Search

Acinar cell carcinoma (ACC) of the pancreas is a malignant tumor of the exocrine cell lineage with a poor prognosis. Due to its rare incidence and technical difficulties, few authentic human cell lines are currently available, hampering detailed investigations of ACC. Therefore, we applied the organoid culture technique to various types of specimens, such as bile, biopsy, and resected tumor, obtained from a single ACC patient. Despite the initial propagation, none of these organoids achieved long-term proliferation or tolerated cryopreservation, confirming the challenging nature of establishing ACC cell lines. Nevertheless, the biopsy-derived early passage organoid developed subcutaneous tumors in immunodeficient mice. The xenograft tumor histologically resembled the original tumor and gave rise to infinitely propagating organoids with solid features and high levels of trypsin secretion. Moreover, the organoid stained positive for carboxylic ester hydrolase, a specific ACC marker, but negative for the duct cell marker CD133 and the endocrine lineage marker synaptophysin. Hence, we concluded the derivation of a novel ACC cell line of the pure exocrine lineage, designated HS-1. Genomic analysis revealed extensive copy number alterations and mutations in EP400 in the original tumor, which were enriched in primary organoids. HS-1 displayed homozygous deletion of CDKN2A, which might underlie xenograft formation from organoids. Although resistant to standard cytotoxic agents, the cell line was highly sensitive to the proteasome inhibitor bortezomib, as revealed by an in vitro drug screen and in vivo validation. In summary, we document a novel ACC cell line, which could be useful for ACC studies in the future.

Cervical cancer remains a major threat to women's health, especially in countries with limited medical resources, and new drugs are needed to improve patient survival and minimize adverse effects. Here, we examine the effects of a triphenylphosphonium (TPP)-conjugated pyrrole-imidazole polyamide (CCC-h1005) targeting the common homoplasmic mitochondrial DNA (mtDNA) cancer risk variant (ATP6 8860AG) on the survival of cervical cancer cell lines, cisplatin-resistant HeLa cells and patient-derived cervical clear cell carcinoma cells as models of cervical cancer treatment. We found that CCC-h1005 induced death in these cells and suppressed the growth of xenografted HeLa tumors with no severe adverse effects. These results suggest that PIP-TPP designed to target mtDNA cancer risk variants can be used to treat many cervical cancers harboring high copies of the target variant, providing a foundation for clinical trials of this class of molecules for treating cervical cancer and other types of cancers.

Cervical clear cell carcinoma (CCCC) is rare. This report describes the case of CCCC with a serous component. A 22-year-old woman presented with vaginal bleeding. A cervical tumor was discovered: pelvic magnetic resonance imaging revealed a tumor measuring 46 mm. Radical hysterectomy was performed based on the diagnosis of stage IB2 cervical cancer. After histological examination of the specimen revealed a coexisting invasive clear cell carcinoma (95%) and serous carcinoma (5%), five cycles of nedaplatin and irinotecan therapy were administered as postoperative adjuvant chemotherapy. Local recurrence in the vaginal vault was observed at 7 months after surgery. Radiation therapy and chemotherapy were administered. The patient is alive without evidence of recurrence at 26 months after surgery.

Genetically engineered mice (GEM) are the gold standard for cancer modeling. However, strict recapitulation of stepwise carcinogenesis from a single tumor-initiating epithelial cell among genetically intact cells in adults is not feasible with the currently available techniques using GEM. In previous studies, we partially overcame this challenge by physically isolating organs from adult animals, followed by genetic engineering in organoids and subcutaneous inoculation in nude mice. Despite the establishment of suitable ex vivo carcinogenesis models for diverse tissues, tumor development remained ectopic and occurred under immunodeficient conditions. Further refinement was, therefore, necessary to establish ideal models. Given the poor prognosis and few models owing to the lack of gall bladder (GB)-specific Cre strain, we assumed that the development of authentic models would considerably benefit GB cancer research. Here, we established a novel model using GB organoids with mutant Kras and Trp53 loss generated in vitro by lentiviral Cre transduction and CRISPR/Cas9 gene editing, respectively. Organoid-derived subcutaneous tumor fragments were sutured to the outer surface of the GB in syngeneic mice, which developed orthotopic tumors that resembled human GB cancer in histological and transcriptional features. This model revealed the infiltration of similar subsets of immune cells in both subcutaneous and orthotopic tumors, confirming the appropriate immune environment during carcinogenesis. In addition, we accurately validated the in vivo efficacy of gemcitabine, a common therapeutic agent for GB cancer, in large cohorts. Taken together, this model may serve as a promising avatar of patients with GB cancer in drug discovery and precision medicine.

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

KRAS, an oncogene, is frequently activated by mutations in many cancers. Kras-driven adenocarcinoma development in the lung, pancreas, and biliary tract has been extensively studied using gene targeting in mice. By taking the organoid- and allograft-based genetic approach to these organs, essentially the same results as in vivo models were obtained in terms of tumor development. To verify the applicability of this approach to other organs, we investigated whether the combination of Kras activation and Pten inactivation, which gives rise to endometrial tumors in mice, could transform murine endometrial organoids in the subcutis of immunodeficient mice. We found that in KrasG12D-expressing endometrial organoids, Pten knockdown did not confer tumorigenicity, but Cdkn2a knockdown or Trp53 deletion led to the development of carcinosarcoma (CS), a rare, aggressive tumor comprising both carcinoma and sarcoma. Although they originated from epithelial cells, some CS cells expressed both epithelial and mesenchymal markers. Upon inoculation in immunodeficient mice, tumor-derived round organoids developed carcinoma or CS, whereas spindle-shaped organoids formed monophasic sarcoma only, suggesting an irreversible epithelial-mesenchymal transition during the transformation of endometrial cells and progression. As commonly observed in mutant Kras-driven tumors, the deletion of the wild-type Kras allele was identified in most induced tumors, whereas some epithelial cells in CS-derived organoids were unexpectedly negative for KrasG12D. Collectively, we showed that the oncogenic potential of KrasG12D and the histological features of derived tumors are context-dependent and varies according to the organ type and experimental settings. Our findings provide novel insights into the mechanisms underlying tissue-specific Kras-driven tumorigenesis.

Cervical clear cell carcinoma (cCCC) constitutes an extremely rare subtype of cervical cancer. Consequently, its pathogenesis remains largely unknown, with no cell lines established from primary tumors. Here, we report the first establishment of cCCC organoids, from biopsy samples of a 23‐year‐old patient diagnosed with cCCC. By applying a protocol that we recently optimized for gynecological tumors, we were able to propagate a patient‐derived cell line (PDC) for more than 6 months as organoids. This PDC tolerated cryopreservation and proliferated either as spheroids or adherent cells, and developed xenografts in immunodeficient mice, ensuring robust utility as a cell line. Intriguingly, the resected tumor focally contained serous carcinoma (SC) in a tiny protruding lesion. Both organoids and derivative xenografts resembled the CCC component of the original tumor in histology, immunostaining profile, and genome‐wide copy number changes, including focal gain of MET. Genomic analysis revealed that both organoids and the CCC component harbored only a few mutations, of which 2 mutations were shared in common. In contrast, the SC component showed a mutator‐phenotype and prominent genome instability along with biallelic inactivation of TP53, but none of them were found in organoids or the CCC component. The PDC proved sensitive to major chemotherapeutic agents and MET inhibitors. These observations clearly indicated that the PDC, designated as YMC7, can be used as a novel cCCC cell line and provide novel insights into the pathogenesis of mixed cervical adenocarcinoma. As a valuable resource for rare cancer, it will likely contribute to investigations in many fields.

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.

Genetically engineered mice have been the gold standard in modeling tumor development. Recent studies have demonstrated that genetically engineered organoids can develop subcutaneous tumors in immunocompromised mice, at least for organs that prefer predominant driver mutations for tumorigenesis. To further substantiate this concept, the fallopian tube (FT), a major cell of origin of ovarian high-grade serous carcinoma (HGSC), which almost invariably carries TP53 mutations, was investigated for p53 inactivation-driven tumorigenesis. Murine FT organoids subjected to lentiviral Cre-mediated Trp53 deletion did not develop tumors. However, subsequent suppression of Pten and simultaneous induction of mutant Pik3ca led to the development of carcinoma in situ and HGSC-like tumors, respectively, whereas concurrent deletion of Apc resulted in the development of benign cysts, mirroring frequent activation of the PI3K/AKT axis and the marginal impact of Wnt pathway activation in HGSC. Consistent with the frequent activation of the RAS pathway in HGSC, mutant Kras cooperated with Trp53 deletion for the development of tumors, which unexpectedly contained sarcoma cells in addition to carcinoma cells, despite the epithelial origin of the inoculated organoids. This finding is in sharp contrast with the exclusive adenocarcinoma development from gastrointestinal organoids with the same genotype reported in previous studies, suggesting a tissue-specific epithelial-mesenchymal transition program. In tumor-derived organoids, the Cre-mediated recombination rate reached 100% for Trp53 but not for the other genes, highlighting the advantage of p53 inactivation in FT tumorigenesis. The Trp53 wildtype FT organoids expressing the mutant Kras developed sarcoma and carcinoma upon Cdkn2a suppression and Tgfbr2 deletion, respectively, revealing novel pro-tumorigenic genetic cooperation and critical roles of TGF-β signaling for epithelial-mesenchymal transition in FT-derived tumorigenesis. Collectively, the organoid-based approach represents a shortcut to tumorigenesis and provides novel insights into the relationships among genotype, cell type, and tumor phenotype underlying tumorigenesis. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Biliary tract cancer (BTC) is one of the most devastating types of malignant neoplasms worldwide. However, the mechanisms underlying the development and progression of BTC remain unresolved. BTC includes extrahepatic bile duct carcinoma (EBDC), gallbladder carcinoma (GBC) and ampulla of Vater carcinoma (AVC), named according to the location of the tumor. Although genetic alterations of intrahepatic cholangiocarcinoma have been investigated, those of EBDC, GBC and AVC have not yet been fully understood. The present study analyzed somatic mutations of 50 cancer-associated genes in 27 Japanese BTC cells, including: 11 EBDC, 14 GBC and 2 AVC. Next-generation sequencing using an Ion AmpliSeq Cancer Panel identified a total of 44 somatic mutations across 14 cancer-associated genes. Among the 44 mutations, 42 were judged as pathological mutations. Frequent mutations were identified in tumor protein 53 (TP53) (14/27), SMAD family member 4 (SMAD4) (6/27), phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit α (PIK3CA) (6/27), and Kirsten rat sarcoma (KRAS) (6/27); no significant differences were identified between EBDC and GBC tissues. Notably, the frequency of the PIK3CA mutation was higher when compared with previous reports. This result may suggest that the activation of the PIK3CA-protein kinase B signaling pathway, in addition to the abrogation of p53, SMAD4 and RAS mitogen-activated protein kinase may have a crucial role in the carcinogenesis of Japanese BTC. These findings may be useful for the development of personalized therapies for BTC.

Ovarian clear cell carcinoma (OCCC) is an aggressive ovarian cancer with a higher frequency in Japan and often becomes chemorefractory disease. Reliable genetic diagnosis is essential to affirm the success of precision medicine for OCCC treatment. The aim of this study is, therefore, to identify novel mutations in OCCCs and develop a feasible clinical next generation sequencing (NGS) approach using formalin-fixed paraffin-embedded (FFPE) rather than preferable but not always available fresh frozen (FF) samples.We optimized and evaluated exome analyses of 409 cancer-related genes using FFPE and FF DNA and analyzed NGS data to identify somatic mutations in Japanese OCCCs.Sufficient and good quality DNAs from FFPE samples were extracted from 18 (FIGO Stage I: 12) out of 29 pairs of matched normal and OCCC for NGS (63%). The fine quality of extracted DNAs depended on the length of storage period (2years storage). We also identified 45 somatic mutations in 34 genes including unreported variants from those FFPE DNA, in which somatic mutations in the PIK3CA gene was the most common (28%) as previously reported. Seven genes (PIK3CA, ARID1A, CTNNB1, CSMD3, LPHN3, LRP1B, and TP53) were mutated in at least two independent OCCCs. FF samples from 3 out of those 18 OCCCs were available and 13 out of 14 FFPE somatic mutations were confirmed.We successfully identified novel genetic alterations in Japanese OCCCs and demonstrated a feasible clinical diagnostic procedure using targeted NGS for OCCC FFPE samples.

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.

Ovarian cancer (OC) is one of the leading causes of female cancer death. Recent studies have documented its extensive variations as a disease entity, in terms of cell or tissue of origin, pre-cancerous lesions, common mutations, and therapeutic responses, leading to the notion that OC is a generic term referring to a whole range of different cancer subtypes. Despite such heterogeneity, OC treatment is stereotypic; aggressive surgery followed by conventional chemotherapy could result in chemo-resistant diseases. Whereas molecular-targeted therapies will become shortly available for a subset of OC, there still remain many patients without effective drugs, requiring development of groundbreaking therapeutic agents. In preclinical studies for drug discovery, cancer cell lines used to be the gold standard, but now this has declined due to frequent failure in predicting therapeutic responses in patients. In this regard, patient-derived cells and tumors are gaining more attention in precise and physiological modeling of in situ tumors, which could also pave the way to implementation of precision medicine. In this article, we comprehensively overviewed the current status of various platforms for patient-derived OC models. We highly appreciate the potentials of organoid culture in achieving high success rate and retaining tumor heterogeneity.

Inactivation of the Adenomatous polyposis coli (APC) gene is an initiating and the most relevant event in most sporadic cases of colorectal cancer, providing a rationale for using Apc-mutant mice as the disease model. Whereas carcinogenesis has been observed only at the organism level, the recent development of the organoid culture technique has enabled long-term propagation of intestinal stem cells in a physiological setting, raising the possibility that organoids could serve as an alternative platform for modeling colon carcinogenesis. Indeed, it is demonstrated in the present study that lentivirus-based RNAi-mediated knockdown of Apc in intestinal organoids gave rise to subcutaneous tumors upon inoculation in immunodeficient mice. Reconstitution of common genetic aberrations in organoids resulted in development of various lesions, ranging from aberrant crypt foci to full-blown cancer, recapitulating multi-step colorectal tumorigenesis. Due to its simplicity and utility, similar organoid-based approaches have been applied to both murine and human cells in many investigations, to gain mechanistic insight into tumorigenesis, to validate putative tumor suppressor genes or oncogenes, and to establish preclinical models for drug discovery. In this review article, we provide a multifaceted overview of these types of approaches that will likely accelerate and advance research on colon cancer.

The metaplastic epithelium of the transformation zone (TZ) including the squamocolumnar junction (SCJ) of the uterine cervix is a prime target of human papilloma virus (HPV) infection and subsequent cancer development. Due to the lack of adequate in vitro models for SCJ, however, investigations into its physiological roles and vulnerability to carcinogenesis have been limited. By using Matrigel-based three-dimensional culture techniques, we propagated organoids derived from the normal SCJ region, along with metaplastic squamous cells in the TZ. Consisting predominantly of squamous cells, organoids basically exhibited a dense structure. However, at least in some organoids, a small but discrete population of mucin-producing endocervix cells co-existed adjacent to the squamous cell population, virtually recapitulating the configuration of SCJ in a TZ background. In addition, transcriptome analysis confirmed a higher expression level of many SCJ marker genes in organoids, compared to that in the immortalized cervical cell lines of non-SCJ origin. Thus, the obtained organoids appear to mimic cervical SCJ cells and, in particular, metaplastic squamous cells from the TZ, likely providing a novel platform in which HPV-driven cervical cancer development could be investigated.

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 due to time-consuming work required. Besides, liver-specific gene manipulation mostly resulted in concurrent development of hepatocellular carcinoma, another type of liver cancer, and gall bladder-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 gall bladder were transduced with lentiviral vectors to reconstitute genetic alterations common in biliary tract cancers, followed by inoculation in immunodeficient mice. Whereas any single genetic alteration did not induce tumors, mutant Kras and repression of major tumor suppressors cooperated for tumor development within two months. Induced lesions varied among normal, dysplastic and papillary lesions to adenocarcinoma, recapitulating multi-step 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, the present study will likely contribute to gaining mechanistic insights into biliary carcinogenesis and providing valuable resources for drug discovery.

Lentivirus-based gene delivery works efficiently for the majority of mammalian cells cultured under standard two-dimensional conditions. By contrast, intestinal epithelial organoids embedded into three-dimensional extracellular matrix appear to be resistant to lentiviral transduction. We observed that Matrigel, a matrix that reconstitutes a basement membrane and is indispensable for cell survival and proliferation, prevents lentiviruses from binding to intestinal cells. In this chapter, we describe a simple method of a highly efficient gene transduction into intestinal organoids. This method involves organoid dispersion into single intestinal epithelial cells, mixing these individual cells with lentiviral particles, plating on Matrigel, and subsequent re-embedding into Matrigel. Under these conditions, the majority of the cells are exposed to the virus in the absence of the matrix barrier while remaining attached to the matrix. Using a GFP-labeled lentivirus, we demonstrate that this method allows for highly efficient infection of intestinal organoids after overnight incubation of Matrigel-attached cells with lentiviral particles.

Lentivirus-based gene delivery works efficiently for the majority of mammalian cells cultured under standard two-dimensional conditions. By contrast, intestinal epithelial organoids embedded into three-dimensional extracellular matrix appear to be resistant to lentiviral transduction. We observed that Matrigel, a matrix that reconstitutes a basement membrane and is indispensable for cell survival and proliferation, prevents lentiviruses from binding to intestinal cells. In this chapter, we describe a simple method of a highly efficient gene transduction into intestinal organoids. This method involves organoid dispersion into single intestinal epithelial cells, mixing these individual cells with lentiviral particles, plating on Matrigel, and subsequent re-embedding into Matrigel. Under these conditions, the majority of the cells are exposed to the virus in the absence of the matrix barrier while remaining attached to the matrix. Using a GFP-labeled lentivirus, we demonstrate that this method allows for highly efficient infection of intestinal organoids after overnight incubation of Matrigel-attached cells with lentiviral particles.

Despite extensive efforts to target mutated RAS proteins, anticancer agents capable of selectively killing tumour cells harbouring KRAS mutations have remained unavailable. Here we demonstrate the direct targeting of KRAS mutant DNA using a synthetic alkylating agent (pyrrole-imidazole polyamide indole-seco-CBI conjugate; KR12) that selectively recognizes oncogenic codon 12 KRAS mutations. KR12 alkylates adenine N3 at the target sequence, causing strand cleavage and growth suppression in human colon cancer cells with G12D or G12V mutations, thus inducing senescence and apoptosis. In xenograft models, KR12 infusions induce significant tumour growth suppression, with low host toxicity in KRAS-mutated but not wild-type tumours. This newly developed approach may be applicable to the targeting of other mutant driver oncogenes in human tumours., KRAS遺伝子変異を持ったがんを標的とした新規のアルキル化剤の開発について. 京都大学プレスリリース. 2015-05-01.