bims-instec 2023-08-13 papers

BMC Biol. 2023 Aug 08. 21(1): 169

A stromal lineage maintains crypt structure and villus homeostasis in the intestinal stem cell niche.

Jinnan Xiang, Jigang Guo, Shaoyang Zhang, Hongguang Wu, Ye-Guang Chen, Junping Wang, Baojie Li, Huijuan Liu.

BACKGROUND: The nutrient-absorbing villi of small intestines are renewed and repaired by intestinal stem cells (ISCs), which reside in a well-organized crypt structure. Genetic studies have shown that Wnt molecules secreted by telocytes, Gli1+ stromal cells, and epithelial cells are required for ISC proliferation and villus homeostasis. Intestinal stromal cells are heterogeneous and single-cell profiling has divided them into telocytes/subepithelial myofibroblasts, myocytes, pericytes, trophocytes, and Pdgfralow stromal cells. Yet, the niche function of these stromal populations remains incompletely understood.
RESULTS: We show here that a Twist2 stromal lineage, which constitutes the Pdgfralow stromal cell and trophocyte subpopulations, maintains the crypt structure to provide an inflammation-restricting niche for regenerating ISCs. Ablating Twist2 lineage cells or deletion of one Wntless allele in these cells disturbs the crypt structure and impairs villus homeostasis. Upon radiation, Wntless haplo-deficiency caused decreased production of anti-microbial peptides and increased inflammation, leading to defective ISC proliferation and crypt regeneration, which were partially rescued by eradication of commensal bacteria. In addition, we show that Wnts secreted by Acta2+ subpopulations also play a role in crypt regeneration but not homeostasis.
CONCLUSIONS: These findings suggest that ISCs may require different niches for villus homeostasis and regeneration and that the Twist2 lineage cells may help to maintain a microbe-restricted environment to allow ISC-mediated crypt regeneration.

Keywords: ISC; Inflammation; Mesenchymal; Niche; Paneth cell; Wntless

DOI: https://doi.org/10.1186/s12915-023-01667-2

Br J Cancer. 2023 Aug 11.

Combined blockade of mTOR and p21-activated kinases pathways prevents tumour growth in KRAS-mutated colorectal cancer.

Stefania Belli, Ada Pesapane, Alberto Servetto, Daniela Esposito, Fabiana Napolitano, Claudia Maria Ascione, Alessandra Allotta, Nicola Zambrano, Federica Zito Marino, Renato Franco, Teresa Troiani, Luigi Formisano, Roberto Bianco.

BACKGROUND: The identification of novel therapeutic strategies for metastatic colorectal cancer (mCRC) patients harbouring KRAS mutations represents an unmet clinical need. In this study, we aimed to clarify the role of p21-activated kinases (Paks) as therapeutic target for KRAS-mutated CRC.
METHODS: Paks expression and activation levels were evaluated in a cohort of KRAS-WT or -mutated CRC patients by immunohistochemistry. The effects of Paks inhibition on tumour cell proliferation and signal transduction were assayed by RNAi and by the use of three pan-Paks inhibitors (PF-3758309, FRAX1036, GNE-2861), evaluating CRC cells, spheroids and tumour xenografts' growth.
RESULTS: Paks activation positively correlated with KRAS mutational status in both patients and cell lines. Moreover, genetic modulation or pharmacological inhibition of Paks led to a robust impairment of KRAS-mut CRC cell proliferation. However, Paks prolonged blockade induced a rapid tumour adaptation through the hyper-activation of the mTOR/p70S6K pathway. The addition of everolimus (mTOR inhibitor) prevented the growth of KRAS-mut CRC tumours in vitro and in vivo, reverting the adaptive tumour resistance to Paks targeting.
CONCLUSIONS: In conclusion, our results suggest the simultaneous blockade of mTOR and Pak pathways as a promising alternative therapeutic strategy for patients affected by KRAS-mut colorectal cancer.

DOI: https://doi.org/10.1038/s41416-023-02390-z

Cell Mol Gastroenterol Hepatol. 2023 Aug 08. pii: S2352-345X(23)00144-3. [Epub ahead of print]

Hypoxia primes human ISCs for interleukin-dependent rescue of stem cell activity.

Kristina R Rivera, R Jarrett Bliton, Joseph Burclaff, Michael J Czerwinski, Jintong Liu, Jessica M Trueblood, Caroline M Hinesley, Keith A Breau, Halston E Deal, Shlok Joshi, Vladimir A Pozdin, Ming Yao, Amanda L Ziegler, Anthony T Blikslager, Michael A Daniele, Scott T Magness.

BACKGROUND & AIMS: Hypoxia in the intestinal epithelium can be caused by acute ischemic events or chronic inflammation where immune cell infiltration produces 'inflammatory hypoxia' starving the mucosa of oxygen. The epithelium has the capacity to regenerate after some ischemic and inflammatory conditions suggesting intestinal stem cells (ISCs) are highly tolerant to acute and chronic hypoxia; however, the impact of hypoxia on human ISC (hISC) function has not been reported. Here we present a new microphysiological system (MPS) to investigate how hypoxia affects hISCs from healthy donors and test the hypothesis that prolonged hypoxia modulates how hISCs respond to inflammation-associated interleukins.
METHODS: HISCs were exposed to <1.0% oxygen in the MPS for 6-, 24-, 48- & 72-hrs. Viability, HIF1α response, transcriptomics, cell cycle dynamics, and response to cytokines were evaluated in hISCs under hypoxia. HIF-stabilizers and inhibitors were screened to evaluate HIF- dependent responses.
RESULTS: The MPS enables precise, real-time control and monitoring of oxygen levels at the cell surface. Under hypoxia, hISCs maintain viability until 72-hrs, and exhibit peak HIF1α at 24-hrs.hISC activity was reduced at 24-hrs but recovered at 48-hrs. Hypoxia induced increases in the proportion of hISCs in G1 and expression changes in 16 interleukin receptors. PHD inhibition failed to reproduce hypoxia-dependent IL-receptor expression patterns. hISC activity increased when treated IL1ß, IL2, IL4, IL6, IL10, IL13, and IL25 and rescued hISC activity caused by 24- hrs of hypoxia.
CONCLUSIONS: Hypoxia pushes hISCs into a dormant but reversible proliferative state and primes hISCs to respond to a subset of interleukins that preserves hISC activity. These findings have important implications for understanding intestinal epithelial regeneration mechanisms caused by inflammatory hypoxia.

Keywords: Cytokines; Inflammatory hypoxia; Intestinal stem cells; Oxygen Sensor; Stem cell priming; microphysiological system

DOI: https://doi.org/10.1016/j.jcmgh.2023.07.012

STAR Protoc. 2023 Aug 03. pii: S2666-1667(23)00448-3. [Epub ahead of print]4(3): 102481

Protocol for establishing a coculture with fibroblasts and colorectal cancer organoids.

Svenja Wallisch, Sylvia Karin Neef, Lukas Denzinger, Dina Mönch, Jana Koch, Julia Marzi, Thomas Mürdter, Nicole Janssen.

The tumor microenvironment is essential for mediating drug resistance and tumor progression. Here, we present a coculture system, which enables drug testing of colorectal cancer organoids and fibroblasts without additional matrix components such as Matrigel or basement membrane extracts. First, we describe steps to use a readout for high-throughput drug testing using a luminescence-based viability assay. Second, we detail a readout that uses flow cytometry to distinguish toxic effects on either colorectal cancer organoids or fibroblasts.

Keywords: Cancer; Cell Biology; Cell Culture; Organoids

DOI: https://doi.org/10.1016/j.xpro.2023.102481

Cell Metab. 2023 Jul 30. pii: S1550-4131(23)00264-4. [Epub ahead of print]

The diapause-like colorectal cancer cells induced by SMC4 attenuation are characterized by low proliferation and chemotherapy insensitivity.

In response to adverse environmental conditions, embryonic development may reversibly cease, a process termed diapause. Recent reports connect this phenomenon with the non-genetic responses of tumors to chemotherapy, but the mechanisms involved are poorly understood. Here, we establish a multifarious role for SMC4 in the switching of colorectal cancer cells to a diapause-like state. SMC4 attenuation promotes the expression of three investment phase glycolysis enzymes increasing lactate production while also suppressing PGAM1. Resultant high lactate levels increase ABC transporter expression via histone lactylation, rendering tumor cells insensitive to chemotherapy. SMC4 acts as co-activator of PGAM1 transcription, and the coordinate loss of SMC4 and PGAM1 affects F-actin assembly, inducing cytokinesis failure and polyploidy, thereby inhibiting cell proliferation. These insights into the mechanisms underlying non-genetic chemotherapy resistance may have significant implications for the field, advancing our understanding of aerobic glycolysis functions in tumor and potentially informing future therapeutic strategies.

Keywords: SMC4; chemotherapy insensitivity; colorectal cancer; diapause-like state; glycolysis; histone lactylation; polyploidy

DOI: https://doi.org/10.1016/j.cmet.2023.07.005