Gastric Cancer Organoids Research Articles

Independent Signaling of Hepatoma Derived Growth Factor and Tumor Necrosis Factor-Alpha in Human Gastric Cancer Organoids Infected by Helicobacter pylori.

We prepared three-dimensional (3-D) organoids of human stomach cancers and examined the correlation between the tumorigenicity and cytotoxicity of Helicobacter pylori (H. pylori). In addition, the effects of hepatoma-derived growth factor (HDGF) and tumor necrosis factor (TNFα) on the growth and invasion activity of H. pylori-infected gastric cancer organoids were examined. Cytotoxin-associated gene A (CagA)-green fluorescence protein (GFP)-labeled H. pylori was used to trace the infection in gastric organoids. The cytotoxicity of Cag encoded toxins from different species of H. pylori did not affect the proliferation of each H. pylori-infected cancer organoid. To clarify the role of HDGF and TNFα secreted from H. pylori-infected cancer organoids, we prepared recombinant HDGF and TNFα and measured the cytotoxicity and invasion of gastric cancer organoids. HDGF controlled the growth of each organoid in a species-specific manner of H. pylori, but TNFα decreased the cell viability in H. pylori-infected cancer organoids. Furthermore, HDGF controlled the invasion activity of H. pylori-infected cancer organoid in a species-dependent manner. However, TNFα decreased the invasion activities of most organoids. We found different signaling of cytotoxicity and invasion of human gastric organoids in response to HDGF and TNFα during infection by H. pylori. Recombinant HDGF and TNFα inhibited the development and invasion of H. pylori-infected gastric cancer differently. Thus, we propose that HDGF and TNFα are independent signals for development of H. pylori-infected gastric cancer. The signaling of growth factors in 3-D organoid culture systems is different from those in two-dimensional cancer cells.

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

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

Murine and Human Gastric Tissue Establishes Organoids after 48 Hours of Cold Ischemia Time during Shipment.

An inadequate supply of fresh tissue is a major limitation of three-dimensional patient-derived gastric organoid research. We propose that tissue procurement for organoid culture could be increased by developing a cold storage shipment protocol for fresh surgical tissues. Sixty stomach specimens from C57BL/6J mice were resected, of which forty-five were stored in Hank's Balanced Salt (HBSS), University of Wisconsin (UW), or Histidine-Tryptophan-Ketoglutarate (HTK) solutions for subsequent organoid culture. Stomachs were dissociated and processed into gastric organoids as fresh tissue or after transport at 4 °C for 24 or 48 h. All gastric organoid cultures were established and maintained for 10 passages. Cold storage for 24 or 48 h did not significantly affect organoid viability. Although cold storage was associated with decreased organoid growth rate, there were no differences in viability, cytotoxic dose response, or LGR5 and TROY stem cell gene expression compared to organoids prepared from fresh tissue. As a proof of concept, six human gastric cancer organoids were established after 24 or 48 h of storage. Patient-derived gastric organoids from mouse and human gastric tissue can be established after 48 h of cold ischemia. Our method, which only requires ice packs, standard shipping containers, and HBSS is feasible and reliable. This method does not affect the reliability of downstream dose-response assays and maintains organoid viability for at least 10 passages. The shipment of fresh tissue for organoid procurement could serve to enhance multicenter collaboration and achieve more elaborate or controlled organoid experimentation.

Establishment of gastric cancer organoid and its application in individualized therapy.

The application of cancer organoids is of great value in individualized therapy as an embodiment of the tumor of a patient, a gastric cancer organoid model was established and its application in individualized drug screening was explored. The primary tumor tissues of 3 patients with gastric cancer who underwent primary surgery at the Fourth Department of General Surgery of the First Hospital of Lanzhou University (Lanzhou, China) between July and August 2021 were selected and digested with mixed enzymes to prepare cell suspensions. Of these, two were cultured by mixing with Matrigel, while the cells from the third patient were placed in a 24-well ultra-low adhesion plate for suspension organoid culture. After intensive organoid growth, they were digested, passaged, cryopreserved and thawed for further analyses. The formation of gastric cancer organoids was observed under an inverted microscope. One case was selected, and organoids were compared with the original tumor tissue via H&E and immunohistochemical staining to evaluate the consistency of the two. Finally, paclitaxel, oxaliplatin and fluorouracil were administered to the organoids to verify the value of screening individualized drugs. It was indicated that the passage and cryopreservation of gastric cancer organoids were successfully established in all three cases. The H&E and immunohistochemical staining results suggested that the structure and protein expression of the organoids were highly similar to those of the source tumor tissue. The use of established gastric cancer organoids for individualized chemotherapy drug screening is of high clinical value. Gastric cancer organoids with high similarity to the original tissue may be successfully constructed by the suspension growth culture method. The established organoids may serve as an effective model for individualized drug screening.

Clinical value and influencing factors of establishing stomach cancer organoids by endoscopic biopsy.

To establish an in vitro study model of gastric cancer, gastric cancer organoid culture system, by biopsy under gastroscope, and to explore and analyze the related factors affecting the success rate of culture, to provide a better in vitro model for the study of gastric cancer. Twenty-six patients with advanced gastric cancer were collected. Organoids were cultured by biopsy under gastroscope. Paraffin sections were made and HE staining was used to compare the consistency of gastroscopic pathological morphology and organoids. To explore the influencing factors of cultivating gastric cancer organoids in combination with clinical data. A total of 26 cases were collected by gastroscopy, and 12 cases of gastric cancer organoids were successfully cultured after identification, with a success rate of about 48%. Its histopathological morphology was highly consistent with that of gastric cancer. According to the pathological type, 21 cases were poorly differentiated adenocarcinoma and 12 cases were successful. Four cases of signet ring cell carcinoma failed. According to the location of the lesion, the success rate of sampling and culture of gastric antrum was significantly lower, which may be related to antral edema and anatomical characteristics of gastric antrum. Some of the failed cases are related to the quality of sampling, technique and contamination of tissue cells. We have successfully established gastric cancer organoids through endoscopic biopsy, and analyzed the factors affecting the success rate of culture from various angles, to improve and enhance the organoid culture technology and provide a better platform for tumor research.

Abstract 6186: Identification of MYOF as a novel biomarker by using oxaliplatin-resistant gastric cancer organoid model

Abstract Background: The prognosis of gastric cancer (GC) is improving due to the development of chemotherapy, however, intrinsic or acquired resistance to anti-cancer drugs is still a major clinical challenge. Oxaliplatin (L-OHP) is one of the anti-cancer drugs used as first-line treatment for GC. As with other anti-cancer drugs, although acquired resistance to L-OHP is a major problem, no biomarkers are clinically available for L-OHP response. Recently, cancer stem cells (CSCs) were shown to be involved in the acquisition of drug resistance. Hence, the major goal is to discover novel predictive biomarkers and overcome L-OHP resistance in GC in relation to CSCs. Methods: We used the organoid model; a culture method that allows 3D in vitro culturing of tissue-like structures and is presumed to contain many stem/progenitor cells. We established three independent L-OHP-resistant gastric cancer organoids (GCOs) by adding increasing doses of L-OHP in the culture medium over time. Gene expression profiles of the pairs of parental and resistant organoids were evaluated using microarray analysis. We analyzed the microarray data of L-OHP-resistant GCOs with our previous study from 5-fluorouracil (5-FU)-resistant GCOs (GSE154127). We validated the upregulated genes in the L-OHP-resistant GCOs by qRT-PCR. To confirm the use of upregulated genes as a novel biomarker, we immunohistochemically evaluated the expression levels in GC tissue samples. Furthermore, we analyzed the effects of knockdown using both GC cell lines and GCOs. Results: Through the comparison of the data, we detected myoferlin (MYOF) to be a candidate gene responsible only for L-OHP resistance. qRT-PCR results confirmed the results of the microarray and found that high expression of MYOF correlated significantly with the IC50 of L-OHP in GCOs. Immunohistochemistry analysis for MYOF using 132 GC cases showed that high expression of MYOF was significantly associated with poor prognosis, T grade, N grade, and lymphatic invasion, and showed MYOF to be an independent prognostic indicator, especially in the GC patients treated with platinum-based chemotherapy. The knockdown or inhibition of MYOF by WJ460 in GC cell line and GCOs enhanced L-OHP sensitivity while it reduced cell growth, spheroid/organoid formation, migration, invasion, and in vivo tumorigenesis. Conclusion: We highlight that MYOF is highly involved in L-OHP resistance and tumor progression in GC. Our results indicate that MYOF could be a promising biomarker in GC to predict L-OHP sensitivity and tumor progression and a potential candidate gene as a therapeutic target. Citation Format: Kenji Harada, Naoya Sakamoto, Shoichi Ukai, Tsuyoshi Takashima, Ryota Maruyama, Daiki Taniyama, Kazuaki Tanabe, Hideki Ohdan, Wataru Yasui. Identification of MYOF as a novel biomarker by using oxaliplatin-resistant gastric cancer organoid model [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 6186.

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.

Escape from cell-cell and cell-matrix adhesion dependence underscores disease progression in gastric cancer organoid models

ObjectiveCell-cell (CC) and cell-matrix (CM) adhesions are essential for epithelial cell survival, yet dissociation-induced apoptosis is frequently circumvented in malignant cells.DesignWe explored CC and CM dependence in 58 gastric cancer...

Essential Roles of TDO2 in Gastric Cancer: TDO2 Is Associated with Cancer Progression, Patient Survival, PD-L1 Expression, and Cancer Stem Cells

Introduction: Tryptophan metabolism has been shown to be involved in tumor development. Two main tryptophan-degrading enzymes, tryptophan 2,3-dioxygenase (TDO2) and indoleamine 2,3-dioxygenase 1 (IDO1), may potently promote cancer cell survival and distant metastasis in diverse types of cancer, such as lung and breast cancer. IDO1 overexpression is an independent prognosticator in gastric cancer (GC). This work aimed to uncover the expression of TDO2 and its clinicopathologic significance in GC. Methods: TDO2 expression was evaluated in public data of The Cancer Genome Atlas cohort STAD and in two different GC cohorts. Correlation between TDO2 and immune cell infiltrates as well as PD-L1 tumor staining was investigated. The biofunction of TDO2 was examined with MTT, colony formation, and spheroid formation assays by RNA interference. Results: TDO2 expression was correlated with both progressive disease and clinical outcome, and its expression was an independent predictor of prognosis in GC. TDO2 expression was correlated with infiltration of immune cells and tumor expression of PD-L1. Inhibition of TDO2 expression suppressed cell proliferation, colony formation, and cell invasion of GC cells. Additionally, suppression of TDO2 expression inhibited spheroid body-formation and viability of GC organoids. Conclusion: Our data show that TDO2 might be a crucial marker for predicting prognosis and targeted therapy in GC.

Bifunctional Fusion Membrane‐Based Hydrogel Enhances Antitumor Potency of Autologous Cancer Vaccines by Activating Dendritic Cells

AbstractAutologous tumor cell vaccine is an individualized tumor treatment strategy to elicit tumor‐specific immune responses. However, it is difficult to exert efficacy in “cold” tumors, which lack tumor‐infiltrating T cells and are not sensitive to immunotherapy. Here, a dendritic cells (DCs) activation hydrogel is constructed based on bifunctional fusion membrane nanoparticles (FM‐NPs) composed of autologous tumor cell membranes and Mycobacterium phlei membrane extracts. The FM‐NPs not only provide tumor antigens but also can activate DCs. Afterward, the FM‐NPs are loaded in an injectable alginate hydrogel together with granulocyte‐macrophage colony‐stimulating factor (GM‐CSF). GM‐CSF releases rapidly and recruits DCs to hydrogel, while FM‐NPs retain in hydrogel to continuously interconnected with DCs, enriching mature DCs loaded with tumor antigens. In the 4T1 mouse breast tumor model, this hydrogel promotes the maturation of DCs in tumor‐draining lymph nodes and induces sufficient effector memory T cells that migrate to the tumor microenvironment. This successfully converts the “cold” tumors to “hot”, thus exerting a significant antitumor effect synergized with the PD‐1 antibody. It is also verified the inhibitory effect of FM‐NPs using human gastric cancer organoids. Altogether, these findings demonstrate the application potential for the bifunctional fusion membrane‐based hydrogel as an autologous tumor vaccine by activating DCs.

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.

Gastric Cancer Cell-Derived Kynurenines Hyperactive Regulatory T Cells to Promote Chemoresistance via the IL-10/STAT3/BCL2 Signaling Pathway.

Chemotherapy resistance is frequently observed in gastric cancer patients and is associated with poor prognosis; tryptophan (Trp) catabolism has been recognized as a key metabolic regulator of many types of cancer progression. Regulatory T cells (Tregs) and Trp metabolite kynurenine (Kyn) were analyzed using tumor tissues. Chemotherapy resistance induced by IL-10 or Treg was detected by flow cytometry assay. The activation of STAT3/BCL2 signaling pathways in gastric cells cocultured by Treg was illustrated by western blotting. Patients' Treg and human gastric cancer organoid model were established to examine the anticancer effects of STAT3 inhibitor. We found that a higher level of IL-10 secreted by Kyn-induced Tregs was responsible for the 5-fluorouracil-induced resistance of gastric cancer cell lines. STAT3 and BCL2 knockout significantly abrogated Treg supernatant- or IL-10-induced chemoresistance in SGC7901 and BGC823 cell lines. Furthermore, STAT3 inhibitor significantly reduced the organoid and clonogenicity of organoids cocultured with Treg. Our data suggested that tumor-derived Kyn may hyperactivate Tregs and induce chemoresistance through the IL-10/STAT3/BCL2 signaling pathway.

METTL3 promotes oxaliplatin resistance of gastric cancer CD133+ stem cells by promoting PARP1 mRNA stability.

Oxaliplatin is the first-line regime for advanced gastric cancer treatment, while its resistance is a major problem that leads to the failure of clinical treatments. Tumor cell heterogeneity has been considered as one of the main causes for drug resistance in cancer. In this study, the mechanism of oxaliplatin resistance was investigated through in vitro human gastric cancer organoids and gastric cancer oxaliplatin-resistant cell lines and in vivo subcutaneous tumorigenicity experiments. The in vitro and in vivo results indicated that CD133+ stem cell-like cells are the main subpopulation and PARP1 is the central gene mediating oxaliplatin resistance in gastric cancer. It was found that PARP1 can effectively repair DNA damage caused by oxaliplatin by means of mediating the opening of base excision repair pathway, leading to the occurrence of drug resistance. The CD133+ stem cells also exhibited upregulated expression of N6-methyladenosine (m6A) mRNA and its writer METTL3 as showed by immunoprecipitation followed by sequencing and transcriptome analysis. METTTL3 enhances the stability of PARP1 by recruiting YTHDF1 to target the 3'-untranslated Region (3'-UTR) of PARP1 mRNA. The CD133+ tumor stem cells can regulate the stability and expression of m6A to PARP1 through METTL3, and thus exerting the PARP1-mediated DNA damage repair ability. Therefore, our study demonstrated that m6A Methyltransferase METTL3 facilitates oxaliplatin resistance in CD133+ gastric cancer stem cells by Promoting PARP1 mRNA stability which increases base excision repair pathway activity.

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

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.

Hedgehog transcriptional effector GLI mediates mTOR-Induced PD-L1 expression in gastric cancer organoids.

Tumors evade immune surveillance by expressing Programmed Death-Ligand 1 (PD-L1), subsequently inhibiting CD8+ cytotoxic T lymphocyte function. Response of gastric cancer to immunotherapy is relatively low. Our laboratory has reported that Helicobacter pylori-induced PD-L1 expression within the gastric epithelium is mediated by the Hedgehog (Hh) signaling pathway. The PI3K/AKT/mTOR pathway is activated in gastric cancer and may have immunomodulatory potential. We hypothesize that Hh signaling mediates mTOR-induced PD-L1 expression. Patient-derived organoids (PDOs) were generated from gastric biopsies and resected tumor tissues. Autologous organoid/immune cell co-cultures were used to study the immunosuppressive function of MDSCs. NanoString Digital Spatial Profiling (DSP) of immune-related protein markers using FFPE slide-mounted tissues from gastric cancer patients was performed. DSP analysis showed infiltration of immunosuppressive MDSCs expressing Arg1, CD66b, VISTA and IDO1 within cancer tissues. Orthotopic transplantation of patient derived organoids (PDOs) resulted in the engraftment of organoids and the development of histology similar to that observed in the patient's tumor tissue. PDO/immune cell co-cultures revealed that PD-L1-expressing organoids were unresponsive to nivolumab in vitro in the presence of PMN-MDSCs. Depletion of PMN-MDSCs within these co-cultures sensitized the organoids to anti-PD-1/PD-L1-induced cancer cell death. Rapamycin decreased phosphorylated S6K, Gli2 and PD-L1 expression in PDO/immune cell co-cultures. Transcriptional regulation of PD-L1 by GLI1 and GLI2 was blocked by rapamycin. In conclusion, the PDO/immune cell co-cultures may be used to study immunosuppressive MDSC function within the gastric tumor microenvironment. The mTOR signaling pathway mediates GLI-induced PD-L1 expression in gastric cancer.

RETRACTED ARTICLE: Ethaselen synergizes with oxaliplatin in tumor growth inhibition by inducing ROS production and inhibiting TrxR1 activity in gastric cancer

BackgroundOxaliplatin is one of the most commonly used chemotherapeutic agent for the treatment of various cancers, including gastric cancer. It has, however, a narrow therapeutic index due to its toxicity and the occurrence of drug resistance. Hence, it is of great significance to develop novel therapies to potentiate the anti-tumor effect and reduce the toxicity of oxaliplatin. In our previous study, we demonstrated that ethaselen (BBSKE), an inhibitor of thioredoxin reductase, effectively inhibited the growth of gastric cancer cells and promoted apoptosis in vitro. In the present study, we investigated whether BBSKE can potentiate the anti-tumor effect of oxaliplatin in gastric cancer in vivo and vitro.MethodsCellular apoptosis and ROS levels were analyzed by flow cytometry. Thioredoxin reductase 1 (TrxR1) activity in gastric cancer cells, organoid and tumor tissues was determined by using the endpoint insulin reduction assay. Western blot was used to analyze the expressions of the indicated proteins. Nude mice xenograft models were used to test the effects of BBSKE and oxaliplatin combinations on gastric cancer cell growth in vivo. In addition, we also used the combined treatment of BBSKE and oxaliplatin in three cases of gastric cancer Patient-Derived organoid (GC-PDO) to detect the anti-tumor effect.ResultsWe found that BBSKE significantly enhanced oxaliplatin-induced growth inhibition in gastric cancer cells by inhibiting TrxR1 activity. Because of the inhibition of TrxR1 activity, BBSKE synergized with oxaliplatin to enhance the production of ROS and activate p38 and JNK signaling pathways which eventually induced apoptosis of gastric cancer cells. In vivo, we also found that BBSKE synergized with oxaliplatin to suppress the gastric cancer tumor growth in xenograft nude mice model, accompanied by the reduced TrxR1 activity. Remarkably, we found that BBSKE attenuated body weight loss evoked by oxaliplatin treatment. We also used three cases of GC-PDO and found that the combined treatment of BBSKE and oxaliplatin dramatically inhibited the growth and viability of GC-PDO with increased ROS level, decreased TrxR1 activity and enhanced apoptosis.ConclusionsThis study elucidates the underlying mechanisms of synergistic effect of BBSKE and oxaliplatin, and suggests that the combined treatment has potential value in gastric cancer therapy.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13046-021-02052-z.

Establishment of oxaliplatin-resistant gastric cancer organoids: importance of myoferlin in the acquisition of oxaliplatin resistance.

The attainment of drug resistance in gastric cancer (GC) is a problematic issue. Although many studies have shown that cancer stem cells (CSCs) play an important role in the acquisition of drug resistance, there is no clinically available biomarker for predicting oxaliplatin (L-OHP) resistance in relation to CSCs. Organoid technology, a novel 3D cell culture system, allows harboring of patient-derived cancer cells containing abundant CSCs using niche factors in a dish. In this study, we established L-OHP-resistant gastric cancer organoids (GCOs) and evaluated their gene expression profile using microarray analysis. We validated the upregulated genes in the L-OHP-resistant GCOs compared to their parental GCOs to find a gene responsible for L-OHP resistance by qRT-PCR, immunohistochemistry,in vitro, and in vivo experiments. We found myoferlin (MYOF) to be a candidate gene through microarray analysis. The results from cell viability assays and qRT-PCR showed that high expression of MYOF correlated significantly with the IC50 of L-OHP in GCOs. Immunohistochemistry of MYOF in GC tissue samples revealed that high expression of MYOF was significantly associated with poor prognosis, T grade, N grade, and lymphatic invasion, and showed MYOF to be an independent prognostic indicator, especially in the GC patients treated with platinum-based chemotherapy. The knockdown of MYOF repressed L-OHP resistance, cell growth, stem cell features, migration, invasion, and in vivo tumor growth. Our results suggest that MYOF is highly involved in L-OHP resistance and tumor progression in GC. MYOF could be a promising biomarker and therapeutic target for L-OHP-resistant GC cases.

Abstract 123: A CRISPR/Cas9-engineered ARID1A-deficient human gastric cancer organoid model reveals essential and non-essential modes of oncogenic transformation

Abstract Mutations in ARID1A, encoding a subunit of the BAF chromatin remodeling complex, rank amongst the most common molecular aberrations in human cancer. However, oncogenic consequences of ARID1A mutation in human cells remain poorly defined due to lack of forward human genetic model systems. Here, CRISPR/Cas9 knockout of ARID1A in primary TP53-/- human gastric organoids induced morphologic dysplasia, tumorigenicity and mucinous differentiation but impaired Wnt/β-catenin signaling. Genetic Wnt pathway activation rescued mucinous differentiation, but not hyperproliferation, suggesting alternative essential pathways of ARID1A-mediated transformation. ARID1A loss induced transcriptional regulatory modules characteristic of MSI and EBV subtype human gastric cancer, including FOXM1 regulation of multiple mitotic regulatory genes and BIRC5/survivin. Convergently, high-throughput compound screening indicated selective vulnerability of ARID1A-deficient organoids to BIRC5/survivin inhibition, functionally implicating BIRC5/survivin as an essential mediator of proliferation following initial ARID1A loss. However, CRISPR inactivation of ARID1A in established gastric cancer cell lines did not elicit sensitivity to BIRC5/survivin inhibitors, suggesting that this pathway may be selectively required during the early establishment of tumorigenesis. Overall, we define distinct pathways downstream of oncogenic ARID1A mutation, with non-essential Wnt-inhibited mucinous differentiation in parallel with essential transcriptional FOXM1/BIRC5-stimulated proliferation, indicating the general utility of organoid-based forward genetic cancer analysis in human cells. Citation Format: Yuan-Hung Lo, Kevin S. Kolahi, Yuhong Du, Chiung-Ying Chang, Andrey Krokhotin, Ajay Nair, Walter D. Sobba, Kasper Karlsson, Sunny J. Jones, Teri A. Longacre, Amanda T. Mah, Alexandra Sockell, Jose A. Seoane, Jin Chen, Jonathan S. Weissman, Christina Curtis, Andrea Califano, Haian Fu, Gerald R. Crabtree, Calvin J. Kuo. A CRISPR/Cas9-engineered ARID1A-deficient human gastric cancer organoid model reveals essential and non-essential modes of oncogenic transformation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 123.