Category: Publications

Abstract

Intestinal stem cell research has greatly aided our understanding of the biology of intestinal self-renewal but has also shed light on the role of cancer stem cells (CSCs) in carcinogenesis, cancer growth, and dissemination. With new possibilities for CSC targeting, there is a need to have established techniques for quantifying (cancer) stem cell clonogenicity, particularly in organoid cultures. Here, we describe a detailed methodology for the isolation and expansion of mouse intestinal crypts from three different locations-the colon, proximal, and distal small intestine. In addition, we describe techniques that allow the measurement of stem cell clonogenicity and its manipulation using two approaches-organoid counting and immunohistochemistry.

Keywords: Cancer stem cells; Clonogenicity; Colorectal cancer; Immunohistochemistry; Isolation; Organoids.

Authors

Prashanthi Ramesh, Aleksandar Buryanov Kirov, David Johannes Huels, Jan Paul Medema

Link

https://doi.org/10.1007/7651_2018_179

Abstract

Evasion of apoptosis is a hallmark of cancer, which is frequently mediated by upregulation of the antiapoptotic BCL-2 family proteins. In colorectal cancer (CRC), previous work has highlighted differential antiapoptotic protein dependencies determined by the stage of the disease. While intestinal stem cells (ISCs) require BCL-2 for adenoma outgrowth and survival during transformation, ISC-specific MCL1 deletion results in disturbed intestinal homeostasis, eventually contributing to tumorigenesis. Colon cancer stem cells (CSCs), however, no longer require BCL-2 and depend mainly on BCL-XL for their survival. We therefore hypothesized that a shift in antiapoptotic protein reliance occurs in ISCs as the disease progresses from normal to adenoma to carcinoma. By targeting antiapoptotic proteins with specific BH3 mimetics in organoid models of CRC progression, we found that BCL-2 is essential only during ISC transformation while MCL1 inhibition did not affect adenoma outgrowth. BCL-XL, on the other hand, was crucial for stem cell survival throughout the adenoma-to-carcinoma sequence. Furthermore, we identified that the limited window of BCL-2 reliance is a result of its downregulation by miR-17-5p, a microRNA that is upregulated upon APC-mutation driven transformation. Here we show that BCL-XL inhibition effectively impairs adenoma outgrowth in vivo and enhances the efficacy of chemotherapy. In line with this dependency, expression of BCL-XL, but not BCL-2 or MCL1, directly correlated to the outcome of chemotherapy-treated CRC patients. Our results provide insights to enable the rational use of BH3 mimetics in CRC management, particularly underlining the therapeutic potential of BCL-XL targeting mimetics in both early and late-stage disease.

Authors

Rene Jackstadt, Sander R van Hooff, Joshua D Leach, Xabier Cortes-Lavaud, Jeroen O Lohuis, Rachel A Ridgway, Valérie M Wouters, Jatin Roper, Timothy J Kendall, Campbell S Roxburgh, Paul G Horgan, Colin Nixon, Craig Nourse, Matthias Gunzer, William Clark, Ann Hedley, Omer H Yilmaz, Mamunur Rashid, Peter Bailey, Andrew V Biankin, Andrew D Campbell, David J Adams, Simon T Barry, Colin W Steele, Jan Paul Medema, Owen J Sansom

Link

https://doi.org/10.1016/j.ccell.2019.08.003

Abstract

Evasion of apoptosis is a hallmark of cancer, which is frequently mediated by upregulation of the antiapoptotic BCL-2 family proteins. In colorectal cancer (CRC), previous work has highlighted differential antiapoptotic protein dependencies determined by the stage of the disease. While intestinal stem cells (ISCs) require BCL-2 for adenoma outgrowth and survival during transformation, ISC-specific MCL1 deletion results in disturbed intestinal homeostasis, eventually contributing to tumorigenesis. Colon cancer stem cells (CSCs), however, no longer require BCL-2 and depend mainly on BCL-XL for their survival. We therefore hypothesized that a shift in antiapoptotic protein reliance occurs in ISCs as the disease progresses from normal to adenoma to carcinoma. By targeting antiapoptotic proteins with specific BH3 mimetics in organoid models of CRC progression, we found that BCL-2 is essential only during ISC transformation while MCL1 inhibition did not affect adenoma outgrowth. BCL-XL, on the other hand, was crucial for stem cell survival throughout the adenoma-to-carcinoma sequence. Furthermore, we identified that the limited window of BCL-2 reliance is a result of its downregulation by miR-17-5p, a microRNA that is upregulated upon APC-mutation driven transformation. Here we show that BCL-XL inhibition effectively impairs adenoma outgrowth in vivo and enhances the efficacy of chemotherapy. In line with this dependency, expression of BCL-XL, but not BCL-2 or MCL1, directly correlated to the outcome of chemotherapy-treated CRC patients. Our results provide insights to enable the rational use of BH3 mimetics in CRC management, particularly underlining the therapeutic potential of BCL-XL targeting mimetics in both early and late-stage disease.

Authors

Prashanthi Ramesh, Tamsin R. M. Lannagan, Rene Jackstadt, Lidia Atencia Taboada, Nico Lansu, Pratyaksha Wirapati, Sander R. van Hooff, Danielle Dekker, Jessica Pritchard, Aleksandar B. Kirov, Sanne M. van Neerven, Sabine Tejpar, Geert J. P. L. Kops, Owen J. Sansom & Jan Paul Medema

Link

https://doi.org/10.1038/s41418-021-00816-w

Abstract

Obesity is a strong risk factor for cancer progression, posing obesity-related cancer as one of the leading causes of death. Nevertheless, the molecular mechanisms that endow cancer cells with metastatic properties in patients affected by obesity remain unexplored.

Here, we show that IL-6 and HGF, secreted by tumor neighboring visceral adipose stromal cells (V-ASCs), expand the metastatic colorectal (CR) cancer cell compartment (CD44v6 + ), which in turn secretes neurotrophins such as NGF and NT-3, and recruits adipose stem cells within tumor mass. Visceral adipose-derived factors promote vasculogenesis and the onset of metastatic dissemination by activation of STAT3, which inhibits miR-200a and enhances ZEB2 expression, effectively reprogramming CRC cells into a highly metastatic phenotype. Notably, obesity-associated tumor microenvironment provokes a transition in the transcriptomic expression profile of cells derived from the epithelial consensus molecular subtype (CMS2) CRC patients towards a mesenchymal subtype (CMS4). STAT3 pathway inhibition reduces ZEB2 expression and abrogates the metastatic growth sustained by adipose-released proteins. Together, our data suggest that targeting adipose factors in colorectal cancer patients with obesity may represent a therapeutic strategy for preventing metastatic disease.

Authors

Simone Di Franco, Paola Bianca, Davide Stefano Sardina, Alice Turdo, Miriam Gaggianesi, Veronica Veschi, Annalisa Nicotra, Laura Rosa Mangiapane, Melania Lo Iacono, Irene Pillitteri, Sander van Hooff, Federica Martorana, Gianmarco Motta, Eliana Gulotta, Vincenzo Luca Lentini, Emanuele Martorana, Micol Eleonora Fiori, Salvatore Vieni, Maria Rita Bongiorno, Giorgio Giannone, Dario Giuffrida, Lorenzo Memeo, Lorenzo Colarossi, Marzia Mare, Paolo Vigneri, Matilde Todaro, Ruggero De Maria, Jan Paul Medema & Giorgio Stassi

Link

https://doi.org/10.1038/s41467-021-25333-9

Abstract

The rapid renewal of the epithelial gut lining is fuelled by stem cells that reside at the base of intestinal crypts. The signal transduction pathways and morphogens that regulate intestinal stem cell self-renewal and differentiation have been extensively characterised. In contrast, although extracellular matrix (ECM) components form an integral part of the intestinal stem cell niche, their direct influence on the cellular composition is less well understood. We set out to systematically compare the effect of two ECM classes, the interstitial matrix and the basement membrane, on the intestinal epithelium. We found that both collagen I and laminin-containing cultures allow growth of small intestinal epithelial cells with all cell types present in both cultures, albeit at different ratios. The collagen cultures contained a subset of cells enriched in fetal-like markers. In contrast, laminin increased Lgr5+ stem cells and Paneth cells, and induced crypt-like morphology changes. The transition from a collagen culture to a laminin culture resembled gut development in vivo. The dramatic ECM remodelling was accompanied by a local expression of the laminin receptor ITGA6 in the crypt-forming epithelium. Importantly, deletion of laminin in the adult mouse resulted in a marked reduction of adult intestinal stem cells. Overall, our data support the hypothesis that the formation of intestinal crypts is induced by an increased laminin concentration in the ECM.

Keywords: Crypt morphology; Extracellular matrix; Intestinal stem cells; Laminin.

© 2022. Published by The Company of Biologists Ltd.

Authors

Rana Ramadan, Valérie M Wouters, Sanne M van Neerven, Nina E de Groot, Tania Martins Garcia, Vanessa Muncan, Olivia D Franklin, Michelle Battle, Karen Sue Carlson, Joshua Leach, Owen J Sansom, Olivier Boulard, Mathias Chamaillard, Louis Vermeulen, Jan Paul Medema, David J Huels

Link

https://doi.org/10.1016/j.xpro.2021.101050