portal-vein-injection-colorectal-cancer-organoids-to-study-liver

Portal Vein Injection of Colorectal Cancer Organoids to Study the Liver Metastasis Stroma

Hepatic metastasis of colorectal cancer (CRC) is a leading cause of cancer-related death. Cancer-associated fibroblasts (CAFs), a major component of the ...

South Australian Health and Medical Research Institute (SAHMRI)

INACTIVE - University of Adelaide

Gastric adenocarcinoma and proximal polyposis of the stomach (GAPPS) is an autosomal-dominant cancer-predisposition syndrome with a significant risk of gastric, but not colorectal, adenocarcinoma. We mapped the gene to 5q22 and found loss of the wild-type allele on 5q in fundic gland polyps from affected individuals. Whole-exome and -genome sequencing failed to find causal mutations but, through Sanger sequencing, we identified point mutations in APC promoter 1B that co-segregated with disease in all six families. The mutations reduced binding of the YY1 transcription factor and impaired activity of the APC promoter 1B in luciferase assays. Analysis of blood and saliva from carriers showed allelic imbalance of APC, suggesting that these mutations lead to decreased allele-specific expression in vivo. Similar mutations in APC promoter 1B occur in rare families with familial adenomatous polyposis (FAP). Promoter 1A is methylated in GAPPS and sporadic FGPs and in normal stomach, which suggests that 1B transcripts are more important than 1A in gastric mucosa. This might explain why all known GAPPS-affected families carry promoter 1B point mutations but only rare FAP-affected families carry similar mutations, the colonic cells usually being protected by the expression of the 1A isoform. Gastric polyposis and cancer have been previously described in some FAP-affected individuals with large deletions around promoter 1B. Our finding that GAPPS is caused by point mutations in the same promoter suggests that families with mutations affecting the promoter 1B are at risk of gastric adenocarcinoma, regardless of whether or not colorectal polyps are present.

Point Mutations in Exon 1B of APC Reveal Gastric Adenocarcinoma and Proximal Polyposis of the Stomach as a Familial Adenomatous Polyposis Variant.

Serrated colorectal cancer (CRC) accounts for approximately 25% of cases and includes tumours that are among the most treatment resistant and with worst outcomes. This CRC subtype is associated with activating mutations in the mitogen-activated kinase pathway gene, , and epigenetic modifications termed the CpG Island Methylator Phenotype, leading to epigenetic silencing of key tumour suppressor genes. It is still not clear which (epi-)genetic changes are most important in neoplastic progression and we begin to address this knowledge gap herein.

Genetic editing of colonic organoids provides a molecularly distinct and orthotopic preclinical model of serrated carcinogenesis.

A retrospective case-controlled analysis was performed to identify drug candidates in the current use that may prevent colorectal cancer, outside of aspirin. A total of 37,510 patients aged ≥20 years were assessed to identify subjects who had been diagnosed with colorectal cancer by colonoscopy without a previous diagnosis of colorectal cancer, inflammatory bowel disease (IBD), or gastrointestinal symptoms; 1,560 patients were identified who were diagnosed with colorectal cancer by colonoscopy. The patients with colorectal cancer were matched with 1,560 age, gender, family history of colorectal cancer and comorbidity-matched control patients who were not diagnosed with colorectal cancer at colonoscopy. The medication histories were compared between the two groups. Next, candidate drugs that were more frequently used by the control patients were selected and their effects on human colorectal cancer cell lines  and an inflammation-induced mouse model of colorectal cancer were tested. Putative colorectal cancer preventative agents were identified, including aspirin, vitamin D, vitamin B, vitamin C, vitamin E, xanthine oxidase inhibitor, alpha-blockers, angiotensin receptor blocker, nateglinide, probiotics, thienopyridine, folic acid, nitrovasodilators, bisphosphonates, calcium channel blockers, steroids, and statins ( < 0.05). Alpha-blockers and xanthine oxidase inhibitors were selected for further study because these agents have not been analyzed previously as factors that may affect colorectal cancer outcomes.  doxazosin (alpha-blocker), but not febuxostat (xanthine oxidase inhibitor), suppressed the proliferation of human colorectal cancer cells. Doxazosin also decreased tumorigenesis in an AOM/DSS mouse colorectal cancer model. Alpha-blockers may prevent colorectal cancer.

Alpha-Blockers As Colorectal Cancer Chemopreventive: Findings from a Case-Control Study, Human Cell Cultures, and  Preclinical Testing.

The tumour microenvironment, also termed the tumour stroma or tumour mesenchyme, includes fibroblasts, immune cells, blood vessels and the extracellular matrix and substantially influences the initiation, growth and dissemination of gastrointestinal cancer. Cancer-associated fibroblasts (CAFs) are one of the critical components of the tumour mesenchyme and not only provide physical support for epithelial cells but also are key functional regulators in cancer, promoting and retarding tumorigenesis in a context-dependent manner. In this Review, we outline the emerging understanding of gastrointestinal CAFs with a particular emphasis on their origin and heterogeneity, as well as their function in cancer cell proliferation, tumour immunity, angiogenesis, extracellular matrix remodelling and drug resistance. Moreover, we discuss the clinical implications of CAFs as biomarkers and potential targets for prevention and treatment of patients with gastrointestinal cancer.

Cancer-associated fibroblasts in gastrointestinal cancer.

Cancer-associated fibroblasts (CAFs) were originally presumed to represent a homogeneous population uniformly driving tumorigenesis, united by their morphology and peritumoural location. Our understanding of CAFs has since been shaped by sophisticated in vitro and in vivo experiments, pathological association and, more recently, ablation, and it is now widely appreciated that CAFs form a group of highly heterogeneous cells with no single overarching marker. Studies have demonstrated that the CAF population contains different subtypes based on the expression of marker proteins with the capacity to promote or inhibit cancer, with their biological role as accomplices or adversaries dependent on many factors, including the cancer stage. So, while CAFs have been endlessly shown to promote the growth, survival and spread of tumours via improvements in functionality and an altered secretome, they are also capable of retarding tumorigenesis via largely unknown mechanisms. It is important to reconcile these disparate results so that the functions of, or factors produced by, tumour-promoting subtypes can be specifically targeted to improve cancer patient outcomes. This review will dissect out CAF complexity and CAF-directed cancer treatment strategies in order to provide a case for future, rational therapies.

Cancer-associated fibroblasts-heroes or villains?

Cancer-associated fibroblasts (CAF) constitute a major component of the tumor microenvironment. Recent observations in genetically engineered mouse models and clinical studies have suggested that there may exist at least two functionally different populations of CAFs, that is, cancer-promoting CAFs (pCAF) and cancer-restraining CAFs (rCAF). Although various pCAF markers have been identified, the identity of rCAFs remains unknown because of the lack of rCAF-specific marker(s). In this study, we found that Meflin, a glycosylphosphatidylinositol-anchored protein that is a marker of mesenchymal stromal/stem cells and maintains their undifferentiated state, is expressed by pancreatic stellate cells that are a source of CAFs in pancreatic ductal adenocarcinoma (PDAC).  hybridization analysis of 71 human PDAC tissues revealed that the infiltration of Meflin-positive CAFs correlated with favorable patient outcome. Consistent herewith, Meflin deficiency led to significant tumor progression with poorly differentiated histology in a PDAC mouse model. Similarly, genetic ablation of Meflin-positive CAFs resulted in poor differentiation of tumors in a syngeneic transplantation model. Conversely, delivery of a Meflin-expressing lentivirus into the tumor stroma or overexpression of Meflin in CAFs suppressed the growth of xenograft tumors. Lineage tracing revealed that Meflin-positive cells gave rise to α-smooth muscle actin-positive CAFs that are positive or negative for Meflin, suggesting a mechanism for generating CAF heterogeneity. Meflin deficiency or low expression resulted in straightened stromal collagen fibers, which represent a signature for aggressive tumors, in mouse or human PDAC tissues, respectively. Together, the data suggest that Meflin is a marker of rCAFs that suppress PDAC progression. SIGNIFICANCE: Meflin marks and functionally contributes to a subset of cancer-associated fibroblasts that exert antitumoral effects. http://cancerres.aacrjournals.org/content/canres/79/20/5367/F1.large.jpg.

Meflin-Positive Cancer-Associated Fibroblasts Inhibit Pancreatic Carcinogenesis.

Pathological Wnt pathway activation is a conserved hallmark of colorectal cancer. Wnt-activating mutations can be divided into: i) ligand-independent (LI) alterations in intracellular signal transduction proteins (, β-catenin), causing constitutive pathway activation and ii) ligand-dependent (LD) mutations affecting the synergistic R-Spondin axis (, -fusions) acting through amplification of endogenous Wnt signal transmembrane transduction. Our aim was to exploit differential Wnt target gene expression to generate a mutation-agnostic biomarker for LD tumours.

Exploiting differential Wnt target gene expression to generate a molecular biomarker for colorectal cancer stratification.

Patients with colorectal cancer with peritoneal metastases (CRPMs) have limited treatment options and the lowest colorectal cancer survival rates. We aimed to determine whether organoid testing could help guide precision treatment for patients with CRPMs, as the clinical utility of prospective, functional drug screening including nonstandard agents is unknown.

Medium-throughput Drug Screening of Patient-derived Organoids from Colorectal Peritoneal Metastases to Direct Personalized Therapy.

Cancer-associated fibroblasts (CAFs), key constituents of the tumor microenvironment, either promote or restrain tumor growth. Attempts to therapeutically target CAFs have been hampered by our incomplete understanding of these functionally heterogeneous cells. Key growth factors in the intestinal epithelial niche, bone morphogenetic proteins (BMPs), also play a critical role in colorectal cancer (CRC) progression. However, the crucial proteins regulating stromal BMP balance and the potential application of BMP signaling to manage CRC remain largely unexplored.

The Balance of Stromal BMP Signaling Mediated by GREM1 and ISLR Drives Colorectal Carcinogenesis.

The stem/progenitor cells of the developing intestine are biologically distinct from their adult counterparts. Here, we examine the microenvironmental cues that regulate the embryonic stem/progenitor population, focusing on the role of Notch pathway factor delta-like protein-1 (DLK1). mRNA-seq analyses of intestinal mesenchymal cells (IMCs) collected from embryonic day 14.5 (E14.5) or adult IMCs and a novel coculture system with E14.5 intestinal epithelial organoids were used. Following addition of recombinant DLK1 (rDLK) or  siRNA (), epithelial characteristics were compared using imaging, replating efficiency assays, qPCR, and immunocytochemistry. The intestinal phenotypes of littermate  and  mice were compared using immunohistochemistry. Using transcriptomic analyses, we identified morphogens derived from the embryonic mesenchyme that potentially regulate the developing epithelial cells, to focus on Notch family candidate DLK1. Immunohistochemistry indicated that DLK1 was expressed exclusively in the intestinal stroma at E14.5 at the top of emerging villi, decreased after birth, and shifted to the intestinal epithelium in adulthood. In coculture experiments, addition of rDLK1 to adult IMCs inhibited organoid differentiation, whereas  knockdown in embryonic IMCs increased epithelial differentiation to secretory lineage cells.  mice had restricted Ki67 cells in the villi base and increased secretory lineage cells compared with  embryos. Mesenchyme-derived DLK1 plays an important role in the promotion of epithelial stem/precursor expansion and prevention of differentiation to secretory lineages in the developing intestine. Using a novel coculture system, transcriptomics, and transgenic mice, we investigated differential molecular signaling between the intestinal epithelium and mesenchyme during development and in the adult. We show that the Notch pathway factor delta-like protein-1 (DLK1) is stromally produced during development and uncover a new role for DLK1 in the regulation of intestinal epithelial stem/precursor expansion and differentiation to secretory lineages.

Stromal DLK1 promotes proliferation and inhibits differentiation of the intestinal epithelium during development.

The intestinal stroma provides an important microenvironment for immune cell activation. The perturbation of this tightly regulated process can lead to excessive inflammation. We know that upregulated Toll-like receptor 4 (TLR4) in the intestinal epithelium plays a key role in the inflammatory condition of preterm infants, such as necrotizing enterocolitis (NEC). However, the surrounding stromal contribution to excessive inflammation in the pre-term setting awaits careful dissection. Ex vivo co-culture of embryonic day 14.5 (E14.5) or adult murine intestinal stromal cells with exogenous monocytes was undertaken. We also performed mRNAseq analysis of embryonic and adult stromal cells treated with vehicle control or lipopolysaccharide (LPS), followed by pathway and network analyses of differentially regulated transcripts. Cell characteristics were compared using flow cytometry and pHrodo red phagocytic stain, candidate gene analysis was performed via siRNA knockdown and gene expression measured by qPCR and ELISA. Embryonic stromal cells promote the differentiation of co-cultured monocytes to CD11bCD11c mononuclear phagocytes, that in turn express decreased levels of CD103. Global mRNAseq analysis of stromal cells following LPS stimulation identified TLR signaling components as the most differentially expressed transcripts in the immature compared to adult setting. We show that CD14 expressed by CD11bCD45 embryonic stromal cells is a key inducer of TLR mediated inflammatory cytokine production and phagocytic activity of monocyte derived cells. We utilise transcriptomic analyses and functional ex vivo modelling to improve our understanding of unique molecular cues provided by the immature intestinal stroma.

Delineating proinflammatory microenvironmental signals by ex vivo modeling of the immature intestinal stroma.

Bone morphogenetic protein (BMP) signaling is required for early forebrain development and cortical formation. How the endogenous modulators of BMP signaling regulate the structural and functional maturation of the developing brain remains unclear. Here, we show that expression of the BMP antagonist Grem1 marks committed layer V and VI glutamatergic neurons in the embryonic mouse brain. Lineage tracing of Grem1-expressing cells in the embryonic brain was examined by administration of tamoxifen to pregnant Grem1creERT; Rosa26LSLTdtomato mice at 13.5 days post coitum (dpc), followed by collection of embryos later in gestation. In addition, at 14.5 dpc, bulk mRNA-seq analysis of differentially expressed transcripts between FACS-sorted Grem1-positive and -negative cells was performed. We also generated Emx1-cre-mediated Grem1 conditional knockout mice (Emx1-Cre;Grem1flox/flox) in which the Grem1 gene was deleted specifically in the dorsal telencephalon. Grem1Emx1cKO animals had reduced cortical thickness, especially layers V and VI, and impaired motor balance and fear sensitivity compared with littermate controls. This study has revealed new roles for Grem1 in the structural and functional maturation of the developing cortex.

Article	Year
Point Mutations in Exon 1B of APC Reveal Gastric Adenocarcinoma and Proximal Polyposis of the Stomach as a Familial Adenomatous Polyposis Variant. American journal of human genetics\| PubMed ID: 27087319	2016
Genetic editing of colonic organoids provides a molecularly distinct and orthotopic preclinical model of serrated carcinogenesis. Gut\| PubMed ID: 29666172	2019
Alpha-Blockers As Colorectal Cancer Chemopreventive: Findings from a Case-Control Study, Human Cell Cultures, and Preclinical Testing. Cancer prevention research (Philadelphia, Pa.)\| PubMed ID: 30700439	2019
Cancer-associated fibroblasts in gastrointestinal cancer. Nature reviews. Gastroenterology & hepatology\| PubMed ID: 30778141	2019
Cancer-associated fibroblasts-heroes or villains? British journal of cancer\| PubMed ID: 31289350	2019
Meflin-Positive Cancer-Associated Fibroblasts Inhibit Pancreatic Carcinogenesis. Cancer research\| PubMed ID: 31439548	2019
Exploiting differential Wnt target gene expression to generate a molecular biomarker for colorectal cancer stratification. Gut\| PubMed ID: 31563876	2020
Medium-throughput Drug Screening of Patient-derived Organoids from Colorectal Peritoneal Metastases to Direct Personalized Therapy. Clinical cancer research : an official journal of the American Association for Cancer Research\| PubMed ID: 32376656	2020
The Balance of Stromal BMP Signaling Mediated by GREM1 and ISLR Drives Colorectal Carcinogenesis. Gastroenterology\| PubMed ID: 33197448	2021
Stromal DLK1 promotes proliferation and inhibits differentiation of the intestinal epithelium during development. American journal of physiology. Gastrointestinal and liver physiology\| PubMed ID: 33470182	2021
Delineating proinflammatory microenvironmental signals by ex vivo modeling of the immature intestinal stroma. Scientific reports\| PubMed ID: 33785826	2021
The BMP antagonist gremlin 1 contributes to the development of cortical excitatory neurons, motor balance and fear responses. Development (Cambridge, England)\| PubMed ID: 34184027	2021

Susan L. Woods

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