佐谷 秀行

Pancreatic cancer is a highly intractable malignancy that necessitates personalized treatment strategies. Conventional patient-derived models, such as three-dimensional organoids, are often limited by intellectual property constraints and high costs. In this study, we developed an affordable adherent culture system for patient-derived pancreatic cancer cells using a proprietary medium and laminin-coated dishes. Primary cultures were successfully established from 28 patients with pancreatic ductal adenocarcinoma, exceeding a 90% success rate. Validation of eight samples confirmed maintenance of epithelial cell adhesion molecule expression and preservation of oncogenic KRAS mutations. Transcriptomic profiling revealed consistent upregulation of a six-gene signature (FAP, IGFBP5, PRRX1, SPARC, WNT5A, and ADAMTS12), which is associated with malignancy. In vitro drug sensitivity assays revealed interpatient heterogeneity with preliminary clinical associations. In conclusion, this simplified platform provides high-purity cancer cells and serves as a functional precision medicine tool. Beyond conventional chemotherapy, this platform has the potential to support applications ranging from biomarker validation and exploratory preclinical testing of novel therapeutics, including immune checkpoint inhibitors and antibody–drug conjugates. This optimization can lead to personalized therapeutic strategies for pancreatic cancer.

Redox regulation is a key mechanism supporting tumor survival and an attractive therapeutic target. In this study, we screened 1161 FDA-approved compounds to identify agents that induce reactive oxygen species (ROS) accumulation in head and neck squamous cell carcinoma (HNSCC) cells. Pimozide, a dopamine D2 receptor antagonist, emerged as the most potent ROS inducer. It selectively suppresses the growth of HNSCC cells with high oxidative stress resistance while exhibiting only modest effects on less resistant cells and normal keratinocytes. Notably, pimozide exhibited anti-tumor effects as a monotherapy and in combination with paclitaxel at clinically relevant doses. Mechanistic analysis revealed that pimozide rapidly induced ROS accumulation via a mechanism distinct from its known action on dopamine D2 receptors and STAT3/5. To identify markers of ROS-induced responses, we examined ROS-responsive genes and found that early growth response 1 (EGR1) was selectively induced in sensitive cells and correlated with pimozide responsiveness. Functional analysis revealed that EGR1 knockdown suppressed pimozide-induced cytotoxicity, suggesting its role as a functional pharmacodynamic marker of pimozide sensitivity. In a patient-derived xenograft model of HNSCC, pimozide significantly reduced the tumor burden alone and in combination with paclitaxel. While tumor volume reduction in the combination group was not statistically greater than that in the monotherapy group, fluorescence immunohistochemistry revealed a marked decrease in undifferentiated tumor cells, indicating enhanced therapeutic effects of combination treatment. Taken together, these findings indicate that pimozide is a promising candidate for repurposing as a novel therapeutic agent against HNSCC.

In the last decade, newly developed drugs have significantly improved the prognosis of patients with multiple myeloma (MM). However, most patients relapse sooner or later, and thus MM remains an incurable hematological malignancy. In addition, serious adverse events occasionally hamper the continuation of treatment. Exploitation of new drugs that potentiate antitumor activities and alleviate the adverse effects of existing drugs is needed. Here, we found through drug repositioning that ambroxol hydrochloride (ambroxol) induces apoptosis of MM cells. Interestingly, turnover and reporter assays revealed that ambroxol inhibits the late stage of autophagy. Transmission electron microscopy observation also revealed that MM cells treated with ambroxol accumulated autophagic vacuoles in the cytoplasm, further supporting the inhibition of late-stage autophagy. Existing anti-MM drugs demonstrate various effects on autophagy; panobinostat, a histone deacetylase inhibitor, induces autophagy, whereas bortezomib and lenalidomide do not. When administered together, ambroxol and panobinostat exhibited a synergistic antimyeloma effect, likely due to ambroxol inhibiting the activation of panobinostat-induced autophagy while downregulating MCL-1 expression. In the KMS11 xenograft model, ambroxol significantly delayed tumor growth when administered alone; when co-administered with panobinostat, ambroxol synergistically enhanced the panobinostat-induced inhibition of tumor growth. Interestingly, concomitant use of ambroxol and panobinostat alleviated panobinostat-induced diarrhea. Gene set enrichment and pathway analyses also revealed that ambroxol increased the expression of genes related to autophagy inhibition and unfolded protein response. These results suggested that autophagy is a promising therapeutic target for MM.

While the fallopian tube epithelium (FTE) is known to be composed of various differentiated cells such as secretory and ciliated cells, the upstream regulatory mechanisms of cell differentiation that are essential for tissue homeostasis remain under investigation. In this study, we established human FTE organoids and identified quiescent cells within the early organoid formation by observing cellular proliferation heterogeneity. We also analyzed two single-cell transcriptomic data to trace the differentiation trajectory in human FTE, and found that the gene LCN2 serves as a marker gene of early stage of the trajectory. Genetically manipulated FTE organoids indicated that LCN2 inhibits ferroptosis and promotes cell survival under oxidative stress. In addition, the FTE organoids introduced p53 dysfunction, the common genetic characteristics of high-grade serous carcinoma, showed upregulated LCN2 expression and enhanced ferroptosis resistance. This study provides insights into the LCN2-mediated protective mechanism of human FTE quiescent cells and its potential role in tumorigenesis.

BACKGROUND: Benzaldehyde (BA) is an aromatic aldehyde found in fruits that has been studied as a potential anticancer agent on the basis of its ability to inhibit transformation in mouse embryo cells and to suppress metastasis in mice. METHODS: We investigated the cytotoxic effects of BA on cancer cells, and probed its effects on intracellular signaling pathways. The anticancer effects of BA in vivo were studied by using a mouse orthotopic transplantation model of pancreatic cancer. RESULTS: BA inhibited the growth of osimertinib- or radiation-resistant cancer cells as well as the interaction between 14-3-3ζ and its client proteins. The interaction of 14-3-3ζ with the Ser28-phosphorylated form of histone H3 (H3S28ph) was implicated in treatment resistance and the transcriptional regulation of genes related to epithelial-mesenchymal transition and stemness, including E2F2, SRSF1, and ID1. Treatment of mice with a BA derivative inhibited pancreatic tumor growth and lung metastasis, as well as suppressed a state of epithelial-mesenchymal plasticity (EMP) of tumor cells. CONCLUSION: The interaction between 14-3-3ζ and H3S28ph plays a key role in EMP and treatment resistance in cancer. The ability of BA to inhibit this and other interactions of 14-3-3ζ offers the potential to overcome treatment resistance and to suppress metastasis.

BACKGROUND AND PURPOSE: Accurate recurrence risk evaluation in patients with stage II and III colorectal cancer (CRC) remains difficult. Traditional histopathological methods frequently fall short in predicting outcomes after adjuvant chemotherapy. This study aims to evaluate the use of comprehensive genomic profiling combined with machine learning for prognostic risk stratification in patients with CRC. METHODS: A machine learning model was developed using a training cohort of 52 patients with stage II/III CRC who underwent curative surgery at Fujita Health University Hospital. Genomic DNA was isolated from formalin-fixed, paraffin-embedded tissue sections and analyzed with a 160 cancer-related gene panel. The random forest algorithm was used to determine key genes affecting recurrence-free survival. The model was validated by developing a risk score with internal and external cohorts, including 44 patients from Keio University Hospital. RESULTS: Six key genes (KRAS, KIT, SMAD4, ARID2, NF1, and FBXW7) were determined as significant prognostic risk predictors. A risk score system integrating these genes with clinicopathological factors effectively stratified patients in both internal (p < 0.001) and external cohorts (p = 0.017). CONCLUSIONS: This study reveals that machine learning, combined with comprehensive genomic profiling, significantly improves prognostic risk stratification in patients with stage II/III CRC after adjuvant chemotherapy. This approach provides a promising tool for individualized treatment strategies, warranting further validation with larger cohorts.

Abstract CD44, a multifunctional cell surface protein, has emerged as a pivotal regulator in cancer stem cell (CSC) biology, orchestrating processes such as stemness, metabolic reprogramming, and therapeutic resistance. Recent studies have identified a critical role of CD44 in ferroptosis resistance by stabilizing SLC7A11 (xCT), a key component of the antioxidant defense system, enabling CSCs to evade oxidative stress and sustain tumorigenic potential. Additionally, CD44 regulates intracellular iron metabolism and redox balance, further supporting CSC survival and adaptation to stressful microenvironments. Therapeutic strategies targeting CD44, including ferroptosis inducers and combination therapies, have shown significant potential in preclinical and early clinical settings. Innovations such as CD44-mediated nanocarriers and metabolic inhibitors present novel opportunities to disrupt CSC-associated resistance mechanisms. Furthermore, the dynamic plasticity of CD44 isoforms governed by transcriptional, post-transcriptional, and epigenetic regulation underscores the importance of context-specific therapeutic approaches. This review highlights the multifaceted roles of CD44 in CSC biology, focusing on its contribution to ferroptosis resistance, iron metabolism, and redox regulation. Targeting CD44 offers a promising avenue for overcoming therapeutic resistance and improving the outcomes of refractory cancers. Future studies are needed to refine these strategies and enable their clinical translation.

Background/Objective: Ovarian cancer (OC) is one of the most lethal gynecological cancers, having a worldwide mortality rate of 66% in 2020. The overall 5-year relative survival rate is only 21% for distant stages, due to the lack of early diagnosis. Epithelial OC, the most common high-grade serous carcinoma, carries p53 mutations in most cases. However, we found that the immediate early response 5 gene (IER5), a p53 target gene, is overexpressed in ovarian cancer cells. The molecular mechanism underlying the role of IER5 in OC has not been well studied. We previously reported that IER5 promotes the dephosphorylation and activation of heat shock factor-1 (HSF1), the master regulator of proteostasis, which induces heat shock protein (HSP) expression. Methods/Results: Here we show that Ier5 mRNA expression is higher in ovarian cancer cells (MOV, ID8G, and HM-1) compared to normal ovarian cells. We also show that OC cells floating in the ascites have higher Ier5 expression than the parental strain. Knockdown of Ier5 suppressed HSP upregulation and proliferation of OC, while overexpression of IER5 promoted HSP upregulation. Knockdown of Hsf1 showed results similar to Ier5 knockdown. Conclusions: These results indicate that the IER5-HSF1 pathway contributes to the proliferation and peritoneal dissemination of OC cells. We also found that higher expression of IER5 family genes is related to poorer prognosis of OC patients, suggesting the potential of the IER5 gene family as diagnostic markers for OC, as well as potential therapeutic targets.

BACKGROUND: Colorectal cancer (CRC) is a substantial global health concern due to its limited treatment options, especially for oxaliplatin (L-OHP) regimen resistance. This study used organoid-based screening methodologies to evaluate drug responses in CRC while validating the approach with patient-derived CRC organoids and investigating potential biomarkers. METHODS: Patient-derived organoids were created from CRC surgical specimens, and drug screening were performed. Selected organoids with high and low L-OHP sensitivity underwent next-generation sequencing (NGS), and in vivo experiments using xenotransplantation were used to validate in vitro results. Moreover, the clinical application of homologous recombination deficiency (HRD) as a biomarker was investigated. RESULTS: Organoid drug screening revealed differences in L-OHP sensitivity among 34 patient-derived CRC organoids, and NGS deemed HRD as a potential biomarker. In vivo experiments validated the correlation between HRD status and L-OHP sensitivity, and clinical data suggested the potential of HRD as a biomarker for recurrence-free survival in patients treated with L-OHP. Additionally, HRD exhibited potential as a biomarker for other platinum agents and poly (ADP-ribose) polymerase inhibitors in CRC. CONCLUSIONS: The study underscores HRD as a potential biomarker for predicting L-OHP sensitivity, expanding its application to other drugs in CRC. Organoid screening is reliable, providing insights into the intricate association between genetic features and treatment responses.

Developmental toxicity testing is essential to identify substances that may harm embryonic development. This study aimed to establish a protocol for evaluating developmental toxicity using human induced pluripotent stem cells (iPSCs) by analyzing cellular activity and gene expression changes. Two ICH S5(R3) positive substances, valproic acid (VPA), which is a substance previously detected as positive by other test methods, and thalidomide (Thalido), were examined during early trichoderm differentiation without fetal bovine serum. RNA-seq analysis identified seven candidate genes, including TP63, associated with altered expression following exposure to VPA or Thalido. These genes were implicated in pathways related to tissue development, cell growth, and molecular interactions. While the assay effectively detected VPA and Thalido, its limitations include testing only soluble substances and focusing on early differentiation stages. Nevertheless, the protocol demonstrates potential for the classification and evaluation of emerging modality drugs based on physical properties such as solubility, polarity, and pH. Integration with AI analysis may enhance its capacity to uncover genetic variations and evaluate previously uncharacterized substances. This study provides a foundation for alternative developmental toxicity testing methods, with further refinements in the culture method expected to improve accuracy and applicability in regulatory toxicology.

Colorectal cancer (CRC) is well characterized in terms of genetic mutations and the mechanisms by which they contribute to carcinogenesis. Mutations in APC, TP53, and KRAS are common in CRC, indicating key roles for these genes in tumor development and progression. However, for certain tumors with low frequencies of these mutations that are defined by tumor location and molecular phenotypes, a carcinogenic mechanism dependent on BRAF mutations has been proposed. We here analyzed targeted sequence data linked to clinical information for CRC, focusing on tumors with a high tumor mutation burden (TMB) in order to identify the characteristics of associated mutations, their relations to clinical features, and the mechanisms of carcinogenesis in tumors lacking the major driver oncogenes. Analysis of overall mutation frequencies confirmed that APC, TP53, and KRAS mutations were the most prevalent in our cohort. Compared with other tumors, TMB-high tumors were more frequent on the right side of the colon, had lower KRAS and higher BRAF mutation frequencies as well as a higher microsatellite instability (MSI) score, and showed a greater contribution of a mutational signature associated with MSI. Ranking of variant allele frequencies to identify genes that play a role early in carcinogenesis suggested that mutations in genes related to the DNA damage response (such as ATM and POLE) and to MSI (such as MSH2 and MSH6) may precede BRAF mutations associated with activation of the serrated pathway in TMB-high tumors. Our results thus indicate that TMB-high tumors suggest that mutations of genes related to mismatch repair and the DNA damage response may contribute to activation of the serrated pathway in CRC.

Effective therapies have yet to be established for pancreatic ductal adenocarcinomas (PDAC) even though it is the most aggressive cancer. In the present study, PDAC was analyzed using novel rat mAbs against membrane proteins in conjunction with flow cytometry and immunohistochemistry. Human epidermal growth receptor (HER)1-4, mesenchymal to epithelial transition factor (MET), sphingosine-1-phospahate receptor 1 (S1PR1), l-type amino acid transporter 1 (LAT1), system x- c transporter (xCT), alanine-serine-cysteine transporter (ASCT2), cationic amino acid transporter 1 (CAT1) and variant CD44 (CD44v) were expressed at high frequencies in both in vitro and in vivo PDAC. Internalization of membrane proteins by mAbs and growth inhibition by toxin-linked mAbs were demonstrated in many PDAC cell lines, and mAbs against S1PR1, ASCT2, HER3 and CD44v inhibited the growth of xenografted MIA PaCa-2 PDAC cells. Furthermore, CD44v-high PDAC showed high mRNA expression of HER1-3, MET and CD44v, and was correlated with poor prognosis. Taken together, our results suggest that CD44v, S1PR1, HER3, MET and the above-mentioned cancer-associated amino acid transporters might be promising targets for the diagnosis and treatment of PDAC.

Cell cycle progression requires periodic gene expression through splicing control. However, the splicing factor that directly controls this cell cycle-dependent splicing remains unknown. Cell cycle-dependent expression of the AURKB (aurora kinase B) gene is essential for chromosome segregation and cytokinesis. We previously reported that RNPS1 is essential to maintain precise splicing in AURKB intron 5. Here we show that RNPS1 plays this role in PSAP complex with PNN and SAP18, but not ASAP complex with ACIN1 and SAP18. Whole-transcriptome sequencing of RNPS1- and PNN-deficient cells indicated that RNPS1, either alone or as PSAP complex, is an essential splicing factor for a subset of introns. Remarkably, protein expression of RNPS1, but not PNN, is coordinated with cyclical splicing in PSAP-controlled introns including AURKB intron 5. The ubiquitin-proteasome pathway is involved in the periodic decrease of RNPS1 protein level. RNPS1 is a key factor that controls periodic splicing during the cell cycle.

The autophagy-lysosome pathway plays an indispensable role in the protein quality control by degrading abnormal organelles and proteins including a-synuclein (aSyn) associated with the pathogenesis of Parkinson's disease (PD). However, the activation of this pathway is mainly by targeting lysosomal enzymic activity. Here, we focused on the autophagosome-lysosome fusion process around the microtubule-organizing center (MTOC) regulated by lysosomal positioning. Through high-throughput chemical screening, we identified 6 out of 1,200 clinically approved drugs enabling the lysosomes to accumulate around the MTOC with autophagy flux enhancement. We further demonstrated that these compounds induce the lysosomal clustering through a JIP4-TRPML1-dependent mechanism. Among them, the lysosomal-clustering compound albendazole promoted the autophagy-dependent degradation of Triton-X-insoluble, proteasome inhibitor-induced aggregates. In a cellular PD model, albendazole boosted insoluble aSyn degradation. Our results revealed that lysosomal clustering can facilitate the breakdown of protein aggregates, suggesting that lysosome-clustering compounds may offer a promising therapeutic strategy against neurodegenerative diseases characterized by the presence of aggregate-prone proteins.

Abstract Refractory high-grade serous carcinoma (HGSC) is one of the most severe clinical problems in gynecology, but we have barely grasped the underlying mechanism of aggressiveness and chemo-resistance. Previous study has shown that homologous recombination genes, BRCA1/2-unmutated HGSC patients possess the potential to be in refractory condition, and their primary response to chemotherapy was heterogenous; thus, there can be subtype-specific determinant factors hatching refractoriness in BRCA1/2-unmutated HGSC patients. To explore the determinant factors, we performed gene set variation analysis and comprehensive log-rank tests on TCGA advanced serous ovarian cancer datasets. As a result, regulation of small GTPase signaling prominently correlated with poor prognosis uniquely in BRCA1/2-unmutated HGSC patients (p-value &amp;lt; 0.001), and eventually, pathway clustering analysis by variational Bayesian Gaussian mixture model discovered aberrant RAS/PI3K crosstalk as peculiar poor prognostic signature of BRCA1/2-unmutated HGSC patients. To elucidate the mechanism of poor progression triggered by RAS/PI3K, we utilized our syngeneic HGSC organoid models newly established from murine fallopian tube epithelium by making Myc overexpressing, and knocking out Trp53 and Rb1; as a supplement, the propriety of these genetic alterations to develop HGSC models was backed up by genetic profiles of high-grade serous ovarian cancer patients in AACR Project GENIE. After that, we knocked out Nf1 and Pten whose deletions were detected relatively with high frequency in HGSC to induce aberrant RAS/PI3K crosstalk in the primitive HGSC model. In-vivo evaluation and growth comparison indicated that aberrant RAS/PI3K crosstalk transformed Brca1/2-unmutated HGSC modeling cells into aggressive phenotypes such as poor survival (p-value &amp;lt; 0.01) and high growth rate (p-value &amp;lt; 0.001). Chemo-sensitivity assay also showed that the genetic manipulation made Brca1/2-unmutated HGSC modeling cells more tolerant to anti-tumor agents like carboplatin (p-value &amp;lt; 0.001), paclitaxel (p-value &amp;lt; 0.01), and olaparib (p-value &amp;lt; 0.01). To investigate biological processes that induce the aggressive and chemo-resistant phenotype, we performed gene set enrichment analysis among our HGSC models. The result showed that aberrant RAS/PI3K crosstalk induces cell cycle acceleration and downregulation of apoptosis in the HGSC cells, which backed up aggressive phenotype and chemo-resistance in our HGSC models. In conclusion, aberrant RAS/PI3K crosstalk transforms Brca1/2-unmutated HGSC modeling cells more aggressive and chemo-resistant, which was well recapitulated by our HGSC modeling organoids. The integration of our computational approach and experimental models will lead us to detect peculiar therapeutic targets against refractory HGSC. Citation Format: Tomohiro Tamura, Kenta Masuda, Shimpei Nagai, Keiyo Imaeda, Juntaro Yamasaki, Eiji Sugihara, Hiroyuki Nobusue, Yuji Otsuki, Rui Yamaguchi, Kazuhiro Sakurada, Wataru Yamagami, Hideyuki Saya, Osamu Nagano. Integration of human omics analysis and new syngeneic tumor organoid models reveals that aberrant RAS/PI3K crosstalk triggers refractoriness in high-grade serous carcinoma [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 1254.

OBJECTIVE: The prognosis of glioblastoma (GBM) correlates with residual tumor volume after surgery. In fluorescence-guided surgery, 5-aminolevulinic acid (ALA) has been used to maximize resection while avoiding neurological morbidity. However, not all tumor cells, particularly glioma stem cells (GSCs), display 5-ALA-mediated protoporphyrin IX (PpIX) fluorescence (5-ALA fluorescence). The authors searched for repositioned drugs that affect mitochondrial functions and energy metabolism, identifying berberine (BBR) as a potential enhancer of 5-ALA fluorescence. In this study, they investigated whether BBR can enhance 5-ALA fluorescence in GSCs and whether BBR can be applied to clinical practice as a 5-ALA fluorescence enhancer. METHODS: The effects of BBR on 5-ALA fluorescence in glioma and GSCs were evaluated by flow cytometry (fluorescence-activated cell sorting [FACS]) analysis. As 5-ALA is metabolized for heme synthesis, the effects of BBR on mRNA expressions of 7 enzymes in the heme-synthesis pathway were analyzed. Enzymes showing significantly higher expression than control in all cells were identified and protein analysis was performed. To examine clinical availability, the detectability and cytotoxicity of BBR in tumor-transplanted mice were analyzed. RESULTS: Fluorescence microscopy revealed much more intense 5-ALA fluorescence in both GSCs and non-stem cells with 5-ALA and BBR than with 5-ALA alone. FACS showed that BBR greatly enhanced 5-ALA fluorescence compared with 5-ALA alone, and enhancement was much higher for GSCs than for glioma cells. Among the 7 enzymes examined, BBR upregulated mRNA expressions of ALA synthetase 1 (ALAS1) more highly in all cells, and activated ALAS1 through deregulating ALAS1 activity inhibited by the negative feedback of heme. An in vivo study showed that 5-ALA fluorescence with 5-ALA and BBR was significantly stronger than with 5-ALA alone, and the sensitivity and specificity of BBR-enhanced fluorescence were both 100%. In addition, BBR did not show any cytotoxicity for normal brain tissue surrounding the tumor mass. CONCLUSIONS: BBR enhanced 5-ALA-mediated PpIX fluorescence by upregulating and activating ALAS1 through deregulation of negative feedback inhibition by heme. BBR is a clinically used drug with no side effects. BBR is expected to significantly augment fluorescence-guided surgery and photodynamic therapy.

BACKGROUND AND PURPOSE: Neoadjuvant chemotherapy (NAC) is a well-established standard practice in invasive bladder cancer (BCa), however patient selection remains challenging. High expression of vasohibin-1 (VASH1), an endogenous regulator of angiogenesis, has been reported in high-grade and advanced BCa; however, its prognostic value for chemotherapy outcomes remains unexplored. In this study, we sought to identify biomarkers of chemotherapy response focusing on the relationship between angiogenesis and tissue hypoxia. METHODS: Forty Japanese patients with BCa who underwent NAC and radical cystectomy were included in the present analysis. We compared the immunohistochemical expression of CD34, VASH1, and carbonic anhydrase 9 (CA9) between patients who achieved tumor clearance at operation (ypT0) and those with residual disease after cystectomy. RESULTS: There were 19 patients in the ypT0 group, while the remaining 21 patients had residual tumors at operation. Patients in the ypT0 group had high microvessel density (p = 0.031), high VASH1 density (p < 0.001), and stronger CA9 staining (p = 0.046) than their counterparts. Multivariate analysis identified microvessel and VASH1 density as independent predictive factors for pathological ypT0 disease (p = 0.043 and 0.002, respectively). The 5-year recurrence-free survival rate was higher in the high VASH1 density group than in the low VASH1 density group (66.3% vs. 33.3%, p = 0.036). CONCLUSION: VASH1 density is a potential therapeutic biomarker for chemotherapy response in BCa.

Malignant ascites accompanied by peritoneal dissemination contain various factors and cell populations as well as cancer cells; however, how the tumor microenvironment is shaped in ascites remains unclear. Single-cell proteomic profiling and a comprehensive proteomic analysis are conducted to comprehensively characterize malignant ascites. Here, we find defects in immune effectors along with immunosuppressive cell accumulation in ascites of patients with gastric cancer (GC) and identify five distinct subpopulations of CD45(-)/EpCAM(-) cells. Mesothelial cells with mesenchymal features in CD45(-)/EpCAM(-) cells are the predominant source of chemokines involved in immunosuppressive myeloid cell (IMC) recruitment. Moreover, mesothelial-mesenchymal transition (MMT)-induced mesothelial cells strongly express extracellular matrix (ECM)-related genes, including tenascin-C (TNC), enhancing metastatic colonization. These findings highlight the definite roles of the mesenchymal cell population in the development of a protumorigenic microenvironment to promote peritoneal dissemination.

BACKGROUND: Extramammary Paget's disease (EMPD) is a rare intraepithelial adenocarcinoma that mainly affects the anogenital and axillary regions. Although its etiology has not been fully elucidated, there is evidence that androgen receptors (AR) are expressed in most cases of EMPD. However, the role of androgen signaling in the pathogenesis of EMPD remains unclear. OBJECTIVE: To evaluate the role of androgen signaling in tumor growth of AR-positive EMPD. METHODS: Patient-derived organoids were established and cultured from two AR-positive EMPD patients: one man and one woman. Cultured organoids were treated with androgen agonists and/or antagonists, then subjected to analysis of changes in organoid proliferation, as well as changes in androgen signaling pathway-specific genes. RESULTS: Organoid cultures were established from each EMPD sample. These organoids were immunohistologically and genetically identical to the original tumor. For each organoid sample, viable cell number increased in response to androgen exposure. The mRNA level of Fkbp5, a known AR target gene, increased in a concentration-dependent manner in organoids exposed to the synthetic androgen R1881. Conversely, the AR inhibitor darolutamide suppressed the viable cell number in a concentration-dependent manner. The mRNA expression levels of MKI67 and Fkbp5 were also suppressed by darolutamide. CONCLUSION: Our results indicate that androgen signaling is a key pathway involved in the growth of AR-positive EMPD. Therefore, androgen signaling inhibition may be a novel treatment option for EMPD patients who require systemic therapy.

The in vitro growth transformation of primary B cells by Epstein-Barr virus (EBV) is the initial step in the development of posttransplant lymphoproliferative disorder (PTLD). We performed electron microscopic analysis and immunostaining of primary B cells infected with wild-type EBV. Interestingly, the nucleolar size was increased by two days after infection. A recent study found that nucleolar hypertrophy, which is caused by the induction of the IMPDH2 gene, is required for the efficient promotion of growth in cancers. In the present study, RNA-seq revealed that the IMPDH2 gene was significantly induced by EBV and that its level peaked at day 2. Even without EBV infection, the activation of primary B cells by the CD40 ligand and interleukin-4 increased IMPDH2 expression and nucleolar hypertrophy. Using EBNA2 or LMP1 knockout viruses, we found that EBNA2 and MYC, but not LMP1, induced the IMPDH2 gene during primary infections. IMPDH2 inhibition by mycophenolic acid (MPA) blocked the growth transformation of primary B cells by EBV, leading to smaller nucleoli, nuclei, and cells. Mycophenolate mofetil (MMF), which is a prodrug of MPA that is approved for use as an immunosuppressant, was tested in a mouse xenograft model. Oral MMF significantly improved the survival of mice and reduced splenomegaly. Taken together, these results indicate that EBV induces IMPDH2 expression through EBNA2-dependent and MYC-dependent mechanisms, leading to the hypertrophy of the nucleoli, nuclei, and cells as well as efficient cell proliferation. Our results provide basic evidence that IMPDH2 induction and nucleolar enlargement are crucial for B cell transformation by EBV. In addition, the use of MMF suppresses PTLD. IMPORTANCE EBV infections cause nucleolar enlargement via the induction of IMPDH2, which are essential for B cell growth transformation by EBV. Although the significance of IMPDH2 induction and nuclear hypertrophy in the tumorigenesis of glioblastoma has been reported, EBV infection brings about the change quickly by using its transcriptional cofactor, EBNA2, and MYC. Moreover, we present here, for the novel, basic evidence that an IMPDH2 inhibitor, namely, MPA or MMF, can be used for EBV-positive posttransplant lymphoproliferative disorder (PTLD).

Boron neutron capture therapy (BNCT) is a high-LET particle radiotherapy clinically tested for treating malignant gliomas. Boronophenylalanine (BPA), a boron-containing phenylalanine derivative, is selectively transported into tumor cells by amino acid transporters, making it an ideal agent for BNCT. In this study, we investigated whether the amino acid 5-aminolevulinic acid (ALA) could sensitize glioma stem cells (GSCs) to BNCT by enhancing the uptake of BPA. Using human and mouse GSC lines, pre-incubation with ALA increased the intracellular accumulation of BPA dose-dependent. We also conducted in vivo experiments by intracerebrally implanting HGG13 cells in mice and administering ALA orally 24 h before BPA administration (ALA + BPA-BNCT). The ALA preloading group increased the tumor boron concentration and improved the tumor/blood boron concentration ratio, resulting in improved survival compared to the BPA-BNCT group. Furthermore, we found that the expression of amino acid transporters was upregulated following ALA treatment both in vitro and in vivo, particularly for ATB0,+. This suggests that ALA may sensitize GSCs to BNCT by upregulating the expression of amino acid transporters, thereby enhancing the uptake of BPA and improving the effectiveness of BNCT. These findings have important implications for strategies to improve the sensitivity of malignant gliomas to BPA-BNCT.

The tumor microenvironment (TME) plays a key role in cancer development and progression, as well as contributes to the therapeutic resistance and metastasis of cancer cells. The TME is heterogeneous and consists of multiple cell types, including cancer-associated fibroblasts (CAFs), endothelial cells, and immune cells, as well as various extracellular components. Recent studies have revealed cross talk between cancer cells and CAFs as well as between CAFs and other TME cells, including immune cells. Signaling by transforming growth factor-β, derived from CAFs, has recently been shown to induce remodeling of tumor tissue, including the promotion of angiogenesis and immune cell recruitment. Immunocompetent mouse cancer models that recapitulate interactions of cancer cells with the TME have provided insight into the TME network and support the development of new anticancer therapeutic strategies. Recent studies based on such models have revealed that the antitumor action of molecularly targeted agents is mediated in part by effects on the tumor immune environment. In this review, we focus on cancer cell–TME interactions in heterogeneous tumor tissue, and we provide an overview of the basis for anticancer therapeutic strategies that target the TME, including immunotherapy.

BACKGROUND: The significance of BRCA alterations has been implicated in the development of metastatic castration-resistant prostate cancer (PC). The details of the frequency and significance of BRCA alterations in localized PC remain unknown. In this study, we investigated the frequency and clinical significance of BRCA alterations in localized PCs using an in-house next-generation sequencer (NGS) system. METHODS: DNA was extracted from formalin-fixed paraffin-embedded tissues of surgical specimens from 126 patients with clinically localized PC who underwent radical prostatectomy. The mutation information of 164 cancer genes was analyzed using the PleSSision-Rapid test. Both copy number (CN) variation and loss of heterozygosity of various genes, such as BRCA1 and BRCA2, were estimated and reported. RESULTS: Next-generation sequencer analyses revealed that the BRCA2 CN was decreased in 17 patients (13.5%) and the BRCA1 CN in six (4.8%) patients. NGS-based CN values were shown to be highly correlated with droplet digital PCR-based CN values. Tissue-specific BRCA expression investigated using the Human Protein Atlas showed that the decreased CN of BRCA2, but not BRCA1, is responsible for the decreased BRCA activity in PC. Ten of the 22 patients with decreased BRCA2 CN were presumed to have somatic heterozygous deletion. There were no observed associations between the heterozygous deletion of BRCA2 and various clinicopathological parameters. Furthermore, three of 10 patients developed biochemical recurrence within 3 months after surgery. Multivariate analyses revealed that the initial prostate-specific antigen levels and BRCA2 CN were independent factors for biochemical recurrence. CONCLUSION: Our results suggest that a decrease in BRCA2 CN may be used as a biomarker for predicting recurrence after surgery in localized PC. Early screening for somatic alterations in BRCA2 using NGS may help to broadly predict the risk of PC progression.

Drug resistance contributes to poor therapeutic response in urothelial carcinoma (UC). Metabolomic analysis suggested metabolic reprogramming in gemcitabine-resistant urothelial carcinoma cells, whereby increased aerobic glycolysis and metabolic stimulation of the pentose phosphate pathway (PPP) promoted pyrimidine biosynthesis to increase the production of the gemcitabine competitor deoxycytidine triphosphate (dCTP) that diminishes its therapeutic effect. Furthermore, we observed that gain-of-function of isocitrate dehydrogenase 2 (IDH2) induced reductive glutamine metabolism to stabilize Hif-1α expression and consequently stimulate aerobic glycolysis and PPP bypass in gemcitabine-resistant UC cells. Interestingly, IDH2-mediated metabolic reprogramming also caused cross resistance to CDDP, by elevating the antioxidant defense via increased NADPH and glutathione production. Downregulation or pharmacological suppression of IDH2 restored chemosensitivity. Since the expression of key metabolic enzymes, such as TIGAR, TKT, and CTPS1, were affected by IDH2-mediated metabolic reprogramming and related to poor prognosis in patients, IDH2 might become a new therapeutic target for restoring chemosensitivity in chemo-resistant urothelial carcinoma.

Excess stroma and cancer-associated fibroblasts (CAFs) enhance cancer progression and faciliate immune evasion. Insights into the mechanisms by which the stroma manipulates the immune microenvironment could help improve cancer treatment. Here, we aimed to elucidate potential approaches for stromal reprogramming and improved cancer immunotherapy. Platelet-derived growth factor C (PDGFC) and D expression were significantly associated with a poor prognosis in patients with gastric cancer (GC), and PDGF receptor beta (PDGFRβ) was predominantly expressed in diffuse-type GC stroma. CAFs stimulated with PDGFs exhibited markedly increased expression of CXCL1, CXCL3, CXCL5 and CXCL8, which are involved in polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) recruitment. Fibrotic GC xenograft tumors exhibited increased PMN-MDSC accumulation and decreased lymphocyte infiltration, as well as resistance to anti-PD-1. Single-cell RNA sequencing and spatial transcriptomics revealed that PDGFRα/β blockade reversed the immunosuppressive microenvironment through stromal modification. Finally, combining PDGFRα/β blockade and anti-PD-1 treatment synergistically suppressed the growth of fibrotic tumors. These findings highlight the impact of stromal reprogramming on immune reactivation and the potential for combined immunotherapy for patients with fibrotic cancer.

BACKGROUND: The coronavirus disease 2019 (COVID-19) pandemic has rapidly and dramatically influenced healthcare across Japan. However, the influence of the COVID-19 pandemic on the number of newly diagnosed cancer, surgical treatment, and diagnostic examination for cancer types have not been completely investigated all over Japan. This study aimed to analyze the number of cases before and during the COVID-19 pandemic. METHODS: This retrospective study was a survey that asked to provide the number of cases diagnosed with gastric, colorectal, lung, breast, and cervical cancer between January 2019 and December 2020. The survey was sent to tertiary healthcare hospitals, including national cancer institutions, university hospitals, and general hospitals, all over Japan. Data obtained from 105 of 486 surveyed hospitals were evaluated, and the number of cases in each quarter in 2020 was compared with that in the equivalent quarter in 2019. RESULTS: In the second quarter (Q2), significant reductions were observed in the median number of newly diagnosed cases from 2019 to 2020: gastric cancer, 26.7% (43 vs. 32, p <  0.001); colorectal cancer, 17.9% (52 vs. 40, p <  0.001); lung cancer, 12.3% (53.5 vs. 47, p <  0.001); and breast cancer, 13.1% (43 vs. 35.5, p <  0.001). A significant reduction of 11.4% (9 vs. 8, p = 0.03) was observed in the third quarter (Q3) for cervical cancer. In Q2, the number of cases decreased by 30.9% (25 vs. 15, p <  0.001) for stage I gastric cancer, by 27.3% (12 vs. 9, p <  0.001) for stage I colorectal cancer, and by 17.6% (13 vs. 10, p <  0.001) for stage II breast cancer. The magnitude of reduction was significant for the localized stages of gastric, colorectal, and breast cancer according to diagnostic examinations in Q2 and surgical and endoscopic treatment in Q3 rather than that for lung or cervical cancer. CONCLUSIONS: COVID-19 has prolonged collateral effects on cancer care, including examination, diagnosis, and surgery, with significant effects on gastric cancer, followed by colorectal, lung, breast, and cervical cancer in Japan.

We previously established mouse models of biliary tract cancer (BTC) based on the injection of cells with biliary epithelial stem cell properties derived from KRAS(G12V)-expressing organoids into syngeneic mice. The resulting mouse tumors appeared to recapitulate the pathological features of human BTC. Here we analyzed images of hematoxylin and eosin (H&E) staining for both the mouse tumor tissue and human cholangiocarcinoma tissue by pixel-level clustering with machine learning. A pixel-clustering model that was established via training with mouse images revealed homologies of tissue structure between the mouse and human tumors, suggesting similarities in tumor characteristics independent of animal species. Analysis of the human cholangiocarcinoma tissue samples with the model also revealed that the entropy distribution of cancer regions was higher than that of noncancer regions, with the entropy of pixels thus allowing discrimination between these two types of regions. Histograms of entropy tended to be broader for noncancer regions of late-stage human cholangiocarcinoma. These analyses indicate that our mouse BTC models are appropriate for investigation of BTC carcinogenesis and may support the development of new therapeutic strategies. In addition, our pixel-level clustering model is highly versatile and may contribute to the development of a new BTC diagnostic tool.

Although the immunological memory produced by BNT162b2 vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been well studied and established, further information using different racial cohorts is necessary to understand the overall immunological response to vaccination. We evaluated memory B and T cell responses to the severe acute respiratory syndrome coronavirus 2 spike protein before and after the third booster using a Japanese cohort. Although the Ab titer against the spike receptor-binding domain (RBD) decreased significantly 8 mo after the second vaccination, the number of memory B cells continued to increase, whereas the number of memory T cells decreased slowly. Memory B and T cells from unvaccinated infected patients showed similar kinetics. After the third vaccination, the Ab titer increased to the level of the second vaccination, and memory B cells increased at significantly higher levels before the booster, whereas memory T cells recovered close to the second vaccination levels. In memory T cells, the frequency of CXCR5+CXCR3+CCR6- circulating follicular Th1 was positively correlated with RBD-specific Ab-secreting B cells. For the response to variant RBDs, although 60-80% of memory B cells could bind to the omicron RBD, their avidity was low, whereas memory T cells show an equal response to the omicron spike. Thus, the persistent presence of memory B and T cells will quickly upregulate Ab production and T cell responses after omicron strain infection, which prevents severe illness and death due to coronavirus disease 2019.

Novel therapeutic targets are needed to better treat osteosarcoma, which is the most common bone malignancy. We previously developed mouse osteosarcoma cells, designated AX (accelerated bone formation) cells from bone marrow stromal cells. AX cells harbor both wild-type and mutant forms of p53 (R270C in the DNA-binding domain, which is equivalent to human R273C). In this study, we showed that mutant p53 did not suppress the transcriptional activation function of wild-type p53 in AX cells. Notably, AXT cells, which are cells derived from tumors originating from AX cells, lost wild-type p53 expression, were devoid of the intact transcription activation function, and were resistant to doxorubicin. ChIP-seq analyses revealed that this mutant form of p53 bound to chromatin in the vicinity of the transcription start sites of various genes but exhibited a different binding profile from wild-type p53. The knockout of mutant p53 in AX and AXT cells by CRISPR-Cas9 attenuated tumor growth but did not affect the invasion of these cells. In addition, depletion of mutant p53 did not prevent metastasis in vivo. Therefore, the therapeutic potency targeting R270C (equivalent to human R273C) mutant p53 is limited in osteosarcoma. However, considering the heterogeneous nature of osteosarcoma, it is important to further evaluate the biological and clinical significance of mutant p53 in various cases.

Memory T cell responses have been analyzed only in small cohorts of COVID-19 vaccines. Herein, we aimed to assess anti-SARS-CoV-2 cellular immunity in a large cohort using QuantiFERON assays, which are IFN-γ release assays (IGRAs) based on short-term whole blood culture. The study included 571 individuals receiving the viral spike (S) protein-expressing BNT162b2 mRNA vaccine. QuantiFERON assays revealed antigen-specific IFN-γ production in most individuals 8 weeks after the second dose. Simultaneous flow cytometric assays to detect T cells expressing activation-induced markers (AIMs) performed for 28 randomly selected individuals provided data correlating with the QuantiFERON data. Simultaneous IFN-γ enzyme-linked immunospot and AIM assays for another subset of 31 individuals, based on short-term peripheral blood mononuclear cell culture, also indicated a correlation between IFN-γ production and AIM positivity. These observations indicated the acquisition of T cell memory responses and supported the usability of IGRAs to assess cellular immunity. The QuantiFERON results were weakly correlated with serum IgG titers against the receptor-binding domain of the S protein and were associated with pre-vaccination infection and adverse reactions after the second dose. The present study revealed cellular immunity after COVID-19 vaccination, providing insights into the effects and adverse reactions of vaccination.

Although the immunological memory produced by BNT162b2 vaccination against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been well studied and established, further information using different racial cohorts is necessary to understand the overall immunological response to vaccination. We evaluated memory B and T cell responses to the severe acute respiratory syndrome coronavirus 2 spike protein before and after the third booster using a Japanese cohort. Although the Ab titer against the spike receptor-binding domain (RBD) decreased significantly 8 mo after the second vaccination, the number of memory B cells continued to increase, whereas the number of memory T cells decreased slowly. Memory B and T cells from unvaccinated infected patients showed similar kinetics. After the third vaccination, the Ab titer increased to the level of the second vaccination, and memory B cells increased at significantly higher levels before the booster, whereas memory T cells recovered close to the second vaccination levels. In memory T cells, the frequency of CXCR5+CXCR3+CCR6- circulating follicular Th1 was positively correlated with RBD-specific Ab-secreting B cells. For the response to variant RBDs, although 60-80% of memory B cells could bind to the omicron RBD, their avidity was low, whereas memory T cells show an equal response to the omicron spike. Thus, the persistent presence of memory B and T cells will quickly upregulate Ab production and T cell responses after omicron strain infection, which prevents severe illness and death due to coronavirus disease 2019.

Peritoneal dissemination of ovarian cancer (OC) correlates with poor prognosis, but the mechanisms underlying the escape of OC cells from the intraperitoneal immune system have remained unknown. We here identify pigment epithelium-derived factor (PEDF) as a promoting factor of OC dissemination, which functions through induction of CD206+ Interleukin-10 (IL-10)-producing macrophages. High PEDF gene expression in tumors is associated with poor prognosis in OC patients. Concentrations of PEDF in ascites and serum are significantly higher in OC patients than those with more benign tumors and correlated with early recurrence of OC patients, suggesting that PEDF might serve as a prognostic biomarker. Bromodomain and extraterminal (BET) inhibitors reduce PEDF expression and limit both OC cell survival and CD206+ macrophage induction in the peritoneal cavity. Our results thus implicate PEDF as a driver of OC dissemination and identify a BET protein-PEDF-IL-10 axis as a promising therapeutic target for OC.