RESUMO
Cancer evolution is predominantly studied by focusing on differences in the genetic characteristics of malignant cells within tumors. However, the spatiotemporal dynamics of clonal outgrowth that underlie evolutionary trajectories remain largely unresolved. Here, we sought to unravel the clonal dynamics of colorectal cancer (CRC) expansion in space and time by using a color-based clonal tracing method. This method involves lentiviral red-green-blue (RGB) marking of cell populations, which enabled us to track individual cells and their clonal outgrowth during tumor initiation and growth in a xenograft model. We found that clonal expansion largely depends on the location of a clone, as small clones reside in the center and large clones mostly drive tumor growth at the border. These dynamics are recapitulated in a computational model, which confirms that the clone position within a tumor rather than cell-intrinsic features, is crucial for clonal outgrowth. We also found that no significant clonal loss occurs during tumor growth and clonal dispersal is limited in most models. Our results imply that, in addition to molecular features of clones such as (epi-)genetic differences between cells, clone location and the geometry of tumor growth are crucial for clonal expansion. Our findings suggest that either microenvironmental signals on the tumor border or differences in physical properties within the tumor, are major contributors to explain heterogeneous clonal expansion. Thus, this study provides further insights into the dynamics of solid tumor growth and progression, as well as the origins of tumor cell heterogeneity in a relevant model system.
Assuntos
Neoplasias Colorretais/patologia , Animais , Linhagem da Célula , Células Clonais , Neoplasias Colorretais/genética , Feminino , Xenoenxertos , Humanos , Camundongos , Camundongos Nus , Transplante de Neoplasias , Análise Espaço-TemporalRESUMO
Patient-derived xenograft (PDX) models have become an important asset in translational cancer research. However, to provide a robust preclinical platform, PDXs need to accommodate the tumor heterogeneity that is observed in patients. Colorectal cancer (CRC) can be stratified into four consensus molecular subtypes (CMS) with distinct biological and clinical features. Surprisingly, using a set of CRC patients, we revealed the partial representation of tumor heterogeneity in PDX models. The epithelial subtypes, the largest subgroups of CRC subtype, were very ineffective in establishing PDXs, indicating the need for further optimization to develop an effective personalized therapeutic approach to CRC. Moreover, we showed that tumor cell proliferation was associated with successful PDX establishment and able to distinguish patient with poor clinical outcomes within CMS2 group.
Assuntos
Neoplasias Colorretais/patologia , Modelos Animais de Doenças , Xenoenxertos , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Humanos , CamundongosRESUMO
Tumor heterogeneity represents an ongoing challenge in the field of cancer therapy. Heterogeneity is evident between cancers from different patients (inter-tumor heterogeneity) and within a single tumor (intra-tumor heterogeneity). The latter includes phenotypic diversity such as cell surface markers, (epi)genetic abnormality, growth rate, apoptosis and other hallmarks of cancer that eventually drive disease progression and treatment failure. Cancer stem cells (CSCs) have been put forward to be one of the determining factors that contribute to intra-tumor heterogeneity. However, recent findings have shown that the stem-like state in a given tumor cell is a plastic quality. A corollary to this view is that stemness traits can be acquired via (epi)genetic modification and/or interaction with the tumor microenvironment (TME). Here we discuss factors contributing to this CSC heterogeneity and the potential implications for cancer therapy.
Assuntos
Neoplasias/genética , Neoplasias/patologia , Células-Tronco Neoplásicas/patologia , Diferenciação Celular , Progressão da Doença , Epigênese Genética , Transição Epitelial-Mesenquimal , Heterogeneidade Genética , Humanos , Modelos Biológicos , Neoplasias/metabolismo , Células-Tronco Neoplásicas/metabolismo , Fenótipo , Microambiente TumoralRESUMO
Retroviral and transposon-based insertional mutagenesis (IM) screens are widely used for cancer gene discovery in mice. Exploiting the full potential of IM screens requires methods for high-throughput sequencing and mapping of transposon and retroviral insertion sites. Current protocols are based on ligation-mediated PCR amplification of junction fragments from restriction endonuclease-digested genomic DNA, resulting in amplification biases due to uneven genomic distribution of restriction enzyme recognition sites. Consequently, sequence coverage cannot be used to assess the clonality of individual insertions. We have developed a novel method, called shear-splink, for the semiquantitative high-throughput analysis of insertional mutations. Shear-splink employs random fragmentation of genomic DNA, which reduces unwanted amplification biases. Additionally, shear-splink enables us to assess clonality of individual insertions by determining the number of unique ligation points (LPs) between the adapter and genomic DNA. This parameter serves as a semiquantitative measure of the relative clonality of individual insertions within heterogeneous tumors. Mixing experiments with clonal cell lines derived from mouse mammary tumor virus (MMTV)-induced tumors showed that shear-splink enables the semiquantitative assessment of the clonality of MMTV insertions. Further, shear-splink analysis of 16 MMTV- and 127 Sleeping Beauty (SB)-induced tumors showed enrichment for cancer-relevant insertions by exclusion of irrelevant background insertions marked by single LPs, thereby facilitating the discovery of candidate cancer genes. To fully exploit the use of the shear-splink method, we set up the Insertional Mutagenesis Database (iMDB), offering a publicly available web-based application to analyze both retroviral- and transposon-based insertional mutagenesis data.
Assuntos
DNA de Neoplasias/genética , Bases de Dados Genéticas , Vírus do Tumor Mamário do Camundongo , Mutagênese Insercional , Infecções por Retroviridae/genética , Infecções Tumorais por Vírus/genética , Animais , Análise Mutacional de DNA/métodos , CamundongosRESUMO
In colorectal cancer (CRC), a subpopulation of tumor cells, called cancer stem cell (CSC) fraction, is suggested to be responsible for tumor initiation, growth, and metastasis. The search for a reliable marker to identify these CSCs is ongoing as current markers, like CD44 and CD133, are more broadly expressed and therefore are not highly selective and currently also lack function in CSC biology. Here, we analyzed whether the Wnt target Lgr5, which has earlier been identified as a marker for murine intestinal stem cells, could potentially serve as a functional marker for CSCs. Fluorescence-activated cell sorting-based detection of Lgr5, using three newly developed antibodies, on primary colorectal tumor cells revealed a clear subpopulation of Epcam+ Lgr5+ cells. Similarly, primary CRC-derived spheroid cultures, known to be enriched for CSCs, contain high levels of Lgr5+ cells, which decrease upon in vitro differentiation of these CSCs. Selection of the Lgr5(high) CRC cells identified the clonogenic fraction in vitro as well as the tumorigenic population in vivo. Finally, we confirm that Lgr5 expression is dependent on the Wnt pathway and show that Lgr5 overexpression induces clonogenic growth. We thus provide evidence that Lgr5 is, next to a functional intestinal stem cell marker, a selective marker for human colorectal CSCs.
Assuntos
Anticorpos Monoclonais Murinos/química , Biomarcadores Tumorais/metabolismo , Neoplasias Colorretais/patologia , Células-Tronco Neoplásicas/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Anticorpos Monoclonais Murinos/imunologia , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/imunologia , Diferenciação Celular , Separação Celular , Transformação Celular Neoplásica/metabolismo , Mapeamento de Epitopos , Citometria de Fluxo , Técnica Indireta de Fluorescência para Anticorpo , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Masculino , Camundongos , Camundongos Nus , Mucina-2/metabolismo , Transplante de Neoplasias , Células-Tronco Neoplásicas/fisiologia , Ratos , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/imunologia , Esferoides Celulares/metabolismo , Células Tumorais Cultivadas , Via de Sinalização WntRESUMO
Colorectal cancer (CRC) is a highly heterogeneous disease both from a molecular and clinical perspective. Several distinct molecular entities, such as microsatellite instability (MSI), have been defined that make up biologically distinct subgroups with their own clinical course. Recent data indicated that CRC can be best segregated into four groups called consensus molecular subtypes (CMS1-4), each of which has a unique biology and gene expression pattern. In order to develop improved, subtype-specific therapies and to gain insight into the molecular wiring and origin of these subtypes, reliable models are needed. This study was designed to determine the heterogeneity and identify the presence of CMSs in a large panel of CRC cell lines, primary cultures and patient-derived xenografts (PDX). We provide a repository encompassing this heterogeneity and moreover describe that a large part of the models can be robustly assigned to one of the four CMSs, independent of the stromal contribution. We subsequently validate our CMS stratification by functional analysis which for instance shows mesenchymal enrichment in CMS4 and metabolic dysregulation in CMS3. Finally, we observe a clear difference in sensitivity to chemotherapy-induced apoptosis, specifically between CMS2 and CMS4. This relates to the in vivo efficacy of chemotherapy, which delays outgrowth of CMS2, but not CMS4 xenografts. Combined our data indicate that molecular subtypes are faithfully modelled in CRC cell cultures and PDXs, representing tumour cell intrinsic and stable features. This repository provides researchers with a platform to study CRC using the existing heterogeneity.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias Colorretais/genética , Modelos Biológicos , Neoplasias Experimentais/genética , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Biomarcadores Tumorais/metabolismo , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Fluoruracila/farmacologia , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Nus , Neoplasias Experimentais/tratamento farmacológico , Neoplasias Experimentais/metabolismo , Oxaliplatina/farmacologia , Relação Estrutura-Atividade , Células Tumorais CultivadasRESUMO
The heterogeneous nature of colorectal cancer (CRC) complicates prognosis and is suggested to be a determining factor in the efficacy of adjuvant therapy for individual patients. Based on gene expression profiling, CRC is currently classified into four consensus molecular subtypes (CMSs), characterized by specific biological programs, thus suggesting the existence of unifying developmental drivers for each CMS Using human organoid cultures, we investigated the role of such developmental drivers at the premalignant stage of distinct CRC subtypes and found that TGFß plays an important role in the development of the mesenchymal CMS4, which is of special interest due to its association with dismal prognosis. We show that in tubular adenomas (TAs), which progress to classical CRCs, the dominating response to TGFß is death by apoptosis. By contrast, induction of a mesenchymal phenotype upon TGFß treatment prevails in a genetically engineered organoid culture carrying a BRAF(V) (600E) mutation, constituting a model system for sessile serrated adenomas (SSAs). Our data indicate that TGFß signaling is already active in SSA precursor lesions and that TGFß is a critical cue for directing SSAs to the mesenchymal, poor-prognosis CMS4 of CRC.
Assuntos
Adenoma/patologia , Carcinogênese , Neoplasias Colorretais/patologia , Transdução de Sinais , Fator de Crescimento Transformador beta/metabolismo , Humanos , OrganoidesRESUMO
Approximately 5-10% of metastatic colorectal cancers harbor a BRAF-V600E mutation, which is correlated with resistance to EGFR-targeted therapies and worse clinical outcome. Vice versa, targeted inhibition of BRAF-V600E with the selective inhibitor PLX 4032 (Vemurafenib) is severely limited due to feedback re-activation of EGFR in these tumors. Mounting evidence indicates that upregulation of the ErbB-3 signaling axis may occur in response to several targeted therapeutics, including Vemurafenib, and NRG-1ß-dependent re-activation of the PI3K/AKT survival pathway has been associated with therapy resistance.Here we show that colon CSCs express, next to EGFR and ErbB-2, also significant amounts of ErbB-3 on their membrane. This expression is functional as NRG-1ß strongly induces AKT/PKB and ERK phosphorylation, cell proliferation, clonogenic growth and promotes resistance to Vemurafenib in BRAF-V600E mutant colon CSCs. This resistance was completely dependent on ErbB-3 expression, as evidenced by knockdown of ErbB-3. More importantly, resistance could be alleviated with therapeutic antibody blocking ErbB-3 activation, which impaired NRG-1ß-driven AKT/PKB and ERK activation, clonogenic growth in vitro and tumor growth in xenograft models. In conclusion, our findings suggest that targeting ErbB-3 receptors could represent an effective therapeutic approach in BRAF-V600E mutant colon cancer.
Assuntos
Neoplasias do Colo/patologia , Resistencia a Medicamentos Antineoplásicos/fisiologia , Células-Tronco Neoplásicas/metabolismo , Neuregulina-1/metabolismo , Proteínas Proto-Oncogênicas B-raf/genética , Receptor ErbB-3/metabolismo , Animais , Antineoplásicos/farmacologia , Proliferação de Células , Neoplasias do Colo/genética , Neoplasias do Colo/metabolismo , Citometria de Fluxo , Humanos , Imuno-Histoquímica , Indóis/farmacologia , Camundongos , Camundongos Nus , Células-Tronco Neoplásicas/patologia , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sulfonamidas/farmacologia , Vemurafenib , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
The design of tissue culture conditions that faithfully reproduce the characteristics of cells in their native environment remains one of the main challenges of cancer stem cell (CSC) biology. Here we describe a detailed methodology for the isolation and expansion of both human colon CSCs and mouse intestinal adenoma together with a brief differentiation and coculture method that proved to be valuable to study the concept of CSCs plasticity.
Assuntos
Células-Tronco Neoplásicas/fisiologia , Adenoma/patologia , Animais , Desdiferenciação Celular , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células , Separação Celular , Células Cultivadas , Técnicas de Cocultura , Neoplasias do Colo/patologia , Meios de Cultivo Condicionados , Células Alimentadoras , Humanos , CamundongosRESUMO
Gene signatures derived from cancer stem cells (CSCs) predict tumor recurrence for many forms of cancer. Here, we derived a gene signature for colorectal CSCs defined by high Wnt signaling activity, which in agreement with previous observations predicts poor prognosis. Surprisingly, however, we found that elevated expression of Wnt targets was actually associated with good prognosis, while patient tumors with low expression of Wnt target genes segregated with immature stem cell signatures. We discovered that several Wnt target genes, including ASCL2 and LGR5, become silenced by CpG island methylation during progression of tumorigenesis, and that their re-expression was associated with reduced tumor growth. Taken together, our data show that promoter methylation of Wnt target genes is a strong predictor for recurrence of colorectal cancer, and suggest that CSC gene signatures, rather than reflecting CSC numbers, may reflect differentiation status of the malignant tissue.