GPR31-dependent dendrite protrusion of intestinal CX3CR1+ cells by bacterial metabolites.

Small intestinal mononuclear cells that express CX3CR1 (CX3CR1+ cells) regulate immune responses1-5. CX3CR1+ cells take up luminal antigens by protruding their dendrites into the lumen1-4,6. However, it remains unclear how dendrite protrusion by CX3CR1+ cells is induced in the intestine. Here we show in mice that the bacterial metabolites pyruvic acid and lactic acid induce dendrite protrusion via GPR31 in CX3CR1+ cells. Mice that lack GPR31, which was highly and selectively expressed in intestinal CX3CR1+ cells, showed defective dendrite protrusions of CX3CR1+ cells in the small intestine. A methanol-soluble fraction of the small intestinal contents of specific-pathogen-free mice, but not germ-free mice, induced dendrite extension of intestinal CX3CR1+ cells in vitro. We purified a GPR31-activating fraction, and identified lactic acid. Both lactic acid and pyruvic acid induced dendrite extension of CX3CR1+ cells of wild-type mice, but not of Gpr31b-/- mice. Oral administration of lactate and pyruvate enhanced dendrite protrusion of CX3CR1+ cells in the small intestine of wild-type mice, but not in that of Gpr31b-/- mice. Furthermore, wild-type mice treated with lactate or pyruvate showed an enhanced immune response and high resistance to intestinal Salmonella infection. These findings demonstrate that lactate and pyruvate, which are produced in the intestinal lumen in a bacteria-dependent manner, contribute to enhanced immune responses by inducing GPR31-mediated dendrite protrusion of intestinal CX3CR1+ cells.

Induction of lung lesions by bronchial administration using bronchoscope technique in mice.

This study aimed to establish an exposure method that can induce homogeneous lesions with minimal inter-individual variability. The distribution of lesions induced by bleomycin (BLM) administration was also analyzed. C57BL mice were intrabronchially administered 20 µL of BLM (3 mg/mL) using a bronchoscope in the left or right bronchus. The mice were sacrificed 14 days after administration, and their lungs were evaluated histopathologically. BLM-induced inflammatory lesions were widely observed in the lungs. In the left bronchus-treated group, lesions were uniformly observed throughout the lobe, and no individual differences were noted. Meanwhile, in the right bronchus-treated group, individual differences in the distribution of the pulmonary lesions were observed. The distribution of lesions differed among the four lobes of the right lung owing to their anatomical features. Administration into the left bronchus is recommended for highly homogeneous lung exposure and for establishing models that contribute to highly accurate toxicity and efficacy evaluations.

Feeding a High-Fat Diet for a Limited Duration Increases Cancer Incidence in a Breast Cancer Model.

High-fat intake by young Asian women impacts the risk of breast cancer. Understanding the underlying molecular mechanisms may be essential for disease prevention in Asia as well as globally. We aimed to examine the effects of corn oil- and animal fat-based high-fat diets (32.9 and 31.4%, respectively, of fat energy ratio as compared to 12.3% in the standard diet) on mammary carcinogenesis and alterations in gene expression and epigenetic statuses in the mammary gland during the growth stages in a rat model. An increased incidence of carcinomas was observed after the cessation of high-fat feeding. In addition, rapid tumor growth and elevations in Celsr2 expression, which may be a result of DNA hypomethylation patterns in the 3' untranslated region of the gene were noted in the animal fat group. In the human breast carcinoma cell line MCF7, a marginal decrease in cell viability was observed following the knockdown of Celsr2, suggesting that the animal fat-associated risk of cancer is partly due to the deregulation of mammary cell proliferation via non-metabolic gene functions. The present results will contribute to the development of strategies for controlling the food-associated risk of breast cancer, particularly in younger age groups.

Long-term evaluation of E6011, an anti-fractalkine monoclonal antibody, in patients with rheumatoid arthritis inadequately responding to biological disease-modifying antirheumatic drugs.

OBJECTIVES: The objective of the study is to evaluate the long-term safety and efficacy of E6011, a humanized anti-fractalkine monoclonal antibody, in patients with rheumatoid arthritis with an inadequate response to biological disease-modifying antirheumatic drugs. METHODS: In the double-blind treatment phase (24 weeks), placebo or E6011 400 mg was administered until Week 10. Thereafter, E6011 200 mg or 400 mg was administered to Week 22. Subjects who completed the evaluation at Week 24 of the treatment phase were rolled over into the extension phase. The extension phase lasted until Week 104, and all subjects received E6011 400 mg or 200 mg every 2 weeks in an open-label manner until Week 102. RESULTS: A total of 47 subjects completed the double-blind treatment phase and were rolled over into the extension phase. In total, 46 (97.9%) subjects experienced any adverse events, and the incidence of treatment-related adverse events was 57.4%. No clear efficacy trend in the American College of Rheumatology 20% response rates was observed. CONCLUSIONS: E6011 was well tolerated in active rheumatoid arthritis patients who had shown an inadequate response to biologic disease-modifying antirheumatic drugs, but no clear benefit in the American College of Rheumatology 20% response rates was observed. Further studies are needed to clarify the clinical benefit of E6011.

Long-term safety and efficacy of E6011, an anti-fractalkine monoclonal antibody, in patients with rheumatoid arthritis inadequately responding to methotrexate.

OBJECTIVES: To evaluate the long-term safety and efficacy of E6011, a humanized anti-fractalkine monoclonal antibody, in patients with rheumatoid arthritis (RA) with an inadequate response to methotrexate (MTX). METHODS: Active RA patients with an inadequate response to MTX were randomly assigned to the E6011 or placebo group and received the study drug subcutaneously every 2 weeks during a 24-week double-blind study period. Subjects who completed evaluations at Week 24 were rolled over into the extension phase and received open-label E6011 (200 or 400 mg) every 2 weeks until Week 102. The safety analysis was conducted up to Week 104, and the efficacy analysis was conducted up to Week 84. RESULTS: A total of 169 subjects completed the double-blind treatment phase and were rolled over into the extension phase. In total, 167 (98.8%) subjects experienced any adverse events, and the incidence of treatment-related adverse events was 56.2%. The American College of Rheumatology 20 response rates were observed between 40 and 70% during the extension phase. CONCLUSIONS: E6011 was safe and well tolerated with no notable safety concerns up to 102 weeks in RA patients with an inadequate response to MTX.

Role of Fractalkine-CX3CR1 Axis in Acute Rejection of Mouse Heart Allografts Subjected to Ischemia Reperfusion Injury.

Transplantation outcomes are affected by the increase in rejection associated with ischemia reperfusion injury (IRI). Fractalkine (FKN), a chemokine for recruitment of CX3CR1+ leukocytes, contributes to the pathogenesis of various inflammatory diseases. Herein, we evaluated the importance of the FKN-CX3CR1 axis during IRI-related rejections using a mouse heterotopic heart transplantation model. FKN expression and graft survival was compared between wild-type C57BL/6 recipients transplanted with BALB/c hearts preserved for 8 (WT-IRI) and 0.5 h (WT-control) at 4°C. Graft survival of WT-IRI was shorter than that of WT-control. FKN was expressed on the vascular endothelium in WT-IRI allografts, but minimally in WT-control. The role of the FKN-CX3CR1 axis in IRI-related rejection was directly investigated using the transplant model with CX3CR1-deficient recipients (CX3CR1 KO-IRI) or treatment with anti-mouse FKN monoclonal antibodies. Graft survival of CX3CR1 KO-IRI was longer than that of WT-IRI; antibody treatment prolonged graft survival. The contribution of CX3CR1+ monocytes to IRI-related rejection was evaluated by adoptive transfer to CX3CR1 KO-IRI. Adoptive transfer of CX3CR1+ monocytes attenuated the effect of prolonged graft survival in CX3CR1 KO-IRI. Overall, the FKN-CX3CR1 axis plays a major role during IRI-related rejection; its blockade has the potential to improve the outcomes of deceased donor transplantation.

Single-cell DNA and RNA sequencing reveals the dynamics of intra-tumor heterogeneity in a colorectal cancer model.

BACKGROUND: Intra-tumor heterogeneity (ITH) encompasses cellular differences in tumors and is related to clinical outcomes such as drug resistance. However, little is known about the dynamics of ITH, owing to the lack of time-series analysis at the single-cell level. Mouse models that recapitulate cancer development are useful for controlled serial time sampling. RESULTS: We performed single-cell exome and transcriptome sequencing of 200 cells to investigate how ITH is generated in a mouse colorectal cancer model. In the model, a single normal intestinal cell is grown into organoids that mimic the intestinal crypt structure. Upon RNAi-mediated downregulation of a tumor suppressor gene APC, the transduced organoids were serially transplanted into mice to allow exposure to in vivo microenvironments, which play relevant roles in cancer development. The ITH of the transcriptome increased after the transplantation, while that of the exome decreased. Mutations generated during organoid culture did not greatly change at the bulk-cell level upon the transplantation. The RNA ITH increase was due to the emergence of new transcriptional subpopulations. In contrast to the initial cells expressing mesenchymal-marker genes, new subpopulations repressed these genes after the transplantation. Analyses of colorectal cancer data from The Cancer Genome Atlas revealed a high proportion of metastatic cases in human subjects with expression patterns similar to the new cell subpopulations in mouse. These results suggest that the birth of transcriptional subpopulations may be a key for adaptation to drastic micro-environmental changes when cancer cells have sufficient genetic alterations at later tumor stages. CONCLUSIONS: This study revealed an evolutionary dynamics of single-cell RNA and DNA heterogeneity in tumor progression, giving insights into the mesenchymal-epithelial transformation of tumor cells at metastasis in colorectal cancer.

The potential of organoids in toxicologic pathology: Histopathological and immunohistochemical evaluation of a mouse normal tissue-derived organoid-based carcinogenesis model.

Recently, we introduced an organoid-based chemical carcinogenesis model using mouse normal tissue-derived organoids. In the present review article, the histopathological and immunohistochemical characteristics of mouse normal tissue-derived organoids and tumors derived from these organoids after their in vitro treatment with genotoxic carcinogens and injection into nude mouse are reviewed. In organoids treated in vitro with genotoxic carcinogens, we confirmed macroscopic tumorigenicity and histopathological findings, including neoplastic characteristics, such as multilayered epithelia and/or invasion of epithelia into the surrounding interstitium. In contrast glandular/cystic structures with monolayered epithelia were clearly demarcated from the surrounding Matrigel/interstitium in the untreated control groups. In addition to macroscopic tumorigenicity, these microscopic epithelial changes, which are characteristic of the early stages of carcinogenesis, are included in the requirements for carcinogenicity-positive judgement of the organoid-based carcinogenesis model. Immunohistochemistry of cytokeratins (CKs), used to determine the origin of epithelia and distribution of extraductal invasive lesions, or oncogenic kinases, which reflect molecular activation in epithelia following chemical treatment, is helpful for accurate diagnosis and molecular evaluation in the early stages of carcinogenesis. This information improves our biological understanding of organoid-based chemical carcinogenesis models.

Characterization of the large-scale Japanese patient-derived xenograft (J-PDX) library.

The use of patient-derived xenografts (PDXs) has recently attracted attention as a drug discovery platform with a high predictive clinical efficacy and a preserved tumor heterogeneity. Given the racial differences in genetic variations, it would be desirable to establish a PDX library from Japanese cancer patients on a large scale. We thus tried to construct the Japanese PDX (J-PDX) library with a detailed clinical information for further clinical utilization. Between August 2018 and May 2020, a total of 1126 cancer specimens from 1079 patients were obtained at the National Cancer Center Hospital and National Cancer Center Hospital East, Japan, and were immediately transplanted to immunodeficient mice at the National Cancer Center Research Institute. A total of 298 cross-cancer PDXs were successfully established. The time to engraftment varied greatly by cancer subtypes, especially in the first passage. The engraftment rate was strongly affected by the clinical stage and survival time of the original patients. Approximately 1 year was needed from tumor collection to the time when coclinical trials were conducted to test the clinical utility. The 1-year survival rates of the patients who were involved in establishing the PDX differed significantly, from 95.6% for colorectal cancer to 56.3% for lung cancer. The J-PDX library consisting of a wide range of cancer subtypes has been successfully established as a platform for drug discovery and development in Japan. When conducting coclinical trials, it is necessary to consider the target cancer type, stage, and engraftment rate in light of this report.

Phase 1 study on the safety and efficacy of E6011, antifractalkine antibody, in patients with Crohn's disease.

BACKGROUND AND AIM: E6011 is a humanized monoclonal antibody targeting fractalkine (FKN), a CX3C chemokine, which regulates leukocyte trafficking during inflammation. We evaluated the safety and pharmacokinetic profile of E6011 in patients with Crohn's disease (CD) and also performed preliminary pharmacodynamic (PD) and efficacy assessments. METHODS: This study included a 12-week multiple ascending dose (MAD) phase (2, 5, 10, and 15 mg/kg intravenously every 2 weeks, n = 6, 8, 7, and 7, respectively) and a 40-week Extension phase (n = 12) at the same dose as the MAD phase. Serum E6011, serum total FKN (free soluble FKN and E6011-FKN complex) as a PD marker and CD activity index were evaluated. The primary outcome was safety assessment in the MAD phase. RESULTS: Twenty-seven (96%) of 28 patients had previously been treated with anti-tumor necrosis factor α agents. During the MAD phase, adverse events (AEs) occurred in 18 (64%). The most common AE was nasopharyngitis (five patients, 18%). No severe AEs occurred. Serious AEs occurred in three patients, progression of CD in two, and anemia in one. Serum E6011 concentrations increased dose-dependently after infusion and reached a plateau around 4-6 weeks. Serum total FKN rose simultaneously. Five (18%) patients developed anti-E6011 antibodies during the study. Overall, clinical response and clinical remission were observed at Week 12 in 40% (10/25) and 16% (4/25) of active CD patients, respectively. CONCLUSION: E6011 was well-tolerated and might be effective in CD patients. These findings need to be clarified in a randomized controlled study.

Serum APOA4 Pharmacodynamically Represents Administered Recombinant Human Hepatocyte Growth Factor (E3112).

BACKGROUND: Hepatocyte growth factor (HGF) is an endogenously induced bioactive molecule that has strong anti-apoptotic and tissue repair activities. In this research, we identified APOA4 as a novel pharmacodynamic (PD) marker of the recombinant human HGF (rh-HGF), E3112. METHODS: rh-HGF was administered to mice, and their livers were investigated for the PD marker. Candidates were identified from soluble proteins and validated by using human hepatocytes in vitro and an animal disease model in vivo, in which its c-Met dependency was also ensured. RESULTS: Among the genes induced or highly enhanced after rh-HGF exposure in vivo, a soluble apolipoprotein, Apoa4, was found to be induced by rh-HGF in the murine liver. By using primary cultured human hepatocytes, the significant induction of human APOA4 was observed at the mRNA and protein levels, and it was inhibited in the presence of a c-Met inhibitor. Although mice constitutively expressed Apoa4 mRNA in the small intestine and the liver, the liver was the primary organ affected by administered rh-HGF to strongly induce APOA4 in a dose- and c-Met-dependent manner. Serum APOA4 levels were increased after rh-HGF administration, not only in normal mice but also in anti-Fas-induced murine acute liver failure (ALF), which confirmed the pharmacodynamic nature of APOA4. CONCLUSIONS: APOA4 was identified as a soluble PD marker of rh-HGF with c-Met dependency. It should be worthwhile to clinically validate its utility through clinical trials with healthy subjects and ALF patients.

A phase 2 study of E6011, an anti-Fractalkine monoclonal antibody, in patients with rheumatoid arthritis inadequately responding to biological disease-modifying antirheumatic drugs.

OBJECTIVES: To evaluate the safety and efficacy of E6011, a novel humanized anti-fractalkine monoclonal antibody, in patients with active rheumatoid arthritis (RA) with an inadequate response to biological disease-modifying antirheumatic drugs (DMARDs). METHODS: Active RA patients inadequately responding to biological DMARDs were randomly assigned to placebo or E6011 400-mg group at a 1:1 ratio, and administered E6011 at weeks 0, 1, 2, and subsequently every 2 weeks. Primary endpoint was American College of Rheumatology (ACR)20 response at week 12. RESULTS: Of 64, 33 received placebo, 31 received E6011 400-mg. The ACR20 response rate at week 12 (non-responder imputation) was 27.3% and 22.6% in the placebo and E6011 groups, respectively. ACR50, ACR70 response rates at week 12 were 3.0%, 0% in the placebo and 9.7%, 3.2% in the E6011 group. Exploratory PK exposure analysis revealed that the effect of E6011 tended to be clearer in patients with higher serum trough E6011 concentration. E6011 was well tolerated with no notable safety concerns. CONCLUSIONS: E6011 400-mg was well tolerated but had no clear efficacy at week 12 in RA patients with inadequate response to biologics. Further investigations are warranted to determine the optimal clinical dose and evaluation period for E6011.

An organoid-based carcinogenesis model induced by in vitro chemical treatment.

Animal carcinogenesis models induced by environmental chemicals have been widely used for basic and applied cancer research. However, establishment of in vitro or ex vivo models is essential for molecular mechanistic elucidation of early events in carcinogenesis, leading to clarification of the total mode of action. In the present study, to establish an organoid-based chemical carcinogenesis model, mouse organoids were treated in vitro with 4 genotoxic chemicals, e.g. ethyl methanesulfonate (EMS), acrylamide (AA), diethylnitrosamine (DEN) and 7,12-dimethylbenz[a]anthracene (DMBA) to examine their tumorigenicity after injection to nude mice. The four chemicals were reported to induce lung, liver or mammary carcinomas in mouse models. DMBA-treated mammary tissue-derived organoids with Trp53 heterozygous knockout exhibited tumorigenicity, but not those with wild-type Trp53, reflecting previous reports of corresponding animal models. Treatment of lung organoids with or without Trp53 knockout with EMS or AA resulted in carcinogenic histopathological characteristics, and the activation of oncogenic kinases was demonstrated in the nodules from the nude mouse subcutis. DEN-treated liver (biliary tract) organoids also had an increased number of similar changes. In conclusion, an ex vivo model for chemical carcinogenesis was established using normal mouse tissue-derived organoids. This model will be applied to detect early molecular events, leading to clarification of the mode of action of chemical carcinogenesis.

Organoid-based ex vivo reconstitution of Kras-driven pancreatic ductal carcinogenesis.

The organoid culture technique has been recently applied to modeling carcinogenesis in several organs. To further explore its potential and gain novel insights into tumorigenesis, we here investigated whether pancreatic ductal adenocarcinoma (PDA) could be generated as subcutaneous tumors in immunocompromised nude mice, by genetic engineering of normal organoids. As expected, acute induction of KrasG12Din vitro occasionally led to development of tiny nodules compatible with early lesions known as pancreatic intraepithelial neoplasia (PanIN). KrasG12D-expressing cells were enriched after inoculation in the subcutis, yet proved rather declined during culture, suggesting that its advantage might depend on surrounding environments. Depletion of growth factors or concurrent Trp53 deletion resulted in its robust enrichment, invariably leading to development of PanIN or large high-grade adenocarcinoma, respectively, consistent with in vivo mouse studies for the same genotype. Progression from PanIN was also recapitulated by subsequent knockdown of common tumor suppressors, whereas the impact of Tgfbr2 deletion was only partially recapitulated, illustrating genotype-dependent requirement of the pancreatic niche for tumorigenesis. Intriguingly, analysis of tumor-derived organoids revealed that KrasG12D-expressing cells with spontaneous deletion of wild-type Kras were positively selected and exhibited an aging-related mutation signature in nude mice, mirroring the pathogenesis of human PDA, and that the sphere-forming potential and orthotopic tumorigenicity in syngenic mice were significantly augmented. These observations highlighted the relevance of the subcutis of nude mice in promoting PDA development despite its ectopic nature. Taken together, pancreatic carcinogenesis could be considerably recapitulated with organoids, which would probably serve as a novel disease model.

Monoclonal antibodies specific for podocalyxin expressed on human induced pluripotent stem cells.

Human induced pluripotent stem cells (hiPSCs) are useful starting materials for the generation of cell therapy products, due to their pluripotency and ability to self-renew. Quality control of hiPSCs is extremely important in creating a stable supply of hPSC-derived products. Previously we identified an hiPSC-specific lectin probe, rBC2LCN, which binds specifically to α1,2-fucosylated glycan and recognizes podocalyxin (PODXL) as a glycoprotein ligand. In this study, we produced monoclonal antibodies (mAbs) specific for α1,2-fucosylated PODXL expressed on hiPSCs. PODXL was recombinantly expressed in fucosyltransferase 1 (FUT1)-transfected HEK293, followed by immunization into mice. Monoclonal antibodies, which bind to PODXL/FUT1-transfected cells, but not to cells transfected with only one of PODXL or FUT1, were screened by flow cytometry. The two mAbs generated (179-6B8C9 and 179-7E12E10), termed α1,2-fucosylated PODXL-specific mAbs (FpMabs), showed binding specificity to PODXL/FUT1-transfected cells. The FpMabs bound to hiPSCs but never to human adipose-derived mesenchymal stem cells, human dermal fibroblasts, or hiPSC-derived mesoderm. Altogether, FpMabs are highly specific probes for hiPSCs, which might be a powerful tool for the characterization of hiPSCs used in regenerative medicine.

Blockade of the fractalkine-CX3CR1 axis ameliorates experimental colitis by dislodging venous crawling monocytes.

Chemokine systems modulate inflammatory and immune responses in inflammatory bowel disease (IBD). The colons of IBD patients show increased levels of fractalkine (FKN) and high numbers of FKN receptor-positive (CX3CR1+) cells; however, the FKN-CX3CR1 axis's role in intestinal inflammation, especially in intravascular leukocyte behaviors, still remains unclear. Here, we show that interruption of the FKN-CX3CR1 axis by anti-FKN monoclonal antibody (mAb) ameliorates murine colitis through regulation of intravascular monocyte behaviors in murine colitis models. FKN expression was detectable in vascular endothelium and CX3CR1+ macrophages accumulated in the mucosal lamina propria and submucosa of the inflamed colons. CD115+ monocytes tethered to the venous endothelium and expressed pro-inflammatory mediators. The anti-FKN mAb improved colitis symptoms, markedly reduced pro-inflammatory factors in the colon, maintained blood vessel integrity and reduced tethered monocytes in the inflamed veins. Intravital imaging revealed that CD115+Gr-1low/- monocytes crawled on the apical surfaces of venous endothelium, and anti-FKN mAb rapidly dislodged the crawling monocytes and inhibited their patrolling behavior. These findings suggest that the FKN-CX3CR1 axis triggers the patrolling behavior of crawling monocytes on the venous endothelium of inflamed colons, and accelerates the subsequent leukocyte activation and infiltration by locally producing inflammatory cytokines and chemokines. The mAb also ameliorated symptoms in another IBD model, T-cell-transferred colitis. Blocking the FKN-CX3CR1 axis with an anti-FKN mAb considerably inhibits the colitis-triggered inflammatory cascades, which may be an alternative strategy to treat IBD.

A yolk sac tumor of the pancreas and derived xenograft model effectively responded to VIP chemotherapy.

BACKGROUND: Yolk sac tumors (YSTs) of the pancreas are extremely rare, and no drug responsiveness data are available regarding YSTs. METHODS: We report a pancreatic YST in a 70-year-old woman, and its chemotherapeutic responsiveness based on clinical records and evaluation of a patient-derived xenograft (PDX) line of the YST. RESULTS: The YST was an 11-cm, solid mass located in the pancreatic tail. Histologically, the tumor showed medullary proliferation of tumor cells, with a variety of growth patterns including microcystic/reticular, endodermal sinus, and hepatoid patterns. Immunohistochemically, the tumor cells were positive for Sall4, glypican-3, and alpha-fetoprotein. We administered VIP (etoposide, ifosfamide, cisplatin) chemotherapy for a recurrent liver tumor, and obtained complete pathological remission. A drug-response assay using the PDX line from this YST revealed that both VIP and gemcitabine effectively inhibit tumor growth. CONCLUSIONS: These results suggest that differential diagnosis of YST from adenocarcinoma is important for selecting appropriate chemotherapy.

Epigenetic priming sensitizes gastric cancer cells to irinotecan and cisplatin by restoring multiple pathways.

BACKGROUND: Gastric cancer is heavily influenced by aberrant DNA methylation that alters multiple cancer-related pathways, and may respond to DNA demethylating agents, such as 5-aza-2'-deoxycytidine (5-aza-dC). Here, we aimed to analyze whether 5-aza-dC can sensitize gastric cancer cells to clinically used cytotoxic drugs. METHODS: Ten gastric cancer cell lines were treated with 5-aza-dC for 72 h and their growth was analyzed by conducting WST assay. In vivo effect of the drugs was analyzed using xenografts of OCUM-2 M/SN38 cells. Genome-wide expression and DNA methylation analyses were conducted using microarrays, and biological functions were identified through ingenuity pathway analysis. RESULTS: The cell lines most resistant to SN38 (an active metabolite of irinotecan), CDDP, PTX, and 5-FU, were identified. 5-Aza-dC pre-treatment of the resistant cell lines decreased the IC50 values for SN38 (TMK1, 226.4 nM to 32.91 nM; 44As3, 128.2 nM to 19.32 nM; OCUM2 M/SN38, 74.43 nM to 16.47 nM) and CDDP (TMK1, 5.05 µM to 2.26 µM; OCUM2 M, 10.79 µM to 2.77 µM), but not PTX and 5-FU. The reactivation of apoptosis-related genes, such as RUNX3, PYCARD, TNF, FAS, and FASLG, was induced by pre-treatment with 5-aza-dC, and the DNA demethylation of promoter CpG islands of RUNX3 and PYCARD was confirmed. In a xenograft model with OCUM2 M/SN38, treatment with 5-aza-dC before irinotecan showed markedly enhanced tumor suppression. CONCLUSION: Epigenetic priming with 5-aza-dC can improve the sensitivity of gastric cancer cells to SN38 and CDDP.

Correction to: Epigenetic priming sensitizes gastric cancer cells to irinotecan and cisplatin by restoring multiple pathways.

Recently published the priming.

Kras-driven heterotopic tumor development from hepatobiliary organoids.

Cancers arising from the biliary tract are refractory to conventional therapies, requiring the development of novel therapeutics. However, only a limited number of genetically engineered mouse models have been created, partly because of time-consuming work required. Besides, liver-specific gene manipulation mostly resulted in concurrent development of hepatocellular carcinoma, another type of liver cancer, and gallbladder-restricted gene targeting is still not feasible. Consequently, establishment of cancer type-specific disease modeling remains a technical challenge. To address this issue, we took an alternative cell-based approach to quickly induce tumorigenesis ex vivo. Specifically, murine primary organoids from liver and gallbladder were transduced with lentiviral vectors to reconstitute genetic alterations common in biliary tract cancers, followed by inoculation in immunodeficient mice. Although any single genetic alteration did not induce tumors, mutant Kras and repression of major tumor suppressors cooperated for tumor development within 2 months. Induced lesions varied among normal, dysplastic and papillary lesions to adenocarcinoma, recapitulating multistep tumorigenesis even in a heterotopic situation. We further demonstrated that two putative oncogenes in intrahepatic cholangiocellular carcinoma, mutant Pik3ca and FGFR2-AHCYL1 fusion, were rather modest drivers for liver-derived organoids, probably requiring additional mutations or hepatic niche to robustly induce full-blown tumors. Thus, we showed that cancer cells could be readily generated from primary cells in the biliary tract, at least in cases where genetic factors play dominant roles. Collectively, this study will likely contribute to gaining mechanistic insights into biliary carcinogenesis and providing valuable resources for drug discovery.