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KRAS is the most frequently mutated oncogene in human cancer, and its activating mutations represent long-sought therapeutic targets. Programmable nucleases, particularly the CRISPR-Cas9 system, provide an attractive tool for genetically targeting KRAS mutations in cancer cells. Here, we show that cleavage of a panel of KRAS driver mutations suppresses growth in various human cancer cell lines, revealing their dependence on mutant KRAS. However, analysis of the remaining cell population after long-term Cas9 expression unmasked the occurence of oncogenic KRAS escape variants that were resistant to Cas9-cleavage. In contrast, the use of an adenine base editor to correct oncogenic KRAS mutations progressively depleted the targeted cells without the appearance of escape variants and allowed efficient and simultaneous correction of a cancer-associated TP53 mutation. Oncogenic KRAS and TP53 base editing was possible in patient-derived cancer organoids, suggesting that base editor approaches to correct oncogenic mutations could be developed for functional interrogation of vulnerabilities in a personalized manner for future precision oncology applications.Significance:Repairing KRAS mutations with base editors can be used for providing a better understanding of RAS biology and may lay the foundation for improved treatments for KRAS-mutant cancers.

Supplementary Data from Efficient Correction of Oncogenic KRAS and TP53 Mutations through CRISPR Base Editing

Abstract Purpose Robotic-assisted minimally invasive esophagectomy (RAMIE) has become one standard approach for the operative treatment of esophageal tumors at specialized centers. Here, we report the results of a prospective multicenter registry for standardized RAMIE. Methods The German da Vinci Xi registry trial included all consecutive patients who underwent RAMIE at five tertiary university centers between Oct 17, 2017, and Jun 5, 2020. RAMIE was performed according to a standard technique using an intrathoracic circular stapled esophagogastrostomy. Results A total of 220 patients were included. The median age was 64 years. Total minimally invasive RAMIE was accomplished in 85.9%; hybrid resection with robotic-assisted thoracic approach was accomplished in an additional 11.4%. A circular stapler size of ≥28 mm was used in 84%, and the median blood loss and operative time were 200 (IQR: 80–400) ml and 425 (IQR: 335–527) min, respectively. The rate of anastomotic leakage was 13.2% (n=29), whereas the two centers with >70 cases each had rates of 7.0% and 12.0%. Pneumonia occurred in 19.5% of patients, and the 90-day mortality was 3.6%. Cumulative sum analysis of the operative time indicated the end of the learning curve after 22 cases. Conclusions High-quality multicenter registry data confirm that RAMIE is a safe procedure and can be reproduced with acceptable leak rates in a multicenter setting. The learning curve is comparably low for experienced robotic surgeons.

Recent studies have shed light on the signaling pathways required for gastric tissue maintenance and how aberrations in these key pathways lead to gastric cancer development. Although it has been shown that the WNT pathway is important for gastric epithelial homeostasis, the identity and source of the responsible canonical WNT ligands remain unknown. Furthermore, it is unclear how gastric cancer acquires WNT niche independence - an important early step in tumorigenesis. Using human and mouse gastric organoids andin vivomouse models, we found that mesenchymal WNT2B and WNT7B maintain gastric epithelium in homeostasis. Next, mouse genetic studies and single-cell multi-omics analyses revealed that activation of MAPK signaling induces secretion of WNT7B in the epithelium itself. We further confirmed that in human gastric cancer, MAPK pathway activation through HER2 overexpression or copy number gains ofWNT2confers WNT independence. Importantly, the epithelium-intrinsic WNT expression could be therapeutically inhibited. Taken together, our results reveal that normal gastric epithelial turnover relies on WNT ligands secreted by niche mesenchymal cells, while transformation involves acquisition of a WNT secretory phenotype in the epithelium - representing a potential target for therapeutic interventions.

Introduction: Patients with advanced gastric cancer (AGC) frequently show peritoneal carcinomatosis (PC). PC reduces life expectancy and quality of life. Cytoreductive surgery (CRS) with hyperthermic intraperitoneal chemotherapy (HIPEC) has been shown to improve overall survival. Nevertheless, it has been reported that CRS and HIPEC are accompanied by an increase in postoperative complications. The purpose of this study was to investigate the complications associated with CRS and HIPEC and overall and disease-free survival. Methods: Patients with AGC and PC, who received complete CRS and HIPEC, were included in the HIPEC group (n = 15). Patients with AGC but without PC, who received resection of the primary tumor alone, constituted the control group (n = 43). Results: Patients enrolled in the HIPEC group presented with a median PCI of 7. In comparison with the control group, no differences were found in patient characteristics, risk factors, pathological findings, and operative procedures. Twenty-five percentage of the patients in both groups suffered from serious postoperative complications (CDC ≥3a). Surgical and medical complications, rate of reoperation, and mortality did not differ. Also, the recurrence pattern, median survival, and 1- and 2-year survival rates showed no differences. Conclusion: CRS and HIPEC do not lead to an increased postoperative morbidity and mortality in AGC with PC. Albeit the poorer prognosis of patients with PC, survival of both groups was comparable.

Pathological complete response (pCR) has been correlated with overall survival in several cancer entities including colorectal cancer. Novel total neoadjuvant treatment (TNT) in rectal cancer has achieved pathological complete response in one-third of the patients. To define better treatment options for nonresponding patients, we used patient-derived organoids (PDOs) as avatars of the patient’s tumor to apply both photon- and proton-based irradiation as well as single and combined chemo(radio)therapeutic treatments. While response to photon and proton therapy was similar, PDOs revealed heterogeneous responses to irradiation and different chemotherapeutic drugs. Radiotherapeutic response of the PDOs was significantly correlated with their ability to repair irradiation-induced DNA damage. The classical combination of 5-FU and irradiation could not sensitize radioresistant tumor cells. Ataxia-telangiectasia mutated (ATM) kinase was activated upon radiation, and by inhibition of this central sensor of DNA damage, radioresistant PDOs were resensitized. The study underlined the capability of PDOs to define nonresponders to irradiation and could delineate therapeutic approaches for radioresistant patients.

Interactions between tumour cells and the surrounding microenvironment contribute to tumour progression, metastasis and recurrence1–3. Although mosaic analyses in Drosophila have advanced our understanding of such interactions4,5, it has been difficult to engineer parallel approaches in vertebrates. Here we present an oncogene-associated, multicolour reporter mouse model—the Red2Onco system—that allows differential tracing of mutant and wild-type cells in the same tissue. By applying this system to the small intestine, we show that oncogene-expressing mutant crypts alter the cellular organization of neighbouring wild-type crypts, thereby driving accelerated clonal drift. Crypts that express oncogenic KRAS or PI3K secrete BMP ligands that suppress local stem cell activity, while changes in PDGFRloCD81+ stromal cells induced by crypts with oncogenic PI3K alter the WNT signalling environment. Together, these results show how oncogene-driven paracrine remodelling creates a niche environment that is detrimental to the maintenance of wild-type tissue, promoting field transformation dominated by oncogenic clones. By inducing changes in surrounding tissue, mutant intestinal stem cells create an unfavourable niche environment that gives them a competitive advantage over non-mutant neighbours.

Sarcopenia is a known risk factor for adverse outcomes after esophageal cancer (EC) surgery. Robot-assisted minimally invasive esophagectomy (RAMIE) offers numerous advantages, including reduced morbidity and mortality. However, no evidence exists to date comparing the development of sarcopenia after RAMIE and open esophagectomy (OE). The objective was to evaluate whether the development of sarcopenia within the first postoperative year after esophagectomy is associated with the surgical approach: RAMIE versus OE.A total of 168 patients with EC were analyzed who either underwent total robotic or fully open Ivor Lewis esophagectomy in a propensity score-matched analysis. Sarcopenia was assessed using the skeletal muscle index (cmAfter 1-to-1 propensity score matching for confounders, 67 patients were allocated to RAMIE and OE groups, respectively. Skeletal muscle index in the OE group was significantly lower compared with the RAMIE group at the third (43.2 ± 7.6 cmTo our knowledge, this study is the first to suggest a substantial benefit of RAMIE compared with open esophagectomy in terms of postoperative sarcopenia. These results add further evidence to support the implementation of the robotic approach in multimodal therapy of EC.

Drug combination therapies for cancer treatment show high efficacy but often induce severe side effects, resulting in dose or cycle number reduction. We investigated the impact of neoadjuvant chemotherapy (neoCTx) adaptions on treatment outcome in 59 patients with pancreatic ductal adenocarcinoma (PDAC). Resections with tumor-free margins were significantly more frequent when full-dose neoCTx was applied. We determined if patient-derived organoids (PDOs) can be used to personalize poly-chemotherapy regimens by pharmacotyping of treatment-naïve and post-neoCTx PDAC PDOs. Five out of ten CTx-naïve PDO lines exhibited a differential response to either the FOLFIRINOX or the Gem/Pac regimen. NeoCTx PDOs showed a poor response to the neoadjuvant regimen that had been administered to the respective patient in 30% of cases. No significant difference in PDO response was noted when comparing modified treatments in which the least effective single drug was removed from the complete regimen. Drug testing of CTx-naïve PDAC PDOs and neoCTx PDOs may be useful to guide neoadjuvant and adjuvant regimen selection, respectively. Personalizing poly-chemotherapy regimens by omitting substances with low efficacy could potentially result in less severe side effects, thereby increasing the fraction of patients receiving a full course of neoadjuvant treatment. © 2022 The Authors. The Journal of Pathology published by John WileySons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Simple Summary Radiotherapy is part of the standard of care for many solid tumors. In pancreatic ductal adenocarcinoma (PDAC), good responses to radiotherapy can only be observed in a minority of patients. In our study, we used PDAC patient-derived organoids (PDO) to investigate alternative radiotherapy approaches for PDAC, such as proton irradiation and combined radiochemotherapy (RCT). Although only very distinct differences in treatment response could be identified, we show the utility of PDOs in translational proton research and found synergistic effects of combined treatments with chemotherapy and proton irradiation in individual PDOs. To optimize neoadjuvant radiochemotherapy of pancreatic ductal adenocarcinoma (PDAC), the value of new irradiation modalities such as proton therapy needs to be investigated in relevant preclinical models. We studied individual treatment responses to RCT using patient-derived PDAC organoids (PDO). Four PDO lines were treated with gemcitabine, 5-fluorouracile (5FU), photon and proton irradiation and combined RCT. Therapy response was subsequently measured via viability assays. In addition, treatment-naive PDOs were characterized via whole exome sequencing and tumorigenicity was investigated in NMRI Foxn1(nu/nu) mice. We found a mutational pattern containing common mutations associated with PDAC within the PDOs. Although we could unravel potential complications of the viability assay for PDOs in radiobiology, distinct synergistic effects of gemcitabine and 5FU with proton irradiation were observed in two PDO lines that may lead to further mechanistical studies. We could demonstrate that PDOs are a powerful tool for translational proton radiation research. CA extern

Pancreatic cancer is a devastating malignancy with minimal treatment options. Standard-of-care therapy, including surgery and chemotherapy, is unsatisfactory, and therapies harnessing the immune system have been unsuccessful in clinical trials. Resistance to therapy and disease progression are mediated by the tumor microenvironment, which contains excessive amounts of extracellular matrix and stromal cells, acting as a barrier to drug delivery. There is a lack of pre-clinical pancreatic cancer models that reconstruct the extracellular, cellular, and biomechanical elements of tumor tissues to assess responses toward immunotherapy. To address this limitation and explore the effects of immunotherapy in combination with chemotherapy, we developed a multicellular 3D cancer model using a star-shaped poly(ethylene glycol)-heparin hydrogel matrix. Human pancreatic cancer cells, cancer-associated fibroblasts, and myeloid cells were grown encapsulated in hydrogels to mimic key components of tumor tissues, and cell responses toward treatment were assessed. Combining the CD11b agonist ADH-503 with anti-PD-1 immunotherapy and chemotherapy led to a significant reduction in tumor cell viability, proliferation, metabolic activity, immunomodulation, and secretion of immunosuppressive and tumor growth-promoting cytokines. This article is protected by copyright. All rights reserved.

KRAS is the most frequently mutated oncogene in human cancer, and its activating mutations represent long-sought therapeutic targets. Programmable nucleases, particularly the CRISPR-Cas9 system, provide an attractive tool for genetically targeting KRAS mutations in cancer cells. Here, we show that cleavage of a panel of KRAS driver mutations suppresses growth in various human cancer cell lines, revealing their dependence on mutant KRAS. However, analysis of the remaining cell population after long-term Cas9 expression unmasked the occurence of oncogenic KRAS escape variants that were resistant to Cas9-cleavage. In contrast, the use of an adenine base editor to correct oncogenic KRAS mutations progressively depleted the targeted cells without the appearance of escape variants and allowed efficient and simultaneous correction of a cancer-associated TP53 mutation. Oncogenic KRAS and TP53 base editing was possible in patient-derived cancer organoids, suggesting that base editor approaches to correct oncogenic mutations could be developed for functional interrogation of vulnerabilities in a personalized manner for future precision oncology applications. Significance: Repairing KRAS mutations with base editors can be used for providing a better understanding of RAS biology and may lay the foundation for improved treatments for KRAS-mutant cancers.

Several genetic and environmental factors increase gastric cancer (GC) risk, with Helicobacter pylori being the main environmental agent. GC is thought to emerge through a sequence of morphological changes that have been elucidated on the molecular level. New technologies have shed light onto pathways that are altered in GC, involving mutational and epigenetic changes and altered signaling pathways. Using various new model systems and innovative approaches, the relevance of such alterations for the emergence and progression of GC has been validated. Here, we highlight the key strategies and the resulting achievements. A major step is the characterization of epithelial stem cell behavior in the healthy stomach. These data, obtained through new reporter mouse lines and lineage tracing, enabled insights into the processes that control cellular proliferation, self-renewal, and differentiation of gastric stem cells. It has become evident that these cells and pathways are often deregulated in carcinogenesis. Second, insights into how H pylori colonizes gastric glands, directly interacts with stem cells, and alters cellular and genomic integrity, as well as the characterization of tissue responses to infection, provide a comprehensive picture of how this bacterium contributes to gastric carcinogenesis. Third, the development of stem cell- and tissue-specific reporter mice have driven our understanding of the signals and mutations that promote different types of GC and now also enable the study of more advanced, metastasized stages. Finally, organoids from human tissue have allowed insights into gastric carcinogenesis by validating mutational and signaling alterations in human primary cells and opening a route to predicting responses to personalized treatment.

Adult stem cells constantly react to local changes to ensure tissue homeostasis. In the main body of the stomach, chief cells produce digestive enzymes; however, upon injury, they undergo rapid proliferation for prompt tissue regeneration. Here, we identified p57

Weltweit erkranken jahrlich 572.000 Menschen an einem Osophaguskarzinom, 509.000 Patienten sterben daran. Die Osophagektomie ist der entscheidende Faktor in der kurativen multimodalen Therapie des lokal fortgeschrittenen Osophaguskarzinoms. Die roboterassisistierte minimal-invasive Osophagektomie (RAMIE) ist als Weiterentwicklung der minimal-invasiven Osophagektomie (MIE) anzusehen, reduziert wie diese im Vergleich zum offenen Vorgehen das operative Trauma sowie die postoperative Morbiditat. Sie verringert die Limitationen der MIE durch eine 3-dimensionale Sicht, eine wiederhergestellte Auge-Hand-Achse, die Filterung des naturlichen Tremors und eine hervorragende Bewegungsfreiheit der Instrumente, deren Gelenke dem menschlichen Handgelenk nachempfunden sind. In diesem Kapitel beschreiben wir unsere Technik der roboterassistierten minimal-invasiven Osophagektomie im Detail und benennen notwendige Voraussetzungen zur erfolgreichen Implementierung.

Bacterial sensing by intestinal tumor cells contributes to tumor growth through cell-intrinsic activation of the calcineurin-NFAT axis, but the role of this pathway in other intestinal cells remains unclear. Here, we found that myeloid-specific deletion of calcineurin in mice activated protective CD8

Gastric cancer ranks as the fifth most common human malignancy and the third leading cause of cancer related deaths. Depending on tumor stage, endoscopic or surgical resection supported by perioperative chemotherapy is the only curative option for patients. Due to late clinical manifestation and missing reliable biomarkers, early detection is challenging and overall survival remains poor. Organoids are cell aggregates cultured in three-dimensions that grow with similar characteristics as their tissue-of-origin. Due to their self-renewal and proliferative capacity, organoids can be maintained long term in culture and expanded in many cases in an unlimited fashion. Patient-derived organoid (PDO) libraries function as living biobanks, allowing the in depth analysis of tissue specific function, development and disease. The recent successful establishment of gastric cancer PDOs opens up new perspectives for multiple translational clinical applications. Here, we review different adult stem cell derived gastric organoid model systems and focus on their establishment, phenotypic and genotypic characterizations as well as their use in predicting therapy response.

Electroporation is a common method for transfection with different kinds of molecules by electrical permeabilization of the plasma membrane. With the increasing use of organoids as a culturing method for primary patient material in the last years, efficient transfer methods of components for genetic engineering in this 3D culture system are in need. Especially for organoids, the efficiency of genetic manipulations depends on a successful transfection. Thus, this protocol was developed to facilitate the electroporation of organoids and to prove its universal functionality in different entities. Human colorectal, pancreatic, hepatic and gastric cancer organoids were successfully electroporated with small and large plasmids in comparison. Based on GFP encoding vectors, the transfection efficiency was determined by FACS. No extensive preparation of the cells or special, cost-intensive electroporation buffers are necessary, and the protocol can be performed within one day.

Jahrlich erkranken beinahe eine Million Menschen an einem Magenkarzinom. Die 5‑Jahres-Uberlebensrate betragt in der westlichen Welt nur 20–25 %. Dies ist hauptsachlich dadurch begrundet, dass bei mehr als 70 % der Patienten bereits ein lokal fortgeschrittenes und/oder metastasiertes Karzinom diagnostiziert wird. Die komplette Resektion des Primartumors inklusive D2-Lymphadenektomie ist die einzig unimodale kurative Therapie. Bei resektablen Tumoren erhoht die Kombination mit perioperativer Chemotherapie die 5‑Jahres-Uberlebensraten um knapp 15 %. Zunehmende medikamentose Optionen inklusive neuer zielgerichteter Substanzen ermoglichen in der palliativen Situation eine Lebensverlangerung bei gleichzeitiger Verbesserung von Lebensqualitat und Symptomkontrolle. Karzinome des gastroosophagealen Ubergangs unterscheiden sich von Magenkarzinomen hinsichtlich ihrer Atiologie, Diagnostik und teilweise auch in der Therapie; sie bedurfen daher einer separaten Darstellung.

Jahrlich erkranken weltweit gut eine Million Menschen an Karzinomen des Magens sowie des gastroosophagealen Ubergangs („adenocarcinomas of the esophagogastric junction“, AEG). Sie gehoren weltweit zu den haufigsten Krebstodesursachen. Die komplette Resektion des Primartumors inklusive onkologisch radikaler Lymphadenektomie ist die einzig kurative Therapie fur die meisten Tumorstadien. Das interdisziplinare Vorgehen richtet sich dabei nach der Stadieneinteilung, das operative zusatzlich nach der exakten Tumorlokalisation sowie der histologischen Differenzierung. Wahrend sich die Risikoprofile und verschiedene epidemiologische Charakteristika unterscheiden, weisen AEG und Adenokarzinome des Magens ahnliche Vorlauferlasionen und molekulare Veranderungen auf. Plattenepithelkarzinome des Osophagus unterscheiden sich in der Atiologie, den molekularen Alterationen und in den therapeutischen Ansatzen; sie bedurfen daher einer separaten Darstellung.

The gastrointestinal tract is characterized by a self-renewing epithelium fueled by adult stem cells residing at the bottom of the intestinal crypt and gastric glands. Their activity and proliferation is strongly dependent on complex signaling pathways involving other crypt/gland cells as well as surrounding stromal cells. In recent years organoids are becoming increasingly popular as a new and powerful tool to study developmental or other biological processes. Organoids retain morphological and molecular patterns of the tissue they are derived from, are self-organizing, relatively simple to handle and accessible to genetic engineering. This review focuses on the developmental processes and signaling molecules involved in epithelial homeostasis and how a profound knowledge of these mechanisms allowed the establishment of a three dimensional organoid culture derived from adult gastrointestinal stem cells.

Summary The gastric corpus epithelium is the thickest part of the gastrointestinal tract and is rapidly turned over. Several markers have been proposed for gastric corpus stem cells in both isthmus and base regions. However, the identity of isthmus stem cells (IsthSCs) and the interaction between distinct stem cell populations is still under debate. Here, based on unbiased genetic labeling and biophysical modeling, we show that corpus glands are compartmentalized into two independent zones, with slow-cycling stem cells maintaining the base and actively cycling stem cells maintaining the pit-isthmus-neck region through a process of “punctuated” neutral drift dynamics. Independent lineage tracing based on Stmn1 and Ki67 expression confirmed that rapidly cycling IsthSCs maintain the pit-isthmus-neck region. Finally, single-cell RNA sequencing (RNA-seq) analysis is used to define the molecular identity and lineage relationship of a single, cycling, IsthSC population. These observations define the identity and functional behavior of IsthSCs., Graphical Abstract, Highlights • Marker-free lineage tracing reveals two types of stem cells in the gastric corpus • Actively cycling isthmus stem cells follow “punctuated” neutral drift dynamics • Stmn1 and Ki67 lineage tracing confirms the active cycling of isthmus stem cells • Single-cell RNA-seq defines identity and lineage relationship of isthmus stem cells, Using lineage-tracing assays and single-cell gene expression profiling, Han et al. show that the mouse gastric corpus gland is compartmentalized, with base and isthmus regions supported by two separate independent stem cell populations. Isthmus stem cells are rapidly cycling, with a broad expression signature, and undergo “punctuated” neutral drift dynamics.

Precision medicine might be the response to the recent questioning of the use of metformin as an anticancer drug in colorectal cancer (CRC). Thus, in order to establish properly its benefits, metformin application needs to be assayed on the different progression stages of CRC. In this way, intestinal organoids imply a more physiological tool, representing a new therapeutic opportunity for CRC personalized treatment to assay tumor stage-dependent drugs. The previously reported lipid metabolism-related axis, Acyl-CoA synthetases/ Stearoyl-CoA desaturase (ACSLs/SCD), stimulates colon cancer progression and metformin is able to rescue the invasive and migratory phenotype conferred to cancer cells upon this axis overexpression. Therefore, we checked ACSL/SCD axis status, its regulatory miRNAs and the effect of metformin treatment in intestinal organoids with the most common acquired mutations in a sporadic CRC (CRC-like organoids) as a model for specific and personalized treatment. Despite ACSL4 expression is upregulated progressively in CRC-like organoids, metformin is able to downregulate its expression, especially in the first two stages (I, II). Besides, organoids are clearly more sensitive in the first stage (Apc mutated) to metformin than current chemotherapeutic drugs such as fluorouracil (5-FU). Metformin performs an independent "Warburg effect" blockade to cancer progression and is able to reduce crypt stem cell markers expression such as LGR5+. These results suggest a putative increased efficiency of the use of metformin in early stages of CRC than in advanced disease.

Background. Organoid cultures of human pancreatic ductal adenocarcinoma (PDAC) have become a promising tool for tumor subtyping and individualized chemosensitivity testing. PDACs have recently been grouped into different molecular subtypes with clinical impact based on cytokeratin-81 (KRT81) and hepatocyte nuclear factor 1A (HNF1A). However, a suitable antibody for HNF1A is currently unavailable. The present study is aimed at establishing subtyping in PDAC organoids using an alternative marker. Methods. A PDAC organoid biobank was generated from human primary tumor samples containing 22 lines. Immunofluorescence staining was established and done for 10 organoid lines for cystic fibrosis transmembrane conductance regulator (CFTR) and KRT81. Quantitative real-time PCR (qPCR) was performed for CFTR and HNF1A. A chemotherapeutic drug response analysis was done using gemcitabine, 5-FU, oxaliplatin, and irinotecan. Results. A biobank of patient-derived PDAC organoids was established. The efficiency was 71% (22/31) with 68% for surgical resections and 83% for fine needle aspirations. Organoids could be categorized into the established quasimesenchymal, exocrine-like, and classical subtypes based on KRT81 and CFTR immunoreactivity. CFTR protein expression was confirmed on the transcript level. CFTR and HNF1A transcript expression levels positively correlated (n=10; r=0.927; p=0.001). PDAC subtypes of the primary tumors and the corresponding organoid lines were identical for most of the cases analyzed (6/7). Treatment with chemotherapeutic drugs revealed tendencies but no significant differences regarding drug responses. Conclusions. Human PDAC organoids can be classified into known subtypes based on KRT81 and CFTR immunoreactivity. CFTR and HNF1A mRNA levels correlated well. Furthermore, subtype-specific immunoreactivity matched well between PDAC organoids and the respective primary tumor tissue. Subtyping of human PDACs using CFTR might constitute an alternative to HNF1A and should be further investigated.

Organoids are three-dimensional culture systems that resemble their organ of origin, are genetically stable, and can phenocopy diseases. They enable modeling of various cancer entities such as gastric or colorectal cancer, in addition to other gastrointestinal tract diseases such as inflammatory bowel disease. Genetic engineering tools like CRISPR/Cas9 allow their manipulation to repair mutations or unravel gene functions. Individual patient-derived organoids allow to test therapies in vitro before their in vivo application, bringing personalized medicine to a next level. Organoid biobanks can be used to conduct drug screenings and validate biomarkers. Interactions with microbiota can be investigated in realistic in vitro models. Transplantability of genetically engineered organoids opens up new avenues in the tissue engineering research field. Organoid cultures thus represent a versatile system to model diseases and test therapeutic interventions.

Precision medicine might be the response to the recent questioning of the use of metformin as an anticancer drug in colorectal cancer (CRC). Thus, in order to establish properly its benefits, metformin application needs to be assayed on the different progression stages of CRC. In this way, intestinal organoids imply a more physiological tool, representing a new therapeutic opportunity for CRC personalized treatment to assay tumor stage-dependent drugs. The previously reported lipid metabolism-related axis, Acyl-CoA synthetases/ Stearoyl-CoA desaturase (ACSLs/SCD), stimulates colon cancer progression and metformin is able to rescue the invasive and migratory phenotype conferred to cancer cells upon this axis overexpression. Therefore, we checked ACSL/SCD axis status, its regulatory miRNAs and the effect of metformin treatment in intestinal organoids with the most common acquired mutations in a sporadic CRC (CRC-like organoids) as a model for specific and personalized treatment. Despite ACSL4 expression is upregulated progressively in CRC-like organoids, metformin is able to downregulate its expression, especially in the first two stages (I, II). Besides, organoids are clearly more sensitive in the first stage (Apc mutated) to metformin than current chemotherapeutic drugs such as fluorouracil (5-FU). Metformin performs an independent “Warburg effect” blockade to cancer progression and is able to reduce crypt stem cell markers expression such as LGR5+. These results suggest a putative increased efficiency of the use of metformin in early stages of CRC than in advanced disease.

The adult mouse subependymal zone provides a niche for mammalian neural stem cells (NSCs). However, the molecular signature, self-renewal potential, and fate behavior of NSCs remain poorly defined. Here we propose a model in which the fate of active NSCs is coupled to the total number of neighboring NSCs in a shared niche. Using knock-in reporter alleles and single-cell RNA sequencing, we show that the Wnt target Tnfrsf19/Troy identifies both active and quiescent NSCs. Quantitative analysis of genetic lineage tracing of individual NSCs under homeostasis or in response to injury reveals rapid expansion of stem-cell number before some return to quiescence. This behavior is best explained by stochastic fate decisions, where stem-cell number within a shared niche fluctuates over time. Fate mapping proliferating cells using a Ki67(iresCreER) allele confirms that active NSCs reversibly return to quiescence, achieving long-term self-renewal. Our findings suggest a niche-based mechanism for the regulation of NSC fate and number.

The gastric corpus epithelium is the thickest part of the gastro-intestinal tract and is characterized by rapid tissue turnover. Several markers have been proposed for gastric corpus stem cells in both isthmus and base regions. However, the identity of isthmus stem cells (IsthSCs) and the interaction between distinct stem cell populations is still under debate. Here, based on unbiased genetic labeling and biophysical modeling, we show that corpus glands are compartmentalized into two independent zones, with actively-cycling stem cells maintaining the pit-isthmus-neck region and slow-cycling reserve stem cells maintaining the base. Independent lineage tracing based on Stmn1 and Ki67 expression confirmed that rapidly-cycling IsthSCs maintain the pit-isthmus-neck of corpus glands. Finally, single cell RNA-seq analysis is used to define the molecular identity and lineage relationship of a single, cycling IsthSC population. These observations define the identity and functional behavior of a single population of actively-cycling IsthSCs.

Summary The mammalian adult small intestinal epithelium is a rapidly self-renewing tissue that is maintained by a pool of cycling stem cells intermingled with Paneth cells at the base of crypts. These crypt base stem cells exclusively express Lgr5 and require Wnt3 or, in its absence, Wnt2b. However, the Frizzled (Fzd) receptor that transmits these Wnt signals is unknown. We determined the expression profile of Fzd receptors in Lgr5+ stem cells, their immediate daughter cells, and Paneth cells. Here we show Fzd7 is enriched in Lgr5+ stem cells and binds Wnt3 and Wnt2b. Conditional deletion of the Fzd7 gene in adult intestinal epithelium leads to stem cell loss in vivo and organoid death in vitro. Crypts of conventional Fzd7 knockout mice show decreased basal Wnt signaling and impaired capacity to regenerate the epithelium following deleterious insult. These observations indicate that Fzd7 is required for robust Wnt-dependent processes in Lgr5+ intestinal stem cells., Graphical Abstract, Highlights • In the intestinal crypt, Fzd7 expression is enriched in the CBC stem cells • Fzd7 gene deletion leads to the loss of CBC stem cells • Fzd7 is necessary for optimal CBC stem cell function • Fzd7 binds Wnt growth factors that govern CBC function, Stem cells maintain normal turnover and repair of the adult intestine epithelium. Intestinal epithelium stem cells require Wnt growth factors for these processes. Vincan and colleagues report here that Frizzled7 functions as a Wnt receptor in these cells. In the absence of Frizzled7, Wnt-dependent processes in the intestinal epithelium were compromised. Closely related Frizzleds could not compensate for Frizzled7 loss.

Background & Aims Patterns of genetic alterations characterize different molecular subtypes of human gastric cancer. We aimed to establish mouse models of these subtypes. Methods We searched databases to identify genes with unique expression in the stomach epithelium, resulting in the identification of Anxa10. We generated mice with tamoxifen-inducible Cre recombinase (CreERT2) in the Anxa10 gene locus. We created 3 mouse models with alterations in pathways that characterize the chromosomal instability (CIN) and the genomically stable (GS) subtypes of human gastric cancer: Anxa10-CreERT2;KrasG12D/+;Tp53R172H/+;Smad4fl/f (CIN mice), Anxa10-CreERT2;Cdh1fl/fl;KrasG12D/+;Smad4fl/fl (GS-TGBF mice), and Anxa10-CreERT2;Cdh1fl/fl;KrasG12D/+;Apcfl/fl (GS-Wnt mice). We analyzed tumors that developed in these mice by histology for cell types and metastatic potential. We derived organoids from the tumors and tested their response to chemotherapeutic agents and the epithelial growth factor receptor signaling pathway inhibitor trametinib. Results The gastric tumors from the CIN mice had an invasive phenotype and formed liver and lung metastases. The tumor cells had a glandular morphology, similar to human intestinal-type gastric cancer. The gastric tumors from the GS–TGFB mice were poorly differentiated with diffuse morphology and signet ring cells, resembling human diffuse-type gastric cancer. Cells from these tumors were invasive, and mice developed peritoneal carcinomatosis and lung metastases. GS-Wnt mice developed adenomatous tooth-like gastric cancer. Organoids derived from tumors of GS-TGBF and GS-Wnt mice were more resistant to docetaxel, whereas organoids from the CIN tumors were more resistant to trametinib. Conclusions Using a stomach-specific CreERT2 system, we created mice that develop tumors with morphologic similarities to subtypes of human gastric cancer. These tumors have different patterns of local growth, metastasis, and response to therapeutic agents. They can be used to study different subtypes of human gastric cancer., Graphical abstract

The intestine has developed over the last few years into a prime model system for adult stem cell research. Intestinal cells have an average lifetime of 5 days, moving within this time from the bottom of intestinal crypts to the top of villi. This rapid self-renewal capacity combined with an easy to follow (mostly) unidirectional movement of cells offers an ideal site to conduct adult stem cell research. The delineation of the active pathways in the intestinal epithelium together with the development of molecular techniques to prove stemness laid the grounds for the identification of the intestinal stem cell. In vitro systems and transgenic mouse models broaden our knowledge on the role of the stem cell niche and those cells that reestablish homeostasis after perturbation of the system. These insights expedited also research on the role of normal adult stem cells in cancer initiation and the factors influencing the maintenance of cancer stem cells.

SummaryProliferation of the self-renewing epithelium of the gastric corpus occurs almost exclusively in the isthmus of the glands, from where cells migrate bidirectionally toward pit and base. The isthmus is therefore generally viewed as the stem cell zone. We find that the stem cell marker Troy is expressed at the gland base by a small subpopulation of fully differentiated chief cells. By lineage tracing with a Troy-eGFP-ires-CreERT2 allele, single marked chief cells are shown to generate entirely labeled gastric units over periods of months. This phenomenon accelerates upon tissue damage. Troy+ chief cells can be cultured to generate long-lived gastric organoids. Troy marks a specific subset of chief cells that display plasticity in that they are capable of replenishing entire gastric units, essentially serving as quiescent “reserve” stem cells. These observations challenge the notion that stem cell hierarchies represent a “one-way street.”

// Yuliya Kurlishchuk 1, 2 , Bozhena Vynnytska-Myronovska 1, 2, 6 , Philipp Grosse-Gehling 1 , Yaroslav Bobak 2 , Friederike Manig 1, 3 , Oleg Chen 1, 2 , Sebastian R. Merker 4 , Thomas Henle 3 , Steffen Lock 1 , Daniel E. Stange 4 , Oleh Stasyk 2 , Leoni A. Kunz-Schughart 1, 5 1 OncoRay–National Center for Radiation Research in Oncology, Faculty of Medicine and University Hospital Carl Gustav Carus, TU Dresden and Helmholtz-Zentrum Dresden-Rossendorf, Institute of Radiooncology, Dresden, Germany 2 Department of Cell Signaling, Institute of Cell Biology, National Academy of Sciences of Ukraine, Lviv, Ukraine 3 Institute of Food Chemistry, TU Dresden, Dresden, Germany 4 Department of Gastrointestinal, Thoracic and Vascular Surgery, University Hospital Carl Gustav Carus, TU Dresden, Dresden, Germany 5 Department of Oncology, University of Oxford, Old Road Campus Research Building, Oxford, UK 6 Current address: Clinic of Urology and Pediatric Urology, Saarland University Medical Center, Homburg/Saar, Germany Correspondence to: Leoni A. Kunz-Schughart, email: leoni.kunz-schughart@oncoray.de Keywords: arginine deprivation, ASS1 expression, colorectal cancer (CRC), CRC spheroids, normal colon organoids Received: January 27, 2016 Accepted: September 19, 2016 Published: September 28, 2016 ABSTRACT The moderate anticancer effect of arginine deprivation in clinical trials has been linked to an induced argininosuccinate synthetase (ASS1) expression in initially ASS1-negative tumors, and ASS1-positive cancers are anticipated as non-responders. Our previous studies indicated that arginine deprivation and low doses of the natural arginine analog canavanine can enhance radioresponse. However, the efficacy of the proposed combination in the presence of extracellular citrulline, the substrate for arginine synthesis by ASS1, remains to be elucidated, in particular for malignant cells with positive and/or inducible ASS1 as in colorectal cancer (CRC). Here, the physiological citrulline concentration of 0.05 mM was insufficient to overcome cell cycle arrest and radiosensitization triggered by arginine deficiency. Hyperphysiological citrulline (0.4 mM) did not entirely compensate for the absence of arginine and significantly decelerated cell cycling. Similar levels of canavanine-induced apoptosis were detected in the absence of arginine regardless of citrulline supplementation both in 2-D and advanced 3-D assays, while normal colon epithelial cells in organoid/colonosphere culture were unaffected. Notably, canavanine tremendously enhanced radiosensitivity of arginine-starved 3-D CRC spheroids even in the presence of hyperphysiological citrulline. We conclude that the novel combinatorial targeting strategy of metabolic-chemo-radiotherapy has great potential for the treatment of malignancies with inducible ASS1 expression.

Crosstalk between the Notch and wingless-type MMTV integration site (WNT) signaling pathways has been investigated for many developmental processes. However, this negative correlation between Notch and WNT/β-catenin signaling activity has been studied primarily in normal developmental and physiological processes in which negative feedback loops for both signaling pathways are intact. We found that Notch1 signaling retained the capability of suppressing the expression of WNT target genes in colorectal cancers even when β-catenin destruction by the adenomatous polyposis coli (APC) complex was disabled. Activation of Notch1 converted high-grade adenoma into low-grade adenoma in an Apcmin mouse colon cancer model and suppressed the expression of WNT target genes in human colorectal cancer cells through epigenetic modification recruiting histone methyltransferase SET domain bifurcated 1 (SETDB1). Extensive microarray analysis of human colorectal cancers also showed a negative correlation between the Notch1 target gene, Notch-regulated ankyrin repeat protein 1 (NRARP), and WNT target genes. Notch is known to be a strong promoter of tumor initiation, but here we uncovered an unexpected suppressive role of Notch1 on WNT/β-catenin target genes involved in colorectal cancer.

Cancer Stem Cells in Color One of the liveliest debates in contemporary cancer research centers on whether cancer stem cells (CSCs) exist and, if so, how these cells are defined phenotypically. CSCs are hypothesized to be a small population of cells within a tumor that are endowed with the unique capacity to drive tumor growth—a scenario that in principle would offer important therapeutic opportunities. By studying mice expressing multicolor reporter genes, Schepers et al. (p. 730 , published online 1 August) were able to visualize and monitor the fate of a candidate stem cell for intestinal adenomas, an early stage of cancer. This “lineage tracing” analysis suggests that tumor cells expressing the intestinal crypt stem cell marker Lgr5 (leucine-rich repeat containing G protein–coupled receptor 5) are the cells that fuel the growth of intestinal adenomas.

Two types of stem cells are currently defined in small intestinal crypts: cycling crypt base columnar (CBC) cells and quiescent ‘+4’ cells. Here, we combine transcriptomics with proteomics to define a definitive molecular signature for Lgr5 + CBC cells. Transcriptional profiling of FACS‐sorted Lgr5 + stem cells and their daughters using two microarray platforms revealed an mRNA stem cell signature of 384 unique genes. Quantitative mass spectrometry on the same cell populations identified 278 proteins enriched in intestinal stem cells. The mRNA and protein data sets showed a high level of correlation and a combined signature of 510 stem cell‐enriched genes was defined. Spatial expression patterns were further characterized by mRNA in‐situ hybridization, revealing that approximately half of the genes were expressed in a gradient with highest levels at the crypt bottom, while the other half was expressed uniquely in Lgr5 + stem cells. Lineage tracing using a newly established knock‐in mouse for one of the signature genes, Smoc2 , confirmed its stem cell specificity. Using this resource, we find—and confirm by independent approaches—that the proposed quiescent/‘+4’ stem cell markers Bmi1 , Tert , Hopx and Lrig1 are robustly expressed in CBC cells.

Hair Today, Skin Tomorrow The epidermis of mammals contains hair follicles, sebaceous glands, and interfollicular epidermis, but it has not been clear how the development and repair of these structures is regulated. Snippert et al. (p. 1385 ) show that a stem-cell cluster in the hair follicle, characterized by the expression of Lgr6, a close homolog of the Lgr5 marker for stem cells in the small intestine and colon, resides directly above the hair bulge and gives rise to all cell lineages of the skin. Skin wounds in adult mice are repaired by Lgr6 stem cells in the hair follicles that flank the damage. After hair morphogenesis, Lgr6 stem cells give rise to epidermal and sebaceous gland lineages to generate fully differentiated new skin.

SummaryThe study of gastric epithelial homeostasis and cancer has been hampered by the lack of stem cell markers and in vitro culture methods. The Wnt target gene Lgr5 marks stem cells in the small intestine, colon, and hair follicle. Here, we investigated Lgr5 expression in the stomach and assessed the stem cell potential of the Lgr5+ve cells by using in vivo lineage tracing. In neonatal stomach, Lgr5 was expressed at the base of prospective corpus and pyloric glands, whereas expression in the adult was predominantly restricted to the base of mature pyloric glands. Lineage tracing revealed these Lgr5+ve cells to be self-renewing, multipotent stem cells responsible for the long-term renewal of the gastric epithelium. With an in vitro culture system, single Lgr5+ve cells efficiently generated long-lived organoids resembling mature pyloric epithelium. The Lgr5 stem cell marker and culture method described here will be invaluable tools for accelerating research into gastric epithelial renewal, inflammation/infection, and cancer.

SummaryThe small intestinal epithelium is the most rapidly self-renewing tissue of mammals. Proliferative cells are confined to crypts, while differentiated cell types predominantly occupy the villi. We recently demonstrated the existence of a long-lived pool of cycling stem cells defined by Lgr5 expression and intermingled with post-mitotic Paneth cells at crypt bottoms. We have now determined a gene signature for these Lgr5 stem cells. One of the genes within this stem cell signature is the Wnt target Achaete scute-like 2 (Ascl2). Transgenic expression of the Ascl2 transcription factor throughout the intestinal epithelium induces crypt hyperplasia and ectopic crypts on villi. Induced deletion of the Ascl2 gene in adult small intestine leads to disappearance of the Lgr5 stem cells within days. The combined results from these gain- and loss-of-function experiments imply that Ascl2 controls intestinal stem cell fate.

Background & Aims Prominin-1(Prom1)/CD133 is used, alone or in combination with other cell surface markers, to identify and isolate stem cells from various adult tissues. We recently identified leucine-rich-repeat-containing G-protein-coupled receptor 5 (Lgr5) as a marker of the intestinal stem cells from which all cellular lineages of the gastrointestinal epithelium are derived. To determine whether there is a relationship between these markers, we investigated the intestinal expression pattern of Prom1/CD133 and created knock-in mice to visualize and trace Prom1 + cells. Methods We analyzed Prom1 mRNA and protein expression among stem cells within intestinal crypts. Prom1/CD133 knock-in mice ( Prom1 -mCherry-IRES-CreERT2 KI) were generated that express a fusion of red fluorescent protein mCherry with the C-terminus of Prom1. The knock-in allele also contains the tamoxifen-inducible CreERT2 recombinase, allowing for genetic tracing of progeny derived from Prom1-positive cells. Results In the small intestine, Prom1 mRNA was detected throughout the lower half of crypts and was not restricted to the rare stem cells that are sandwiched between Paneth cells. Prom1 protein was detected at the apical membranes of Lgr5 + intestinal stem cells, but also on the transit-amplifying progenitors located above the Paneth cells. Analyses of the Prom1 -mCherry-IRES-CreERT2 KI mice showed that Prom1 is not exclusively expressed in Lgr5 + intestinal stem cells but marks a much larger stem cell/transit-amplifying progenitor compartment. Conclusions Prom-1 marks intestinal stem cells, as well as transit-amplifying progenitors, so it is not a specific marker for Lgr5 + intestinal stem cells.

The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. We have recently demonstrated the presence of about six cycling Lgr5(+) stem cells at the bottoms of small-intestinal crypts. Here we describe the establishment of long-term culture conditions under which single crypts undergo multiple crypt fission events, while simultanously generating villus-like epithelial domains in which all differentiated cell types are present. Single sorted Lgr5(+) stem cells can also initiate these cryptvillus organoids. Tracing experiments indicate that the Lgr5(+) stem-cell hierarchy is maintained in organoids. We conclude that intestinal cryptvillus units are self-organizing structures, which can be built from a single stem cell in the absence of a non-epithelial cellular niche.

Defensins are endogenous antimicrobial peptides that protect the intestinal mucosa against bacterial invasion. It has been suggested that deficient defensin expression may underlie the chronic inflammation of Crohn disease (CD). The DNA copy number of the beta-defensin gene cluster on chromosome 8p23.1 is highly polymorphic within the healthy population, which suggests that the defective beta-defensin induction in colonic CD could be due to low beta-defensin-gene copy number. Here, we tested this hypothesis, using genomewide DNA copy number profiling by array-based comparative genomic hybridization and quantitative polymerase-chain-reaction analysis of the human beta-defensin 2 (HBD-2) gene. We showed that healthy individuals, as well as patients with ulcerative colitis, have a median of 4 (range 2-10) HBD-2 gene copies per genome. In a surgical cohort with ileal or colonic CD and in a second large cohort with inflammatory bowel diseases, those with ileal resections/disease exhibited a normal median HBD-2 copy number of 4, whereas those with colonic CD had a median of only 3 copies per genome (P=.008 for the surgical cohort; P=.032 for the second cohort). Overall, the copy number distribution in colonic CD was shifted to lower numbers compared with controls (P=.002 for both the surgical cohort and the cohort with inflammatory bowel diseases). Individuals withor = 3 copies have a significantly higher risk of developing colonic CD than did individuals withor = 4 copies (odds ratio 3.06; 95% confidence interval 1.46-6.45). An HBD-2 gene copy number of4 was associated with diminished mucosal HBD-2 mRNA expression (P=.033). In conclusion, a lower HBD-2 gene copy number in the beta-defensin locus predisposes to colonic CD, most likely through diminished beta-defensin expression.