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1.
Development ; 148(14)2021 07 15.
Artigo em Inglês | MEDLINE | ID: mdl-34184027

RESUMO

Bone morphogenetic protein (BMP) signaling is required for early forebrain development and cortical formation. How the endogenous modulators of BMP signaling regulate the structural and functional maturation of the developing brain remains unclear. Here, we show that expression of the BMP antagonist Grem1 marks committed layer V and VI glutamatergic neurons in the embryonic mouse brain. Lineage tracing of Grem1-expressing cells in the embryonic brain was examined by administration of tamoxifen to pregnant Grem1creERT; Rosa26LSLTdtomato mice at 13.5 days post coitum (dpc), followed by collection of embryos later in gestation. In addition, at 14.5 dpc, bulk mRNA-seq analysis of differentially expressed transcripts between FACS-sorted Grem1-positive and -negative cells was performed. We also generated Emx1-cre-mediated Grem1 conditional knockout mice (Emx1-Cre;Grem1flox/flox) in which the Grem1 gene was deleted specifically in the dorsal telencephalon. Grem1Emx1cKO animals had reduced cortical thickness, especially layers V and VI, and impaired motor balance and fear sensitivity compared with littermate controls. This study has revealed new roles for Grem1 in the structural and functional maturation of the developing cortex.


Assuntos
Proteína Morfogenética Óssea 1/antagonistas & inibidores , Encéfalo/fisiologia , Medo/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Neurônios Motores/metabolismo , Transdução de Sinais , Animais , Comportamento Animal , Proteína Morfogenética Óssea 1/genética , Encéfalo/embriologia , Diferenciação Celular , Proliferação de Células , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/fisiologia , Células-Tronco , Transcriptoma
2.
Br J Cancer ; 128(7): 1333-1343, 2023 03.
Artigo em Inglês | MEDLINE | ID: mdl-36717674

RESUMO

BACKGROUND: Colorectal cancer (CRC) primary tumours are molecularly classified into four consensus molecular subtypes (CMS1-4). Genetically engineered mouse models aim to faithfully mimic the complexity of human cancers and, when appropriately aligned, represent ideal pre-clinical systems to test new drug treatments. Despite its importance, dual-species classification has been limited by the lack of a reliable approach. Here we utilise, develop and test a set of options for human-to-mouse CMS classifications of CRC tissue. METHODS: Using transcriptional data from established collections of CRC tumours, including human (TCGA cohort; n = 577) and mouse (n = 57 across n = 8 genotypes) tumours with combinations of random forest and nearest template prediction algorithms, alongside gene ontology collections, we comprehensively assess the performance of a suite of new dual-species classifiers. RESULTS: We developed three approaches: MmCMS-A; a gene-level classifier, MmCMS-B; an ontology-level approach and MmCMS-C; a combined pathway system encompassing multiple biological and histological signalling cascades. Although all options could identify tumours associated with stromal-rich CMS4-like biology, MmCMS-A was unable to accurately classify the biology underpinning epithelial-like subtypes (CMS2/3) in mouse tumours. CONCLUSIONS: When applying human-based transcriptional classifiers to mouse tumour data, a pathway-level classifier, rather than an individual gene-level system, is optimal. Our R package enables researchers to select suitable mouse models of human CRC subtype for their experimental testing.


Assuntos
Neoplasias Colorretais , Humanos , Animais , Camundongos , Neoplasias Colorretais/patologia , Modelos Animais de Doenças , Transdução de Sinais
3.
Gastroenterology ; 162(3): 890-906, 2022 03.
Artigo em Inglês | MEDLINE | ID: mdl-34883119

RESUMO

BACKGROUND & AIMS: Cancer-associated fibroblasts (CAFs) play an important role in colorectal cancer (CRC) progression and predict poor prognosis in CRC patients. However, the cellular origins of CAFs remain unknown, making it challenging to therapeutically target these cells. Here, we aimed to identify the origins and contribution of colorectal CAFs associated with poor prognosis. METHODS: To elucidate CAF origins, we used a colitis-associated CRC mouse model in 5 different fate-mapping mouse lines with 5-bromodeoxyuridine dosing. RNA sequencing of fluorescence-activated cell sorting-purified CRC CAFs was performed to identify a potential therapeutic target in CAFs. To examine the prognostic significance of the stromal target, CRC patient RNA sequencing data and tissue microarray were used. CRC organoids were injected into the colons of knockout mice to assess the mechanism by which the stromal gene contributes to colorectal tumorigenesis. RESULTS: Our lineage-tracing studies revealed that in CRC, many ACTA2+ CAFs emerge through proliferation from intestinal pericryptal leptin receptor (Lepr)+ cells. These Lepr-lineage CAFs, in turn, express melanoma cell adhesion molecule (MCAM), a CRC stroma-specific marker that we identified with the use of RNA sequencing. High MCAM expression induced by transforming growth factor ß was inversely associated with patient survival in human CRC. In mice, stromal Mcam knockout attenuated orthotopically injected colorectal tumoroid growth and improved survival through decreased tumor-associated macrophage recruitment. Mechanistically, fibroblast MCAM interacted with interleukin-1 receptor 1 to augment nuclear factor κB-IL34/CCL8 signaling that promotes macrophage chemotaxis. CONCLUSIONS: In colorectal carcinogenesis, pericryptal Lepr-lineage cells proliferate to generate MCAM+ CAFs that shape the tumor-promoting immune microenvironment. Preventing the expansion/differentiation of Lepr-lineage CAFs or inhibiting MCAM activity could be effective therapeutic approaches for CRC.


Assuntos
Fibroblastos Associados a Câncer/patologia , Fibroblastos Associados a Câncer/fisiologia , Carcinogênese/patologia , Linhagem da Célula , Neoplasias Colorretais/patologia , Células-Tronco Mesenquimais/fisiologia , Actinas/genética , Actinas/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Antígeno CD146/genética , Antígeno CD146/metabolismo , Carcinogênese/genética , Carcinogênese/metabolismo , Diferenciação Celular , Proliferação de Células , Neoplasias Colorretais/metabolismo , Modelos Animais de Doenças , Feminino , Humanos , Mucosa Intestinal/patologia , Antígeno Ki-67/metabolismo , Masculino , Camundongos , Camundongos Transgênicos , Pessoa de Meia-Idade , Organoides/patologia , Organoides/fisiologia , Prognóstico , Receptores para Leptina/genética , Receptores para Leptina/metabolismo , Análise de Sequência de RNA , Taxa de Sobrevida , Microambiente Tumoral
4.
Gut ; 71(12): 2502-2517, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-35477539

RESUMO

OBJECTIVE: Stroma-rich tumours represent a poor prognostic subtype in stage II/III colon cancer (CC), with high relapse rates and limited response to standard adjuvant chemotherapy. DESIGN: To address the lack of efficacious therapeutic options for patients with stroma-rich CC, we stratified our human tumour cohorts according to stromal content, enabling identification of the biology underpinning relapse and potential therapeutic vulnerabilities specifically within stroma-rich tumours that could be exploited clinically. Following human tumour-based discovery and independent clinical validation, we use a series of in vitro and stroma-rich in vivo models to test and validate the therapeutic potential of elevating the biology associated with reduced relapse in human tumours. RESULTS: By performing our analyses specifically within the stroma-rich/high-fibroblast (HiFi) subtype of CC, we identify and validate the clinical value of a HiFi-specific prognostic signature (HPS), which stratifies tumours based on STAT1-related signalling (High-HPS v Low-HPS=HR 0.093, CI 0.019 to 0.466). Using in silico, in vitro and in vivo models, we demonstrate that the HPS is associated with antigen processing and presentation within discrete immune lineages in stroma-rich CC, downstream of double-stranded RNA and viral response signalling. Treatment with the TLR3 agonist poly(I:C) elevated the HPS signalling and antigen processing phenotype across in vitro and in vivo models. In an in vivo model of stroma-rich CC, poly(I:C) treatment significantly increased systemic cytotoxic T cell activity (p<0.05) and reduced liver metastases (p<0.0002). CONCLUSION: This study reveals new biological insight that offers a novel therapeutic option to reduce relapse rates in patients with the worst prognosis CC.


Assuntos
Biomarcadores Tumorais , Neoplasias do Colo , Humanos , Biomarcadores Tumorais/genética , Células Estromais/patologia , Recidiva Local de Neoplasia/prevenção & controle , Recidiva Local de Neoplasia/patologia , Neoplasias do Colo/patologia , Prognóstico
5.
Gastroenterology ; 160(4): 1224-1239.e30, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-33197448

RESUMO

BACKGROUND & AIMS: Cancer-associated fibroblasts (CAFs), key constituents of the tumor microenvironment, either promote or restrain tumor growth. Attempts to therapeutically target CAFs have been hampered by our incomplete understanding of these functionally heterogeneous cells. Key growth factors in the intestinal epithelial niche, bone morphogenetic proteins (BMPs), also play a critical role in colorectal cancer (CRC) progression. However, the crucial proteins regulating stromal BMP balance and the potential application of BMP signaling to manage CRC remain largely unexplored. METHODS: Using human CRC RNA expression data, we identified CAF-specific factors involved in BMP signaling, then verified and characterized their expression in the CRC stroma by in situ hybridization. CRC tumoroids and a mouse model of CRC hepatic metastasis were used to test approaches to modify BMP signaling and treat CRC. RESULTS: We identified Grem1 and Islr as CAF-specific genes involved in BMP signaling. Functionally, GREM1 and ISLR acted to inhibit and promote BMP signaling, respectively. Grem1 and Islr marked distinct fibroblast subpopulations and were differentially regulated by transforming growth factor ß and FOXL1, providing an underlying mechanism to explain fibroblast biological dichotomy. In patients with CRC, high GREM1 and ISLR expression levels were associated with poor and favorable survival, respectively. A GREM1-neutralizing antibody or fibroblast Islr overexpression reduced CRC tumoroid growth and promoted Lgr5+ intestinal stem cell differentiation. Finally, adeno-associated virus 8 (AAV8)-mediated delivery of Islr to hepatocytes increased BMP signaling and improved survival in our mouse model of hepatic metastasis. CONCLUSIONS: Stromal BMP signaling predicts and modifies CRC progression and survival, and it can be therapeutically targeted by novel AAV-directed gene delivery to the liver.


Assuntos
Proteínas Morfogenéticas Ósseas/metabolismo , Neoplasias Colorretais/patologia , Imunoglobulinas/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Neoplasias Hepáticas/secundário , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Fibroblastos Associados a Câncer/metabolismo , Carcinogênese/patologia , Diferenciação Celular , Linhagem Celular Tumoral , Neoplasias Colorretais/mortalidade , Progressão da Doença , Feminino , Hepatócitos/metabolismo , Humanos , Imunoglobulinas/genética , Estimativa de Kaplan-Meier , Masculino , Camundongos , Pessoa de Meia-Idade , Prognóstico , Transdução de Sinais , Microambiente Tumoral , Regulação para Cima , Ensaios Antitumorais Modelo de Xenoenxerto
6.
Am J Physiol Gastrointest Liver Physiol ; 320(4): G506-G520, 2021 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-33470182

RESUMO

The stem/progenitor cells of the developing intestine are biologically distinct from their adult counterparts. Here, we examine the microenvironmental cues that regulate the embryonic stem/progenitor population, focusing on the role of Notch pathway factor delta-like protein-1 (DLK1). mRNA-seq analyses of intestinal mesenchymal cells (IMCs) collected from embryonic day 14.5 (E14.5) or adult IMCs and a novel coculture system with E14.5 intestinal epithelial organoids were used. Following addition of recombinant DLK1 (rDLK) or Dlk1 siRNA (siDlk1), epithelial characteristics were compared using imaging, replating efficiency assays, qPCR, and immunocytochemistry. The intestinal phenotypes of littermate Dlk1+/+ and Dlk1-/- mice were compared using immunohistochemistry. Using transcriptomic analyses, we identified morphogens derived from the embryonic mesenchyme that potentially regulate the developing epithelial cells, to focus on Notch family candidate DLK1. Immunohistochemistry indicated that DLK1 was expressed exclusively in the intestinal stroma at E14.5 at the top of emerging villi, decreased after birth, and shifted to the intestinal epithelium in adulthood. In coculture experiments, addition of rDLK1 to adult IMCs inhibited organoid differentiation, whereas Dlk1 knockdown in embryonic IMCs increased epithelial differentiation to secretory lineage cells. Dlk1-/- mice had restricted Ki67+ cells in the villi base and increased secretory lineage cells compared with Dlk1+/+ embryos. Mesenchyme-derived DLK1 plays an important role in the promotion of epithelial stem/precursor expansion and prevention of differentiation to secretory lineages in the developing intestine.NEW & NOTEWORTHY Using a novel coculture system, transcriptomics, and transgenic mice, we investigated differential molecular signaling between the intestinal epithelium and mesenchyme during development and in the adult. We show that the Notch pathway factor delta-like protein-1 (DLK1) is stromally produced during development and uncover a new role for DLK1 in the regulation of intestinal epithelial stem/precursor expansion and differentiation to secretory lineages.


Assuntos
Proteínas de Ligação ao Cálcio/metabolismo , Comunicação Celular , Diferenciação Celular , Proliferação de Células , Células-Tronco Embrionárias/enzimologia , Células Epiteliais/enzimologia , Mucosa Intestinal/enzimologia , Células Estromais/enzimologia , Animais , Proteínas de Ligação ao Cálcio/deficiência , Proteínas de Ligação ao Cálcio/genética , Linhagem da Célula , Células Cultivadas , Técnicas de Cocultura , Regulação da Expressão Gênica no Desenvolvimento , Mucosa Intestinal/embriologia , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Organoides , Via Secretória , Transdução de Sinais , Nicho de Células-Tronco , Transcriptoma
7.
Gut ; 69(6): 1092-1103, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-31563876

RESUMO

OBJECTIVE: Pathological Wnt pathway activation is a conserved hallmark of colorectal cancer. Wnt-activating mutations can be divided into: i) ligand-independent (LI) alterations in intracellular signal transduction proteins (Adenomatous polyposis coli, ß-catenin), causing constitutive pathway activation and ii) ligand-dependent (LD) mutations affecting the synergistic R-Spondin axis (RNF43, RSPO-fusions) acting through amplification of endogenous Wnt signal transmembrane transduction. Our aim was to exploit differential Wnt target gene expression to generate a mutation-agnostic biomarker for LD tumours. DESIGN: We undertook harmonised multi-omic analysis of discovery (n=684) and validation cohorts (n=578) of colorectal tumours collated from publicly available data and the Stratification in Colorectal Cancer Consortium. We used mutation data to establish molecular ground truth and subdivide lesions into LI/LD tumour subsets. We contrasted transcriptional, methylation, morphological and clinical characteristics between groups. RESULTS: Wnt disrupting mutations were mutually exclusive. Desmoplastic stromal upregulation of RSPO may compensate for absence of epithelial mutation in a subset of stromal-rich tumours. Key Wnt negative regulator genes were differentially expressed between LD/LI tumours, with targeted hypermethylation of some genes (AXIN2, NKD1) occurring even in CIMP-negative LD cancers. AXIN2 mRNA expression was used as a discriminatory molecular biomarker to distinguish LD/LI tumours (area under the curve >0.93). CONCLUSIONS: Epigenetic suppression of appropriate Wnt negative feedback loops is selectively advantageous in LD tumours and differential AXIN2 expression in LD/LI lesions can be exploited as a molecular biomarker. Distinguishing between LD/LI tumour types is important; patients with LD tumours retain sensitivity to Wnt ligand inhibition and may be stratified at diagnosis to clinical trials of Porcupine inhibitors.


Assuntos
Neoplasias Colorretais/diagnóstico , Transdução de Sinais/genética , Proteína Wnt1/metabolismo , Idoso , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Marcadores Genéticos/genética , Humanos , Masculino , Pessoa de Meia-Idade , Proteína Wnt1/genética
8.
Gut ; 68(4): 684-692, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-29666172

RESUMO

OBJECTIVE: Serrated colorectal cancer (CRC) accounts for approximately 25% of cases and includes tumours that are among the most treatment resistant and with worst outcomes. This CRC subtype is associated with activating mutations in the mitogen-activated kinase pathway gene, BRAF, and epigenetic modifications termed the CpG Island Methylator Phenotype, leading to epigenetic silencing of key tumour suppressor genes. It is still not clear which (epi-)genetic changes are most important in neoplastic progression and we begin to address this knowledge gap herein. DESIGN: We use organoid culture combined with CRISPR/Cas9 genome engineering to sequentially introduce genetic alterations associated with serrated CRC and which regulate the stem cell niche, senescence and DNA mismatch repair. RESULTS: Targeted biallelic gene alterations were verified by DNA sequencing. Organoid growth in the absence of niche factors was assessed, as well as analysis of downstream molecular pathway activity. Orthotopic engraftment of complex organoid lines, but not BrafV600E alone, quickly generated adenocarcinoma in vivo with serrated features consistent with human disease. Loss of the essential DNA mismatch repair enzyme, Mlh1, led to microsatellite instability. Sphingolipid metabolism genes are differentially regulated in both our mouse models of serrated CRC and human CRC, with key members of this pathway having prognostic significance in the human setting. CONCLUSION: We generate rapid, complex models of serrated CRC to determine the contribution of specific genetic alterations to carcinogenesis. Analysis of our models alongside patient data has led to the identification of a potential susceptibility for this tumour type.


Assuntos
Adenocarcinoma/genética , Adenocarcinoma/patologia , Colo/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Organoides/patologia , Proteínas Proto-Oncogênicas B-raf/genética , Adenocarcinoma/metabolismo , Alelos , Colo/metabolismo , Neoplasias Colorretais/metabolismo , Ilhas de CpG/genética , Reparo de Erro de Pareamento de DNA , Análise Mutacional de DNA , Progressão da Doença , Epigenômica , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Humanos , Modelos Genéticos , Mutação , Organoides/metabolismo , Fenótipo , Proteínas Proto-Oncogênicas B-raf/metabolismo
9.
Am J Hum Genet ; 98(5): 830-842, 2016 05 05.
Artigo em Inglês | MEDLINE | ID: mdl-27087319

RESUMO

Gastric adenocarcinoma and proximal polyposis of the stomach (GAPPS) is an autosomal-dominant cancer-predisposition syndrome with a significant risk of gastric, but not colorectal, adenocarcinoma. We mapped the gene to 5q22 and found loss of the wild-type allele on 5q in fundic gland polyps from affected individuals. Whole-exome and -genome sequencing failed to find causal mutations but, through Sanger sequencing, we identified point mutations in APC promoter 1B that co-segregated with disease in all six families. The mutations reduced binding of the YY1 transcription factor and impaired activity of the APC promoter 1B in luciferase assays. Analysis of blood and saliva from carriers showed allelic imbalance of APC, suggesting that these mutations lead to decreased allele-specific expression in vivo. Similar mutations in APC promoter 1B occur in rare families with familial adenomatous polyposis (FAP). Promoter 1A is methylated in GAPPS and sporadic FGPs and in normal stomach, which suggests that 1B transcripts are more important than 1A in gastric mucosa. This might explain why all known GAPPS-affected families carry promoter 1B point mutations but only rare FAP-affected families carry similar mutations, the colonic cells usually being protected by the expression of the 1A isoform. Gastric polyposis and cancer have been previously described in some FAP-affected individuals with large deletions around promoter 1B. Our finding that GAPPS is caused by point mutations in the same promoter suggests that families with mutations affecting the promoter 1B are at risk of gastric adenocarcinoma, regardless of whether or not colorectal polyps are present.


Assuntos
Adenocarcinoma/genética , Proteína da Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/genética , Pólipos Adenomatosos/genética , Éxons/genética , Mutação Puntual/genética , Neoplasias Gástricas/genética , Desequilíbrio Alélico/genética , Variações do Número de Cópias de DNA/genética , Exoma/genética , Feminino , Mucosa Gástrica/metabolismo , Ligação Genética/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Perda de Heterozigosidade , Masculino , Linhagem , Regiões Promotoras Genéticas/genética
10.
Oncoimmunology ; 13(1): 2330194, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38516270

RESUMO

Colorectal cancer (CRC) is the third most prevalent cancer worldwide with a high mortality rate (20-30%), especially due to metastasis to adjacent organs. Clinical responses to chemotherapy, radiation, targeted and immunotherapies are limited to a subset of patients making metastatic CRC (mCRC) difficult to treat. To understand the therapeutic modulation of immune response in mCRC, we have used a genetically engineered mouse model (GEMM), "KPN", which resembles the human 'CMS4'-like subtype. We show here that transforming growth factor (TGF-ß1), secreted by KPN organoids, increases cancer cell proliferation, and inhibits splenocyte activation in vitro. TGF-ß1 also inhibits activation of naive but not pre-activated T cells, suggesting differential effects on specific immune cells. In vivo, the inhibition of TGF-ß inflames the KPN tumors, causing infiltration of T cells, monocytes and monocytic intermediates, while reducing neutrophils and epithelial cells. Co-inhibition of TGF-ß and PD-L1 signaling further enhances cytotoxic CD8+T cells and upregulates innate immune response and interferon gene signatures. However, simultaneous upregulation of cancer-related metabolic genes correlated with limited control of tumor burden and/or progression despite combination treatment. Our study illustrates the importance of using GEMMs to predict better immunotherapies for mCRC.


Assuntos
Neoplasias do Colo , Neoplasias Retais , Camundongos , Animais , Humanos , Fator de Crescimento Transformador beta1 , Fator de Crescimento Transformador beta/metabolismo , Interferons , Antígeno B7-H1/genética , Linfócitos T Citotóxicos/metabolismo
11.
Clin Cancer Res ; 30(8): 1518-1529, 2024 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-38493804

RESUMO

PURPOSE: The current approach for molecular subtyping of colon cancer relies on gene expression profiling, which is invasive and has limited ability to reveal dynamics and spatial heterogeneity. Molecular imaging techniques, such as PET, present a noninvasive alternative for visualizing biological information from tumors. However, the factors influencing PET imaging phenotype, the suitable PET radiotracers for differentiating tumor subtypes, and the relationship between PET phenotypes and tumor genotype or gene expression-based subtyping remain unknown. EXPERIMENTAL DESIGN: In this study, we conducted 126 PET scans using four different metabolic PET tracers, [18F]fluorodeoxy-D-glucose ([18F]FDG), O-(2-[18F]fluoroethyl)-l-tyrosine ([18F]FET), 3'-deoxy-3'-[18F]fluorothymidine ([18F]FLT), and [11C]acetate ([11C]ACE), using a spectrum of five preclinical colon cancer models with varying genetics (BMT, AKPN, AK, AKPT, KPN), at three sites (subcutaneous, orthograft, autochthonous) and at two tumor stages (primary vs. metastatic). RESULTS: The results demonstrate that imaging signatures are influenced by genotype, tumor environment, and stage. PET imaging signatures exhibited significant heterogeneity, with each cancer model displaying distinct radiotracer profiles. Oncogenic Kras and Apc loss showed the most distinctive imaging features, with [18F]FLT and [18F]FET being particularly effective, respectively. The tissue environment notably impacted [18F]FDG uptake, and in a metastatic model, [18F]FET demonstrated higher uptake. CONCLUSIONS: By examining factors contributing to PET-imaging phenotype, this study establishes the feasibility of noninvasive molecular stratification using multiplex radiotracer PET. It lays the foundation for further exploration of PET-based subtyping in human cancer, thereby facilitating noninvasive molecular diagnosis.


Assuntos
Neoplasias do Colo , Fluordesoxiglucose F18 , Humanos , Didesoxinucleosídeos , Tomografia por Emissão de Pósitrons/métodos , Neoplasias do Colo/diagnóstico por imagem , Neoplasias do Colo/genética , Compostos Radiofarmacêuticos
12.
Cancer Res Commun ; 4(2): 588-606, 2024 02 29.
Artigo em Inglês | MEDLINE | ID: mdl-38358352

RESUMO

Neutrophils are a highly heterogeneous cellular population. However, a thorough examination of the different transcriptional neutrophil states between health and malignancy has not been performed. We utilized single-cell RNA sequencing of human and murine datasets, both publicly available and independently generated, to identify neutrophil transcriptomic subtypes and developmental lineages in health and malignancy. Datasets of lung, breast, and colorectal cancer were integrated to establish and validate neutrophil gene signatures. Pseudotime analysis was used to identify genes driving neutrophil development from health to cancer. Finally, ligand-receptor interactions and signaling pathways between neutrophils and other immune cell populations in primary colorectal cancer and metastatic colorectal cancer were investigated. We define two main neutrophil subtypes in primary tumors: an activated subtype sharing the transcriptomic signatures of healthy neutrophils; and a tumor-specific subtype. This signature is conserved in murine and human cancer, across different tumor types. In colorectal cancer metastases, neutrophils are more heterogeneous, exhibiting additional transcriptomic subtypes. Pseudotime analysis implicates IL1ß/CXCL8/CXCR2 axis in the progression of neutrophils from health to cancer and metastasis, with effects on T-cell effector function. Functional analysis of neutrophil-tumoroid cocultures and T-cell proliferation assays using orthotopic metastatic mouse models lacking Cxcr2 in neutrophils support our transcriptional analysis. We propose that the emergence of metastatic-specific neutrophil subtypes is driven by the IL1ß/CXCL8/CXCR2 axis, with the evolution of different transcriptomic signals that impair T-cell function at the metastatic site. Thus, a better understanding of neutrophil transcriptomic programming could optimize immunotherapeutic interventions into early and late interventions, targeting different neutrophil states. SIGNIFICANCE: We identify two recurring neutrophil populations and demonstrate their staged evolution from health to malignancy through the IL1ß/CXCL8/CXCR2 axis, allowing for immunotherapeutic neutrophil-targeting approaches to counteract immunosuppressive subtypes that emerge in metastasis.


Assuntos
Neoplasias Colorretais , Neutrófilos , Animais , Camundongos , Humanos , Recidiva Local de Neoplasia/metabolismo , Transdução de Sinais/genética , Neoplasias Colorretais/genética , Análise de Célula Única
13.
Nat Commun ; 15(1): 646, 2024 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-38245513

RESUMO

Bioengineered probiotics enable new opportunities to improve colorectal cancer (CRC) screening, prevention and treatment. Here, first, we demonstrate selective colonization of colorectal adenomas after oral delivery of probiotic E. coli Nissle 1917 (EcN) to a genetically-engineered murine model of CRC predisposition and orthotopic models of CRC. We next undertake an interventional, double-blind, dual-centre, prospective clinical trial, in which CRC patients take either placebo or EcN for two weeks prior to resection of neoplastic and adjacent normal colorectal tissue (ACTRN12619000210178). We detect enrichment of EcN in tumor samples over normal tissue from probiotic-treated patients (primary outcome of the trial). Next, we develop early CRC intervention strategies. To detect lesions, we engineer EcN to produce a small molecule, salicylate. Oral delivery of this strain results in increased levels of salicylate in the urine of adenoma-bearing mice, in comparison to healthy controls. To assess therapeutic potential, we engineer EcN to locally release a cytokine, GM-CSF, and blocking nanobodies against PD-L1 and CTLA-4 at the neoplastic site, and demonstrate that oral delivery of this strain reduces adenoma burden by ~50%. Together, these results support the use of EcN as an orally-deliverable platform to detect disease and treat CRC through the production of screening and therapeutic molecules.


Assuntos
Adenoma , Neoplasias Colorretais , Animais , Humanos , Camundongos , Adenoma/diagnóstico , Adenoma/terapia , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/genética , Neoplasias Colorretais/terapia , Escherichia coli/genética , Estudos Prospectivos , Salicilatos , Método Duplo-Cego
14.
Nat Genet ; 56(3): 458-472, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38351382

RESUMO

Molecular stratification using gene-level transcriptional data has identified subtypes with distinctive genotypic and phenotypic traits, as exemplified by the consensus molecular subtypes (CMS) in colorectal cancer (CRC). Here, rather than gene-level data, we make use of gene ontology and biological activation state information for initial molecular class discovery. In doing so, we defined three pathway-derived subtypes (PDS) in CRC: PDS1 tumors, which are canonical/LGR5+ stem-rich, highly proliferative and display good prognosis; PDS2 tumors, which are regenerative/ANXA1+ stem-rich, with elevated stromal and immune tumor microenvironmental lineages; and PDS3 tumors, which represent a previously overlooked slow-cycling subset of tumors within CMS2 with reduced stem populations and increased differentiated lineages, particularly enterocytes and enteroendocrine cells, yet display the worst prognosis in locally advanced disease. These PDS3 phenotypic traits are evident across numerous bulk and single-cell datasets, and demark a series of subtle biological states that are currently under-represented in pre-clinical models and are not identified using existing subtyping classifiers.


Assuntos
Neoplasias Colorretais , Humanos , Neoplasias Colorretais/patologia , Prognóstico , Diferenciação Celular/genética , Fenótipo , Biomarcadores Tumorais/genética , Perfilação da Expressão Gênica
15.
J Exp Clin Cancer Res ; 43(1): 64, 2024 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-38424636

RESUMO

Colorectal cancer (CRC) is a heterogenous malignancy underpinned by dysregulation of cellular signaling pathways. Previous literature has implicated aberrant JAK/STAT3 signal transduction in the development and progression of solid tumors. In this study we investigate the effectiveness of inhibiting JAK/STAT3 in diverse CRC models, establish in which contexts high pathway expression is prognostic and perform in depth analysis underlying phenotypes. In this study we investigated the use of JAK inhibitors for anti-cancer activity in CRC cell lines, mouse model organoids and patient-derived organoids. Immunohistochemical staining of the TransSCOT clinical trial cohort, and 2 independent large retrospective CRC patient cohorts was performed to assess the prognostic value of JAK/STAT3 expression. We performed mutational profiling, bulk RNASeq and NanoString GeoMx® spatial transcriptomics to unravel the underlying biology of aberrant signaling. Inhibition of signal transduction with JAK1/2 but not JAK2/3 inhibitors reduced cell viability in CRC cell lines, mouse, and patient derived organoids (PDOs). In PDOs, reduced Ki67 expression was observed post-treatment. A highly significant association between high JAK/STAT3 expression within tumor cells and reduced cancer-specific survival in patients with high stromal invasion (TSPhigh) was identified across 3 independent CRC patient cohorts, including the TrasnSCOT clinical trial cohort. Patients with high phosphorylated STAT3 (pSTAT3) within the TSPhigh group had higher influx of CD66b + cells and higher tumoral expression of PDL1. Bulk RNAseq of full section tumors showed enrichment of NFκB signaling and hypoxia in these cases. Spatial deconvolution through GeoMx® demonstrated higher expression of checkpoint and hypoxia-associated genes in the tumor (pan-cytokeratin positive) regions, and reduced lymphocyte receptor signaling in the TME (pan-cytokeratin- and αSMA-) and αSMA (pan-cytokeratin- and αSMA +) areas. Non-classical fibroblast signatures were detected across αSMA + regions in cases with high pSTAT3. Therefore, in this study we have shown that inhibition of JAK/STAT3 represents a promising therapeutic strategy for patients with stromal-rich CRC tumors. High expression of JAK/STAT3 proteins within both tumor and stromal cells predicts poor outcomes in CRC, and aberrant signaling is associated with distinct spatially-dependant differential gene expression.


Assuntos
Neoplasias Colorretais , Humanos , Animais , Camundongos , Estudos Retrospectivos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Transdução de Sinais , Hipóxia , Queratinas/metabolismo , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Linhagem Celular Tumoral
16.
Reproduction ; 146(6): 581-91, 2013 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24051059

RESUMO

The mechanisms that regulate the induction of term or preterm delivery (PTD) are not fully understood. Infection is known to play a role in the induction of pro-inflammatory cascades in uteroplacental tissues associated with preterm pathological parturition. Similar but not identical cascades are evident in term labour. In the current study, we used a mouse model to evaluate the role of prokineticins in term and preterm parturition. Prokineticins are multi-functioning secreted proteins that signal through G-protein-coupled receptors to induce gene expression, including genes important in inflammatory responses. Expression of prokineticins (Prok1 and Prok2) was quantified in murine uteroplacental tissues by QPCR in the days preceding labour (days 16-19). Prok1 mRNA expression increased significantly on D18 in fetal membranes (compared with D16) but not in uterus or placenta. Intrauterine injection of PROK1 on D17 induced fetal membrane mRNA expression of the pro-inflammatory mediators Il6, Il1b, Tnf, Cxcl2 and Cxcl5, which are not normally up-regulated until D19 of pregnancy. However, intrauterine injection of PROK1 did not result in PTD. As expected, injection of lipopolysaccharide (LPS) induced PTD, but this was not associated with changes in expression of Prok1 or its receptor (Prokr1) in fetal membranes. These results suggest that although Prok1 exhibits dynamic mRNA regulation in fetal membranes preceding labour and induces a pro-inflammatory response when injected into the uterus on D17, it is insufficient to induce PTD. Additionally, prokineticin up-regulation appears not to be part of the LPS-induced inflammatory response in mouse fetal membranes.


Assuntos
Membranas Extraembrionárias/efeitos dos fármacos , Inflamação/induzido quimicamente , Fator de Crescimento do Endotélio Vascular Derivado de Glândula Endócrina/farmacologia , Animais , Membranas Extraembrionárias/imunologia , Membranas Extraembrionárias/metabolismo , Feminino , Inflamação/genética , Inflamação/metabolismo , Mediadores da Inflamação/metabolismo , Injeções , Lipopolissacarídeos/farmacologia , Masculino , Camundongos , Gravidez , Nascimento Prematuro , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Regulação para Cima/efeitos dos fármacos , Útero , Fator de Crescimento do Endotélio Vascular Derivado de Glândula Endócrina/genética , Fator de Crescimento do Endotélio Vascular Derivado de Glândula Endócrina/metabolismo
17.
bioRxiv ; 2023 Dec 10.
Artigo em Inglês | MEDLINE | ID: mdl-38106050

RESUMO

Targeting cancer stem cells (CSCs) is crucial for effective cancer treatment 1 . However, the molecular mechanisms underlying resistance to LGR5 + CSCs depletion in colorectal cancer (CRC) 2,3 remain largely elusive. Here, we unveil the existence of a primitive cell state dubbed the oncofetal (OnF) state, which works in tandem with the LGR5 + stem cells (SCs) to fuel tumor evolution in CRC. OnF cells emerge early during intestinal tumorigenesis and exhibit features of lineage plasticity. Normally suppressed by the Retinoid X Receptor (RXR) in mature SCs, the OnF program is triggered by genetic deletion of the gatekeeper APC. We demonstrate that diminished RXR activity unlocks an epigenetic circuity governed by the cooperative action of YAP and AP1, leading to OnF reprogramming. This high-plasticity state is inherently resistant to conventional chemotherapies and its adoption by LGR5 + CSCs enables them to enter a drug-tolerant state. Furthermore, through phenotypic tracing and ablation experiments, we uncover a functional redundancy between the OnF and stem cell (SC) states and show that targeting both cellular states is essential for sustained tumor regression in vivo . Collectively, these findings establish a mechanistic foundation for developing effective combination therapies with enduring impact on CRC treatment.

18.
bioRxiv ; 2023 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-37034712

RESUMO

Osteoarthritis (OA), which carries an enormous disease burden across the world, is characterised by irreversible degeneration of articular cartilage (AC), and subsequently bone. The cellular cause of OA is unknown. Here, using lineage tracing in mice, we show that the BMP-antagonist Gremlin 1 (Grem1) marks a novel chondrogenic progenitor (CP) cell population in the articular surface that generates joint cartilage and subchondral bone during development and adulthood. Notably, this CP population is depleted in injury-induced OA, and with age. OA is also induced by toxin-mediated ablation of Grem1 CP cells in young mice. Transcriptomic analysis and functional modelling in mice revealed articular surface Grem1-lineage cells are dependent on Foxo1; ablation of Foxo1 in Grem1-lineage cells led to early OA. This analysis identified FGFR3 signalling as a therapeutic target, and injection of its activator, FGF18, caused proliferation of Grem1-lineage CP cells, increased cartilage thickness, and reduced OA pathology. We propose that OA arises from the loss of CP cells at the articular surface secondary to an imbalance in progenitor cell homeostasis and present a new progenitor population as a locus for OA therapy.

19.
Nat Commun ; 14(1): 6909, 2023 10 31.
Artigo em Inglês | MEDLINE | ID: mdl-37907525

RESUMO

Osteoarthritis (OA) is characterised by an irreversible degeneration of articular cartilage. Here we show that the BMP-antagonist Gremlin 1 (Grem1) marks a bipotent chondrogenic and osteogenic progenitor cell population within the articular surface. Notably, these progenitors are depleted by injury-induced OA and increasing age. OA is also caused by ablation of Grem1 cells in mice. Transcriptomic and functional analysis in mice found that articular surface Grem1-lineage cells are dependent on Foxo1 and ablation of Foxo1 in Grem1-lineage cells caused OA. FGFR3 signalling was confirmed as a promising therapeutic pathway by administration of pathway activator, FGF18, resulting in Grem1-lineage chondrocyte progenitor cell proliferation, increased cartilage thickness and reduced OA. These findings suggest that OA, in part, is caused by mechanical, developmental or age-related attrition of Grem1 expressing articular cartilage progenitor cells. These cells, and the FGFR3 signalling pathway that sustains them, may be effective future targets for biological management of OA.


Assuntos
Cartilagem Articular , Osteoartrite , Camundongos , Animais , Osteoartrite/genética , Osteoartrite/metabolismo , Células-Tronco/metabolismo , Células Cultivadas , Perfilação da Expressão Gênica , Osteogênese , Cartilagem Articular/metabolismo , Condrócitos/metabolismo , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo
20.
bioRxiv ; 2023 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-37066243

RESUMO

Bioengineered probiotics enable new opportunities to improve colorectal cancer (CRC) screening, prevention and treatment strategies. Here, we demonstrate the phenomenon of selective, long-term colonization of colorectal adenomas after oral delivery of probiotic E. coli Nissle 1917 (EcN) to a genetically-engineered murine model of CRC predisposition. We show that, after oral administration, adenomas can be monitored over time by recovering EcN from stool. We also demonstrate specific colonization of EcN to solitary neoplastic lesions in an orthotopic murine model of CRC. We then exploit this neoplasia-homing property of EcN to develop early CRC intervention strategies. To detect lesions, we engineer EcN to produce a small molecule, salicylate, and demonstrate that oral delivery of this strain results in significantly increased levels of salicylate in the urine of adenoma-bearing mice, in comparison to healthy controls. We also assess EcN engineered to locally release immunotherapeutics at the neoplastic site. Oral delivery to mice bearing adenomas, reduced adenoma burden by ∻50%, with notable differences in the spatial distribution of T cell populations within diseased and healthy intestinal tissue, suggesting local induction of robust anti-tumor immunity. Together, these results support the use of EcN as an orally-delivered platform to detect disease and treat CRC through its production of screening and therapeutic molecules.

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