RESUMO
Advanced solid cancers are complex assemblies of tumor, immune, and stromal cells characterized by high intratumoral variation. We use highly multiplexed tissue imaging, 3D reconstruction, spatial statistics, and machine learning to identify cell types and states underlying morphological features of known diagnostic and prognostic significance in colorectal cancer. Quantitation of these features in high-plex marker space reveals recurrent transitions from one tumor morphology to the next, some of which are coincident with long-range gradients in the expression of oncogenes and epigenetic regulators. At the tumor invasive margin, where tumor, normal, and immune cells compete, T cell suppression involves multiple cell types and 3D imaging shows that seemingly localized 2D features such as tertiary lymphoid structures are commonly interconnected and have graded molecular properties. Thus, while cancer genetics emphasizes the importance of discrete changes in tumor state, whole-specimen imaging reveals large-scale morphological and molecular gradients analogous to those in developing tissues.
Assuntos
Adenocarcinoma , Neoplasias Colorretais , Humanos , Adenocarcinoma/patologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/patologia , Processamento de Imagem Assistida por Computador , Oncogenes , Microambiente TumoralRESUMO
Colorectal cancer exhibits dynamic cellular and genetic heterogeneity during progression from precursor lesions toward malignancy. Analysis of spatial multi-omic data from 31 human colorectal specimens enabled phylogeographic mapping of tumor evolution that revealed individualized progression trajectories and accompanying microenvironmental and clonal alterations. Phylogeographic mapping ordered genetic events, classified tumors by their evolutionary dynamics, and placed clonal regions along global pseudotemporal progression trajectories encompassing the chromosomal instability (CIN+) and hypermutated (HM) pathways. Integrated single-cell and spatial transcriptomic data revealed recurring epithelial programs and infiltrating immune states along progression pseudotime. We discovered an immune exclusion signature (IEX), consisting of extracellular matrix regulators DDR1, TGFBI, PAK4, and DPEP1, that charts with CIN+ tumor progression, is associated with reduced cytotoxic cell infiltration, and shows prognostic value in independent cohorts. This spatial multi-omic atlas provides insights into colorectal tumor-microenvironment co-evolution, serving as a resource for stratification and targeted treatments.
Assuntos
Neoplasias Colorretais , Instabilidade de Microssatélites , Microambiente Tumoral , Humanos , Instabilidade Cromossômica/genética , Neoplasias Colorretais/patologia , Perfilação da Expressão Gênica , Quinases Ativadas por p21/genética , Filogenia , Mutação , Progressão da Doença , PrognósticoRESUMO
Immune responses to cancer are highly variable, with mismatch repair-deficient (MMRd) tumors exhibiting more anti-tumor immunity than mismatch repair-proficient (MMRp) tumors. To understand the rules governing these varied responses, we transcriptionally profiled 371,223 cells from colorectal tumors and adjacent normal tissues of 28 MMRp and 34 MMRd individuals. Analysis of 88 cell subsets and their 204 associated gene expression programs revealed extensive transcriptional and spatial remodeling across tumors. To discover hubs of interacting malignant and immune cells, we identified expression programs in different cell types that co-varied across tumors from affected individuals and used spatial profiling to localize coordinated programs. We discovered a myeloid cell-attracting hub at the tumor-luminal interface associated with tissue damage and an MMRd-enriched immune hub within the tumor, with activated T cells together with malignant and myeloid cells expressing T cell-attracting chemokines. By identifying interacting cellular programs, we reveal the logic underlying spatially organized immune-malignant cell networks.
Assuntos
Neoplasias Colorretais/imunologia , Neoplasias Colorretais/patologia , Proteínas Morfogenéticas Ósseas/metabolismo , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Compartimento Celular , Linhagem Celular Tumoral , Quimiocinas/metabolismo , Estudos de Coortes , Neoplasias Colorretais/genética , Reparo de Erro de Pareamento de DNA/genética , Células Endoteliais/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Imunidade , Inflamação/patologia , Monócitos/patologia , Células Mieloides/patologia , Neutrófilos/patologia , Células Estromais/metabolismo , Linfócitos T/metabolismo , Transcrição GênicaRESUMO
Colorectal cancers (CRCs) arise from precursor polyps whose cellular origins, molecular heterogeneity, and immunogenic potential may reveal diagnostic and therapeutic insights when analyzed at high resolution. We present a single-cell transcriptomic and imaging atlas of the two most common human colorectal polyps, conventional adenomas and serrated polyps, and their resulting CRC counterparts. Integrative analysis of 128 datasets from 62 participants reveals adenomas arise from WNT-driven expansion of stem cells, while serrated polyps derive from differentiated cells through gastric metaplasia. Metaplasia-associated damage is coupled to a cytotoxic immune microenvironment preceding hypermutation, driven partly by antigen-presentation differences associated with tumor cell-differentiation status. Microsatellite unstable CRCs contain distinct non-metaplastic regions where tumor cells acquire stem cell properties and cytotoxic immune cells are depleted. Our multi-omic atlas provides insights into malignant progression of colorectal polyps and their microenvironment, serving as a framework for precision surveillance and prevention of CRC.
Assuntos
Pólipos do Colo/patologia , Neoplasias Colorretais/patologia , Microambiente Tumoral , Imunidade Adaptativa , Adenoma/genética , Adenoma/patologia , Adulto , Idoso , Animais , Carcinogênese/genética , Carcinogênese/patologia , Morte Celular , Diferenciação Celular , Pólipos do Colo/genética , Pólipos do Colo/imunologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/imunologia , Progressão da Doença , Feminino , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Heterogeneidade Genética , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Mutação/genética , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , RNA-Seq , Reprodutibilidade dos Testes , Análise de Célula Única , Microambiente Tumoral/imunologiaRESUMO
Cancer cells enter a reversible drug-tolerant persister (DTP) state to evade death from chemotherapy and targeted agents. It is increasingly appreciated that DTPs are important drivers of therapy failure and tumor relapse. We combined cellular barcoding and mathematical modeling in patient-derived colorectal cancer models to identify and characterize DTPs in response to chemotherapy. Barcode analysis revealed no loss of clonal complexity of tumors that entered the DTP state and recurred following treatment cessation. Our data fit a mathematical model where all cancer cells, and not a small subpopulation, possess an equipotent capacity to become DTPs. Mechanistically, we determined that DTPs display remarkable transcriptional and functional similarities to diapause, a reversible state of suspended embryonic development triggered by unfavorable environmental conditions. Our study provides insight into how cancer cells use a developmentally conserved mechanism to drive the DTP state, pointing to novel therapeutic opportunities to target DTPs.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Diapausa , Resistencia a Medicamentos Antineoplásicos , Animais , Antineoplásicos/farmacologia , Autofagia/efeitos dos fármacos , Autofagia/genética , Linhagem Celular Tumoral , Células Clonais , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/metabolismo , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Heterogeneidade Genética/efeitos dos fármacos , Humanos , Irinotecano/farmacologia , Irinotecano/uso terapêutico , Camundongos Endogâmicos NOD , Camundongos SCID , Modelos Biológicos , Transdução de Sinais/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Single-cell RNA sequencing (scRNA-seq) is a powerful tool for defining cellular diversity in tumors, but its application toward dissecting mechanisms underlying immune-modulating therapies is scarce. We performed scRNA-seq analyses on immune and stromal populations from colorectal cancer patients, identifying specific macrophage and conventional dendritic cell (cDC) subsets as key mediators of cellular cross-talk in the tumor microenvironment. Defining comparable myeloid populations in mouse tumors enabled characterization of their response to myeloid-targeted immunotherapy. Treatment with anti-CSF1R preferentially depleted macrophages with an inflammatory signature but spared macrophage populations that in mouse and human expresses pro-angiogenic/tumorigenic genes. Treatment with a CD40 agonist antibody preferentially activated a cDC population and increased Bhlhe40+ Th1-like cells and CD8+ memory T cells. Our comprehensive analysis of key myeloid subsets in human and mouse identifies critical cellular interactions regulating tumor immunity and defines mechanisms underlying myeloid-targeted immunotherapies currently undergoing clinical testing.
Assuntos
Neoplasias do Colo/patologia , Células Mieloides/metabolismo , Análise de Célula Única/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Sequência de Bases/genética , Linfócitos T CD8-Positivos/imunologia , China , Neoplasias do Colo/terapia , Neoplasias Colorretais/patologia , Células Dendríticas/imunologia , Feminino , Humanos , Imunoterapia , Macrófagos/imunologia , Masculino , Camundongos , Pessoa de Meia-Idade , Análise de Sequência de RNA/métodos , Microambiente Tumoral/genética , Microambiente Tumoral/imunologiaRESUMO
Widespread changes to DNA methylation and chromatin are well documented in cancer, but the fate of higher-order chromosomal structure remains obscure. Here we integrated topological maps for colon tumors and normal colons with epigenetic, transcriptional, and imaging data to characterize alterations to chromatin loops, topologically associated domains, and large-scale compartments. We found that spatial partitioning of the open and closed genome compartments is profoundly compromised in tumors. This reorganization is accompanied by compartment-specific hypomethylation and chromatin changes. Additionally, we identify a compartment at the interface between the canonical A and B compartments that is reorganized in tumors. Remarkably, similar shifts were evident in non-malignant cells that have accumulated excess divisions. Our analyses suggest that these topological changes repress stemness and invasion programs while inducing anti-tumor immunity genes and may therefore restrain malignant progression. Our findings call into question the conventional view that tumor-associated epigenomic alterations are primarily oncogenic.
Assuntos
Cromatina/metabolismo , Cromossomos/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Metilação de DNA , Epigênese Genética , Regulação Neoplásica da Expressão Gênica/genética , Divisão Celular , Senescência Celular/genética , Sequenciamento de Cromatina por Imunoprecipitação , Cromossomos/genética , Estudos de Coortes , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Biologia Computacional , Metilação de DNA/genética , Epigenômica , Células HCT116 , Humanos , Hibridização in Situ Fluorescente , Microscopia Eletrônica de Transmissão , Simulação de Dinâmica Molecular , RNA-Seq , Análise Espacial , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismoRESUMO
Lineage tracing provides key insights into the fate of individual cells in complex organisms. Although effective genetic labeling approaches are available in model systems, in humans, most approaches require detection of nuclear somatic mutations, which have high error rates, limited scale, and do not capture cell state information. Here, we show that somatic mutations in mtDNA can be tracked by single-cell RNA or assay for transposase accessible chromatin (ATAC) sequencing. We leverage somatic mtDNA mutations as natural genetic barcodes and demonstrate their utility as highly accurate clonal markers to infer cellular relationships. We track native human cells both in vitro and in vivo and relate clonal dynamics to gene expression and chromatin accessibility. Our approach should allow clonal tracking at a 1,000-fold greater scale than with nuclear genome sequencing, with simultaneous information on cell state, opening the way to chart cellular dynamics in human health and disease.
Assuntos
DNA Mitocondrial/genética , Mitocôndrias/genética , Sequência de Bases , Linhagem da Célula , Cromatina , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Genômica/métodos , Células HEK293 , Células-Tronco Hematopoéticas/fisiologia , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Mutação , Análise de Célula Única , TransposasesRESUMO
Regulatory T (Treg) cells are a barrier for tumor immunity and a target for immunotherapy. Using single-cell transcriptomics, we found that CD4+ T cells infiltrating primary and metastatic colorectal cancer and non-small-cell lung cancer are highly enriched for two subsets of comparable size and suppressor function comprising forkhead box protein P3+ Treg and eomesodermin homolog (EOMES)+ type 1 regulatory T (Tr1)-like cells also expressing granzyme K and chitinase-3-like protein 2. EOMES+ Tr1-like cells, but not Treg cells, were clonally related to effector T cells and were clonally expanded in primary and metastatic tumors, which is consistent with their proliferation and differentiation in situ. Using chitinase-3-like protein 2 as a subset signature, we found that the EOMES+ Tr1-like subset correlates with disease progression but is also associated with response to programmed cell death protein 1-targeted immunotherapy. Collectively, these findings highlight the heterogeneity of Treg cells that accumulate in primary tumors and metastases and identify a new prospective target for cancer immunotherapy.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/imunologia , Hematopoiese Clonal/imunologia , Neoplasias Colorretais/imunologia , Neoplasias Pulmonares/imunologia , Linfócitos T Reguladores/imunologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/secundário , Carcinoma Pulmonar de Células não Pequenas/terapia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Proliferação de Células/genética , Quimioterapia Adjuvante/métodos , Quitinases/metabolismo , Colectomia , Colo/patologia , Colo/cirurgia , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Neoplasias Colorretais/terapia , Conjuntos de Dados como Assunto , Progressão da Doença , Resistencia a Medicamentos Antineoplásicos/imunologia , Feminino , Citometria de Fluxo , Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica/imunologia , Granzimas/metabolismo , Humanos , Inibidores de Checkpoint Imunológico/farmacologia , Inibidores de Checkpoint Imunológico/uso terapêutico , Estimativa de Kaplan-Meier , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/terapia , Masculino , Pessoa de Meia-Idade , Cultura Primária de Células , Receptor de Morte Celular Programada 1/antagonistas & inibidores , RNA-Seq , Análise de Célula Única , Proteínas com Domínio T/metabolismo , Linfócitos T Reguladores/metabolismoRESUMO
Cancer immunotherapies have shown substantial clinical activity for a subset of patients with epithelial cancers. Still, technological platforms to study cancer T-cell interactions for individual patients and understand determinants of responsiveness are presently lacking. Here, we establish and validate a platform to induce and analyze tumor-specific T cell responses to epithelial cancers in a personalized manner. We demonstrate that co-cultures of autologous tumor organoids and peripheral blood lymphocytes can be used to enrich tumor-reactive T cells from peripheral blood of patients with mismatch repair-deficient colorectal cancer and non-small-cell lung cancer. Furthermore, we demonstrate that these T cells can be used to assess the efficiency of killing of matched tumor organoids. This platform provides an unbiased strategy for the isolation of tumor-reactive T cells and provides a means by which to assess the sensitivity of tumor cells to T cell-mediated attack at the level of the individual patient.
Assuntos
Leucócitos Mononucleares/citologia , Linfócitos T/imunologia , Idoso , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Técnicas de Cultura de Células , Técnicas de Cocultura , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Feminino , Humanos , Técnicas In Vitro , Interferon gama/farmacologia , Leucócitos Mononucleares/metabolismo , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Ativação Linfocitária/efeitos dos fármacos , Masculino , Pessoa de Meia-Idade , Linfócitos T/citologia , Linfócitos T/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
In colorectal cancer patients, a high density of cytotoxic CD8+ T cells in tumors is associated with better prognosis. Using a Stat3 loss-of-function approach in two wnt/ß-catenin-dependent autochthonous models of sporadic intestinal tumorigenesis, we unravel a complex intracellular process in intestinal epithelial cells (IECs) that controls the induction of a CD8+ T cell based adaptive immune response. Elevated mitophagy in IECs causes iron(II)-accumulation in epithelial lysosomes, in turn, triggering lysosomal membrane permeabilization. Subsequent release of proteases into the cytoplasm augments MHC class I presentation and activation of CD8+ T cells via cross-dressing of dendritic cells. Thus, our findings highlight a so-far-unrecognized link between mitochondrial function, lysosomal integrity, and MHC class I presentation in IECs and suggest that therapies triggering mitophagy or inducing LMP in IECs may prove successful in shifting the balance toward anti-tumor immunity in colorectal cancer.
Assuntos
Imunidade Adaptativa , Mitofagia , Imunidade Adaptativa/efeitos dos fármacos , Animais , Azoximetano/toxicidade , Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/efeitos dos fármacos , Linfócitos T CD8-Positivos/metabolismo , Permeabilidade da Membrana Celular , Neoplasias Colorretais/mortalidade , Neoplasias Colorretais/patologia , Citocinas/metabolismo , Células Dendríticas/citologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Feminino , Compostos Ferrosos/metabolismo , Humanos , Interferon gama/metabolismo , Interferon gama/farmacologia , Mucosa Intestinal/citologia , Mucosa Intestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Lisossomos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Mitofagia/efeitos dos fármacos , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo , Taxa de SobrevidaRESUMO
Gut microbiota are linked to chronic inflammation and carcinogenesis. Chemotherapy failure is the major cause of recurrence and poor prognosis in colorectal cancer patients. Here, we investigated the contribution of gut microbiota to chemoresistance in patients with colorectal cancer. We found that Fusobacterium (F.) nucleatum was abundant in colorectal cancer tissues in patients with recurrence post chemotherapy, and was associated with patient clinicopathological characterisitcs. Furthermore, our bioinformatic and functional studies demonstrated that F. nucleatum promoted colorectal cancer resistance to chemotherapy. Mechanistically, F. nucleatum targeted TLR4 and MYD88 innate immune signaling and specific microRNAs to activate the autophagy pathway and alter colorectal cancer chemotherapeutic response. Thus, F. nucleatum orchestrates a molecular network of the Toll-like receptor, microRNAs, and autophagy to clinically, biologically, and mechanistically control colorectal cancer chemoresistance. Measuring and targeting F. nucleatum and its associated pathway will yield valuable insight into clinical management and may ameliorate colorectal cancer patient outcomes.
Assuntos
Autofagia , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Fusobacterium nucleatum/fisiologia , Microbioma Gastrointestinal , Animais , Antineoplásicos/uso terapêutico , Capecitabina/uso terapêutico , Neoplasias Colorretais/metabolismo , Resistencia a Medicamentos Antineoplásicos , Xenoenxertos , Camundongos , MicroRNAs/metabolismo , Transplante de Neoplasias , Compostos de Platina/uso terapêutico , Recidiva , Receptores Toll-Like/metabolismo , Microambiente TumoralRESUMO
Investigating therapeutic "outliers" that show exceptional responses to anti-cancer treatment can uncover biomarkers of drug sensitivity. We performed preclinical trials investigating primary murine acute myeloid leukemias (AMLs) generated by retroviral insertional mutagenesis in KrasG12D "knockin" mice with the MEK inhibitor PD0325901 (PD901). One outlier AML responded and exhibited intrinsic drug resistance at relapse. Loss of wild-type (WT) Kras enhanced the fitness of the dominant clone and rendered it sensitive to MEK inhibition. Similarly, human colorectal cancer cell lines with increased KRAS mutant allele frequency were more sensitive to MAP kinase inhibition, and CRISPR-Cas9-mediated replacement of WT KRAS with a mutant allele sensitized heterozygous mutant HCT116 cells to treatment. In a prospectively characterized cohort of patients with advanced cancer, 642 of 1,168 (55%) with KRAS mutations exhibited allelic imbalance. These studies demonstrate that serial genetic changes at the Kras/KRAS locus are frequent in cancer and modulate competitive fitness and MEK dependency.
Assuntos
Antineoplásicos/uso terapêutico , Benzamidas/uso terapêutico , Neoplasias Colorretais/genética , Difenilamina/análogos & derivados , Sistema de Sinalização das MAP Quinases , Proteínas Proto-Oncogênicas p21(ras)/genética , Animais , Antineoplásicos/farmacologia , Benzamidas/farmacologia , Linhagem Celular Tumoral , Evolução Clonal , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/patologia , Difenilamina/farmacologia , Difenilamina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/genética , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Camundongos , Mutação , RetroviridaeRESUMO
Peritoneal metastasis (PM) is often regarded as a less frequent pattern of spread; however, collectively across all spectra of primary tumors, the consequences of PM impact a large population of patients annually. Unlike other modes of metastasis, symptoms at presentation or during the treatment course are common, representing an additional challenge in the management of PM. Early efforts with chemotherapy and incomplete surgical interventions transiently improved symptoms, but durable symptom control and survival extension were rare, which established a perspective of treatment futility for PM through most of the 20th century. Notably, the continued development of better systemic therapy combinations, optimization of cytoreductive surgery (CRS), and rigorous investigation of combining regional therapy-specifically hyperthermic intraperitoneal chemotherapy-with CRS, have resulted in more effective multimodal treatment options for patients with PM. In this article, the authors provide a comprehensive review of the data establishing the contemporary approach for tumors with a high frequency of PM, including appendix, colorectal, mesothelioma, and gastric cancers. The authors also explore the emerging role of adding hyperthermic intraperitoneal chemotherapy to the well established paradigm of CRS and systemic therapy for advanced ovarian cancer, as well as the recent clinical trials identifying the efficacy of poly(adenosine diphosphate ribose) polymerase maintenance therapy. Finally, recent data are included that explore the role of precision medicine technology in PM management that, in the future, may help further improve patient selection, identify the best systemic therapy regimens, detect actionable mutations, and identify new targets for drug development.
Assuntos
Neoplasias Colorretais , Hipertermia Induzida , Neoplasias Peritoneais , Humanos , Neoplasias Peritoneais/terapia , Neoplasias Peritoneais/secundário , Futilidade Médica , Hipertermia Induzida/métodos , Terapia Combinada , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Procedimentos Cirúrgicos de Citorredução/métodos , Neoplasias Colorretais/terapia , Neoplasias Colorretais/patologiaRESUMO
In Rspondin-based 3D cultures, Lgr5 stem cells from multiple organs form ever-expanding epithelial organoids that retain their tissue identity. We report the establishment of tumor organoid cultures from 20 consecutive colorectal carcinoma (CRC) patients. For most, organoids were also generated from adjacent normal tissue. Organoids closely recapitulate several properties of the original tumor. The spectrum of genetic changes within the "living biobank" agrees well with previous large-scale mutational analyses of CRC. Gene expression analysis indicates that the major CRC molecular subtypes are represented. Tumor organoids are amenable to high-throughput drug screens allowing detection of gene-drug associations. As an example, a single organoid culture was exquisitely sensitive to Wnt secretion (porcupine) inhibitors and carried a mutation in the negative Wnt feedback regulator RNF43, rather than in APC. Organoid technology may fill the gap between cancer genetics and patient trials, complement cell-line- and xenograft-based drug studies, and allow personalized therapy design. PAPERCLIP.
Assuntos
Bancos de Espécimes Biológicos , Neoplasias Colorretais/patologia , Ensaios de Seleção de Medicamentos Antitumorais/métodos , Organoides , Neoplasias Colorretais/tratamento farmacológico , Proteínas de Ligação a DNA/metabolismo , Humanos , Proteínas Oncogênicas/metabolismo , Técnicas de Cultura de Órgãos , Organoides/efeitos dos fármacos , Medicina de Precisão , Ubiquitina-Proteína LigasesRESUMO
The adenomatous polyposis coli (APC) tumor suppressor is mutated in the vast majority of human colorectal cancers (CRC) and leads to deregulated Wnt signaling. To determine whether Apc disruption is required for tumor maintenance, we developed a mouse model of CRC whereby Apc can be conditionally suppressed using a doxycycline-regulated shRNA. Apc suppression produces adenomas in both the small intestine and colon that, in the presence of Kras and p53 mutations, can progress to invasive carcinoma. In established tumors, Apc restoration drives rapid and widespread tumor-cell differentiation and sustained regression without relapse. Tumor regression is accompanied by the re-establishment of normal crypt-villus homeostasis, such that once aberrantly proliferating cells reacquire self-renewal and multi-lineage differentiation capability. Our study reveals that CRC cells can revert to functioning normal cells given appropriate signals and provide compelling in vivo validation of the Wnt pathway as a therapeutic target for treatment of CRC.
Assuntos
Proteína da Polipose Adenomatosa do Colo/metabolismo , Neoplasias Colorretais/genética , Modelos Animais de Doenças , Intestino Grosso/patologia , Intestino Delgado/patologia , Proteína da Polipose Adenomatosa do Colo/genética , Animais , Proliferação de Células , Neoplasias Colorretais/patologia , Doxiciclina/administração & dosagem , Genes p53 , Pólipos Intestinais/metabolismo , Pólipos Intestinais/patologia , Intestino Grosso/metabolismo , Intestino Delgado/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas Proto-Oncogênicas p21(ras)/genética , Interferência de RNA , Via de Sinalização WntRESUMO
Colorectal cancer is a leading cause of cancer-related deaths. Mutations in the innate immune sensor AIM2 are frequently identified in patients with colorectal cancer, but how AIM2 modulates colonic tumorigenesis is unknown. Here, we found that Aim2-deficient mice were hypersusceptible to colonic tumor development. Production of inflammasome-associated cytokines and other inflammatory mediators was largely intact in Aim2-deficient mice; however, intestinal stem cells were prone to uncontrolled proliferation. Aberrant Wnt signaling expanded a population of tumor-initiating stem cells in the absence of AIM2. Susceptibility of Aim2-deficient mice to colorectal tumorigenesis was enhanced by a dysbiotic gut microbiota, which was reduced by reciprocal exchange of gut microbiota with healthy wild-type mice. These findings uncover a synergy between a specific host genetic factor and gut microbiota in determining the susceptibility to colorectal cancer. Therapeutic modulation of AIM2 expression and microbiota has the potential to prevent colorectal cancer.
Assuntos
Proliferação de Células , Neoplasias Colorretais/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células-Tronco/patologia , Animais , Azoximetano , Colite/induzido quimicamente , Neoplasias Colorretais/genética , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/patologia , Sulfato de Dextrana , Enterócitos/patologia , Trato Gastrointestinal/microbiologia , Inflamassomos/metabolismo , Camundongos , Mutação , Células-Tronco/metabolismoRESUMO
Colorectal cancer primarily metastasizes to the liver and globally kills over 600,000 people annually. By functionally screening 661 microRNAs (miRNAs) in parallel during liver colonization, we have identified miR-551a and miR-483 as robust endogenous suppressors of liver colonization and metastasis. These miRNAs convergently target creatine kinase, brain-type (CKB), which phosphorylates the metabolite creatine, to generate phosphocreatine. CKB is released into the extracellular space by metastatic cells encountering hepatic hypoxia and catalyzes production of phosphocreatine, which is imported through the SLC6A8 transporter and used to generate ATPfueling metastatic survival. Combinatorial therapeutic viral delivery of miR-551a and miR-483-5p through single-dose adeno-associated viral (AAV) delivery significantly suppressed colon cancer metastasis, as did CKB inhibition with a small-molecule inhibitor. Importantly, human liver metastases express higher CKB and SLC6A8 levels and reduced miR-551a/miR-483 levels relative to primary tumors. We identify the extracellular space as an important compartment for malignant energetic catalysis and therapeutic targeting.
Assuntos
Neoplasias Colorretais/metabolismo , Neoplasias Hepáticas/secundário , MicroRNAs/metabolismo , Metástase Neoplásica/genética , Animais , Linhagem Celular Tumoral , Neoplasias Colorretais/genética , Neoplasias Colorretais/patologia , Creatina Quinase Forma BB/metabolismo , Metabolismo Energético , Matriz Extracelular , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias Hepáticas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Metástase Neoplásica/patologia , Proteínas do Tecido Nervoso/metabolismo , Proteínas da Membrana Plasmática de Transporte de Neurotransmissores/metabolismoRESUMO
Fusobacterium nucleatum (Fn), a bacterium present in the human oral cavity and rarely found in the lower gastrointestinal tract of healthy individuals1, is enriched in human colorectal cancer (CRC) tumours2-5. High intratumoural Fn loads are associated with recurrence, metastases and poorer patient prognosis5-8. Here, to delineate Fn genetic factors facilitating tumour colonization, we generated closed genomes for 135 Fn strains; 80 oral strains from individuals without cancer and 55 unique cancer strains cultured from tumours from 51 patients with CRC. Pangenomic analyses identified 483 CRC-enriched genetic factors. Tumour-isolated strains predominantly belong to Fn subspecies animalis (Fna). However, genomic analyses reveal that Fna, considered a single subspecies, is instead composed of two distinct clades (Fna C1 and Fna C2). Of these, only Fna C2 dominates the CRC tumour niche. Inter-Fna analyses identified 195 Fna C2-associated genetic factors consistent with increased metabolic potential and colonization of the gastrointestinal tract. In support of this, Fna C2-treated mice had an increased number of intestinal adenomas and altered metabolites. Microbiome analysis of human tumour tissue from 116 patients with CRC demonstrated Fna C2 enrichment. Comparison of 62 paired specimens showed that only Fna C2 is tumour enriched compared to normal adjacent tissue. This was further supported by metagenomic analysis of stool samples from 627 patients with CRC and 619 healthy individuals. Collectively, our results identify the Fna clade bifurcation, show that specifically Fna C2 drives the reported Fn enrichment in human CRC and reveal the genetic underpinnings of pathoadaptation of Fna C2 to the CRC niche.
Assuntos
Neoplasias Colorretais , Fusobacterium nucleatum , Animais , Humanos , Camundongos , Adenoma/microbiologia , Estudos de Casos e Controles , Neoplasias Colorretais/microbiologia , Neoplasias Colorretais/patologia , Fezes/microbiologia , Fusobacterium nucleatum/classificação , Fusobacterium nucleatum/genética , Fusobacterium nucleatum/isolamento & purificação , Fusobacterium nucleatum/patogenicidade , Trato Gastrointestinal/metabolismo , Trato Gastrointestinal/microbiologia , Genoma Bacteriano/genética , Boca/microbiologia , FemininoRESUMO
Three-dimensional organoid culture technologies have revolutionized cancer research by allowing for more realistic and scalable reproductions of both tumour and microenvironmental structures1-3. This has enabled better modelling of low-complexity cancer cell behaviours that occur over relatively short periods of time4. However, available organoid systems do not capture the intricate evolutionary process of cancer development in terms of tissue architecture, cell diversity, homeostasis and lifespan. As a consequence, oncogenesis and tumour formation studies are not possible in vitro and instead require the extensive use of animal models, which provide limited spatiotemporal resolution of cellular dynamics and come at a considerable cost in terms of resources and animal lives. Here we developed topobiologically complex mini-colons that are able to undergo tumorigenesis ex vivo by integrating microfabrication, optogenetic and tissue engineering approaches. With this system, tumorigenic transformation can be spatiotemporally controlled by directing oncogenic activation through blue-light exposure, and emergent colon tumours can be tracked in real-time at the single-cell resolution for several weeks without breaking the culture. These induced mini-colons display rich intratumoural and intertumoural diversity and recapitulate key pathophysiological hallmarks displayed by colorectal tumours in vivo. By fine-tuning cell-intrinsic and cell-extrinsic parameters, mini-colons can be used to identify tumorigenic determinants and pharmacological opportunities. As a whole, our study paves the way for cancer initiation research outside living organisms.