RESUMO
Disseminated tumor cells (dTCs) can frequently be detected in the bone marrow (BM) of colorectal cancer (CRC) patients, raising the possibility that the BM serves as a reservoir for metastatic tumor cells. Identification of dTCs in BM aspirates harbors the potential of assessing therapeutic outcome and directing therapy intensity with limited risk and effort. Still, the functional and prognostic relevance of dTCs is not fully established. We have previously shown that CRC cell clones can be traced to the BM of mice carrying patient-derived xenografts. However, cellular interactions, proliferative state and tumorigenicity of dTCs remain largely unknown. Here, we applied a coculture system modeling the microvascular niche and used immunofluorescence imaging of the murine BM to show that primary CRC cells migrate toward endothelial tubes. dTCs in the BM were rare, but detectable in mice with xenografts from most patient samples (8/10) predominantly at perivascular sites. Comparable to primary tumors, a substantial fraction of proliferating dTCs was detected in the BM. However, most dTCs were found as isolated cells, indicating that dividing dTCs rather separate than aggregate to metastatic clones-a phenomenon frequently observed in the microvascular niche model. Clonal tracking identified subsets of self-renewing tumor-initiating cells in the BM that formed tumors out of BM transplants, including one subset that did not drive primary tumor growth. Our results indicate an important role of the perivascular BM niche for CRC cell dissemination and show that dTCs can be a potential source for tumor relapse and tumor heterogeneity.
Assuntos
Medula Óssea/patologia , Neoplasias Colorretais/patologia , Proteínas de Fluorescência Verde/metabolismo , Células-Tronco Mesenquimais/citologia , Células Neoplásicas Circulantes/patologia , Células Tumorais Cultivadas/citologia , Animais , Medula Óssea/metabolismo , Rastreamento de Células , Técnicas de Cocultura , Neoplasias Colorretais/metabolismo , Proteínas de Fluorescência Verde/genética , Células Endoteliais da Veia Umbilical Humana , Humanos , Células-Tronco Mesenquimais/metabolismo , Camundongos , Células Neoplásicas Circulantes/metabolismo , Imagem Óptica , Prognóstico , Nicho de Células-Tronco , Imagem com Lapso de Tempo , Células Tumorais Cultivadas/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: While colorectal cancer (CRC) patients with localized disease have a favorable prognosis, the five-year-survival rate in patients with distant spread is still below 15%. Hence, a detailed understanding of the mechanisms regulating metastasis formation is essential to develop therapeutic strategies targeting metastasized CRC. The notch pathway has been shown to be involved in the metastatic spread of various tumor entities; however, the impact of its target gene HEYL remains unclear so far. METHODS: In this study, we functionally assessed the association between high HEYL expression and metastasis formation in human CRC. Therefore, we lentivirally overexpressed HEYL in two human patient-derived CRC cultures differing in their spontaneous metastasizing capacity and analyzed metastasis formation as well as tumor cell dissemination into the bone marrow after xenotransplantation into NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. RESULTS: HEYL overexpression decreased tumor cell dissemination and the absolute numbers of formed metastases in a sub-renal capsular spontaneous metastasis formation model, addressing all steps of the metastatic cascade. In contrast, metastatic capacity was not decreased following intrasplenic xenotransplantation where the cells are placed directly into the blood circulation. CONCLUSION: These results suggest that HEYL negatively regulates metastasis formation in vivo presumably by inhibiting intravasation of metastasis-initiating cells.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Neoplasias da Medula Óssea/secundário , Neoplasias Colorretais/patologia , Proteínas Repressoras/metabolismo , Esferoides Celulares/patologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Neoplasias da Medula Óssea/genética , Neoplasias da Medula Óssea/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Feminino , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Receptor Notch1/metabolismo , Proteínas Repressoras/genética , Esferoides Celulares/metabolismoRESUMO
Patient-derived cancer xenografts (PDX) are widely used to identify and evaluate novel therapeutic targets, and to test therapeutic approaches in preclinical mouse avatar trials. Despite their widespread use, potential caveats of PDX models remain considerably underappreciated. Here, we demonstrate that EBV-associated B-lymphoproliferations frequently develop following xenotransplantation of human colorectal and pancreatic carcinomas in highly immunodeficient NOD.Cg-Prkdcscid Il2rgtm1Wjl /SzJ (NSG) mice (18/47 and 4/37 mice, respectively), and in derived cell cultures in vitro. Strikingly, even PDX with carcinoma histology can host scarce EBV-infected B-lymphocytes that can fully overgrow carcinoma cells during serial passaging in vitro and in vivo. As serial xenografting is crucial to expand primary tumor tissue for biobanks and cohorts for preclinical mouse avatar trials, the emerging dominance of B-lymphoproliferations in serial PDX represents a serious confounding factor in these models. Consequently, repeated phenotypic assessments of serial PDX are mandatory at each expansion step to verify "bona fide" carcinoma xenografts.
Assuntos
Linfócitos B/transplante , Carcinoma Ductal Pancreático/patologia , Neoplasias Colorretais/patologia , Infecções por Vírus Epstein-Barr/patologia , Transtornos Linfoproliferativos/etiologia , Neoplasias Pancreáticas/patologia , Ensaio de Cápsula Sub-Renal , Animais , Antígenos de Neoplasias/análise , Linfócitos B/patologia , Linfócitos B/virologia , Carcinoma Ductal Pancreático/imunologia , Carcinoma Ductal Pancreático/virologia , Divisão Celular , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/virologia , Meios de Cultura Livres de Soro , Infecções por Vírus Epstein-Barr/imunologia , Xenoenxertos/imunologia , Xenoenxertos/patologia , Humanos , Hospedeiro Imunocomprometido , Antígenos Comuns de Leucócito/análise , Transtornos Linfoproliferativos/patologia , Transtornos Linfoproliferativos/virologia , Camundongos , Camundongos Endogâmicos NOD , Especificidade de Órgãos , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/virologia , Esferoides Celulares , Ensaio de Cápsula Sub-Renal/métodosRESUMO
Lentiviral vectors (LV) are widely used to stably transfer genes into target cells investigating or treating gene functions. In addition, gene transfer into early murine embryos may be improved to efficiently generate transgenic mice. We applied lentiviral gene transfer to generate a mouse model transgenic for SET binding protein-1 (Setbp1) and enhanced green fluorescent protein (eGFP). Neither transgenic founders nor their vector-positive offspring transcribed or expressed the transgenes. Bisulfite sequencing of the internal spleen focus-forming virus (SFFV) promoter demonstrated extensive methylation of all analyzed CpGs in the transgenic mice. To analyze the impact of Setbp1 on epigenetic silencing, embryonic stem cells (ESC) were differentiated into cardiomyocytes (CM) in vitro. In contrast to human promoters in LV, virally derived promoter sequences were strongly methylated during differentiation, independent of the transgene. Moreover, the commonly used SFFV promoter (SFFVp) was highly methylated with remarkable strength and frequency during hematopoietic differentiation in vivo in LV but less in γ-retroviral (γ-RV) backbones. In summary, we conclude that LV using an internal SFFVp are not suitable to generate transgenic mice or perform constitutive expression studies in differentiating cells. Choosing the appropriate promoter is also crucial to allow stable transgene expression in clinical gene therapy.
Assuntos
Proteínas de Transporte/genética , Vetores Genéticos , Lentivirus/genética , Camundongos Transgênicos/genética , Vírus Formadores de Foco no Baço/genética , Células-Tronco/metabolismo , Animais , Diferenciação Celular , Ilhas de CpG/genética , Metilação de DNA , Epigênese Genética , Efeito Fundador , Inativação Gênica , Genes Essenciais , Proteínas de Fluorescência Verde/genética , Humanos , Camundongos , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Regiões Promotoras Genéticas , Análise de Sequência de DNA , Células-Tronco/citologia , TransgenesRESUMO
Accumulating evidence identifies non-genetic mechanisms substantially contributing to drug resistance in cancer patients. Preclinical and clinical data implicate the transcriptional co-activators YAP1 and its paralog TAZ in resistance to multiple targeted therapies, highlighting the strong need for therapeutic strategies overcoming YAP1/TAZ-mediated resistance across tumor entities. Here, we show particularly high YAP1/TAZ activity in MITFlow/AXLhigh melanomas characterized by resistance to MAPK pathway inhibition and broad receptor tyrosine kinase activity. To uncover genetic dependencies of melanoma cells with high YAP1/TAZ activity, we used a genome-wide CRISPR/Cas9 functional screen and identified SLC35B2, the 3'-phosphoadenosine-5'-phosphosulfate transporter of the Golgi apparatus, as an essential gene for YAP1/TAZ-driven drug resistance. SLC35B2 expression correlates with tumor progression, and its loss decreases heparan sulfate expression, reduces receptor tyrosine kinase activity, and sensitizes resistant melanoma cells to BRAF inhibition in vitro and in vivo. Thus, targeting heparan sulfation via SLC35B2 represents a novel approach for breaking receptor tyrosine kinase-mediated resistance to MAPK pathway inhibitors.
Assuntos
Melanoma , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Heparitina Sulfato/metabolismo , Humanos , Melanoma/tratamento farmacológico , Melanoma/genética , Melanoma/patologia , Inibidores de Proteínas Quinases/farmacologia , Receptores Proteína Tirosina Quinases , Fatores de Transcrição , Proteínas de Sinalização YAPRESUMO
BACKGROUND: Frequent activation of the co-transcriptional factor YAP is observed in a large number of solid tumors. Activated YAP associates with enhancer loci via TEAD4-DNA-binding protein and stimulates cancer aggressiveness. Although thousands of YAP/TEAD4 binding-sites are annotated, their functional importance is unknown. Here, we aim at further identification of enhancer elements that are required for YAP functions. RESULTS: We first apply genome-wide ChIP profiling of YAP to systematically identify enhancers that are bound by YAP/TEAD4. Next, we implement a genetic approach to uncover functions of YAP/TEAD4-associated enhancers, demonstrate its robustness, and use it to reveal a network of enhancers required for YAP-mediated proliferation. We focus on EnhancerTRAM2, as its target gene TRAM2 shows the strongest expression-correlation with YAP activity in nearly all tumor types. Interestingly, TRAM2 phenocopies the YAP-induced cell proliferation, migration, and invasion phenotypes and correlates with poor patient survival. Mechanistically, we identify FSTL-1 as a major direct client of TRAM2 that is involved in these phenotypes. Thus, TRAM2 is a key novel mediator of YAP-induced oncogenic proliferation and cellular invasiveness. CONCLUSIONS: YAP is a transcription co-factor that binds to thousands of enhancer loci and stimulates tumor aggressiveness. Using unbiased functional approaches, we dissect YAP enhancer network and characterize TRAM2 as a novel mediator of cellular proliferation, migration, and invasion. Our findings elucidate how YAP induces cancer aggressiveness and may assist diagnosis of cancer metastasis.
Assuntos
Carcinogênese/genética , Elementos Facilitadores Genéticos , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Animais , Sítios de Ligação , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Proteínas de Ligação a DNA/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Glicoproteínas de Membrana/química , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Fatores de Transcrição de Domínio TEA/genética , Fatores de Transcrição de Domínio TEA/metabolismo , Fatores de Transcrição/metabolismo , TranscriptomaRESUMO
Intra-tumor heterogeneity of tumor-initiating cell (TIC) activity drives colorectal cancer (CRC) progression and therapy resistance. Here, we used single-cell RNA-sequencing of patient-derived CRC models to decipher distinct cell subpopulations based on their transcriptional profiles. Cell type-specific expression modules of stem-like, transit amplifying-like, and differentiated CRC cells resemble differentiation states of normal intestinal epithelial cells. Strikingly, identified subpopulations differ in proliferative activity and metabolic state. In summary, we here show at single-cell resolution that transcriptional heterogeneity identifies functional states during TIC differentiation. Furthermore, identified expression signatures are linked to patient prognosis. Targeting transcriptional states associated to cancer cell differentiation might unravel novel vulnerabilities in human CRC.
RESUMO
Novel treatment options for metastatic colorectal cancer (CRC) are urgently needed to improve patient outcome. Here, we screen a library of non-characterized small molecules against a heterogeneous collection of patient-derived CRC spheroids. By prioritizing compounds with inhibitory activity in a subset of-but not all-spheroid cultures, NCT02 is identified as a candidate with minimal risk of non-specific toxicity. Mechanistically, we show that NCT02 acts as molecular glue that induces ubiquitination of cyclin K (CCNK) and proteasomal degradation of CCNK and its complex partner CDK12. Knockout of CCNK or CDK12 decreases proliferation of CRC cells in vitro and tumor growth in vivo. Interestingly, sensitivity to pharmacological CCNK/CDK12 degradation is associated with TP53 deficiency and consensus molecular subtype 4 in vitro and in patient-derived xenografts. We thus demonstrate the efficacy of targeted CCNK/CDK12 degradation for a CRC subset, highlighting the potential of drug-induced proteolysis for difficult-to-treat types of cancer.
Assuntos
Antineoplásicos/farmacologia , Neoplasias Colorretais/metabolismo , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/metabolismo , Proteólise , Antineoplásicos/química , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Autorrenovação Celular/efeitos dos fármacos , Dano ao DNA , Feminino , Ensaios de Triagem em Larga Escala , Humanos , Complexo de Endopeptidases do Proteassoma/metabolismo , Proteólise/efeitos dos fármacos , Proteômica , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/efeitos dos fármacosRESUMO
OBJECTIVES: Salinomycin is a polyether antibiotic with selective activity against human cancer stem cells. The impact of salinomycin on patient-derived primary human colorectal cancer cells has not been investigated so far. Thus, here we aimed to investigate the activity of salinomycin against tumor initiating cells isolated from patients with colorectal cancer. METHODS: Primary tumor-initiating cells (TIC) isolated from human patients with colorectal liver metastases or from human primary colon carcinoma were exposed to salinomycin and compared to treatment with 5-FU and oxaliplatin. TICs were injected subcutaneously into NOD/SCID mice to induce a patient-derived mouse xenograft model of colorectal cancer. Animals were treated either with salinomycin, FOLFOX regimen, or salinomycin and FOLFOX. Human colorectal cancer cells were used to delineate an underlying molecular mechanism of salinomycin in this tumor entity. RESULTS: Applying TICs isolated from human patients with colorectal liver metastases or from human primary colon carcinoma, we demonstrated that salinomycin exerts increased antiproliferative activity compared to 5-fluorouracil and oxaliplatin treatment. Consistently, salinomycin alone or in combination with FOLFOX exerts superior antitumor activity compared to FOLFOX therapy in a patient-derived mouse xenograft model of colorectal cancer. Salinomycin induces apoptosis of human colorectal cancer cells, accompanied by accumulation of dysfunctional mitochondria and reactive oxygen species. These effects are associated with expressional down-regulation of superoxide dismutase-1 (SOD1) in response to salinomycin treatment. CONCLUSION: Collectively, the results of this pre-clinical study indicate that salinomycin alone or in combination with 5-fluorouracil and oxaliplatin exerts increased antitumoral activity compared to common chemotherapy.
Assuntos
Apoptose/efeitos dos fármacos , Neoplasias Colorretais , Neoplasias Hepáticas , Mitocôndrias , Piranos/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Idoso , Idoso de 80 Anos ou mais , Animais , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Feminino , Células HCT116 , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Neoplasias Hepáticas/secundário , Masculino , Camundongos Endogâmicos NOD , Camundongos SCID , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Mitocôndrias/patologia , Metástase Neoplásica , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
In highly aggressive malignancies like pancreatic cancer (PC), patient-derived tumor models can serve as disease-relevant models to understand disease-related biology as well as to guide clinical decision-making. In this study, we describe a two-step protocol allowing systematic establishment of patient-derived primary cultures from PC patient tumors. Initial xenotransplantation of surgically resected patient tumors (n = 134) into immunodeficient mice allows for efficient in vivo expansion of vital tumor cells and successful tumor expansion in 38% of patient tumors (51/134). Expansion xenografts closely recapitulate the histoarchitecture of their matching patients' primary tumors. Digestion of xenograft tumors and subsequent in vitro cultivation resulted in the successful generation of semi-adherent PC cultures of pure epithelial cell origin in 43.1% of the cases. The established primary cultures include diverse pathological types of PC: Pancreatic ductal adenocarcinoma (86.3%, 19/22), adenosquamous carcinoma (9.1%, 2/22) and ductal adenocarcinoma with oncocytic IPMN (4.5%, 1/22). We here provide a protocol to establish quality-controlled PC patient-derived primary cell cultures from heterogeneous PC patient tumors. In vitro preclinical models provide the basis for the identification and preclinical assessment of novel therapeutic opportunities targeting pancreatic cancer.
Assuntos
Modelos Biológicos , Neoplasias Pancreáticas/patologia , Animais , Linhagem Celular Tumoral , Proliferação de Células , Feminino , Humanos , Masculino , Camundongos , Ensaios Antitumorais Modelo de Xenoenxerto , Neoplasias PancreáticasRESUMO
Colorectal adenomas are precursor lesions of colorectal cancers and represent clonal amplifications of single cells from colonic crypts. DNA methylation patterns specify cell-type identity during cellular differentiation and, therefore, provide opportunities for the molecular analysis of tumors. We have now analyzed DNA methylation patterns in colorectal adenomas and identified three biologically defined subclasses that describe different intestinal crypt differentiation stages. Importantly, colorectal carcinomas could be classified into the same methylation subtypes, reflecting their shared cell types of origin with adenomas. Further data analysis also revealed significantly reduced overall survival for one of the subtypes. Our results provide a concept for understanding the methylation patterns observed in colorectal cancer and provide opportunities for tumor subclassification and patient stratification.
Assuntos
Carcinogênese/genética , Neoplasias Colorretais/patologia , Metilação de DNA , Adenoma/classificação , Adenoma/genética , Adenoma/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais/classificação , Neoplasias Colorretais/genética , Epigenômica , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Fatores de Transcrição/metabolismoRESUMO
Although tumor-initiating cell (TIC) self-renewal has been postulated to be essential in progression and metastasis formation of human pancreatic adenocarcinoma (PDAC), clonal dynamics of TICs within PDAC tumors are yet unknown. Here, we show that long-term progression of PDAC in serial xenotransplantation is driven by a succession of transiently active TICs producing tumor cells in temporally restricted bursts. Clonal tracking of individual, genetically marked TICs revealed that individual tumors are generated by distinct sets of TICs with very little overlap between subsequent xenograft generations. An unexpected functional and phenotypic plasticity of pancreatic TICs in vivo underlies the recruitment of inactive TIC clones in serial xenografts. The observed clonal succession of TIC activity in serial xenotransplantation is in stark contrast to the continuous activity of limited numbers of self-renewing TICs within a fixed cellular hierarchy observed in other epithelial cancers and emphasizes the need to target TIC activation, rather than a fixed TIC population, in PDAC.
Assuntos
Adenocarcinoma/patologia , Células-Tronco Neoplásicas/fisiologia , Neoplasias Pancreáticas/patologia , Animais , Modelos Animais de Doenças , Feminino , Xenoenxertos , Humanos , Masculino , Camundongos Endogâmicos NODRESUMO
A hierarchically organized cell compartment drives colorectal cancer (CRC) progression. Genetic barcoding allows monitoring of the clonal output of tumorigenic cells without prospective isolation. In this study, we asked whether tumor clone-initiating cells (TcICs) were genetically heterogeneous and whether differences in self-renewal and activation reflected differential kinetics among individual subclones or functional hierarchies within subclones. Monitoring genomic subclone kinetics in three patient tumors and corresponding serial xenografts and spheroids by high-coverage whole-genome sequencing, clustering of genetic aberrations, subclone combinatorics, and mutational signature analysis revealed at least two to four genetic subclones per sample. Long-term growth in serial xenografts and spheroids was driven by multiple genomic subclones with profoundly differing growth dynamics and hence different quantitative contributions over time. Strikingly, genetic barcoding demonstrated stable functional heterogeneity of CRC TcICs during serial xenografting despite near-complete changes in genomic subclone contribution. This demonstrates that functional heterogeneity is, at least frequently, present within genomic subclones and independent of mutational subclone differences.
Assuntos
Neoplasias Colorretais/genética , Variações do Número de Cópias de DNA , Células-Tronco Neoplásicas/metabolismo , Esferoides Celulares/metabolismo , Animais , Células Clonais/metabolismo , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/patologia , Análise Mutacional de DNA/métodos , Heterogeneidade Genética , Genômica/métodos , Humanos , Subunidade gama Comum de Receptores de Interleucina/deficiência , Subunidade gama Comum de Receptores de Interleucina/genética , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Mutação , Transplante Heterólogo , Células Tumorais CultivadasRESUMO
Extensive prior research focused on somatic copy-number alterations (SCNAs) affecting cancer genes, yet the extent to which recurrent SCNAs exert their influence through rearrangement of cis-regulatory elements (CREs) remains unclear. Here we present a framework for inferring cancer-related gene overexpression resulting from CRE reorganization (e.g., enhancer hijacking) by integrating SCNAs, gene expression data and information on topologically associating domains (TADs). Analysis of 7,416 cancer genomes uncovered several pan-cancer candidate genes, including IRS4, SMARCA1 and TERT. We demonstrate that IRS4 overexpression in lung cancer is associated with recurrent deletions in cis, and we present evidence supporting a tumor-promoting role. We additionally pursued cancer-type-specific analyses and uncovered IGF2 as a target for enhancer hijacking in colorectal cancer. Recurrent tandem duplications intersecting with a TAD boundary mediate de novo formation of a 3D contact domain comprising IGF2 and a lineage-specific super-enhancer, resulting in high-level gene activation. Our framework enables systematic inference of CRE rearrangements mediating dysregulation in cancer.
Assuntos
Variações do Número de Cópias de DNA/genética , Elementos Facilitadores Genéticos/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Substratos do Receptor de Insulina/genética , Fator de Crescimento Insulin-Like II/genética , Neoplasias/genética , Estudos de Associação Genética , Predisposição Genética para Doença , Humanos , Regiões Promotoras GenéticasRESUMO
Within primary colorectal cancer (CRC) a subfraction of all tumor-initiating cells (TIC) drives long-term progression in serial xenotransplantation. It has been postulated that efficient maintenance of TIC activity in vitro requires serum-free spheroid culture conditions that support a stem-like state of CRC cells. To address whether tumorigenicity is indeed tightly linked to such a stem-like state in spheroids, we transferred TIC-enriched spheroid cultures to serum-containing adherent conditions that should favor their differentiation. Under these conditions, primary CRC cells did no longer grow as spheroids but formed an adherent cell layer, up-regulated colon epithelial differentiation markers, and down-regulated TIC-associated markers. Strikingly, upon xenotransplantation cells cultured under either condition equally efficient formed serially transplantable tumors. Clonal analyses of individual lentivirally marked TIC clones cultured under either culture condition revealed no systematic differences in contributing clone numbers, indicating that phenotypic differentiation does not select for few individual clones adapted to unfavorable culture conditions. Our results reveal that CRC TIC can be propagated under conditions previously thought to induce their elimination. This phenotypic plasticity allows addressing primary human CRC TIC properties in experimental settings based on adherent cell growth.
Assuntos
Diferenciação Celular , Neoplasias Colorretais/patologia , Células-Tronco Neoplásicas/patologia , Animais , Biomarcadores Tumorais/metabolismo , Adesão Celular , Proliferação de Células , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Xenoenxertos , Humanos , Camundongos Endogâmicos NOD , Camundongos Transgênicos , Células-Tronco Neoplásicas/metabolismo , Fenótipo , Cultura Primária de Células , Esferoides Celulares , Fatores de Tempo , Carga Tumoral , Células Tumorais CultivadasRESUMO
Human colon cancer harbors a small subfraction of tumor-initiating cells (TICs) that is assumed to be a functionally homogeneous stem-cell-like population driving tumor maintenance and metastasis formation. We found unexpected cellular heterogeneity within the TIC compartment, which contains three types of TICs. Extensively self-renewing long-term TICs (LT-TICs) maintained tumor formation in serial xenotransplants. Tumor transient amplifying cells (T-TACs) with limited or no self-renewal capacity contributed to tumor formation only in primary mice. Rare delayed contributing TICs (DC-TICs) were exclusively active in secondary or tertiary mice. Bone marrow was identified as an important reservoir of LT-TICs. Metastasis formation was almost exclusively driven by self-renewing LT-TICs. Our results demonstrate that tumor initiation, self-renewal, and metastasis formation are limited to particular subpopulations of TICs in primary human colon cancer. We identify LT-TICs as a quantifiable target for therapies aimed toward eradication of self-renewing tumorigenic and metastatic colon cancer cells.