1 to induce anti-GC cell activity. The expression of miR-200a was downregulated in human GC, correlating with upregulation of a novel miR-200a-targeting long non-coding RNA (LncRNA), PINK1 (PTEN Induced Kinase 1)-AS. RNA immunoprecipitation, RNA-pull down, and RNA fluorescence in situ hybridization assays confirmed that PINK1-AS directly binds to miR-200a. Silencing PINK1-AS in GC cells led to miR-200a accumulation, Gαi1 downregulation, and inhibition of GC cell progression in vitro, whereas PINK1-AS upregulation produced the converse results. Significantly, anti-GC cell activity induced by PINK1-AS shRNA was ameliorated by the expression of miR-200a antisense or the 3'-UTR (untranslated region)-depleted Gαi1. In vivo, the growth of subcutaneous MGC-803 xenografts in nude mice was inhibited by PINK1-AS shRNA, but accelerated by PINK1-AS overexpression. Patient-derived GC xenograft growth in nude mice was largely inhibited after intratumoral injection of PINK1-AS shRNA lentivirus. In conclusion, PINK1-AS promotes Gαi1-driven GC progression by sponging miR-200a.

EPRS/GluRS promotes gastric cancer development via WNT/GSK-3β/β-catenin signaling pathway.

Glutamyl-prolyl-tRNA synthetase (EPRS/GluRS) is primarily part of the multi-synthetase complex that may play a key role in cancer development. However, the biological function, molecular mechanism, and inhibitor of EPRS have not been investigated in gastric cancer (GC). Immunohistochemistry was performed to detect the expression of EPRS in human gastric tumor tissues. Knocking down of EPRS, cell-derived xenograft mouse model, and patient-derived xenograft mouse model was used to identify the biological function of EPRS. Immunoprecipitation was applied to elucidate the interaction between EPRS and SCYL2. Computer docking model and multiple in vitro and in vivo experiments were conducted to discover EPRS inhibitors. Here, we report that EPRS is frequently overexpressed in GC tissues compared to that adjacent controls and its overexpression predicts poor prognosis in GC patients. Functionally, high expression of EPRS positively co-relates with GC development both in vitro and in vivo. Mechanistically, EPRS directly binds with SCYL2 to enhance the activation of WNT/GSK-3β/β-catenin signaling pathway and the accumulation of β-catenin in the nuclear, leading to GC cell proliferation and tumor growth. Moreover, we identified that xanthoangelol (XA) and 4-hydroxyderricin (4-HD) can directly bind to EPRS to block WNT/GSK-3β/β-catenin signaling pathway. More importantly, XA and 4-HD restrain gastric cancer patient-derived xenograft tumor growth and Helicobacter pylori combined with alcohol-induced atrophic gastritis and gastric tumorigenesis. These findings unveil a promising strategy for GC prevention and therapy by targeting EPRS-mediated WNT/GSK-3β/β-catenin cascades. Moreover, XA and 4-HD may be effective reagents used for GC prevention and therapy.

The antitumor capacity of mesothelin-CAR-T cells in targeting solid tumors in mice

Since the approval of chimeric antigen receptor (CAR) T cell therapy targeting CD19 by the FDA, CAR-T cell therapy has received increasing attention as a new method for targeting tumors. Although CAR-T cell therapy has a good effect against hematological malignancies, it has been less effective against solid tumors. In the present study, we selected mesothelin (MSLN/MESO) as a target for CAR-T cells because it is highly expressed by solid tumors but only expressed at low levels by normal tissues. We engineered a third generation MSLN-CAR comprising a single-chain variable fragment (scFv) targeting MSLN (MSLN-scFv), a CD8 transmembrane domain, the costimulatory domains from CD28 and 4-1BB, and the activating domain CD3ζ. In vitro, MSLN-CAR-T cells killed various solid tumor cell lines, demonstrating that it could specifically kill MSLN-positive cells and release cytokines. In vivo, we investigated the effects of MSLN-CAR-T cell therapy against ovarian, breast, and colorectal cancer cell-line-derived xenografts (CDX) and MSLN-positive colorectal and gastric cancer patient-derived xenografts (PDX). MSLN-CAR decreased the growth of MSLN-positive tumors concomitant with significantly increased T cells and cytokine levels compared to the control group. These results indicated that modified MSLN-CAR-T cells could be a promising therapeutic approach for solid tumors.

Abstract PS5-40: CDKN2A loss can be a predictive marker of palbociclib in breast and gastric cancer

Abstract Background: Abnormal cell cycle progression is a characteristic of cancer, and targeting the cell cycle is a strategy for cancer treatment. TCGA reported that 7% and 12% of gastric cancers exhibit CCND1 or CCNE1 alterations, respectively. Besides, Cyclin D1 is overexpressed in 25% to 60% of invasive breast carcinomas, and gene amplification is observed in 10% to 30% of breast cancer cases. Furthermore, CDK4/6 and CDNK2A/B aberrations are frequently observed in gastric (39.6%) and breast (7.6%) cancers. The presence of such abnormalities in cell cycle-related molecules suggests that gastric and breast cancers are good candidates for treatment with cell cycle inhibitors. Palbociclib is a specific inhibitor of CDK4/6, a vital regulator of the G1 checkpoint, and has been approved by the FDA because it provided a significant benefit by extending PFS in a phase III trial for hormone-positive advanced breast cancer. However, the predictive marker of palbociclib is not determined. Even though CDKN2A loss has been considered as a sensitive marker of palbociclib, there is no preclinical evidence to support whether CDKN2A deficient cancer shows sensitivity to palbociclib, especially in gastric and breast cancer. Therefore, we investigated the effects of palbociclib on CDKN2A loss gastric and breast cancer cell lines as well as patient derived-xenograft (PDX) models. Methods: The cytotoxic assay, cell cycle analysis, and western blotting were conducted to determine the anti-tumor effect and action mechanisms of palbociclib on gastric and breast cancer cell lines. Moreover, modulation of CDKN2A expression was conducted by siRNA and plasmid overexpression. These in vitro data were validated in vivo model and gastric cancer PDX models which have CDKN2A loss as well. Results: There is a meaningful correlation between CDKN2A loss and palbociclib sensitivity among gastric and breast cancer cell lines. CDKN2A loss cells showed G1 cell cycle arrest by blocking Rb phosphorylation and inhibited proliferative cell signaling. Moreover, palbociclib promoted senescence rather than apoptosis. The depletion of CDKN2A expression using siRNA increased palbociclib sensitivity with G1 cell cycle arrest accompanied by senescence. In contrast, CDKN2A overexpression in sensitive cells showed insensitivity to palbociclib. The anti-tumor effects of palbociclib on CDKN2A loss breast cancer cells were validated in the xenograft model, and the two different gastric cancer PDX models have CDKN2A loss also showed a significant response to palbociclib as well. Conclusions: CDK4/6 inhibitor palbociclib showed an anti-tumor effect in vitro and in vivo xenograft model of CDKN2A loss gastric and breast cancer. Our results suggest that palbociclib has therapeutic potential for the treatment of not only breast cancer but also gastric cancer, not limited to a hormone-positive breast cancer type. Our results provide a rationale for the future clinical trials of palbociclib in the treatment of breast cancers. Citation Format: Ahrum Min, Yu Jin Kim, Miso Lee, Kyung-Hun Lee, Seock-Ah Im. CDKN2A loss can be a predictive marker of palbociclib in breast and gastric cancer [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-40.