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 ...

 was recently shown to identify a discrete population of stem cells within the isthmus of the oxyntic gland within the gastric corpus. Chief cells at the base of the gastric corpus also express . The relevance of  expression as a marker of specific cell populations within the antral glands of the distal stomach, however, is unknown. Using -CreERT mice, we revealed that  antral cells, distinct from the  population in the corpus, comprise long-lived progenitors that reside within the antral isthmus above  or CCK2R cells.  antral progenitors can serve as an origin of antral tumors induced by loss of Apc or MNU treatment.  antral progenitors, as well as other antral stem/progenitor population, express Cxcr4, and are located in close proximity to Cxcl12 (the Cxcr4 ligand)-expressing endothelium. During antral carcinogenesis, there is an expansion of Cxcr4 epithelial cells as well as the Cxcl12 perivascular niche. Deletion of Cxcl12 in endothelial cells or pharmacological blockade of Cxcr4 inhibits antral tumor growth. Cxcl12/Cxcr4 signaling may be a potential therapeutic target.

CXCR4-expressing  progenitors in the gastric antrum contribute to gastric cancer development.

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.

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
CXCR4-expressing progenitors in the gastric antrum contribute to gastric cancer development. Oncotarget\| PubMed ID: 29340033	2017
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
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

Nobumi Suzuki

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