bims-instec Biomed News
on Intestinal stem cells and chemoresistance in colon cancer and intestinal regeneration
Issue of 2022‒12‒04
eighteen papers selected by
Maria-Virginia Giolito
Free University of Brussels

  1. Keio J Med. 2022 Dec 01.
      Our understanding of the biology of the intestinal epithelium has advanced since the establishment of an organoid culture system. Although organoids have enabled investigation of the mechanism of self-renewal of human intestinal stem cells in vitro, it remains difficult to clarify the behavior of human normal and diseased intestinal epithelium in vivo. Recently, we developed a xenotransplantation system in which human intestinal organoids are engrafted onto epithelium-depleted mouse colons. This xenograft recapitulated the original tissue structures. Upon xenotransplantation, normal colon organoids developed normal colon crypt structures without tumorigenesis, whereas tumor-derived organoids formed colonic tumors resembling the original tumors. The non-tumorigenicity of human intestinal organoids highlights the safety of organoid-based regenerative medicine. As an example of regenerative medicine for short bowel syndrome, we devised a unique organ-repurposing approach to convert colons into small intestines by organoid transplantation. In this approach, the transplanted rat small intestinal organoids not only engrafted onto the rat colons but also remodeled the colon subepithelial structures into a small intestine-like conformation. Luminal flow accelerated the maturation of villi in the small intestine, which promoted the formation of a lymphovascular network mimicking lacteals. In this review, we provide an overview of recent advances in gastrointestinal organoid transplantation and share our understanding of human disease biology and regenerative medicine derived from these studies.
    Keywords:  epithelium replacement; intestinal organoid; organoid transplantation; regenerative medicine; xenograft
  2. Elife. 2022 Nov 29. pii: e80682. [Epub ahead of print]11
      During renewal of the intestine, cells are continuously generated by proliferation. Proliferation and differentiation must be tightly balanced, as any bias toward proliferation results in uncontrolled exponential growth. Yet, the inherently stochastic nature of cells raises the question how such fluctuations are limited. We used time-lapse microscopy to track all cells in crypts of growing mouse intestinal organoids for multiple generations, allowing full reconstruction of the underlying lineage dynamics in space and time. Proliferative behavior was highly symmetric between sister cells, with both sisters either jointly ceasing or continuing proliferation. Simulations revealed that such symmetric proliferative behavior minimizes cell number fluctuations, explaining our observation that proliferating cell number remained constant even as crypts increased in size considerably. Proliferative symmetry did not reflect positional symmetry but rather lineage control through the mother cell. Our results indicate a concrete mechanism to balance proliferation and differentiation with minimal fluctuations that may be broadly relevant for other tissues.
    Keywords:  cell lineage; cell proliferation; developmental biology; fluctuations; intestinal epithelium; mouse; physics of living systems; stem cell dynamics; time-lapse microscopy
  3. Nat Commun. 2022 Nov 28. 13(1): 7316
      Inflammation is a common medical complication in colorectal cancer (CRC) patients, which plays significant roles in tumor progression and immunosuppression. However, the influence of inflammatory conditions on the tumor response to immune checkpoint inhibitors (ICI) is incompletely understood. Here we show that in a patient with high microsatellite instability (MSI-H) CRC and a local inflammatory condition, the primary tumor progresses but its liver metastasis regresses upon Pembrolizumab treatment. In silico investigation prompted by this observation confirms correlation between inflammatory conditions and poor tumor response to PD-1 blockade in MSI-H CRCs, which is further validated in a cohort of 62 patients retrospectively enrolled to our study. Inhibition of local but not systemic immune response is verified in cultures of paired T cells and organoid cells from patients. Single-cell RNA sequencing suggests involvement of neutrophil leukocytes via CD80/CD86-CTLA4 signaling in the suppressive immune microenvironment. In concordance with this finding, elevated neutrophil-to-lymphocyte ratio indicates inhibited immune status and poor tumor response to ICIs. Receiver operating characteristic curve further demonstrates that both inflammatory conditions and a high NLR could predict a poor response to ICIs in MSI- CRCs, and the predictive value could be further increased when these two predictors are combined. Our study thus suggests that inflammatory conditions in MSI-H CRCs correlate with resistance to ICIs through neutrophil leukocyte associated immunosuppression and proposes both inflammatory conditions and high neutrophil-to-lymphocyte ratio as clinical features for poor ICI response.
  4. Cell Rep. 2022 Nov 29. pii: S2211-1247(22)01606-0. [Epub ahead of print]41(9): 111728
      The presence of BRAFV600E in colorectal cancer (CRC) is associated with a higher chance of distant metastasis. Oxidative stress in disseminated tumor cells limits metastatic capacity. To study the relationship between BRAFV600E, sensitivity to oxidative stress, and metastatic capacity in CRC, we use patient-derived organoids (PDOs) and tissue samples. BRAFV600E tumors and PDOs express high levels of glutamate-cysteine ligase (GCL), the rate-limiting enzyme in glutathione synthesis. Deletion of GCL in BRAFV600E PDOs strongly reduces their capacity to form distant liver and lung metastases but does not affect peritoneal metastasis outgrowth. Vice versa, the glutathione precursor N-acetyl-cysteine promotes organ-site-specific metastasis in the liver and the lungs but not in the peritoneum. BRAFV600E confers resistance to pharmacologically induced oxidative stress in vitro, which is partially overcome by treatment with the BRAF-inhibitor vemurafenib. We conclude that GCL-driven glutathione synthesis protects BRAFV600E-expressing tumors from oxidative stress during distant metastasis to the liver and the lungs.
    Keywords:  BRAF(V600E); CP: Cancer; colorectal; glutathione; metastasis; metastatic organotropsim; oxidative stress
  5. J Proteome Res. 2022 Nov 30.
      The development of metastasis severely reduces the life expectancy of patients with colorectal cancer (CRC). Although loss of SMAD4 is a key event in CRC progression, the resulting changes in biological processes in advanced disease and metastasis are not fully understood. Here, we applied a multiomics approach to a CRC organoid model that faithfully reflects the metastasis-supporting effects of SMAD4 inactivation. We show that loss of SMAD4 results in decreased differentiation and activation of pro-migratory and cell proliferation processes, which is accompanied by the disruption of several key oncogenic pathways, including the TGFβ, WNT, and VEGF pathways. In addition, SMAD4 inactivation leads to increased secretion of proteins that are known to be involved in a variety of pro-metastatic processes. Finally, we show that one of the factors that is specifically secreted by SMAD4-mutant organoids─DKK3─reduces the antitumor effects of natural killer cells (NK cells). Altogether, our data provide new insights into the role of SMAD4 perturbation in advanced CRC.
    Keywords:  SMAD4; cancer progression; colorectal cancer; multiomics; secretomics
  6. Gut. 2022 Nov 28. pii: gutjnl-2022-327608. [Epub ahead of print]
      OBJECTIVE: Biological insights into the stepwise development and progression of colorectal cancer (CRC) are imperative to develop tailored approaches for early detection and optimal clinical management of this disease. Here, we aimed to dissect the transcriptional and immunologic alterations that accompany malignant transformation in CRC and to identify clinically relevant biomarkers through spatial profiling of pT1 CRC samples.DESIGN: We employed digital spatial profiling (GeoMx) on eight pT1 CRCs to study gene expression in the epithelial and stromal segments across regions of distinct histology, including normal mucosa, low-grade and high-grade dysplasia and cancer. Consecutive histology sections were profiled by imaging mass cytometry to reveal immune contextures. Finally, publicly available single-cell RNA-sequencing data was analysed to determine the cellular origin of relevant transcripts.
    RESULTS: Comparison of gene expression between regions within pT1 CRC samples identified differentially expressed genes in the epithelium (n=1394 genes) and the stromal segments (n=1145 genes) across distinct histologies. Pathway analysis identified an early onset of inflammatory responses during malignant transformation, typified by upregulation of gene signatures such as innate immune sensing. We detected increased infiltration of myeloid cells and a shift in macrophage populations from pro-inflammatory HLA-DR+CD204- macrophages to HLA-DR-CD204+ immune-suppressive subsets from normal tissue through dysplasia to cancer, accompanied by the upregulation of the CD47/SIRPα 'don't eat me signal'.
    CONCLUSION: Spatial profiling revealed the molecular and immunological landscape of CRC tumourigenesis at early disease stage. We identified biomarkers with strong association with disease progression as well as targetable immune processes that are exploitable in a clinical setting.
    Keywords:  colorectal neogenesis; gene expression; immunogenetics
  7. Aging (Albany NY). 2022 Nov 29. 14
      Colorectal cancer (CRC) is one of the most common malignant tumors in the world, and most colorectal cancer is transformed from colorectal adenoma (CRA). Identifying biomarkers for the early prediction of colorectal cancer would be an important finding. Circular RNA (circRNA) plays a key role in the occurrence and development of tumors, and its biological characteristics make it a potential biomarker for the early diagnosis of diseases. Therefore, we explored the relationship between circRNA and the malignant transformation from colorectal adenoma to colorectal cancer. We constructed inflammation-based tumorigenesis mouse models and performed high-throughput RNA sequencing to determine the expression profile of circRNAs in tissues at different stages of disease. Subsequent STEM analysis showed that with the development of the disease, 30 circRNAs were significantly downregulated, and 10 circRNAs were significantly upregulated. After qRT-PCR and Fish analysis verification, it was clear that mmu_circ_0008035 and mmu_circ_0000420 were significantly and continuously overexpressed in the development of colorectal cancer in our mouse model. Next, through homology analysis of circRNA in human and mouse and validation of clinical normal tissues, adenoma tissues and CRC tissues, we found biomarkers of has_circ0101338 ahashsa_circ0022426 that could predict the malignant transformation of human colorectal inflammation into CRC and have certain diagnostic value. In conclusion, our results may shed light on the mechanism of progression from precancerous adenoma to cancer and provide biomarkers that may be used in the early diagnosis of CRC.
    Keywords:  RNA sequencing; biomarkers; circular RNAs (circRNAs); colorectal adenoma; colorectal cancer; inflammatory cancer transformation
  8. Elife. 2022 Dec 02. pii: e79811. [Epub ahead of print]11
      Efficacy of immunotherapy is limited in patients with colorectal cancer (CRC) because high expression of tumor-derived transforming growth factor (TGF)-β pathway molecules and interferon (IFN)-stimulated genes (ISGs) promotes tumor immune evasion. Here, we identified a long noncoding RNA (lncRNA), VPS9D1-AS1, which was located in ribosomes and amplified TGF-β signaling and ISG expression. We show that high expression of VPS9D1-AS1 was negatively associated with T lymphocyte infiltration in two independent cohorts of CRC. VPS9D1-AS1 served as a scaffolding lncRNA by binding with ribosome protein S3 (RPS3) to increase the translation of TGF-β, TGFBR1, and SMAD1/5/9. VPS9D1-AS1 knockout downregulated OAS1, an ISG gene, which further reduced IFNAR1 levels in tumor cells. Conversely, tumor cells overexpressing VPS9D1-AS1 were resistant to CD8+ T cell killing and lowered IFNAR1 expression in CD8+ T cells. In a conditional overexpression mouse model, VPS9D1-AS1 enhanced tumorigenesis and suppressed the infiltration of CD8+ T cells. Treating tumor-bearing mice with antisense oligonucleotide drugs targeting VPS9D1-AS1 significantly suppressed tumor growth. Our findings indicate that the tumor-derived VPS9D1-AS1/TGF-β/ISG signaling cascade promotes tumor growth and enhances immune evasion and may thus serve as a potential therapeutic target for CRC.
    Keywords:  cancer biology; human; immunology; inflammation; mouse
  9. BMC Genomics. 2022 Dec 01. 23(1): 792
      Somatic mutations drive colorectal cancer (CRC) by disrupting gene regulatory mechanisms. Distinct combinations of mutations can result in unique changes to regulatory mechanisms leading to variability in the efficacy of therapeutics. MicroRNAs are important regulators of gene expression, and their activity can be altered by oncogenic mutations. However, it is unknown how distinct combinations of CRC-risk mutations differentially affect microRNAs. Here, using genetically-modified mouse intestinal organoid (enteroid) models, we identify 12 different modules of microRNA expression patterns across different combinations of mutations common in CRC. We also show that miR-24-3p is aberrantly upregulated in genetically-modified mouse enteroids irrespective of mutational context. Furthermore, we identify an enrichment of miR-24-3p predicted targets in downregulated gene lists from various mutational contexts compared to WT. In follow-up experiments, we demonstrate that miR-24-3p promotes CRC cell survival in multiple cell contexts. Our novel characterization of genotype-specific patterns of miRNA expression offer insight into the mechanisms that drive inter-tumor heterogeneity and highlight candidate microRNA therapeutic targets for the advancement of precision medicine for CRC.
    Keywords:  CRISPR/Cas9; Colorectal cancer; MicroRNA; Organoid; Precision medicine; Tumor heterogeneity
  10. Theranostics. 2022 ;12(17): 7624-7639
      Rationale: Patients with colorectal cancer die mainly due to liver metastases (CRC-LM). Although the tumor microenvironment (TME) plays an important role in tumor development and therapeutic response, our understanding of the individual TME components, especially cancer-associated fibroblasts (CAFs), remains limited. Methods: We analyzed CRC-LM CAFs and cancer cells by single-cell transcriptomics and used bioinformatics for data analysis and integration with related available single-cell and bulk transcriptomic datasets. We validated key findings by RT-qPCR, western blotting, and immunofluorescence. Results: By single-cell transcriptomic analysis of 4,397 CAFs from six CRC-LM samples, we identified two main CAF populations, contractile CAFs and extracellular matrix (ECM)-remodeling/pro-angiogenic CAFs, and four subpopulations with distinct phenotypes. We found that ECM-remodeling/pro-angiogenic CAFs derive from portal resident fibroblasts. They associate with areas of strong desmoplastic reaction and Wnt signaling in low-proliferating tumor cells engulfed in a stiff extracellular matrix. By integrating public single-cell primary liver tumor data, we propose a model to explain how different liver malignancies recruit CAFs of different origins to this organ. Lastly, we found that LTBP2 plays an important role in modulating collagen biosynthesis, ECM organization, and adhesion pathways. We developed fully human antibodies against LTBP2 that depleted LTBP2+ CAFs in vitro. Conclusion: This study complements recent reports on CRC-LM CAF heterogeneity at the single-cell resolution. The number of sequenced CAFs was more than one order of magnitude larger compared to existing data. LTBP2 targeting by antibodies might create opportunities to deplete ECM-remodeling CAFs in CRC-LMs. This might be combined with other therapies, e.g., anti-angiogenic compounds as already done in CRC. Moreover, we showed that in intrahepatic cholangiocarcinoma, in which ECM-remodeling CAF proportion is similar to that of CRC-LM, several genes expressed by ECM-remodeling CAFs, such as LTBP2, were associated with survival.
    Keywords:  CAF; LTBP2.; liver metastasis; single-cell; tumor microenvironment
  11. Life Sci. 2022 Nov 25. pii: S0024-3205(22)00917-1. [Epub ahead of print] 121217
      AIMS: Aberrant overactivation/overexpression of NRF2 is implicated as a driving event in tumor progression, which has been attributed to its mutation or inactivation of the inhibitory protein, KEAP1. However, alternative mechanisms responsible for sustained activation of NRF2 are less understood.MAIN METHODS: Human colon cancer cell lines and tissues obtained from colorectal cancer patients were used. To examine the expression levels of ARD1 and NRF2, Western blot and immunofluorescence analyses were performed. To investigate the potential relevance of NRF2 and ARD1 to human colorectal cancer (CRC), NRF2 and ARD1 were individually silenced in human CRC cells (HCT-116) by transfection with their specific small interfering RNA (siRNA). To determine the functional role of ARD1 in NRF2 regulation, PLA, immunoprecipitation, nano-LC-ESI MS/MS, and in vitro acetylation assays were performed.
    KEY FINDINGS: ARD1 knockdown in human colon cancer cell lines significantly reduced the protein levels of NRF2 without affecting its mRNA expression; however, silencing of NRF2 did not alter ARD1 protein expression. In addition, these two proteins were co-localized and physically interacted with each other both in human colon cancer cells (HCT-116) and human colon tumor tissues. Mechanistically, ARD1 overexpression increased the acetylation levels of NRF2. Moreover, an in vitro acetylation assay and mass spectrometric analysis demonstrated that ARD1 could directly acetylate NRF2. Ectopic expression of mutant forms of ARD1 with defective acetyltransferase activity reduced the stability of NRF2.
    SIGNIFICANCE: In conclusion, ARD1 may potentiate the oncogenic function of NRF2 in human colon cancer by stabilizing this transcription factor.
    Keywords:  ARD1; Acetylation; Colorectal cancer; NRF2; Posttranslational modification
  12. J Mol Cell Biol. 2022 Dec 02. pii: mjac064. [Epub ahead of print]
      Peritoneal metastases (PM) from colorectal cancer (CRC) are associated with poor survival. The extracellular matrix (ECM) plays a fundamental role in modulating the homing of CRC metastases to the peritoneum. The mechanisms underlying the interactions between metastatic cells and the ECM, however, remain poorly understood, and the number of in vitro models available for the study of the peritoneal metastatic process is limited. Here, we show that decellularized ECM of the peritoneal cavity allows the growth of organoids obtained from PM, favoring the development of three-dimensional (3D) nodules that maintain the characteristics of in vivo PM. Organoids preferentially grow on scaffolds obtained from neoplastic peritoneum, which are characterized by greater stiffness than normal scaffolds. A gene expression analysis of organoids grown on different substrates reflected faithfully the clinical and biological characteristics of the organoids. An impact of the ECM on the response to standard chemotherapy treatment for PM was also observed. The ex vivo 3D model, obtained by combining patient-derived decellularized ECM with organoids to mimic the metastatic niche, could be an innovative tool to develop new therapeutic strategies in a biologically relevant context to personalize treatments.
    Keywords:  ECM stiffness; colorectal cancer; decellularized extracellular matrix; engineered disease model; extracellular matrix (ECM); organoids; peritoneal metastasis
  13. Cell Rep Methods. 2022 Nov 21. 2(11): 100337
      Human intestinal organoids (HIOs) derived from human pluripotent stem cells (hPSCs) hold great promise for translational medical applications. A common method to obtain HIOs has been to harvest floating hindgut spheroids arising from hPSCs. As this technique is elegant but burdensome due to the complex protocol and line-to-line variability, a more feasible method is desired. Here, we establish a robust differentiation method into suspension-cultured HIOs (s-HIOs) by seeding dissociated cells on a spheroid-forming plate. This protocol realizes the reliable generation of size-controllable spheroids. Under optimized conditions in a rotating bioreactor, the generated spheroids quickly grow and mature into large s-HIOs with supporting mesenchyme. Upon mesenteric transplantation, s-HIOs further mature and develop complex tissue architecture in vivo. This method demonstrates that intestinal tissue can be generated from iPSC-derived HIOs via suspension induction and bioreactor maturation, establishing a reliable culture platform with wide applications in regenerative medicine.
    Keywords:  hindgut spheroids; human intestinal organoids; human pluripotent stem cells; induced pluripotent stem cells; regenerative medicine; rotating bioreactor; suspension culture; transplantation
  14. Cell Mol Life Sci. 2022 Nov 27. 79(12): 607
      Colorectal cancer (CRC) is a leading cause of cancer-related death worldwide, largely due to the development of colorectal liver metastases (CRLM). For the establishment of CRLM, CRC cells must remodel their tumor-microenvironment (TME), avoid the immune system, invade the underlying stroma, survive the hostile environment of the circulation, extravasate into the liver, reprogram the hepatic microenvironment into a permissive pre-metastatic niche, and finally, awake from a dormant state to grow out into clinically detectable CRLM. These steps form part of the invasion-metastasis cascade that relies on reciprocal interactions between the tumor and its ever-changing microenvironment. Such interplay provides a strong rational for therapeutically targeting the TME. In fact, several TME constituents, such as VEGF, TGF-β coreceptor endoglin, and CXCR4, are already targeted in clinical trials. It is, however, of utmost importance to fully understand the complex interactions in the invasion-metastasis cascade to identify novel potential therapeutic targets and prevent the establishment of CRLM, which may ultimately greatly improve patient outcome.
    Keywords:  Cancer; Circulating tumor cells; Epithelial-mesenchymal transition; Invasion-metastasis cascade; Pre-metastatic niche; Tumor microenvironment
  15. J Vis Exp. 2022 Nov 11.
      Organoids are a reliable method for modeling organ tissue due to their self-organizing properties and retention of function and architecture after propagation from primary tissue or stem cells. This method of organoid generation forgoes single-cell differentiation through multiple passages and instead uses differential centrifugation to isolate mammary epithelial organoids from mechanically and enzymatically dissociated tissues. This protocol provides a streamlined technique for rapidly producing small and large epithelial organoids from both mouse and human mammary tissue in addition to techniques for organoid embedding in collagen and basement extracellular matrix. Furthermore, instructions for in-gel fixation and immunofluorescent staining are provided for the purpose of visualizing organoid morphology and density. These methodologies are suitable for myriad downstream analyses, such as co-culturing with immune cells and ex vivo metastasis modeling via collagen invasion assay. These analyses serve to better elucidate cell-cell behavior and create a more complete understanding of interactions within the tumor microenvironment.
  16. Front Immunol. 2022 ;13 978195
      Chimeric antigen receptor (CAR) T-cells represent a new genetically engineered cell-based immunotherapy tool against cancer. The use of CAR T-cells has revolutionized the therapeutic approach for hematological malignancies. Unfortunately, there is a long way to go before this treatment can be developed for solid tumors, including colorectal cancer. CAR T-cell therapy for colorectal cancer is still in its early stages, and clinical data are scarce. Major limitations of this therapy include high toxicity, relapses, and an impermeable tumor microenvironment for CAR T-cell therapy in colorectal cancer. In this review, we summarize current knowledge, highlight challenges, and discuss perspectives regarding CAR T-cell therapy in colorectal cancer.
    Keywords:  CAR T cells; clinical data; colorectal cancer; immunotherapies; tumor microenvironement; tumor mutational burden