Yoshiyuki Tsukamoto

Genes associated with patient prognosis have been widely investigated as potential biomarkers and therapeutic targets in cancer research. The TCGA project has generated comprehensive molecular and clinical datasets across multiple cancer types, and web-based bioinformatics tools, such as GEPIA, enabled the identification of prognosis-related genes in a cancer type-specific manner. However, functional validation of these genes requires appropriate experimental models. Because expression patterns of prognosis-associated genes vary among patients even within the same cancer type, establishing large and diverse cell line panels that recapitulate patient-derived molecular features is essential. In this study, we performed comprehensive gene-expression profiling of one of the largest GC cell-line panels analyzed under uniform experimental conditions. Unsupervised clustering analysis classified 42 GC cell lines into two distinct groups, Cluster A and Cluster B. Using prognosis-related genes derived from GEPIA, we constructed gene sets and performed Gene Set Enrichment Analysis (GSEA), which demonstrated that Cluster B was significantly enriched for the prognostic gene expression signature for gastric cancer. This study provides a robust platform for functional analyses of prognosis-associated genes and supports biomarker discovery and precision medicine-oriented therapeutic development in gastric cancer.

Mitogen-activated protein kinase kinase (MEK) is a component of an important signaling pathway involved in the development and progression of pancreatic ductal adenocarcinoma (PDAC). However, MEK-targeted therapeutics are not effective, and therefore not indicated, for patients with PDAC. We have found that annexin A8 (ANXA8) is involved in resistance to MEK inhibitor therapy in PDAC. Expression of ANXA8 was induced at both the mRNA and protein levels early by MEK inhibitor treatment in PDAC cells, and the level of ANXA8 mRNA expression was inversely correlated with sensitivity to the MEK inhibitor. Furthermore, downregulation of ANXA8 enhanced the inhibitory effect of the MEK inhibitor on PDAC cell proliferation, suggesting that ANXA8 could be a potential therapeutic target for PDAC. To achieve a therapeutic strategy targeting ANXA8, we have identified all-trans retinoic acid (ATRA) as a compound exerting ANXA8-inhibitory effects in PDAC cells. Combination of the MEK inhibitor and ATRA demonstrated additive antitumor effects in PDAC cells in vitro and in vivo. IHC analysis revealed that ANXA8 was frequently upregulated in PDAC showing poor differentiation relative to PDAC with high or moderate differentiation. Furthermore, patients with ANXA8-positive PDAC were found to have a significantly poorer prognosis than those with ANXA8-negative PDAC. In summary, our findings suggest that ANXA8 plays a role in MEK inhibitor resistance in PDAC and that a combination of MEK inhibition with ANXA8-targeted therapy could be a novel effective strategy for PDAC.

Introduction: We experienced a case of esophageal squamous cell carcinoma (ESCC) exhibiting mucus-negative cytoplasmic vacuoles. The carcinoma cells with vacuoles were positive for p63, a marker of squamous cell carcinoma (SCC). Since the first description by Cramer and Heggeness in 1989, 24 cases of SCC with mucus-negative vacuoles have been reported to date. However, little is known about the clinical course of SCC with vacuoles (SCCVs) and the nature of the cytoplasmic vacuoles themselves, due to its rarity and the lack of suitable models. Methods: We established a novel organoid line – designated ECO_Vac – from residual cancer tissue of a patient with esophageal SCCV (ESCCV) after neoadjuvant chemotherapy, evaluated its applicability as a model of ESCCV, and characterized its cytoplasmic vacuoles by electron microscopic and organelle-specific fluorescent probe uptake assays. Results: ECO_Vac was successfully established and was found to be applicable to in vitro drug assays and experiments involving in vivo tumor formation. We also found that the vacuoles in ESCCV were enlarged autolysosomes. Conclusion: We established a novel organoid line, ECO_Vac, as a useful model for investigating the molecular pathogenesis of ESCCV. By using ECO_Vac, we demonstrated that the cytoplasmic vacuoles in ESCCV were unphysiologically enlarged autolysosomes.

Diffuse Large B-cell Lymphoma (DLBCL), with its intrinsic genetic and epigenetic heterogeneity, exhibits significantly variable clinical outcomes among patients treated with the current standard regimen. Disulfidptosis, a novel form of regulatory cell death triggered by disulfide stress, is characterized by the collapse of cytoskeleton proteins and F-actin due to intracellular accumulation of disulfides. We investigated the expression variations of disulfidptosis-related genes (DRGs) in DLBCL using two publicly available gene expression datasets. The initial analysis of DRGs in DLBCL (GSE12453) revealed differences in gene expression patterns between various normal B cells and DLBCL. Subsequent analysis (GSE31312) identified DRGs strongly associated with prognostic outcomes, revealing eight characteristic DRGs (CAPZB, DSTN, GYS1, IQGAP1, MYH9, NDUFA11, NDUFS1, OXSM). Based on these DRGs, DLBCL patients were stratified into three groups, indicating that (1) DRGs can predict prognosis, and (2) DRGs can help identify novel therapeutic candidates. This study underscores the significant role of DRGs in various biological processes within DLBCL. Assessing the risk scores of individual DRGs allows for more precise stratification of prognosis and treatment strategies for DLBCL patients, thereby enhancing the effectiveness of clinical practice.

Neoadjuvant chemotherapy (NAC) followed by surgery is a standard approach for management of locally advanced esophageal squamous cell carcinoma (ESCC). Patients who do not respond well to NAC have a poor prognosis. Despite extensive research, the mechanisms of chemoresistance in ESCC remain largely unknown. Here, we established paired tumor organoids—designated as PreNAC-O and PostNAC-O—from one ESCC patient before and after NAC, respectively. Although the two organoids did not exhibit significant differences in proliferation, morphology or drug sensitivity in vitro, the tumorigenicity of PostNAC-O in vivo was significantly higher than that of PreNAC-O. Xenografts from PreNAC-O tended to exhibit keratinization, while those from PostNAC-O displayed conspicuous necrotic areas. The tumorigenicity of PostNAC-O xenografts during the chemotherapy was comparable to that of PreNAC-O without treatment. Furthermore, the gene expression profiles of the xenografts suggested that expression of genes involved in the EMT and/or hypoxia response might be related to the tumorigenicity of PostNAC-O. Our data suggested that the tumorigenicity of residual cancer had been enhanced, outweighing the effects of chemotherapy, rather than being attributable to intrinsic chemoresistance. Further studies are required to clarify the extent to which residual cancers share a common mechanism similar to that revealed here.

Neoadjuvant chemotherapy (NAC) followed by surgery is one of the standard therapeutic approaches in Japan for patients with locally advanced esophageal carcinoma. Recently, the JCOG1109 study revealed that NAC with docetaxel, cisplatin and 5‐fluorouracil (5‐FU) (DCF‐NAC) is superior to NAC with cisplatin and 5‐FU, and has now become the standard preoperative chemotherapy. Using a microarray system, we have previously investigated the expression profiles of endoscopic biopsy samples from patients with esophageal squamous cell carcinoma (ESCC) before DCF‐NAC (preNAC) and identified 17 molecules as biomarkers predictive of a pathologically complete response to DCF‐NAC. Here, we re‐grouped our previous dataset based on the histopathological response grade with the addition of several microarray profiles and conducted a re‐analysis using bioinformatic web tools including DAVID, GSEA, UALCAN, and CIBERSORTx. We identified 204 genes that were differentially expressed between the highly resistant and sensitive groups. Some of these differentially expressed genes (DEGs) were related to the immune response and showed higher expression in the sensitive group. UALCAN showed that high expression of 28 of the top 50 DEGs was associated with a favorable prognosis (p < 0.25), and that this reached a significant (p < 0.05) level for 18 of them, suggesting that patients with high expression of these genes might have benefited from chemotherapy and thus had a better outcome. In preNAC biopsy tissues from a DCF‐sensitive case, we demonstrated the presence of cells expressing mRNA for CXCL9, one of the prognosis‐related DEGs. Our results highlight the association of immune‐related expression profile in preNAC ESCC with the DCF‐NAC efficacy.

Neoadjuvant chemotherapy (NAC) followed by surgery is a standard approach for management of locally advanced esophageal squamous cell carcinoma (ESCC). Patients who do not respond well to NAC have a poor prognosis. Despite extensive research, the mechanisms of chemoresistance in ESCC remain largely unknown. Here, we established paired tumor organoids – designated as PreNAC-O and PostNAC-O – from one ESCC patient before and after NAC, respectively. Although the two organoids did not exhibit significant differences in proliferation, morphology or drug sensitivity in vitro, the tumorigenicity of PostNAC-O in vivo was significantly higher than that of PreNAC-O. Xenografts from PreNAC-O tended to exhibit keratinization, while those from PostNAC-O displayed conspicuous necrotic areas. The tumorigenicity of PostNAC-O xenografts during the chemotherapy was comparable to that of PreNAC-O without treatment. Furthermore, the gene expression profiles of the xenografts suggested that expression of genes involved in the EMT and/or hypoxia response might be related to the tumorigenicity of PostNAC-O. Our data suggested that the tumorigenicity of residual cancer had been enhanced, outweighing the effects of chemotherapy, rather than being attributable to intrinsic chemoresistance. Further studies are required to clarify the extent to which residual cancers share a common mechanism similar to that revealed here.