Patient-derived Gastric Cancer Research Articles

Blockade of Src signaling prevented stemness gene expression and proliferation of patient-derived gastric cancer stem cells

ABSTRACT Objectives: Gastric cancer (GC) is one of the most malignant tumors. Mounting studies highlighted gastric cancer stem cells (GCSCs) were responsible for the failure of treatment due to recurrence and drug resistance of advanced GC. However, targeted therapy against GCSC for improving GC prognosis suffered from lack of suitable models and molecular targets in terms of personalized medicine. To address this issue, two patient-derived GC cell lines SD209 and SD292 with cancer stem cells (CSCs) such as phenotype were isolated for establishing targeted therapy aiming at critical metastatic signaling in GC. Materials and Methods: The primary patient-derived GCSCs were established from parts of GC tissues for characterization of stem cells (SCs) phenotype at both cellular and molecular levels. Western blot and Immunohistochemistry (IHC) were performed for identifying the deregulated signaling in GC tissue. Immunofluorescence was used for analyzing proliferating and SC markers in GCSC attached on fibroblast. Acridine orange and propidium iodide analyses were performed for the survival of GCSC in suspensions. Results: In the culture environments of both SD209 and SD292, a lot of mesenchymal fibroblasts spread and crowd together on which a lot of cell clumps, suspected as GCSC, were firmly attached. In the IHC analysis, the GCSC stemness genes CD44 and Ep-CAM increased in tumor tissues of SD209, whereas Nanog-1 and octamer-binding transcription factor 3 (OCT-3) increased in that of SD292. By immunofluorescent analysis of a proliferation marker Ki67, the growth of SD209 and SD292 on mesenchymal fibroblasts was found to be reduced by dasatinib, the inhibitor of the Src kinase whose activity was upregulated in tumor tissues of both GCs. Dasatinib also suppressed the expression of Nanog-1 and OCT-3 in SD292 attached on mesenchymal fibroblasts. Conclusion: This study may provide a base for targeted therapy against GCSCs/GCs progression in future preclinical/clinical settings.

Gastric cancer fibroblasts affect the effect of immunotherapy and patient prognosis by inducing micro-vascular production.

Immunotherapy is critical for treating many cancers, and its therapeutic success is linked to the tumor microenvironment. Although anti-angiogenic drugs are used to treat gastric cancer (GC), their efficacy remains limited. Cancer-associated fibroblast (CAF)-targeted therapies complement immunotherapy; however, the lack of CAF-specific markers poses a challenge. Therefore, we developed a CAF angiogenesis prognostic score (CAPS) system to evaluate prognosis and immunotherapy response in patients with GC, aiming to improve patient stratification and treatment efficacy. We assessed patient-derived GC CAFs for promoting angiogenesis using EdU, cell cycle, apoptosis, wound healing, and angiogenesis analysis. We then identified CAF-angiogenesis-associated differentially-expressed genes, leading to the development of CAPS, which included THBS1, SPARC, EDNRA, and VCAN. We used RT-qPCR to conduct gene-level validation, and eight GEO datasets and the HPA database to validate the CAPS system at the gene and protein levels. Six independent GEO datasets were utilized for validation. Overall survival time was shorter in the high- than the low-CAPS group. Immune microenvironment and immunotherapy response analysis showed that the high-CAPS group had a greater tendency toward immune escape and reduced immunotherapy efficacy than the low-CAPS group. CAPS is closely associated with GC prognosis and immunotherapy outcomes. It is therefore an independent predictor of GC prognosis and immunotherapy efficacy.

Targeting Patient-Derived Orthotopic Gastric Cancers with a Fluorescent Humanized Anti-CEA Antibody

BackgroundGastric cancer poses a major diagnostic and therapeutic challenge as surgical resection provides the only opportunity for a cure. Specific labeling of gastric cancer could distinguish resectable and nonresectable disease and facilitate an R0 resection, which could improve survival.MethodsTwo patient-derived gastric cancer lines, KG8 and KG10, were established from surgical specimens of two patients who underwent gastrectomy for gastric adenocarcinoma. Harvested tumor fragments were implanted into the greater curvature of the stomach to establish patient-derived orthotopic xenograft (PDOX) models. M5A (humanized anti-CEA antibody) or IgG control antibodies were conjugated with the near-infrared dye IRDye800CW. Mice received 50 µg of M5A-IR800 or 50 µg of IgG-IR800 intravenously and were imaged after 72 hr. Fluorescence imaging was performed by using the LI-COR Pearl Imaging System. A tumor-to-background ratio (TBR) was calculated by dividing the mean fluorescence intensity of the tumor versus adjacent stomach tissue.ResultsM5A-IR800 administration resulted in bright labeling of both KG8 and K10 tumors. In the KG8 PDOX models, the TBR for M5A-IR800 was 5.85 (SE ± 1.64) compared with IgG-IR800 at 0.70 (SE ± 0.17). The K10 PDOX models had a TBR of 3.71 (SE ± 0.73) for M5A-IR800 compared with 0.66 (SE ± 0.12) for IgG-IR800.ConclusionsHumanized anti-CEA (M5A) antibodies conjugated to fluorescent dyes provide bright and specific labeling of gastric cancer PDOX models. This tumor-specific fluorescent antibody is a promising potential clinical tool to detect the extent of disease for the determination of resectability as well as to visualize tumor margins during gastric cancer resection.

ImmunoPET imaging of Trop2 expression in solid tumors with nanobody tracers.

The high expression of the transmembrane glycoprotein trophoblast cell-surface antigen 2 (Trop2) was strongly associated with the progression of solid tumors, including pancreatic and gastric cancers. Our study aimed to construct Trop2-specific immuno-positron emission tomography (immunoPET) probes and assess the diagnostic abilities in preclinical pancreatic and gastric cancer models. The expression of Trop2 in pancreatic cancer was determined by single-cell sequencing and immunohistochemistry on tissue microarray (TMA). Flow cytometry was used to screen the expression of Trop2 in pancreatic cancer cell lines. Two nanobodies (i.e., RTD98 and RTD01) targeting Trop2 were developed and labeled with gallium-68 (68Ga, T1/2 = 1.1 h) to construct immunoPET imaging probes. The agents were researched in cell-derived pancreatic and patient-derived gastric cancer models expressing varying Trop2. Single-cell sequencing results showed high expression of Trop2 in pancreatic ductal cells as well as acinar cells and immunohistochemical staining of TMA from pancreatic cancers showed significantly higher expression of Trop2 in cancerous than in paracancerous tissues. ImmunoPET utilizing [68Ga]Ga-NOTA-RTD98 could clearly delineate subcutaneous tumors, both in cell-derived pancreatic cancer models and patient-derived gastric cancer models, superior to imaging using [18F]-FDG or a non-specific probe [68Ga]Ga-NOTA-RTD161. Another probe with improved pharmacokinetics targeting Trop2, [68Ga]Ga-NOTA-RTD01, was further prepared and showed advantageous diagnostic capabilities in preclinical pancreatic cancer models. In the work, we reported two nanobody tracers targeting human Trop2 which may facilitate better use of Trop2-targeted therapeutics by noninvasively displaying expression dynamics of the target.

Novel and efficient method for culturing patient-derived gastric cancer stem cells.

Experimental techniques for patient-derived cancer stem-cell organoids/spheroids can be powerful diagnostic tools for personalized chemotherapy. However, establishing their cultures from gastric cancer remains challenging due to low culture efficiency and cumbersome methods. To propagate gastric cancer cells as highly proliferative stem-cell spheroids in vitro, we initially used a similar method to that for colorectal cancer stem cells, which, unfortunately, resulted in a low success rate (25%, 18 of 71 cases). We scrutinized the protocol and found that the unsuccessful cases were largely caused by the paucity of cancer stem cells in the sampled tissues as well as insufficient culture media. To overcome these obstacles, we extensively revised our sample collection protocol and culture conditions. We then investigated the following second cohort and, consequently, achieved a significantly higher success rate (88%, 29 of 33 cases). One of the key improvements included new sampling procedures for tumor tissues from wider and deeper areas of gastric cancer specimens, which allowed securing cancer stem cells more reproducibly. Additionally, we embedded tumor epithelial pieces separately in both Matrigel and collagen type-I as their preference to the extracellular matrix was different depending on the tumors. We also added a low concentration of Wnt ligands to the culture, which helped the growth of occasional Wnt-responsive gastric cancer stem-cell spheroids without allowing proliferation of the normal gastric epithelial stem cells. This newly improved spheroid culture method may facilitate further studies, including personalized drug-sensitivity tests prior to drug therapy.

Abstract 1753: IKS014, a HER2-targeting antibody drug conjugate incorporating novel bioconjugation and tumor-selective linker technology with improved in vivo efficacy and tolerability

Abstract HER2 has long been a target of high interest for antibody and antibody drug conjugate (ADC) therapeutics due to its well-documented overexpression in breast, gastric and lung cancer. While trastuzumab and ado-trastuzumab emtansine (T-DM1) have become an integral part of treatment paradigms for HER2-positive cancer, the more recent approvals of the fam-trastuzumab deruxtecan (DS-8201) ADC and the Fc-engineered margetuximab antibody have highlighted the potential for continued improvement over existing HER2-targeting therapies. IKS014 is a HER2-directed ADC that incorporates site-directed conjugation and tumor-selective glucuronide-trigger linker technology to reduce systemic off-target toxicity while maximizing efficient intracellular lysosomal payload release, thus holding the promise of a wider therapeutic index. IKS014 was generated by site-specific conjugation via a proprietary beta-glucuronide linker to the microtubule agent MMAF with a drug-to-antibody ratio (DAR) 2. In vitro and in vivo activity was evaluated in HER2-positive preclinical models with varying HER2 expression levels in comparison to benchmark ADCs. Pharmacokinetics (PK) and tolerability studies were conducted in rodent and cynomolgus monkey. IKS014 conjugation resulted in a homogeneous ADC with defined DAR and good physio-chemical properties. In vitro, IKS014 demonstrated dose dependent specific cytotoxicity against Her2-positive cell lines. In JIMT-1 breast cancer xenografts with moderate HER2-expression (HER2 IHC 2+), IKS014 causes complete regressions at a single dose of 5 mg/kg and partial regressions at 1 mg/kg, while T-DM1 results in only tumor growth inhibition even at 15 mg/kg. Anti-tumor efficacy of IKS014 in NCI-N87 (HER2 3+) gastric xenografts is comparable to DS-8201 but superior to T-DM1 at equivalent single doses ranging from 1 to 5 mg/kg. In a HER2-positive patient-derived gastric cancer model (HER2 2+), IKS014 was highly active at 5mg/kg Q2W x2, while T-DM1 was inactive at the same dosing schedule. IKS014 demonstrated stable PK with a terminal half-life of 8.7 days in rat and 4.6 days in monkey, and DAR 2 was maintained for up to 4 weeks. In cynomolgus monkeys, IKS014 was tolerated at 12 mg/kg single dose and 5 mg/kg repeat dose without ocular or lung toxicity findings. IKS014 was highly efficacious against HER2-positive tumor xenografts in vivo, including models with moderate target expression, and compared favorably to clinically validated benchmark ADCs. This improved preclinical efficacy combined with stable PK and good tolerability profile warrants further development of this novel ADC for HER2-positive cancers. Citation Format: Jutta Deckert, Jenny Thirlway, Yun-Hee Park, Ho Young Song, Chul-Woong Chung, Xuesong Wang, Zhenshan Zhang, Robert J. Lutz. IKS014, a HER2-targeting antibody drug conjugate incorporating novel bioconjugation and tumor-selective linker technology with improved in vivo efficacy and tolerability [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 1753.

Abstract 5700: Increasing efficacy of iBET-151, a small molecule inhibitor of BET protein in combination with paclitaxel in gastric cancer

Abstract Epigenetic dysregulation is a common characteristic of cancers, including gastric cancer (GC), which contributes to the development and progression of GC. BRD4 is one of the leaders for epigenetic regulation and is known to regulate the expression of oncogenes such as c-MYC, BCL-2, and CDK4. In a previous study, we evaluated the efficacy of iBET-151 in a large panel of GC cell lines and xenograft mouse models. We found that iBET-151 showed a modest growth-inhibitory effect in GC cells. Recent report showed, lowering the expression of BCL-2 and BCL-xL, which are the targets of BRD4, induces sensitivity to paclitaxel, frequently used antimitotic agent in GC. Therefore, we evaluate iBET-151 plus paclitaxel as a new combination treatment regimen for GC. The synergistic effect and possible mechanisms of iBET-151 with paclitaxel were evaluated in 49 GC cell lines or 10 GC organoids using cell proliferation assay, flow cytometry, western blotting, colony formation, and migration assays. We found that the addition of iBET-151 increased the sensitivity to paclitaxel alone treatment from 15% to 50% in approximately 70% (34/49) of GC cells. iBET-151 plus paclitaxel significantly promoted cell-cycle arrest at G1 or G2/M phase and increased sub-G1 phase and cleaved PARP expression, which correlates to increased cell death. Also, combined treatment suppressed c-Myc, Bcl-2, and Bcl-xL expression in AGS, YCC-1, YCC-32, and YCC-34. We found BET proteins were overexpressed in most patient-derived GC organoids whereas c-Myc, BCL-2, and BCL-xL were expressed at variable levels. We observed that there was a synergistic growth inhibition in 6 out of 10 (60%) organoids. The data suggests iBET-151 and paclitaxel have a synergistic effect not only in GC cell lines but also in GC organoids. In summary, our data show the combination of iBET-151 and paclitaxel resulted in markedly higher anti-tumor effects compared with either treatment alone for GC. With further studies to validate the efficacy and biomarker explorations, the combination of iBET-151 and paclitaxel represents a promising therapeutic strategy in GC. Citation Format: Sun Kyoung Kang, Sang Woo Cho, Woo Sun Kwon, Tae Soo Kim, Hyun Cheol Chung, Sun Young Rha. Increasing efficacy of iBET-151, a small molecule inhibitor of BET protein in combination with paclitaxel in gastric cancer [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 5700.

Activity of KIN-3248, a next-generation pan-FGFR inhibitor, against acquired FGFR-gatekeeper and molecular-brake drug resistance mutations.

461 Background: Oncogenic FGFR (FGFR1, FGFR2, FGFR3, and FGFR4) gene alterations are observed in approximately 7% of all human cancers and present as point mutations, small intragenic deletions, genomic amplifications, or chromosomal rearrangements/ gene fusions. FGFR2 gene fusions and FGFR3 activating alterations are predicted oncogenic drivers in 10-20% of cholangiocarcinoma and 20-35% of urothelial cancers, respectively. While currently approved FGFR inhibitors (e.g., erdafitinib, pemigatinib, infigratinib) and those in development (e.g., futibatinib) provide clinical benefit in these cancer indications, disease progression typically occurs within 6-8 months of starting treatment and is often associated with the emergence of on-target resistance mutations within the FGFR kinase domain. KIN-3248 is a next-generation, irreversible, small molecule, pan-FGFR inhibitor designed to target clinically relevant primary FGFR driver alterations and secondary resistance mutations, including FGFR2 and FGFR3 gatekeeper, molecular brake, and activation loop mutations. Methods: KIN-3248 was evaluated across wild-type FGFR family members and clinically relevant fusions and kinase domain resistance mutations in vitro. In addition, KIN-3248 activity was assessed in FGFR-driven and FGFR inhibitor-resistant human gastrointestinal xenograft tumor models. Results: KIN-3248 exhibited low nanomolar biochemical potency against wild-type FGFR family members as well as mutants associated with resistance to FGFR inhibitors (IC50 3.9 – 24.1 nM). Consistently, KIN-3248 was active in human FGFR2-PHGDH fusion-positive CCLP-1 and FGFR2-OPTN fusion-positive ICC13-7 cholangiocarcinoma cell lines engineered to express wild-type or clinically relevant gatekeeper, molecular brake, and activation loop mutant alleles (EC50 2.4 – 9.9 nM). Lastly, KIN-3248 led to dose-dependent tumor growth inhibition and regressions in FGFR inhibitor-resistant, patient-derived gastric cancer and cholangiocarcinoma models harboring secondary FGFR2 kinase domain mutations. This efficacy was accompanied by both pharmacodynamic biomarker modulation, downstream pathway inhibition, and apoptotic cell death across models in vitro and in vivo. Conclusions: KIN-3248 is a next-generation, irreversible, orally available, small molecule pan-FGFR inhibitor that overcomes clinically observed secondary mutations in FGFR2 and FGFR3 known to drive resistance to both reversible and irreversible FGFR inhibitors and associated with disease progression. Its highly-selective, potent and broad-spectrum activity against mutations in both the FGFR2 and FGFR3 kinase domains – including gatekeeper, molecular brake, and activation loop alterations – is unique among FGFR inhibitors and has the potential to extend the clinical response of cancer patients with FGFR-altered tumors.

Establishment of Patient-Derived Gastric Cancer Organoid Model From Tissue Obtained by Endoscopic Biopsies.

Cancer organoids are three-dimensional mini-organ analogues derived from cancer tissues and have been proposed as models capable of simulating the structure and function of human organs and tissues in vitro. We sought to establish gastric cancer patient-derived organoids (PDOs) from tissues obtained by endoscopic biopsies. Gastric cancer-PDOs were successfully established and cultured from cancer tissues with gastric adenocarcinoma by endoscopic biopsies. To confirm that gastric cancer-PDOs were derived from cancer tissue, the consistency of the original cancer tissue was assessed by histopathological examination. As a result, it was confirmed that the shape and internal structure of gastric cancer-PDO were derived from the original gastric cancer cells, and the tumor specificity of gastric cancer-PDO was confirmed through Periodic acid-Schiff (PAS) and polyclonal carcinoembryonic antigen antibody staining. These results demonstrate that gastric cancer-PDO models show the characteristics of primary tumors and have potential for drug screening and providing a personalized medicine platform.

Disruption of Her2-Induced PD-L1 Inhibits Tumor Cell Immune Evasion in Patient-Derived Gastric Cancer Organoids.

Simple SummaryHER2 may contribute to immune evasion in gastric cancer that is associated with PD-L1 expression. Autologous organoid/immune cell co-cultures serve as an appropriate in vitro model to study the effects of anti-HER2 targeted therapy in combination with anti-PD1 immune checkpoint inhibition and may be used as an ex vivo tool for precision medicine.(1) Background: The expression of programmed death-ligand 1 (PD-L1), which interacts with programmed cell death protein 1 (PD-1) on cytotoxic T lymphocytes (CTLs), enables tumors to escape immunosurveillance. The PD-1/PD-L1 interaction results in the inhibition of CTL proliferation, and effector function, thus promoting tumor cell evasion from immunosurveillance and cancer persistence. Despite 40% of gastric cancer patients exhibiting PD-L1 expression, only a small subset of patients responds to immunotherapy. Human epidermal growth factor receptor2 (HER2) is one of the critical regulators of several solid tumors, including metastatic gastric cancer. Although half of PD-L1-positive gastric tumors co-express HER2, crosstalk between HER2 and PD-1/PD-L1 in gastric cancer remains undetermined. (2) Methods: Human gastric cancer organoids (huTGOs) were generated from biopsied or resected tissues and co-cultured with CTLs and myeloid-derived suppressor cells (MDSCs). Digital Spatial Profiling (DSP) was performed on FFPE tissue microarrays of numerous gastric cancer patients to examine the protein expression of immune markers. (3) Results: Knockdown of HER2 in PD-L1/HER2-positive huTGOs led to a concomitant decrease in PD-L1 expression. Similarly, in huTGOs/immune cell co-cultures, PD-L1 expression decreased in huTGOs and was correlated with an increase in CTL proliferation which enhanced huTGO death. Treatment with Nivolumab exhibited similar effects. However, a combinatorial treatment with Mubritinib and Nivolumab was unable to inhibit HER2 expression in co-cultures containing MDSCs. (4) Conclusions: Our study suggested that co-expression of HER2 and PD-L1 may contribute to tumor cell immune evasion. In addition, autologous organoid/immune cell co-cultures can be exploited to effectively screen responses to a combination of anti-HER2 and immunotherapy to tailor treatment for gastric cancer patients.

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.

Three-Dimensional Ex Vivo Culture for Drug Responses of Patient-Derived Gastric Cancer Tissue.

Gastric cancer (GC) is one of the most common malignancies with high mortality and substantial morbidity. Although the traditional treatment strategies for GC revolve around surgery, radiotherapy, and chemotherapy, none have been able to optimally treat most affected patients. To improve clinical outcomes and overcome potential GC resistance, we established a three-dimensional (3D) culturing platform that accurately predicts drug responses in a time- and cost-effective manner. We collected tumor tissues from patients following surgeries and cultured them for 3 days using our protocol. We first evaluated cell proliferation, viability, and apoptosis using the following markers: Ki67 and cleaved caspase 3 (Cas3). We demonstrated that cell viability was maintained for 72 h in culture and that the tumor microenvironments and vascular integrities of the tissues were intact throughout the culture period. We then administered chemotherapeutics to assess drug responses and found differential sensitivity across different patient-derived tissues, enabling us to determine individualized medication plans. Overall, our study validated this rapid, cost-effective, scalable, and reproducible protocol for GC tissue culture that can be employed for drug response assessments. Our 3D culture platform paves a new way for personalized medication in GC and other tumors and can greatly impact future oncological research.

Wnt Signaling Shapes the Histologic Variation in Diffuse Gastric Cancer

Diffuse-type gastric cancer (GC) is currently subdivided into signet-ring cell carcinoma (SRCC) and non-SRCC, referred to as poorly cohesive carcinoma not otherwise specified (PCC-NOS). Although these subtypes are considered to be independent, they often coexist in the same tumors, raising a question of whether they clonally differ or not. To tackle this question, we established an experimental platform for human diffuse GC that enables accurate modeling of histologic subtypes. Seven patient-derived diffuse GC organoid lines were established, characterized by histopathologic analysis, in situ hybridization, and gene expression analysis. For genetic modeling of diffuse GC, we knocked out CDH1 and/or TP53 in human normal gastric organoids. Green fluorescent protein-labeled GC organoids were xenotransplanted into immune-deficient mice for invivo assessment. PCC-NOS organoids transformed into SRCC-like structures on removal of Wnt and R-spondin from the culture medium. This morphologic change paralleled downregulation of Wnt-target and gastric stem cell genes, including LGR5, and elevation of differentiation markers, such as KRT20 and MUCs. The association between Wnt target gene expression and histologic subtypes was confirmed in 3 patient-derived GC tissues. Invivo, single clone-derived organoids formed tumors that comprised 2 distinct histologic compartments, each corresponding to SRCC and PCC-NOS. The transition from PCC-NOS to SRCC histology reflected the abundance of surrounding R-spondin-expressing fibroblasts. SRCC and PCC-NOS were clonally identical, and their morphology was regulated by extracellular Wnt and R-spondin expression. Our results decoded how genetic mutations and the tumor environment shape pathohistologic and biologic phenotypes in human diffuse GCs.

MiR-185-3p regulates epithelial mesenchymal transition via PI3K/Akt signaling pathway by targeting cathepsin D in gastric cancer cells.

BackgroundRecently research reported that miR-185-3p could serve as an independent prognosis factor in gastric cancer (GC). However, the functional role and underlying mechanism of miR-185-3p in GC and epithelial-mesenchymal transition (EMT) progression remains largely elusive.MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) was carried out to analyze the expression of miR-185-3p and cathepsin D in patient-derived GC samples and various GC cell lines. Scratch assay and Transwell assay were used to evaluate the migration ability. The influence of miR-185-3p on the cell cycle distribution and cell apoptosis was evaluated using flow cytometry. Western blotting assay was performed to detect the expression of EMT associated proteins and the activity of PI3K/Akt signaling pathway. Furthermore, the interaction between miR-185-3p and cathepsin D was explored by dual-luciferase reporter assay.ResultsOur data revealed that miR-185-3p was down-regulated, while cathepsin D was up-regulated in both patient-derived GC samples and GC cells. Apart from inducing apoptosis, overexpression of miR-185-3p also inhibited EMT process and migration of GC cells. Mechanically, we firstly verified that miR-185-3p directly targeted the cathepsin D. Furthermore, miR-185-3p exerted its function on EMT process and migration via inhibiting cathepsin D to mediated PI3K/Akt signaling pathway.ConclusionsOur findings suggested that miR-185-3p targeted cathepsin D inhibiting EMT process via PI3K/Akt signaling, which may serve as a potential prognosis factor and therapeutic target to reduce the malignancy of GCs.

A Gemcitabine Plus 5-Fluorouracil Combination Inhibits Gastric-Cancer Liver Metastasis in a PDOX Model: A Novel Treatment Strategy.

We established a new patient-derived orthotopic xenograft (PDOX) model of gastric cancer liver metastasis and evaluated the efficacy of a novel combination chemotherapy, gemcitabine (GEM) plus 5-fluorouracil (5-FU), compared to a standard regimen of oxaliplatinum (L-OHP) plus 5-FU on the liver metastasis. Patient-derived gastric cancer was established in nude mice from the patient' s surgical tumor specimen. A single tumor fragment was implanted in the liver of nude mice. The mice with tumors were treated by GEM plus 5-FU or L-OHP plus 5-FU. GEM plus 5-FU or L-OHP plus 5-FU significantly and similarly inhibited tumor growth on the liver compared to the untreated control (p=0.007, p=0.02, respectively). GEM plus 5-FU could be a novel future clinical alternative to L-OHP plus 5-FU in gastric cancer patients who cannot tolerate platinum drugs.

1471P 3D patient-derived gastric cancer organoids as a tool for functional precision medicine for gastric cancer patients

1471P 3D patient-derived gastric cancer organoids as a tool for functional precision medicine for gastric cancer patients

Abstract 4170: Decursin inhibits gastric cancer cell growth and autophagic flux by suppressing cathepsin C

Abstract Purpose: Autophagy is a series of processes in which biomolecules are digested and recycled, and it also provides energy for the development and progression of cancer. Thus, autophagy inhibitors can be used to suppress the progression of cancer. In this study, we investigated whether decursin, which is derived from natural products, inhibits the growth and autophagy of cancer cells. Methods: The gastric cancer cell lines SNU216 and NCI-N87 were treated with or without decursin. Cell growth was evaluated by Cell-Counting Kit-8, colony formation, and BrdU assays, as well as by propidium iodide staining, and autophagic flux was evaluated by western blotting, LC3 puncta, GFP-RFP-LC3, and transmission electron microscopy. Finally, tumor spheroids and patient-derived gastric cancer organoids were used to confirm the effect of decursin in three-dimensional culture. Results: Decursin reduced the growth and anchorage-dependent viability of gastric cancer cells. LC3 cleavage and LC3-GFP puncta were increased by decursin in a time- and concentration-dependent manner. However, the increase in autophagosome formation did not lead to an increase in autophagic flux. Decursin downregulated cathepsin C (CTSC), a lysosomal protease, irrespective of cellular pH. In addition, knockdown of CTSC reduced the mRNA and protein levels of E2F3, resulting in cell-cycle arrest. Decursin also reduced cell growth in both tumor spheroids and patient-derived gastric cancer organoids. Conclusion: The results demonstrated that decursin inhibited tumor growth and progression by suppressing CTSC-mediated autophagic flux and cell-cycle progression. Therefore, decursin is a novel inhibitor of autophagic flux, with potential for the treatment of gastric cancer. Citation Format: Solbi Kim, Mina Joo, Nayoung KIM, Myung-Won Lee, Heung Jin Jeon, Hyewon Ryu, Ik-Chan Song, Hyo Jin Lee. Decursin inhibits gastric cancer cell growth and autophagic flux by suppressing cathepsin C [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4170.

Resistance to FGFR1-targeted therapy leads to autophagy via TAK1/AMPK activation in gastric cancer.

Fibroblast growth factor receptor 1 (FGFR1) is frequently dysregulated in various tumors. FGFR inhibitors have shown promising therapeutic value in several preclinical models. However, tumors resistant to FGFR inhibitors have emerged, compromising therapeutic outcomes by demonstrating markedly aggressive metastatic progression; however, the underlying signaling mechanism of resistance remains unknown. We established FGFR inhibitor-resistant cell models using two gastric cancer (GC) cell lines, MGC-803 and BGC-823. RNA-seq was performed to determine the continuous cellular transcriptome changes between parental and resistant cells. We explored the mechanism of resistance to FGFR inhibitor, using a subcutaneous tumor model and GC patient-derived tumor organotypic culture. We observed that FGFR1 was highly expressed in GC and FGFR1 inhibitor-resistant cell lines, demonstrating elevated levels of autophagic activity. These resistant cells were characterized by epithelial-mesenchymal transition (EMT) required to facilitate metastatic outgrowth. In drug-resistant cells, the FGFR1 inhibitor regulated GC cell autophagy via AMPK/mTOR signal activation, which could be blocked using either pharmacological inhibitors or essential gene knockdown. Furthermore, TGF-β-activated kinase 1 (TAK1) amplification and metabolic restrictions led to AMPK pathway activation and autophagy. In vitro and in vivo results demonstrated that the FGFR inhibitor AZD4547 and TAK1 inhibitor NG25 synergistically inhibited proliferation and autophagy in AZD4547-resistant cell lines and patient-derived GC organotypic cultures. We elucidated the molecular mechanisms underlying primary resistance to FGFR1 inhibitors in GC, and revealed that the inhibition of FGFR1 and TAK1 signaling could present a potential novel therapeutic strategy for FGFR1 inhibitor-resistant GC patients.

Combined Conditional Knockdown and Adapted Sphere Formation Assay to Study a Stemness-Associated Gene of Patient-derived Gastric Cancer Stem Cells.

Cancer stem cells (CSCs) are implicated in tumor initiation, development and recurrence after treatment, and have become the center of attention of many studies in the last decades. Therefore, it is important to develop methods to investigate the role of key genes involved in cancer cell stemness. Gastric cancer (GC) is one of the most common and mortal types of cancers. Gastric cancer stem cells (GCSCs) are thought to be the root of gastric cancer relapse, metastasis and drug resistance. Understanding GCSCsbiology is needed to advance the development of targeted therapies and eventually to reduce mortality among patients. In this protocol, we present an experimental design using a conditional knockdown system and an adapted sphere formation assay to study the effect of clusterin on the stemness of patient-derived GCSCs. The protocol can be easily adapted to study both in vitro and in vivo function of stemness-associated genes in different types of CSCs.

Combined Conditional Knockdown and Adapted Sphere Formation Assay to Study a Stemness-Associated Gene of Patient-derived Gastric Cancer Stem Cells

Combined Conditional Knockdown and Adapted Sphere Formation Assay to Study a Stemness-Associated Gene of Patient-derived Gastric Cancer Stem Cells