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Reprogrammed metabolism is a hallmark of colorectal cancer (CRC). CRC cells are geared toward rapid proliferation, requiring nutrients and the removal of cellular waste in nutrient-poor environments. Intestinal stem cells (ISCs), the primary cell of origin for CRCs, must adapt their metabolism along the adenoma-carcinoma sequence to the unique features of their complex microenvironment that include interactions with intestinal epithelial cells, immune cells, stromal cells, commensal microbes, and dietary components. Emerging evidence implicates modifiable risk factors related to the environment, such as diet, as important in CRC pathogenesis. Here, we focus on describing the metabolism of ISCs, diets that influence CRC initiation, CRC genetics and metabolism, and the tumor microenvironment. The mechanistic links between environmental factors, metabolic adaptations, and the tumor microenvironment in enhancing or supporting CRC tumorigenesis are becoming better understood. Thus, greater knowledge of CRC metabolism holds promise for improved prevention and treatment. Expected final online publication date for the

5-fluorouracil (5-FU) is a successful and broadly used anti-cancer therapeutic. A major mechanism of action of 5-FU is thought to be through thymidylate synthase (TYMS) inhibition resulting in dTTP depletion and activation of the DNA damage response. This suggests that 5-FU should synergize with other DNA damaging agents. However, we found that combinations of 5-FU and oxaliplatin or irinotecan failed to display any evidence of synergy in clinical trials, and resulted in sub-additive killing in a panel of colorectal cancer (CRC) cell lines. In seeking to understand this antagonism, we unexpectedly found that an RNA damage response during ribosome biogenesis dominates the drug’s efficacy in tumor types for which 5-FU shows clinical benefit. 5-FU has an inherent bias for RNA incorporation, and blocking this greatly reduced drug-induced lethality, indicating that accumulation of damaged RNA is more deleterious than the lack of new RNA synthesis. Using 5-FU metabolites that specifically incorporate into either RNA or DNA revealed that CRC cell lines and patient-derived colorectal cancer organoids are inherently more sensitive to RNA damage. This difference held true in cell lines from other tissues in which 5-FU has shown clinical utility, whereas cell lines from tumor tissues that lack clinical 5-FU responsiveness typically showed greater sensitivity to the drug’s DNA damage effects. Analysis of changes in the phosphoproteome and ubiquitinome shows RNA damage triggers the selective ubiquitination of multiple ribosomal proteins leading to autophagy-dependent rRNA catabolism and proteasome-dependent degradation of ubiquitinated ribosome proteins. Further, RNA damage response to 5-FU is selectively enhanced by compounds that promote ribosome biogenesis, such as KDM2A inhibitors. These results demonstrate the presence of a strong RNA damage response linked to apoptotic cell death, with clear utility of combinatorially targeting this response in cancer therapy.

Supplementary Figure from Transcriptome-wide In Vitro Effects of Aspirin on Patient-derived Normal Colon Organoids

Supplementary Data from Transcriptome-wide In Vitro Effects of Aspirin on Patient-derived Normal Colon Organoids

Improved biomarkers are needed for early cancer detection, risk stratification, treatment selection, and monitoring treatment response. While proteins can be useful blood-based biomarkers, many have limited sensitivity or specificity for these applications. Long INterspersed Element-1 (LINE-1, L1) open reading frame 1 protein (ORF1p) is a transposable element protein overexpressed in carcinomas and high-risk precursors during carcinogenesis with negligible detectable expression in corresponding normal tissues, suggesting ORF1p could be a highly specific cancer biomarker. To explore the potential of ORF1p as a blood-based biomarker, we engineered ultrasensitive digital immunoassays that detect mid-attomolar (10-17M) ORF1p concentrations in patient plasma samples across multiple cancers with high specificity. Plasma ORF1p shows promise for early detection of ovarian cancer, improves diagnostic performance in a multi-analyte panel, and provides early therapeutic response monitoring in gastric and esophageal cancers. Together, these observations nominate ORF1p as a multi-cancer biomarker with potential utility for disease detection and monitoring.Statement of SignificanceLINE-1 ORF1p transposon protein is pervasively expressed in many cancers and a highly specific biomarker of multiple common, lethal carcinomas and their high-risk precursors in tissue and blood. Ultrasensitive ORF1p assays from as little as 25 μL plasma are novel, rapid, cost-effective tools in cancer detection and monitoring.

BACKGROUND Obesity increases the colorectal cancer risk, in part by elevating colonic proinflammatory cytokines. Curcumin (CUR) and supplemental vitamin B-6 each suppress colonic inflammation. OBJECTIVES We examined whether the combination of CUR and vitamin B-6 amplifies each supplement's effects and thereby suppress obesity-promoted tumorigenesis. METHODS Male Friend Virus B (FVB) mice (4-week-old; n = 110) received 6 weekly injections of azoxymethane beginning 1 week after arrival. Thereafter, they were randomized to receive a low-fat diet (10% energy from fat), a high-fat diet (HFD; 60% energy from fat), a HFD containing 0.2% CUR, a HFD containing supplemental vitamin B-6 (24 mg pyridoxine HCl/kg), or a HFD containing both CUR and supplemental vitamin B-6 (C + B) for 15 weeks. Colonic inflammation, assessed by fecal calprotectin, and tumor metrics were the primary endpoints. The anti-inflammatory efficacy of the combination was also determined in human colonic organoids. RESULTS HFD-induced obesity produced a 2.6-fold increase in plasma IL-6 (P

Immune evasion is a hallmark of cancer and therapies that restore immune surveillance have proven highly effective in cancers with high tumor mutation burden (TMB) (for example, those with microsatellite instability). Whether low TMB cancers, which are largely refractory to immunotherapy, harbor potentially immunogenic neoantigens remains unclear. Here, we show that tumors from all patients with microsatellite stable colorectal cancer express clonal predicted neoantigens despite low TMB. Unexpectedly, these neoantigens are broadly expressed at lower levels compared to those in colorectal cancer with microsatellite instability. Using a versatile platform for modulating neoantigen expression in colorectal cancer organoids and transplantation into the distal colon of mice, we show that low expression precludes productive cross-priming and drives immediate T-cell dysfunction. Notably, experimental or therapeutic rescue of priming rendered T cells capable of controlling tumors with low neoantigen expression. These findings underscore a critical role of neoantigen expression level in immune evasion and therapy response. Jacks and colleagues demonstrate the effects of neoantigen expression level on T-cell priming and immune surveillance during tumor development and progression and explore implications for immunotherapies, using in vivo models of colorectal cancer.

Mechanisms underlying aspirin chemoprevention of colorectal cancer remain unclear. Prior studies have been limited because of the inability of preclinical models to recapitulate human normal colon epithelium or cellular heterogeneity present in mucosal biopsies. To overcome some of these obstacles, we performed in vitro aspirin treatment of colon organoids derived from normal mucosal biopsies to reveal transcriptional networks relevant to aspirin chemoprevention. Colon organoids derived from 38 healthy individuals undergoing endoscopy were treated with 50 μmol/L aspirin or vehicle control for 72 hours and subjected to bulk RNA sequencing. Paired regression analysis using DESeq2 identified differentially expressed genes (DEG) associated with aspirin treatment. Cellular composition was determined using CIBERSORTx. Aspirin treatment was associated with 1,154 significant (q < 0.10) DEGs prior to deconvolution. We provide replication of these findings in an independent population-based RNA-sequencing dataset of mucosal biopsies (BarcUVa-Seq), where a significant enrichment for overlap of DEGs was observed (P < 2.2E−16). Single-cell deconvolution revealed changes in cell composition, including a decrease in transit-amplifying cells following aspirin treatment (P = 0.01). Following deconvolution, DEGs included novel putative targets for aspirin such as TRABD2A (q = 0.055), a negative regulator of Wnt signaling. Weighted gene co-expression network analysis identified 12 significant modules, including two that contained hubs for EGFR and PTGES2, the latter being previously implicated in aspirin chemoprevention. In summary, aspirin treatment of patient-derived colon organoids using physiologically relevant doses resulted in transcriptome-wide changes that reveal altered cell composition and improved understanding of transcriptional pathways, providing novel insight into its chemopreventive properties. Prevention Relevance: Numerous studies have highlighted a role for aspirin in colorectal cancer chemoprevention, though the mechanisms driving this association remain unclear. We addressed this by showing that aspirin treatment of normal colon organoids diminished the transit-amplifying cell population, inhibited prostaglandin synthesis, and dysregulated expression of novel genes implicated in colon tumorigenesis.

The clinical significance of severe acute respiratory syndrome coronavirus 2 (SARS CoV-2) RNA in stool remains uncertain. We found that extrapulmonary dissemination of infection to the gastrointestinal tract, assessed by the presence of SARS-CoV-2 RNA in stool, is associated with decreased coronavirus disease 2019 (COVID-19) survival. Measurement of SARS-CoV-2 RNA in stool may have utility for clinical risk assessment.

Aneuploidy, defined as whole chromosome gains and losses, is associated with poor patient prognosis in many cancer types. However, the condition causes cellular stress and cell cycle delays, foremost in G1 and S phase. Here, we investigate how aneuploidy causes both slow proliferation and poor disease outcome. We test the hypothesis that aneuploidy brings about resistance to chemotherapies because of a general feature of the aneuploid condition—G1 delays. We show that single chromosome gains lead to increased resistance to the frontline chemotherapeutics cisplatin and paclitaxel. Furthermore, G1 cell cycle delays are sufficient to increase chemotherapeutic resistance in euploid cells. Mechanistically, G1 delays increase drug resistance to cisplatin and paclitaxel by reducing their ability to damage DNA and microtubules, respectively. Finally, we show that our findings are clinically relevant. Aneuploidy correlates with slowed proliferation and drug resistance in the Cancer Cell Line Encyclopedia (CCLE) dataset. We conclude that a general and seemingly detrimental effect of aneuploidy, slowed proliferation, provides a selective benefit to cancer cells during chemotherapy treatment.

In response to DNA damage, a synthetic lethal relationship exists between the cell cycle checkpoint kinase MK2 and the tumor suppressor p53. Here, we describe the concept of augmented synthetic lethality (ASL): depletion of a third gene product enhances a pre-existing synthetic lethal combination. We show that loss of the DNA repair protein XPA markedly augments the synthetic lethality between MK2 and p53, enhancing anti-tumor responses alone and in combination with cisplatin chemotherapy. Delivery of siRNA-peptide nanoplexes co-targeting MK2 and XPA to pre-existing p53-deficient tumors in a highly aggressive, immunocompetent mouse model of lung adenocarcinoma improves long-term survival and cisplatin response beyond those of the synthetic lethal p53 mutant/MK2 combination alone. These findings establish a mechanism for co-targeting DNA damage-induced cell cycle checkpoints in combination with repair of cisplatin-DNA lesions in vivo using RNAi nanocarriers, and motivate further exploration of ASL as a generalized strategy to improve cancer treatment., Cell cycle checkpoint kinase, MK2, is in synthetic relationship with p53 in the DNA damage response to chemotherapeutic agents. Here, the authors report XPA as a third gene in which simultaneous targeting of MK2 and XPA further enhances sensitivity to cisplatin in p53-deficient tumours.

SUMMARYIntestinal stem cells (ISCs) depend on niche factors for their proper function. However, the source of these ISC niche factors and how they support ISCs remain controversial due to their redundant expression patterns. Here, we report that the maintenance of ISCs depends on both lymphatic endothelial cells (LECs) and Rspo3+Grem1+ fibroblasts (RGFs). We found that LECs are surrounded by RGFs and located in close proximity to Lgr5+ ISCs. RSPO3 production is restricted to LECs and RGFs and they can partially compensate for each other; however, RSPO3 loss in both of LECs and RGFs drastically compromises ISC numbers, villi length, and repair after irradiation-induced injury. Mechanistically, irradiation-induced damage expands LEC and RGF numbers and enhances the latter’s generation of RSPO3 through IL-1 receptor activation. We propose that LECs represent a novel component of the ISC niche, which together with RGFs, provide essential RSPO3 to sustain ISCs in homeostasis and regeneration.

Description, Small cell size preserves the function of hematopoietic stem cells (HSCs); HSC enlargement during aging causes their dysfunction., Stem cells are remarkably small. Whether small size is important for stem cell function is unknown. We find that hematopoietic stem cells (HSCs) enlarge under conditions known to decrease stem cell function. This decreased fitness of large HSCs is due to reduced proliferation and was accompanied by altered metabolism. Preventing HSC enlargement or reducing large HSCs in size averts the loss of stem cell potential under conditions causing stem cell exhaustion. Last, we show that murine and human HSCs enlarge during aging. Preventing this age-dependent enlargement improves HSC function. We conclude that small cell size is important for stem cell function in vivo and propose that stem cell enlargement contributes to their functional decline during aging.

A cell's phenotype and function are influenced by dynamic interactions with its microenvironment. To examine cellular spatiotemporal activity, we developed SPACECAT-Spatially PhotoActivatable Color Encoded Cell Address Tags-to annotate, track, and isolate cells while preserving viability. In SPACECAT, samples are stained with photocaged fluorescent molecules, and cells are labeled by uncaging those molecules with user-patterned near-UV light. SPACECAT offers single-cell precision and temporal stability across diverse cell and tissue types. Illustratively, we target crypt-like regions in patient-derived intestinal organoids to enrich for stem-like and actively mitotic cells, matching literature expectations. Moreover, we apply SPACECAT to ex vivo tissue sections from four healthy organs and an autochthonous lung tumor model. Lastly, we provide a computational framework to identify spatially-biased transcriptome patterns and enriched phenotypes. This minimally perturbative and broadly applicable method links cellular spatiotemporal and/or behavioral phenotypes with diverse downstream assays, enabling insights into the connections between tissue microenvironments and (dys)function.

A cell’s phenotype and function are influenced by dynamic interactions with its microenvironment. To examine cellular spatiotemporal activity, we developed SPACECAT—Spatially PhotoActivatable Color Encoded Cell Address Tags—to annotate, track, and isolate cells while preserving viability. In SPACECAT, samples are stained with photocaged fluorescent molecules, and cells are labeled by uncaging those molecules with user-patterned near-UV light. SPACECAT offers single-cell precision and temporal stability across diverse cell and tissue types. Illustratively, we target crypt-like regions in patient-derived intestinal organoids to enrich for stem-like and actively mitotic cells, matching literature expectations. Moreover, we apply SPACECAT to ex vivo tissue sections from four healthy organs and an autochthonous lung tumor model. Lastly, we provide a computational framework to identify spatially-biased transcriptome patterns and enriched phenotypes. This minimally perturbative and broadly applicable method links cellular spatiotemporal and/or behavioral phenotypes with diverse downstream assays, enabling insights into the connections between tissue microenvironments and (dys)function., Spatial transcriptomics aims to pair omic data with tissue structure. Here the authors report Spatially PhotoActivatable Colour Encoded Cell Address Tags (SPACECAT) to track and isolate live cells by location; this enables spatially informed downstream assays like scRNA-seq and flow cytometry.

Summary The CD155/TIGIT axis can be co-opted during immune evasion in chronic viral infections and cancer. Pancreatic adenocarcinoma (PDAC) is a highly lethal malignancy, and immune-based strategies to combat this disease have been largely unsuccessful to date. We corroborate prior reports that a substantial portion of PDAC harbors predicted high-affinity MHC class I-restricted neoepitopes and extend these findings to advanced/metastatic disease. Using multiple preclinical models of neoantigen-expressing PDAC, we demonstrate that intratumoral neoantigen-specific CD8+ T cells adopt multiple states of dysfunction, resembling those in tumor-infiltrating lymphocytes of PDAC patients. Mechanistically, genetic and/or pharmacologic modulation of the CD155/TIGIT axis was sufficient to promote immune evasion in autochthonous neoantigen-expressing PDAC. Finally, we demonstrate that the CD155/TIGIT axis is critical in maintaining immune evasion in PDAC and uncover a combination immunotherapy (TIGIT/PD-1 co-blockade plus CD40 agonism) that elicits profound anti-tumor responses in preclinical models, now poised for clinical evaluation.

Purpose: There is an unmet need for identifying novel biomarkers in Barrett's esophagus that could stratify patients with regards to neoplastic progression. We investigate the expression patterns of extracellular matrix (ECM) molecules in Barrett's esophagus and Barrett's esophagus–related neoplasia, and assess their value as biomarkers for the diagnosis of Barrett's esophagus–related neoplasia and to predict neoplastic progression. Experimental Design: Gene-expression analyses of ECM matrisome gene sets were performed using publicly available data on human Barrett's esophagus, Barrett's esophagus–related dysplasia, esophageal adenocarcinoma (ADCA) and normal esophagus. Immunohistochemical expression of basement membrane (BM) marker agrin (AGRN) and p53 was analyzed in biopsies of Barrett's esophagus–related neoplasia from 321 patients in three independent cohorts. Results: Differential gene-expression analysis revealed significant enrichment of ECM matrisome gene sets in dysplastic Barrett's esophagus and ADCA compared with controls. Loss of BM AGRN expression was observed in both Barrett's esophagus–related dysplasia and ADCA. The mean AGRN loss in Barrett's esophagus glands was significantly higher in Barrett's esophagus–related dysplasia and ADCA compared with non-dysplastic Barrett's esophagus (NDBE; P < 0.001; specificity = 82.2% and sensitivity = 96.4%). Loss of AGRN was significantly higher in NDBE samples from progressors compared with non-progressors (P < 0.001) and identified patients who progressed to advanced neoplasia with a specificity of 80.2% and sensitivity of 54.8%. Moreover, the combination of AGRN loss and abnormal p53 staining identified progression to Barrett's esophagus–related advanced neoplasia with a specificity and sensitivity of 86.5% and 58.7%. Conclusions: We highlight ECM changes during Barrett's esophagus progression to neoplasia. BM AGRN loss is a novel diagnostic biomarker that can identify patients with NDBE at increased risk of developing advanced neoplasia.

In vitro, cell cultures are essential tools in the study of intestinal function and disease. For the past few decades, monolayer cellular cultures, such as cancer cell lines or immortalized cell lines, have been widely applied in gastrointestinal research. Recently, the development of three-dimensional cultures known as organoids has permitted the growth of normal crypt-villus units that recapitulate many aspects of intestinal physiology. Organoid culturing has also been applied to study gastrointestinal diseases, intestinal-microbe interactions, and colorectal cancer. These models are amenable to CRISPR gene editing and drug treatments, including high-throughput small-molecule testing. Three-dimensional intestinal cultures have been transplanted into mice to develop versatile in vivo models of intestinal disease, particularly cancer. Limitations of currently available organoid models include cost and challenges in modeling nonepithelial intestinal cells, such as immune cells and the microbiota. Here, we describe the development of organoid models of intestinal biology and the applications of organoids for study of the pathophysiology of intestinal diseases and cancer.

AimsThe histological distinction of intrahepatic cholangiocarcinoma (ICC) from metastatic adenocarcinoma remains a challenge. The primary goal was to evaluate the diagnostic value of morphology and albumin expression in the diagnosis of ICC.MethodsWe evaluated morphological patterns in 120 ICCs and 677 non-hepatic adenocarcinomas and performed in situ hybridisation (ISH) stain for albumin in the former cohort (retrospective cohort). We also identified 119 samples from primary and metastatic lesions, the validation cohort, in which albumin ISH was performed as part of the diagnostic workup. Targeted sequencing was performed on selected cases. We also mined existing expression profiling data including cases from The Cancer Genome Atlas (TCGA) (41 760 unique samples).ResultsIn the retrospective cohort, 45% of ICCs and IDH mutations and FGFR2 fusions) were identified in 31% of ICCs (10 of 32). An analysis of the TCGA data validated the specificity of the albumin assay.ConclusionsThe cholangiolar pattern and albumin RNA ISH distinguishes ICC from metastatic adenocarcinoma with high specificity. Given the high prevalence of targetable mutations in ICC, albumin RNA ISH is an essential component in the workup of tumours of uncertain origin. A specific diagnosis of ICC could trigger molecular testing and uncover targetable genetic alterations.

Tumors display increased uptake and processing of nutrients to fulfill the demands of rapidly proliferating cancer cells. Seminal studies have shown that the proto-oncogene MYC promotes metabolic reprogramming by altering glutamine uptake and metabolism in cancer cells. How MYC regulates the metabolism of other amino acids in cancer is not fully understood. Using high-performance liquid chromatography (HPLC)-tandem mass spectrometry (LC-MS/MS), we found that MYC increased intracellular levels of tryptophan and tryptophan metabolites in the kynurenine pathway. MYC induced the expression of the tryptophan transporters SLC7A5 and SLC1A5 and the enzyme arylformamidase (AFMID), involved in the conversion of tryptophan into kynurenine. SLC7A5, SLC1A5, and AFMID were elevated in colon cancer cells and tissues, and kynurenine was significantly greater in tumor samples than in the respective adjacent normal tissue from patients with colon cancer. Compared with normal human colonic epithelial cells, colon cancer cells were more sensitive to the depletion of tryptophan. Blocking enzymes in the kynurenine pathway caused preferential death of established colon cancer cells and transformed colonic organoids. We found that only kynurenine and no other tryptophan metabolite promotes the nuclear translocation of the transcription factor aryl hydrocarbon receptor (AHR). Blocking the interaction between AHR and kynurenine with CH223191 reduced the proliferation of colon cancer cells. Therefore, we propose that limiting cellular kynurenine or its downstream targets could present a new strategy to reduce the proliferation of MYC-dependent cancer cells.

A decline in stem cell function impairs tissue regeneration during ageing, but the role of the stem-cell-supporting niche in ageing is not well understood. The small intestine is maintained by actively cycling intestinal stem cells that are regulated by the Paneth cell niche(1,2). Here we show that the regenerative potential of human and mouse intestinal epithelium diminishes with age owing to defects in both stem cells and their niche. The functional decline was caused by a decrease in stemness-maintaining Wnt signalling due to production of Notum, an extracellular Wnt inhibitor, in aged Paneth cells. Mechanistically, high activity of mammalian target of rapamycin complex 1 (mTORC1) in aged Paneth cells inhibits activity of peroxisome proliferator activated receptor alpha (PPAR-alpha)(3), and lowered PPAR-a activity increased Notum expression. Genetic targeting of Notum or Wnt supplementation restored function of aged intestinal organoids. Moreover, pharmacological inhibition of Notum in mice enhanced the regenerative capacity of aged stem cells and promoted recovery from chemotherapy-induced damage. Our results reveal a role of the stem cell niche in ageing and demonstrate that targeting of Notum can promote regeneration of aged tissues.

Little is known about how the fibrotic gut stiffening caused by inflammatory bowel diseases (IBD) directs the fate of intestinal stem cells (ISCs). To address this question we first developed a novel long-term culture of quasi-3D gut organoids plated on hydrogel matrix of varying stiffness. Stiffening from 0.6kPa to 9.6kPa significantly reduces Lgr5high ISCs and Ki67+ progenitor cells while promoting their differentiation towards goblet cells. These stiffness-driven events are attributable to YAP nuclear translocation. Matrix stiffening also extends the expression of the stemness marker Olfactomedin 4 (Olfm4) into villus-like regions, mediated by cytoplasmic YAP. We next used single-cell RNA sequencing to generate for the first time the stiffness-regulated transcriptional signatures of ISCs and their differentiated counterparts. These signatures confirm the impact of stiffening on ISC fate and additionally suggest a stiffening-induced switch in metabolic phenotype, from oxidative phosphorylation to glycolysis. Finally, we used colon samples from IBD patients as well as chronic colitis murine models to confirm the in vivo stiffening-induced epithelial deterioration similar to that observed in vitro. Together, these results demonstrate stiffness-dependent ISC reprograming wherein YAP nuclear translocation diminishes ISCs and Ki67+ progenitors and drives their differentiation towards goblet cells, suggesting stiffening as potential target to mitigate gut epithelial deterioration during IBD.

SummaryDoes fibrotic gut stiffening caused by inflammatory bowel diseases (IBD) direct the fate of intestinal stem cells (ISCs)? To address this question we first developed a novel long-term culture of quasi-3D gut organoids plated on hydrogel matrix of varying stiffness. Stiffening from 0.6kPa to 9.6kPa significantly reduces Lgr5high ISCs and Ki67+ progenitor cells while promoting their differentiation towards goblet cells. These stiffness-driven events are attributable to YAP nuclear translocation. Matrix stiffening also extends the expression of the stemness marker Olfactomedin 4 (Olfm4) into villus-like regions, mediated by cytoplasmic YAP. We next used single-cell RNA sequencing to generate for the first time the stiffness-regulated transcriptional signatures of ISCs and their differentiated counterparts. These signatures confirm the impact of stiffening on ISC fate and additionally suggest a stiffening-induced switch in metabolic phenotype, from oxidative phosphorylation to glycolysis. Finally, we used colon samples from IBD patients as well as chronic colitis murine models to confirm the in vivo stiffening-induced epithelial deterioration similar to that observed in vitro. Together, these results demonstrate stiffness-dependent ISC reprograming wherein YAP nuclear translocation diminishes ISCs and Ki67+ progenitors and drives their differentiation towards goblet cells, suggesting stiffening as potential target to mitigate gut epithelial deterioration during IBD.

In this forum piece, we review progress in exploiting diet and nutrition for enhancing tissue regeneration with a particular emphasis on how dietary composition and diet-induced physiology influence adult stem cell biology.

Mechanisms underlying aspirin chemoprevention of colorectal cancer remain unclear. Prior studies have been limited due to the inability of preclinical models to recapitulate human normal colon epithelium or cellular heterogeneity present in mucosal biopsies. To overcome some of these obstacles we performed in vitro aspirin treatment of colon organoids derived from normal mucosal biopsies to reveal transcriptional networks relevant to aspirin chemoprevention. Colon organoids derived from 38 healthy individuals undergoing endoscopy were treated with 50μM aspirin or vehicle control for 72 hours and subjected to bulk RNA-sequencing. Paired regression analysis using DESeq2 identified differentially expressed genes (DEGs) associated with aspirin treatment. Cellular composition was determined using CIBERSORTx. Aspirin treatment was associated with 1,154 significant (q

Food and nutrition have a profound impact on organismal health and diseases, and tissue-specific adult stem cells play a crucial role in coordinating tissue maintenance by responding to dietary cues. Emerging evidence indicates that adult intestinal stem cells (ISCs) actively adjust their fate decisions in response to diets and nutritional states to drive intestinal adaptation. Here, we review the signaling mechanisms mediating the dietary responses imposed by caloric intake and nutritional composition (i.e., macronutrients and micronutrients), fasting-feeding patterns, diet-induced growth factors, and microbiota on ISCs and their relevance to the beginnings of intestinal tumors.

Stem cells are remarkably small in size. Whether small size is important for stem cell function is unknown. We find that murine hematopoietic stem cells (HSCs) enlarge under conditions known to decrease stem cell function. This decreased fitness of large HSCs is due to reduced proliferative potential. Preventing HSC enlargement by inhibiting macromolecule biosynthesis or reducing large HSCs size by shortening G1 averts the loss of stem cell potential under conditions causing stem cell exhaustion. Finally, we show that a fraction of murine and human HSCs enlarge during aging. Preventing this age-dependent enlargement improves HSC function. We conclude that small cell size is important for stem cell function in vivo and propose that stem cell enlargement contributes to their functional decline during aging.One Sentence SummarySize increase drives stem cell aging.

Little is known about how interactions between diet, immune recognition, and intestinal stem cells (ISCs) impact the early steps of intestinal tumorigenesis. Here, we show that a high fat diet (HFD) reduces the expression of the major histocompatibility complex II (MHC-II) genes in ISCs. This decline in ISC MHC-II expression in a HFD correlates with an altered intestinal microbiome composition and is recapitulated in antibiotic treated and germ-free mice on a control diet. Mechanistically, pattern recognition receptor and IFNg signaling regulate MHC-II expression in ISCs. Although MHC-II expression on ISCs is dispensable for stem cell function in organoid cultures in vitro, upon loss of the tumor suppressor gene Apc in a HFD, MHC-II- ISCs harbor greater in vivo tumor-initiating capacity than their MHC-II+ counterparts, thus implicating a role for epithelial MHC-II in suppressing tumorigenesis. Finally, ISC-specific genetic ablation of MHC-II in engineered Apc-mediated intestinal tumor models increases tumor burden in a cell autonomous manner. These findings highlight how a HFD alters the immune recognition properties of ISCs through the regulation of MHC-II expression in a manner that could contribute to intestinal tumorigenesis.

Eosinophilic gastroenteropathies are rare disorders. An 18-year-old girl presented to the hospital with abdominal pain and distension. Abdominal tenderness and grade 2 ascites, serious peripheral eosinophilia and high immunoglobulin E level were found. Laparoscopic antrum biopsy showed subserosal eosinophilic infiltration. Eosinophilic gastroenteropathy was suspected, diet elimination was given, no steroid treatment was used.

Funder: Jane Coffin Childs Memorial Fund for Medical Research; FundRef: http://dx.doi.org/10.13039/100001033, Funder: Swedish Foundation for Strategic Research; FundRef: http://dx.doi.org/10.13039/501100001729, Funder: Knut and Alice Wallenberg Foundation; FundRef: http://dx.doi.org/10.13039/501100004063, Funder: Barbro Osher Pro Suecia Foundation; FundRef: http://dx.doi.org/10.13039/100008483, Funder: Howard Hughes Medical Institute; FundRef: http://dx.doi.org/10.13039/100000011, Funder: NIHR Cambridge BRC, Funder: Lustgarten Foundation; FundRef: http://dx.doi.org/10.13039/100005979, Funder: Stand Up To Cancer; FundRef: http://dx.doi.org/10.13039/100009730, Funder: MIT Center for Precision Cancer Medicine, Funder: Ludwig Center at MIT, Funder: Emerald Foundation, Tumors are composed of many different cell types including cancer cells, fibroblasts, and immune cells. Dissecting functional metabolic differences between cell types within a mixed population can be challenging due to the rapid turnover of metabolites relative to the time needed to isolate cells. To overcome this challenge, we traced isotope-labeled nutrients into macromolecules that turn over more slowly than metabolites. This approach was used to assess differences between cancer cell and fibroblast metabolism in murine pancreatic cancer organoid-fibroblast co-cultures and tumors. Pancreatic cancer cells exhibited increased pyruvate carboxylation relative to fibroblasts, and this flux depended on both pyruvate carboxylase and malic enzyme 1 activity. Consequently, expression of both enzymes in cancer cells was necessary for organoid and tumor growth, demonstrating that dissecting the metabolism of specific cell populations within heterogeneous systems can identify dependencies that may not be evident from studying isolated cells in culture or bulk tissue.

The cellular composition of barrier epithelia is essential to organismal homoeostasis. In particular, within the small intestine, adult stem cells establish tissue cellularity, and may provide a means to control the abundance and quality of specialized epithelial cells. Yet, methods for the identification of biological targets regulating epithelial composition and function, and of small molecules modulating them, are lacking. Here we show that druggable biological targets and small-molecule regulators of intestinal stem cell differentiation can be identified via multiplexed phenotypic screening using thousands of miniaturized organoid models of intestinal stem cell differentiation into Paneth cells, and validated via longitudinal single-cell RNA-sequencing. We found that inhibitors of the nuclear exporter Exportin 1 modulate the fate of intestinal stem cells, independently of known differentiation cues, significantly increasing the abundance of Paneth cells in the organoids and in wild-type mice. Physiological organoid models of the differentiation of intestinal stem cells could find broader utility for the screening of biological targets and small molecules that can modulate the composition and function of other barrier epithelia.

Summary Background The gut microbiome plays an important role in human health and disease. Gnotobiotic animal and in vitro cell-based models provide some informative insights into mechanistic crosstalk. However, there is no existing system for a long-term co-culture of a human colonic mucosal barrier with super oxygen-sensitive commensal microbes, hindering the study of human-microbe interactions in a controlled manner. Methods Here, we investigate the effects of an abundant super oxygen-sensitive commensal anaerobe, Faecalibacterium prausnitzii, on a primary human mucosal barrier using a gut microbiome (GuMI) physiome platform that we designed and fabricated. Findings Long-term continuous co-culture of F. prausnitzii for 2 days with colon epithelia, enabled by continuous flow of completely anoxic apical media and aerobic basal media, results in a strictly anaerobic apical environment fostering the growth of and butyrate production by F. prausnitzii, while maintaining a stable colon epithelial barrier. We identify elevated differentiation and hypoxia-responsive genes and pathways in the platform compared with conventional aerobic static culture of the colon epithelia, attributable to a combination of anaerobic environment and continuous medium replenishment. Furthermore, we demonstrate the anti-inflammatory effects of F. prausnitzii through histone deacetylase (HDAC) and the Toll-like receptor-TLR-nuclear factor κB (NF-κB) axis. Finally, we identify that butyrate largely contributes to the anti-inflammatory effects by downregulating TLR3 and TLR4. Conclusions Our results are consistent with some clinical observations regarding F. prausnitzii, thus motivating further studies using this platform with more complex engineered colon tissues for understanding the interaction between the human colonic mucosal barrier and microbiota, pathogens, or engineered bacteria. Funding US National Institute of Biomedical Imaging and Bioengineering, Boehringer Ingelheim Strategic Hub for Innovative New Therapeutics Concept Exploration (SHINE) Program, US National Institute of Environmental Health Sciences.

Tumors are composed of many different cell types including cancer cells, fibroblasts, and immune cells. Dissecting functional metabolic differences between various cell types within a mixed population can be limited by the rapid turnover of metabolites relative to the time needed to isolate cells. To overcome this challenge, we traced isotope-labeled nutrients into macromolecules that turn over more slowly than metabolites. This approach was used to assess differences between cancer cell and fibroblast metabolism in pancreatic cancer organoid-fibroblast co-cultures and in pancreatic tumors. In these contexts, we find pancreatic cancer cells exhibit increased pyruvate carboxylation relative to fibroblasts, and that this flux depends on both pyruvate carboxylase and malic enzyme 1 activity. Consequently, expression of both enzymes in cancer cells is necessary for organoid and tumor growth, demonstrating that dissecting the metabolism of specific cell populations within heterogeneous systems can identify dependencies that may not be evident from studying isolated cells in culture or bulk tumor tissue.

SummaryAssociation between the microbiome, IBD and liver diseases are known, yet cause and effect remain elusive. By connecting human microphysiological systems of the gut, liver and circulating Treg/Th17 cells, we modeled progression of ulcerative colitis (UC) ex vivo. We show that microbiome-derived short-chain fatty acids (SCFA) may either improve or worsen disease severity, depending on the activation state of CD4 T cells. Employing multiomics, we found SCFA increased production of ketone bodies, glycolysis and lipogenesis, while markedly reducing innate immune activation of the UC gut. However, during acute T cell-mediated inflammation, SCFA exacerbated CD4+T cell effector function, partially through metabolic reprograming, leading to gut barrier disruption and hepatic injury. These paradoxical findings underscore the emerging utility of human physiomimetic technology in combination with systems immunology to study causality and the fundamental entanglement of immunity, metabolism and tissue homeostasis.

β-hydroxybutyrate (β-OHB) provides an essential metabolic energy source during fasting and functions as an epigenetic regulator by lysine β-hydroxybutyrylation (Kbhb) modification of core histones H3 and H4. We report that H3K9bhb, but not H4K12bhb, becomes highly enriched at proximal promoters of genes regulating lipolytic and ketogenic metabolic programs in small intestinal (SI) crypts during fasting and these pathways are transcriptionally activated in Lgr5 + SI stem cells. Similar Kbhb enrichment is observed in Lgr5 + stem cell-enriched epithelial spheroids treated with β-OHB in vitro. Combinatorial chromatin state analysis reveals that H3K9bhb enrichment is associated with active chromatin states and that fasting enriches for an H3K9bhb–H3K27ac signature at active metabolic gene promoters and distal DNA enhancer elements. Intestinal knockout of Hmgcs2 results in a marked loss of H3K9bhb associated fasting-induced gene programs suggesting that local production of β-OHB is responsible for epigenetic reprogramming. We conclude that specific modulation of H3K9bhb in SI crypts is a key epigenetic event in response to fasting.

SUMMARY The small intestine is responsible for nutrient absorption and one of the most important interfaces between the environment and the body. During aging, changes of the epithelium lead to food malabsorption and reduced barrier function, thus increasing disease risk. The drivers of these alterations remain poorly understood. Here, we compare the proteomes of intestinal crypts from mice across different anatomical regions and ages. We find that aging alters epithelial immunity, metabolism, and cell proliferation and is accompanied by region-dependent skewing in the cellular composition of the epithelium. Of note, short-term dietary restriction followed by refeeding partially restores the epithelium by promoting stem cell differentiation toward the secretory lineage. We identify Hmgcs2 (3-hydroxy-3-methylglutaryl-coenzyme A [CoA] synthetase 2), the rate-limiting enzyme for ketogenesis, as a modulator of stem cell differentiation that responds to dietary changes, and we provide an atlas of region- and age-dependent proteome changes of the small intestine., In Brief Using proteomics, Gebert et al. find that aging has region-specific effects on the small intestine epithelium of mice. These effects can be partially reversed by modulating ketone body signaling in intestinal stem cells via dietary interventions., Graphical Abstract

© Springer Science+Business Media, LLC, part of Springer Nature 2020. This protocol describes a multipronged approach that we have created to determine the transcriptional induction of fatty acid oxidation (FAO) genes in Lgr5high intestinal stem cells and a subsequent metabolomics-based approach for assessing fatty acid utilization in the mammalian intestinal crypt. More specifically, we describe methods for crypt isolation followed by a FACS-based purification of stem and progenitor populations and RNA-sequencing analysis. Using this workflow, we can determine both basal gene expression profiles of key metabolic genes as well as corresponding changes in response to altered metabolic states, such as fasting. Subsequently, we describe a complementary metabolomics-based approach that we have developed to assess fatty acid uptake and utilization in the crypt using 13C stable isotope tracing. Combining these approaches, one can gain a better understanding of substrate utilization and the preceding transcriptional changes that accommodate these reactions in physiologic states of low carbohydrate utilization or during overabundance of dietary lipids.

Although the Hippo transcriptional coactivator YAP is considered oncogenic in many tissues, its roles in intestinal homeostasis and colorectal cancer (CRC) remain controversial. Here, we demonstrate that the Hippo kinases LATS1/2 and MST1/2, which inhibit YAP activity, are required for maintaining Wnt signaling and canonical stem cell function. Hippo inhibition induces a distinct epithelial cell state marked by low Wnt signaling, a wound-healing response, and transcription factor Klf6 expression. Notably, loss of LATS1/2 or overexpression of YAP is sufficient to reprogram Lgr5+ cancer stem cells to this state and thereby suppress tumor growth in organoids, patient-derived xenografts, and mouse models of primary and metastatic CRC. Finally, we demonstrate that genetic deletion of YAP and its paralog TAZ promotes the growth of these tumors. Collectively, our results establish the role of YAP as a tumor suppressor in the adult colon and implicate Hippo kinases as therapeutic vulnerabilities in colorectal malignancies.

The CD155/TIGIT axis can be co-opted during immune evasion in chronic viral infections and cancer. Pancreatic adenocarcinoma (PDAC) is a highly lethal malignancy, and immune-based strategies to combat this disease have been largely unsuccessful to date. We corroborate prior reports that a substantial portion of PDAC harbors predicted high affinity MHC class I-restricted neoepitopes and extend these findings to advanced/metastatic disease. Using two novel preclinical models of neoantigen-expressing PDAC, we demonstrate that intratumoral neoantigen-specific CD8+ T cells adopt multiple states of dysfunction, which are similar to tumor-infiltrating lymphocytes of human PDAC patients. Mechanistically, genetic and/or pharmacologic modulation of the CD155/TIGIT axis was sufficient to promote immune evasion in autochthonous neoantigen-expressing PDAC. Finally, we demonstrate that the CD155/TIGIT axis is critical to maintain immune evasion in PDAC and uncover a combination immunotherapy (TIGIT/PD-1 co-blockade plus CD40 agonism) that elicits profound anti-tumor responses in preclinical models, now poised for clinical evaluation.