Different in vivo and in vitro transformation of intestinal stem cells in mismatch repair deficiency

AbstractDNA mismatch repair deficiency (MMRd) in human cancer is associated with high tumor mutational burden (TMB), frameshift mutation-derived neoantigens, increased T cell infiltration, and remarkable responsiveness to immune checkpoint blockade (ICB) therapy. Nevertheless, about half of MMRd tumors do not respond to ICB for unclear reasons. While tumor cell line transplant models of MMRd have reinforced the importance of TMB in immune response, critical questions remain regarding the role of immunosurveillance in the evolution of MMRd tumors induced in vivo. Here, we developed autochthonous mouse models of lung and colon cancer with highly efficient ablation of MMR genes via in vivo CRISPR/Cas9 targeting. Surprisingly, MMRd in these models did not result in increased immunogenicity or response to ICB. Mechanistically, we showed this lack of immunogenicity to be driven by profound intratumoral heterogeneity (ITH). Studies in animals depleted of T cells further demonstrated that immunosurveillance in MMRd tumors has no impact on TMB but shapes the clonal architecture of neoantigens by exacerbating ITH. These results provide important context for understanding immune evasion in cancers with high TMB and have major implications for therapies aimed at increasing TMB.

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Impact of Diet-Induced Obesity on Intestinal Stem Cells: Hyperproliferation but Impaired Intrinsic Function That Requires Insulin/IGF1

Endocrinology ◽

10.1210/en.2014-1112 ◽

2014 ◽

Vol 155 (9) ◽

pp. 3302-3314 ◽

Cited By ~ 48

Author(s):

Amanda T. Mah ◽

Laurianne Van Landeghem ◽

Hannah E. Gavin ◽

Scott T. Magness ◽

P. Kay Lund

Keyword(s):

Stem Cells ◽

Nutrient Intake ◽

Intestinal Stem Cells ◽

Exogenous Insulin ◽

Reporter Mouse ◽

Diet Induced Obesity ◽

Egfp Reporter ◽

The Impact

Abstract Nutrient intake regulates intestinal epithelial mass and crypt proliferation. Recent findings in model organisms and rodents indicate nutrient restriction impacts intestinal stem cells (ISC). Little is known about the impact of diet-induced obesity (DIO), a model of excess nutrient intake on ISC. We used a Sox9-EGFP reporter mouse to test the hypothesis that an adaptive response to DIO or associated hyperinsulinemia involves expansion and hyperproliferation of ISC. The Sox9-EGFP reporter mouse allows study and isolation of ISC, progenitors, and differentiated lineages based on different Sox9-EGFP expression levels. Sox9-EGFP mice were fed a high-fat diet for 20 weeks to induce DIO and compared with littermates fed low-fat rodent chow. Histology, fluorescence activated cell sorting, and mRNA analyses measured impact of DIO on jejunal crypt-villus morphometry, numbers, and proliferation of different Sox9-EGFP cell populations and gene expression. An in vitro culture assay directly assessed functional capacity of isolated ISC. DIO mice exhibited significant increases in body weight, plasma glucose, insulin, and insulin-like growth factor 1 (IGF1) levels and intestinal Igf1 mRNA. DIO mice had increased villus height and crypt density but decreased intestinal length and decreased numbers of Paneth and goblet cells. In vivo, DIO resulted in a selective expansion of Sox9-EGFPLow ISC and percentage of ISC in S-phase. ISC expansion significantly correlated with plasma insulin levels. In vitro, isolated ISC from DIO mice formed fewer enteroids in standard 3D Matrigel culture compared to controls, indicating impaired ISC function. This decreased enteroid formation in isolated ISC from DIO mice was rescued by exogenous insulin, IGF1, or both. We conclude that DIO induces specific increases in ISC and ISC hyperproliferation in vivo. However, isolated ISC from DIO mice have impaired intrinsic survival and growth in vitro that can be rescued by exogenous insulin or IGF1.

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Robust Three-dimensional (3D) Expansion of Bovine Intestinal Stem Cells: A Model for Replacement of Animal Experiments

10.21203/rs.3.rs-164747/v1 ◽

2021 ◽

Author(s):

Bo Ram Lee ◽

Hyeon Yang ◽

Sang In Lee ◽

Inamul Haq ◽

Sun A Ock ◽

...

Keyword(s):

Stem Cells ◽

Small Intestine ◽

Functional Characterization ◽

Animal Experiments ◽

Three Dimensional ◽

Intestinal Stem Cells ◽

Great Promise ◽

Intestinal Organoids

Abstract Background Intestinal organoids offer great promise for disease modelling based host-pathogen interactions and nutritional research for feed efficiency measurement in livestock as well as regenerative medicine for therapeutic purposes. However, very limited studies are available on the functional characterization and three-dimensional (3D) expansion of adult stem cells in livestock species compared to mammals. Therefore, we characterized intestinal stem cells derived from small intestine in adult bovine and cultivated intestinal organoids under in vitro 3D culture system.Results In this study, we successfully established intestinal organoids in bovine. Intestinal organoids were long-term cultivated over several passages of culture without loss of the recapitulating capacity of crypts and they had the specific expression of several specific markers involved in intestinal stem cells, intestinal epithelium and nutrient absorption. In addition, they showed the key functionality with regard to a high permeability for compounds of up to FITC-dextran 4 kDa, while FITC-dextran 40 kDa failed to enter the organoid lumen. Furthermore, the genetic properties of intestinal organoids were highly similar to those of in vivo based on QuantSeq 3’ mRNA-Seq. data.Conclusions Collectively, these results provide a reliable method for efficient isolation of intestinal crypts from small intestine and robust 3D expansion of intestinal stem cells in adult bovine and demonstrate the in vitro 3D organoids mimics the in vivo tissue topology and functionality. Finally, intestinal organoids are potential alternatives to in vivo system and will facilitate the practical use of a model to replace animal experiments in the fields of animal biotechnology for various purposes.

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Accurate diagnosis of mismatch repair deficiency in colorectal cancer using high-quality DNA samples from cultured stem cells

Oncotarget ◽

10.18632/oncotarget.26495 ◽

2018 ◽

Vol 9 (101) ◽

pp. 37534-37548

Author(s):

Tadayoshi Yamaura ◽

Hiroyuki Miyoshi ◽

Hisatsugu Maekawa ◽

Tomonori Morimoto ◽

Takehito Yamamoto ◽

...

Keyword(s):

Colorectal Cancer ◽

Stem Cells ◽

Mismatch Repair ◽

Accurate Diagnosis ◽

Mismatch Repair Deficiency ◽

High Quality ◽

Repair Deficiency

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Explosive mutation accumulation triggered by heterozygous human Pol ε proofreading-deficiency is driven by suppression of mismatch repair

eLife ◽

10.7554/elife.32692 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 16

Author(s):

Karl P Hodel ◽

Richard de Borja ◽

Erin E Henninger ◽

Brittany B Campbell ◽

Nathan Ungerleider ◽

...

Keyword(s):

Mismatch Repair ◽

Dna Polymerase ◽

Mutation Accumulation ◽

Critical Force ◽

Mismatch Repair Deficiency ◽

Tumor Mutation Burden ◽

Repair Deficiency ◽

Mutation Burden ◽

Generation Sequencing

Tumors defective for DNA polymerase (Pol) ε proofreading have the highest tumor mutation burden identified. A major unanswered question is whether loss of Pol ε proofreading by itself is sufficient to drive this mutagenesis, or whether additional factors are necessary. To address this, we used a combination of next generation sequencing and in vitro biochemistry on human cell lines engineered to have defects in Pol ε proofreading and mismatch repair. Absent mismatch repair, monoallelic Pol ε proofreading deficiency caused a rapid increase in a unique mutation signature, similar to that observed in tumors from patients with biallelic mismatch repair deficiency and heterozygous Pol ε mutations. Restoring mismatch repair was sufficient to suppress the explosive mutation accumulation. These results strongly suggest that concomitant suppression of mismatch repair, a hallmark of colorectal and other aggressive cancers, is a critical force for driving the explosive mutagenesis seen in tumors expressing exonuclease-deficient Pol ε.

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Genomic Signature of Mismatch Repair Deficiency in Areca Nut–Related Oral Cancer

Journal of Dental Research ◽

10.1177/0022034520930641 ◽

2020 ◽

Vol 99 (11) ◽

pp. 1252-1261

Author(s):

W.F. Yang ◽

N. Qin ◽

X. Song ◽

C. Jiang ◽

T. Li ◽

...

Keyword(s):

Mismatch Repair ◽

Differential Expression Analysis ◽

The Cancer Genome Atlas ◽

Genomic Signature ◽

Areca Nut ◽

Mismatch Repair Deficiency ◽

Sequencing Data ◽

Repair Deficiency

Areca nut (AN) chewing contributes to an increase of oral squamous cell carcinoma (OSCC) cases in South and Southeast Asia; however, genomic events underlying the carcinogenesis process of AN-related OSCC remain unclear. Here, we comprehensively describe the genomic and transcriptome alterations of 113 Chinese OSCC patients (89 AN related and 24 AN negative) by whole-exome sequencing and RNA sequencing, and we compared the genomic differences between AN-related and AN-negative samples by integrating sequencing data of 325 OSCC patients from The Cancer Genome Atlas database and 50 from a published Taiwanese study. We identified 11 significantly mutated genes for OSCC, including 4 novel ones ( ATG2A, WEE1, DST, and TSC2), of which WEE1 and ATG2A mutated with significantly higher rates in AN-related samples ( P = 0.04 and P = 0.003, respectively). Mutational signature analysis revealed that AN-related OSCCs were specially characterized by the genomic signature of mismatch repair deficiency (dMMR), which could also predict the prognosis status of AN-related OSCC. In addition, an elevated PD-L1 expression was also observed in both AN-related patients ( P = 3.71 × 10−11) and those with a high dMMR level ( P = 1.99 × 10−4). Further differential expression analysis and in vitro experiments confirmed the role of dMMR in the development of OSCC induced by AN exposure. Taken together, this study first revealed the molecular profiles and highlighted the role of dMMR in AN-related OSCC among the Chinese population and identified that AN-related OSCC may represent a potential cohort for effective anti-PD-1/L1 immunotherapy.

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Functional Repair Assay for the Diagnosis of Constitutional Mismatch Repair Deficiency From Non-Neoplastic Tissue

Journal of Clinical Oncology ◽

10.1200/jco.18.00474 ◽

2019 ◽

Vol 37 (6) ◽

pp. 461-470 ◽

Cited By ~ 5

Author(s):

Andrew Y. Shuen ◽

Stella Lanni ◽

Gagan B. Panigrahi ◽

Melissa Edwards ◽

Lisa Yu ◽

...

Keyword(s):

Mismatch Repair ◽

Cell Lines ◽

Neurofibromatosis Type ◽

Lymphoblastoid Cell Lines ◽

Mismatch Repair Deficiency ◽

Cancer Syndrome ◽

Li Fraumeni Syndrome ◽

Repair Deficiency

Purpose Constitutional mismatch repair deficiency (CMMRD) is a highly penetrant cancer predisposition syndrome caused by biallelic mutations in mismatch repair (MMR) genes. As several cancer syndromes are clinically similar, accurate diagnosis is critical to cancer screening and treatment. As genetic diagnosis is confounded by 15 or more pseudogenes and variants of uncertain significance, a robust diagnostic assay is urgently needed. We sought to determine whether an assay that directly measures MMR activity could accurately diagnose CMMRD. Patients and Methods In vitro MMR activity was quantified using a 3′-nicked G-T mismatched DNA substrate, which requires MSH2-MSH6 and MLH1-PMS2 for repair. We quantified MMR activity from 20 Epstein-Barr virus–transformed lymphoblastoid cell lines from patients with confirmed CMMRD. We also tested 20 lymphoblastoid cell lines from patients who were suspected for CMMRD. We also characterized MMR activity from patients with neurofibromatosis type 1, Li-Fraumeni syndrome, polymerase proofreading-associated cancer syndrome, and Lynch syndrome. Results All CMMRD cell lines had low MMR activity (n = 20; mean, 4.14 ± 1.56%) relative to controls (n = 6; mean, 44.00 ± 8.65%; P < .001). Repair was restored by complementation with the missing protein, which confirmed MMR deficiency. All cases of patients with suspected CMMRD were accurately diagnosed. Individuals with Lynch syndrome (n = 28), neurofibromatosis type 1 (n = 5), Li-Fraumeni syndrome (n = 5), and polymerase proofreading-associated cancer syndrome (n = 3) had MMR activity that was comparable to controls. To accelerate testing, we measured MMR activity directly from fresh lymphocytes, which yielded results in 8 days. Conclusion On the basis of the current data set, the in vitro G-T repair assay was able to diagnose CMMRD with 100% specificity and sensitivity. Rapid diagnosis before surgery in non-neoplastic tissues could speed proper therapeutic management.

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Mismatch repair deficiency is not sufficient to elicit tumor immunogenicity

10.21203/rs.3.rs-879464/v1 ◽

2021 ◽

Author(s):

Tyler Jacks ◽

Peter Westcott ◽

Francesc Muyas ◽

Olivia Smith ◽

Haley Hauck ◽

...

Keyword(s):

T Cell ◽

Mismatch Repair ◽

Immune Checkpoint Blockade ◽

Cell Infiltration ◽

Mismatch Repair Deficiency ◽

Dna Mismatch ◽

T Cell Infiltration ◽

Repair Deficiency ◽

Tumor Mutational Burden

Abstract DNA mismatch repair deficiency (MMRd) is associated with high tumor mutational burden (TMB), increased T cell infiltration, and remarkable responsiveness to immune checkpoint blockade (ICB) therapy1. Nevertheless, about half of MMRd tumors do not respond to ICB for unclear reasons. While cell line transplant models of MMRd have reinforced the importance of TMB in immune response2,3, critical questions remain regarding the role of immunosurveillance in the evolution of MMRd tumors induced in vivo. Here, we developed autochthonous mouse models of lung and colon cancer with ablation of MMR via in vivo CRISPR/Cas9 targeting. Surprisingly, MMRd in these models did not increase T cell infiltration or response to ICB. Mechanistically, we showed this lack of immunogenicity to be driven by profound intratumoral heterogeneity. Studies in immune deficient animals further demonstrated that immunosurveillance in MMRd tumors has no impact on TMB but shapes the clonal architecture of neoantigens by exacerbating heterogeneity. These results provide important context for understanding immune evasion in cancers with high TMB and have major implications for therapies aimed at increasing TMB.

Download Full-text