bims-instec Biomed News
on Intestinal stem cells and chemoresistance in colon cancer and intestinal regeneration
Issue of 2023‒03‒05
ten papers selected by
Maria-Virginia Giolito
Free University of Brussels

  1. J Vis Exp. 2023 Feb 10.
      Over the last decade, more sophisticated preclinical colorectal cancer (CRC) models have been established using patient-derived cancer cells and 3D tumoroids. Since patient derived tumor organoids can retain the characteristics of the original tumor, these reliable preclinical models enable cancer drug screening and the study of drug resistance mechanisms. However, CRC related death in patients is mostly associated with the presence of metastatic disease. It is therefore essential to evaluate the efficacy of anti-cancer therapies in relevant in vivo models that truly recapitulate the key molecular features of human cancer metastasis. We have established an orthotopic model based on the injection of CRC patient-derived cancer cells directly into the cecum wall of mice. These tumor cells develop primary tumors in the cecum that metastasize to the liver and lungs, which is frequently observed in patients with advanced CRC. This CRC mouse model can be used to evaluate drug responses monitored by microcomputed tomography (µCT), a clinically relevant small-scale imaging method that can easily identify primary tumors or metastases in patients. Here, we describe the surgical procedure and the required methodology to implant patient-derived cancer cells in the cecum wall of immunodeficient mice.
  2. Aging Cell. 2023 Mar 02. e13802
      The intestinal epithelium consists of cells derived from continuously cycling Lgr5hi intestinal stem cells (Lgr5hi ISCs) that mature developmentally in an ordered fashion as the cells progress along the crypt-luminal axis. Perturbed function of Lgr5hi ISCs with aging is documented, but the consequent impact on overall mucosal homeostasis has not been defined. Using single-cell RNA sequencing, the progressive maturation of progeny was dissected in the mouse intestine, which revealed that transcriptional reprogramming with aging in Lgr5hi ISCs retarded the maturation of cells in their progression along the crypt-luminal axis. Importantly, treatment with metformin or rapamycin at a late stage of mouse lifespan reversed the effects of aging on the function of Lgr5hi ISCs and subsequent maturation of progenitors. The effects of metformin and rapamycin overlapped in reversing changes of transcriptional profiles but were also complementary, with metformin more efficient than rapamycin in correcting the developmental trajectory. Therefore, our data identify novel effects of aging on stem cells and the maturation of their daughter cells contributing to the decline of epithelial regeneration and the correction by geroprotectors.
    Keywords:  gerotherapeutics; intestinal stem cell aging; intestinal stem cells; metformin; rapamycin; single-cell RNA sequencing
  3. J Exp Clin Cancer Res. 2023 Mar 03. 42(1): 56
      BACKGROUND: Colorectal cancer (CRC) can be divided into four consensus molecular subtypes (CMS), each with distinct biological features. CMS4 is associated with epithelial-mesenchymal transition and stromal infiltration (Guinney et al., Nat Med 21:1350-6, 2015; Linnekamp et al., Cell Death Differ 25:616-33, 2018), whereas clinically it is characterized by lower responses to adjuvant therapy, higher incidence of metastatic spreading and hence dismal prognosis (Buikhuisen et al., Oncogenesis 9:66, 2020).METHODS: To understand the biology of the mesenchymal subtype and unveil specific vulnerabilities, a large CRISPR-Cas9 drop-out screen was performed on 14 subtyped CRC cell lines to uncover essential kinases in all CMSs. Dependency of CMS4 cells on p21-activated kinase 2 (PAK2) was validated in independent 2D and 3D in vitro cultures and in vivo models assessing primary and metastatic outgrowth in liver and peritoneum. TIRF microscopy was used to uncover actin cytoskeleton dynamics and focal adhesion localization upon PAK2 loss. Subsequent functional assays were performed to determine altered growth and invasion patterns.
    RESULTS: PAK2 was identified as a key kinase uniquely required for growth of the mesenchymal subtype CMS4, both in vitro and in vivo. PAK2 plays an important role in cellular attachment and cytoskeletal rearrangements (Coniglio et al., Mol Cell Biol 28:4162-72, 2008; Grebenova et al., Sci Rep 9:17171, 2019). In agreement, deletion or inhibition of PAK2 impaired actin cytoskeleton dynamics in CMS4 cells and, as a consequence, significantly reduced invasive capacity, while it was dispensable for CMS2 cells. Clinical relevance of these findings was supported by the observation that deletion of PAK2 from CMS4 cells prevented metastatic spreading in vivo. Moreover, growth in a model for peritoneal metastasis was hampered when CMS4 tumor cells were deficient for PAK2.
    CONCLUSION: Our data reveal a unique dependency of mesenchymal CRC and provide a rationale for PAK2 inhibition to target this aggressive subgroup of colorectal cancer.
    Keywords:  Cellular attachment; Colorectal cancer; Epithelial-mesenchymal transition; Metastasis; PAK family
  4. Cell Mol Gastroenterol Hepatol. 2023 Mar 01. pii: S2352-345X(23)00036-X. [Epub ahead of print]
      BACKGROUND & AIMS: Patient-derived organoid cancer models are generated from epithelial tumor cells and reflect tumor characteristics. However, they lack the complexity of the tumor microenvironment, which is a key driver of tumorigenesis and therapy response. Here, we developed a colorectal cancer organoid model that incorporates matched epithelial cells and stromal fibroblasts.METHODS: Primary fibroblasts and tumor cells were isolated from colorectal cancer specimens. Fibroblasts were characterized for their proteome, secretome and gene expression signatures. Fibroblast/organoid co-cultures were analyzed by immunohistochemistry and compared to their tissue of origin, as well as on gene expression levels compared to standard organoid models. Bioinformatics deconvolution was used to calculate cellular proportions of cell subsets in organoids based on single cell RNA sequencing data.
    RESULTS: Normal primary fibroblasts, isolated from tumor adjacent tissue, and cancer associated fibroblasts retained their molecular characteristics in vitro including higher motility of cancer associated compared to normal fibroblasts. Importantly, both CAFs and NFs supported cancer cell proliferation in 3D co-cultures, without the addition of classical niche factors. Organoids grown together with fibroblasts displayed a larger cellular heterogeneity of tumor cells compared to mono-cultures, and closely resembled the in vivo tumor morphology. Additionally, we observed a mutual crosstalk between tumor cells and fibroblasts in the co-cultures. This was manifested by considerably deregulated pathways such as cell-cell communication and extracellular matrix remodeling in the organoids. Thrombospondin-1 was identified as a critical factor for fibroblast invasiveness.
    CONCLUSION: We developed a physiological tumor/stroma model, which will be vital as a personalized tumor model to study disease mechanisms and therapy response in CRC.
    Keywords:  cancer; co-cultures; colorectal cancer; fibroblasts; organoids
  5. Cancer Discov. 2023 Feb 28. OF1
      Dual inhibition of the MAPK pathway helps boost immune responses to checkpoint inhibitors in patients with previously treated BRAFV600E-mutated metastatic colorectal cancer.
  6. JCO Oncol Pract. 2023 Mar 02. OP2200762
      Immune checkpoint inhibitors (ICIs) have revolutionized the treatment paradigm of mismatch repair-deficient/microsatellite instability-high (MMMR-D/MSI-H) colorectal cancer (CRC). Unique molecular features of MMR-D/MSI-H CRC with frameshift alterations, which result in mutation-associated neoantigen (MANA) generation, create an ideal molecular framework for MANA-driven T-cell priming and antitumor immunity. These biologic characteristics of MMR-D/MSI-H CRC resulted in rapid drug development with ICIs for patients with MMR-D/MSI-H CRC. Observed deep and durable responses with the use of ICIs in advanced-stage disease have stimulated the development of clinical trials with ICIs for patients with early-stage MMR-D/MSI-H CRC. Most recently, neoadjuvant dostarlimab monotherapy for nonoperative management of MMR-D/MSI-H rectal cancer and neoadjuvant NICHE trial with nivolumab and ipilimumab for MMR-D/MSI-H colon cancer resulted in groundbreaking results. Although nonoperative management of patients with MMR-D/MSI-H rectal cancer with ICIs will potentially define our current therapeutic approach, therapeutic goals of neoadjuvant ICI therapy for patients with MMR-D/MSI-H colon cancer may differ given that nonoperative management has not been well established for colon cancer. Herein, we overview recent advancements in ICI-based therapies for patients with early-stage MMR-D/MSI-H colon and rectal cancer and elaborate on the future treatment paradigm of this unique subgroup of CRC.
  7. STAR Protoc. 2023 Feb 07. pii: S2666-1667(23)00063-1. [Epub ahead of print]4(1): 102105
      Inflammatory bowel diseases (IBDs) contribute to the tumorigenesis of colorectal cancer (CRC). Here, we describe a step-by-step protocol for the construction of colitis-associated CRC murine model by sequential utilization of azoxymethane and dextran sulfate sodium. We also detail steps to determine the degree of murine intestinal inflammation and to generate colorectum Swiss roll for further histopathological analyses. This is a convenient and reproducible protocol for colitis-associated CRC murine model by the induction of general chemical reagents. For complete details on the use and execution of this protocol, please refer to Yang et al. (2022).1.
    Keywords:  Cancer; Immunology
  8. Cancer Sci. 2023 Feb 27.
      Protein kinase C delta (PKCδ) is a multifunctional serine-threonine kinase implicated in cell proliferation, differentiation, tumorigenesis, and therapeutic resistance. However, it remains unclear how the molecular mechanism of PKCδ in colorectal cancer (CRC). In this study, we showed that PKCδ acts as a negative regulator of cellular senescence in p53 wild-type (wt-p53) CRC. Immunohistochemical analysis revealed that PKCδ levels in human CRC tissues were higher than those in the surrounding normal tissues. Deletion studies have shown that cell proliferation and tumorigenesis in wt-p53 CRC is sensitive to PKCδ expression. We found that PKCδ activates p21 via a p53-independent pathway and that PKCδ-kinase activity is essential for p21 activity. In addition, both repression of PKCδ expression and inhibition of PKCδ activity induced cellular senescence-like phenotypes, including increased β-gal staining, low Lamin B1 expression, large nucleus size, and senescence-associated secretory phenotype detection. Finally, a kinase inhibitor of PKCδ suppressed senescence-dependent tumorigenicity in a dose-dependent manner. These results offer a mechanistic insight into CRC survival and tumorigenesis and propose a novel therapeutic strategy for wt-p53 CRC.
    Keywords:  cellular senescence; colorectal cancer; kinase inhibitor; protein kinase C delta; tumorigenesis
  9. Front Pharmacol. 2022 ;13 991881
      Objective: Accumulated evidence highlights the biological significance of oxidative stress in tumorigenicity and progression of colorectal cancer (CRC). Our study aimed to establish a reliable oxidative stress-related signature to predict patients' clinical outcomes and therapeutic responses. Methods: Transcriptome profiles and clinical features of CRC patients were retrospectively analyzed from public datasets. LASSO analysis was used to construct an oxidative stress-related signature to predict overall survival, disease-free survival, disease-specific survival, and progression-free survival. Additionally, antitumor immunity, drug sensitivity, signaling pathways, and molecular subtypes were analyzed between different risk subsets through TIP, CIBERSORT, oncoPredict, etc. approaches. The genes in the signature were experimentally verified in the human colorectal mucosal cell line (FHC) along with CRC cell lines (SW-480 and HCT-116) through RT-qPCR or Western blot. Results: An oxidative stress-related signature was established, composed of ACOX1, CPT2, NAT2, NRG1, PPARGC1A, CDKN2A, CRYAB, NGFR, and UCN. The signature displayed an excellent capacity for survival prediction and was linked to worse clinicopathological features. Moreover, the signature correlated with antitumor immunity, drug sensitivity, and CRC-related pathways. Among molecular subtypes, the CSC subtype had the highest risk score. Experiments demonstrated that CDKN2A and UCN were up-regulated and ACOX1, CPT2, NAT2, NRG1, PPARGC1A, CRYAB, and NGFR were down-regulated in CRC than normal cells. In H2O2-induced CRC cells, their expression was notably altered. Conclusion: Altogether, our findings constructed an oxidative stress-related signature that can predict survival outcomes and therapeutic response in CRC patients, thus potentially assisting prognosis prediction and adjuvant therapy decisions.
    Keywords:  antitumor immunity; colorectal cancer; drug sensitivity; oxidative stress; prognosis