RESUMEN
BACKGROUND AND AIMS: This study sought to determine the value of patient-derived organoids (PDOs) from esophago-gastric adenocarcinoma (EGC) for response prediction to neoadjuvant chemotherapy (neoCTx). METHODS: Endoscopic biopsies of patients with locally advanced EGC (n = 120) were taken into culture and PDOs expanded. PDOs' response towards the single substances of the FLOT regimen and the combination treatment were correlated to patients' pathological response using tumor regression grading. A classifier based on FLOT response of PDOs was established in an exploratory cohort (n = 13) and subsequently confirmed in an independent validation cohort (n = 13). RESULTS: EGC PDOs reflected patients' diverse responses to single chemotherapeutics and the combination regimen FLOT. In the exploratory cohort, PDOs response to single 5-FU and FLOT combination treatment correlated with the patients' pathological response (5-FU: Kendall's τ = 0.411, P = 0.001; FLOT: Kendall's τ = 0.694, P = 2.541e-08). For FLOT testing, a high diagnostic precision in receiver operating characteristic (ROC) analysis was reached with an AUCROC of 0.994 (CI 0.980 to 1.000). The discriminative ability of PDO-based FLOT testing allowed the definition of a threshold, which classified in an independent validation cohort FLOT responders from non-responders with high sensitivity (90%), specificity (100%) and accuracy (92%). CONCLUSION: In vitro drug testing of EGC PDOs has a high predictive accuracy in classifying patients' histological response to neoadjuvant FLOT treatment. Taking into account the high rate of successful PDO expansion from biopsies, the definition of a threshold that allows treatment stratification paves the way for an interventional trial exploring PDO-guided treatment of EGC patients.
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Adenocarcinoma , Carbamatos , Pirazinas , Piridinas , Neoplasias Gástricas , Humanos , Neoplasias Gástricas/tratamiento farmacológico , Terapia Combinada , Terapia Neoadyuvante , Adenocarcinoma/tratamiento farmacológico , Organoides , Fluorouracilo/farmacologíaRESUMEN
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.
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Médula Ósea/patología , Neoplasias Colorrectales/patología , Proteínas Fluorescentes Verdes/metabolismo , Células Madre Mesenquimatosas/citología , Células Neoplásicas Circulantes/patología , Células Tumorales Cultivadas/citología , Animales , Médula Ósea/metabolismo , Rastreo Celular , Técnicas de Cocultivo , Neoplasias Colorrectales/metabolismo , Proteínas Fluorescentes Verdes/genética , Células Endoteliales de la Vena Umbilical Humana , Humanos , Células Madre Mesenquimatosas/metabolismo , Ratones , Células Neoplásicas Circulantes/metabolismo , Imagen Óptica , Pronóstico , Nicho de Células Madre , Imagen de Lapso de Tiempo , Células Tumorales Cultivadas/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
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Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Neoplasias de la Médula Ósea/secundario , Neoplasias Colorrectales/patología , Proteínas Represoras/metabolismo , Esferoides Celulares/patología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/metabolismo , Neoplasias de la Médula Ósea/genética , Neoplasias de la Médula Ósea/metabolismo , Línea Celular Tumoral , Proliferación Celular/fisiología , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Femenino , Xenoinjertos , Humanos , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Receptor Notch1/metabolismo , Proteínas Represoras/genética , Esferoides Celulares/metabolismoRESUMEN
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.
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Linfocitos B/trasplante , Carcinoma Ductal Pancreático/patología , Neoplasias Colorrectales/patología , Infecciones por Virus de Epstein-Barr/patología , Trastornos Linfoproliferativos/etiología , Neoplasias Pancreáticas/patología , Ensayo de Capsula Subrrenal , Animales , Antígenos de Neoplasias/análisis , Linfocitos B/patología , Linfocitos B/virología , Carcinoma Ductal Pancreático/inmunología , Carcinoma Ductal Pancreático/virología , División Celular , Neoplasias Colorrectales/inmunología , Neoplasias Colorrectales/virología , Medio de Cultivo Libre de Suero , Infecciones por Virus de Epstein-Barr/inmunología , Xenoinjertos/inmunología , Xenoinjertos/patología , Humanos , Huésped Inmunocomprometido , Antígenos Comunes de Leucocito/análisis , Trastornos Linfoproliferativos/patología , Trastornos Linfoproliferativos/virología , Ratones , Ratones Endogámicos NOD , Especificidad de Órganos , Neoplasias Pancreáticas/inmunología , Neoplasias Pancreáticas/virología , Esferoides Celulares , Ensayo de Capsula Subrrenal/métodosRESUMEN
Linking clinical multi-omics with mechanistic studies may improve the understanding of rare cancers. We leverage two precision oncology programs to investigate rhabdomyosarcoma with FUS/EWSR1-TFCP2 fusions, an orphan malignancy without effective therapies. All tumors exhibit outlier ALK expression, partly accompanied by intragenic deletions and aberrant splicing resulting in ALK variants that are oncogenic and sensitive to ALK inhibitors. Additionally, recurrent CKDN2A/MTAP co-deletions provide a rationale for PRMT5-targeted therapies. Functional studies show that FUS-TFCP2 blocks myogenic differentiation, induces transcription of ALK and truncated TERT, and inhibits DNA repair. Unlike other fusion-driven sarcomas, TFCP2-rearranged tumors exhibit genomic instability and signs of defective homologous recombination. DNA methylation profiling demonstrates a close relationship with undifferentiated sarcomas. In two patients, sarcoma was preceded by benign lesions carrying FUS-TFCP2, indicating stepwise sarcomagenesis. This study illustrates the potential of linking precision oncology with preclinical research to gain insight into the classification, pathogenesis, and therapeutic vulnerabilities of rare cancers.
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Sarcoma , Neoplasias de los Tejidos Blandos , Humanos , Multiómica , Medicina de Precisión , Factores de Transcripción/genética , Sarcoma/genética , Sarcoma/terapia , Sarcoma/diagnóstico , Proteína EWS de Unión a ARN/genética , Neoplasias de los Tejidos Blandos/genética , Neoplasias de los Tejidos Blandos/terapia , Proteínas Tirosina Quinasas Receptoras , Biomarcadores de Tumor/genética , Proteínas de Fusión Oncogénica/genética , Proteína-Arginina N-Metiltransferasas , Proteínas de Unión al ADN/genéticaRESUMEN
BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is a tumour entity with unmet medical need. To assess the therapeutic potential of oncolytic virotherapy (OVT) against PDAC, different oncolytic viruses (OVs) are currently investigated in clinical trials. However, systematic comparisons of these different OVs in terms of efficacy against PDAC and biomarkers predicting therapeutic response are lacking. METHODS: We screened fourteen patient-derived PDAC cultures which reflect the intra- and intertumoural heterogeneity of PDAC for their sensitivity to five clinically relevant OVs, namely serotype 5 adenovirus Ad5-hTERT, herpes virus T-VEC, measles vaccine strain MV-NIS, reovirus jin-3, and protoparvovirus H-1PV. Live cell analysis, quantification of viral genome/gene expression, cell viability as well as cytotoxicity assays and titration of viral progeny were conducted. Transcriptome profiling was employed to identify potential predictive biomarkers for response to OV treatment. FINDINGS: Patient-derived PDAC cultures showed individual response patterns to OV treatment. Twelve of fourteen cultures were responsive to at least one OV, with no single OV proving superior or inferior across all cultures. Known host factors for distinct viruses were retrieved as potential biomarkers. Compared to the classical molecular subtype, the quasi-mesenchymal or basal-like subtype of PDAC was found to be more sensitive to H-1PV, jin-3, and T-VEC. Generally, expression of viral entry receptors did not correlate with sensitivity to OV treatment, with one exception: Expression of Galectin-1 (LGALS1), a factor involved in H-1PV entry, positively correlated with H-1PV induced cell killing. Rather, cellular pathways controlling immunological, metabolic and proliferative signaling appeared to determine outcome. For instance, high baseline expression of interferon-stimulated genes (ISGs) correlated with relative resistance to oncolytic measles virus, whereas low cyclic GMP-AMP synthase (cGAS) expression was associated with exceptional response. Combination treatment of MV-NIS with a cGAS inhibitor improved tumour cell killing in several PDAC cultures and cells overexpressing cGAS were found to be less sensitive to MV oncolysis. INTERPRETATION: Considering the heterogeneity of PDAC and the complexity of biological therapies such as OVs, no single biomarker can explain the spectrum of response patterns. For selection of a particular OV, PDAC molecular subtype, ISG expression as well as activation of distinct signaling and metabolic pathways should be considered. Combination therapies can overcome resistance in specific constellations. Overall, oncolytic virotherapy is a viable treatment option for PDAC, which warrants further development. This study highlights the need for personalised treatment in OVT. By providing all primary data, this study provides a rich source and guidance for ongoing developments. FUNDING: German National Science Foundation (Deutsche Forschungsgemeinschaft, DFG), German Cancer Aid (Deutsche Krebshilfe), German National Academic Scholarship Foundation (Studienstiftung des deutschen Volkes), Survival with Pancreatic Cancer Foundation.
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Biomarcadores de Tumor , Viroterapia Oncolítica , Virus Oncolíticos , Neoplasias Pancreáticas , Humanos , Viroterapia Oncolítica/métodos , Virus Oncolíticos/genética , Neoplasias Pancreáticas/terapia , Neoplasias Pancreáticas/metabolismo , Biomarcadores de Tumor/metabolismo , Carcinoma Ductal Pancreático/terapia , Carcinoma Ductal Pancreático/metabolismo , Perfilación de la Expresión Génica , Línea Celular Tumoral , Supervivencia Celular , Células Tumorales CultivadasRESUMEN
The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive primary immunodeficiency disorder associated with thrombocytopenia, eczema, and autoimmunity. We treated two patients who had this disorder with a transfusion of autologous, genetically modified hematopoietic stem cells (HSC). We found sustained expression of WAS protein expression in HSC, lymphoid and myeloid cells, and platelets after gene therapy. T and B cells, natural killer (NK) cells, and monocytes were functionally corrected. After treatment, the patients' clinical condition markedly improved, with resolution of hemorrhagic diathesis, eczema, autoimmunity, and predisposition to severe infection. Comprehensive insertion-site analysis showed vector integration that targeted multiple genes controlling growth and immunologic responses in a persistently polyclonal hematopoiesis. (Funded by Deutsche Forschungsgemeinschaft and others; German Clinical Trials Register number, DRKS00000330.).
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Terapia Genética , Trasplante de Células Madre Hematopoyéticas , Familia de Proteínas del Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/terapia , Técnicas de Transferencia de Gen , Terapia Genética/efectos adversos , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Humanos , Lactante , Masculino , Mutagénesis Insercional , Trasplante Autólogo , Síndrome de Wiskott-Aldrich/genética , Síndrome de Wiskott-Aldrich/inmunologíaRESUMEN
Hematopoietic stem cells (HSCs) generate all mature blood cells during the whole lifespan of an individual. However, the clonal contribution of individual HSC and progenitor cells in steady-state hematopoiesis is poorly understood. To investigate the activity of HSCs under steady-state conditions, murine HSC and progenitor cells were genetically marked in vivo by integrating lentiviral vectors (LVs) encoding green fluorescent protein (GFP). Hematopoietic contribution of individual marked clones was monitored by determination of lentiviral integration sites using highly sensitive linear amplification-mediated-polymerase chain reaction. A remarkably stable small proportion of hematopoietic cells expressed GFP in LV-injected animals for up to 24 months, indicating stable marking of murine steady-state hematopoiesis. Analysis of the lentiviral integration sites revealed that multiple hematopoietic clones with both myeloid and lymphoid differentiation potential contributed to long-term hematopoiesis. In contrast to intrafemoral vector injection, intravenous administration of LV preferentially targeted short-lived progenitor cells. Myelosuppressive treatment of mice prior to LV-injection did not affect the marking efficiency. Our study represents the first continuous analysis of clonal behavior of genetically marked hematopoietic cells in an unmanipulated system, providing evidence that multiple clones are simultaneously active in murine steady-state hematopoiesis.
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Hematopoyesis/fisiología , Células Madre Hematopoyéticas/citología , Animales , Diferenciación Celular/fisiología , Linaje de la Célula , Técnicas de Transferencia de Gen , Vectores Genéticos , Células Madre Hematopoyéticas/metabolismo , Humanos , Ratones , Ratones Endogámicos C57BLRESUMEN
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.
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Proteínas Portadoras/genética , Vectores Genéticos , Lentivirus/genética , Ratones Transgénicos/genética , Virus Formadores de Foco en el Bazo/genética , Células Madre/metabolismo , Animales , Diferenciación Celular , Islas de CpG/genética , Metilación de ADN , Epigénesis Genética , Efecto Fundador , Silenciador del Gen , Genes Esenciales , Proteínas Fluorescentes Verdes/genética , Humanos , Ratones , Miocitos Cardíacos/citología , Miocitos Cardíacos/metabolismo , Regiones Promotoras Genéticas , Análisis de Secuencia de ADN , Células Madre/citología , TransgenesRESUMEN
Treatment with oncolytic measles vaccines (MV) elicits activation of immune cells, including natural killer (NK) cells. However, we found that MV-activated NK cells show only modest direct cytotoxic activity against tumor cells. To specifically direct NK cells towards tumor cells, we developed oncolytic measles vaccines encoding bispecific killer engagers (MV-BiKE) targeting CD16A on NK cells and carcinoembryonic antigen (CEA) as a model tumor antigen. MV-BiKE are only slightly attenuated compared to parental MV and mediate secretion of functional BiKE from infected tumor cells. We tested MV-BiKE activity in cocultures of colorectal or pancreatic cancer cells with primary human NK cells. MV-BiKE mediate expression of effector cytokines, degranulation and specific anti-tumor cytotoxicity by NK cells. Experiments with patient-derived pancreatic cancer cultures indicate that efficacy of MV-BiKE may vary between individual tumors with differential virus permissiveness. Remarkably, we confirmed MV-BiKE activity in primaryhuman colorectal carcinoma specimens with autochthonous tumor and NK cells.This study provides proof-of-concept for MV-BiKE as a novel immunovirotherapy to harness virus-activated NK cells as anti-tumor effectors.
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Sarampión , Neoplasias Pancreáticas , Vacunas , Humanos , Células Asesinas Naturales , Antígenos de Neoplasias/metabolismo , Neoplasias Pancreáticas/terapia , Neoplasias Pancreáticas/metabolismo , Vacunas/metabolismo , Sarampión/metabolismo , Línea Celular TumoralRESUMEN
Targeted therapies are effective in treating cancer, but success depends on identifying cancer vulnerabilities. In our study, we utilize small RNA sequencing to examine the impact of pathway activation on microRNA (miRNA) expression patterns. Interestingly, we discover that miRNAs capable of inhibiting key members of activated pathways are frequently diminished. Building on this observation, we develop an approach that integrates a low-miRNA-expression signature to identify druggable target genes in cancer. We train and validate our approach in colorectal cancer cells and extend it to diverse cancer models using patient-derived in vitro and in vivo systems. Finally, we demonstrate its additional value to support genomic and transcriptomic-based drug prediction strategies in a pan-cancer patient cohort from the National Center for Tumor Diseases (NCT)/German Cancer Consortium (DKTK) Molecularly Aided Stratification for Tumor Eradication (MASTER) precision oncology trial. In conclusion, our strategy can predict cancer vulnerabilities with high sensitivity and accuracy and might be suitable for future therapy recommendations in a variety of cancer subtypes.
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MicroARNs , Neoplasias , Humanos , Neoplasias/tratamiento farmacológico , Neoplasias/genética , MicroARNs/genética , MicroARNs/metabolismo , Medicina de Precisión , Genómica , TranscriptomaRESUMEN
In-frame BRAF exon 12 deletions are increasingly identified in various tumor types. The resultant BRAFΔß3-αC oncoproteins usually lack five amino acids in the ß3-αC helix linker and sometimes contain de novo insertions. The dimerization status of BRAFΔß3-αC oncoproteins, their precise pathomechanism, and their direct druggability by RAF inhibitors (RAFi) has been under debate. Here, we functionally characterize BRAFΔLNVTAP>F and two novel mutants, BRAFdelinsFS and BRAFΔLNVT>F, and compare them with other BRAFΔß3-αC oncoproteins. We show that BRAFΔß3-αC oncoproteins not only form stable homodimers and large multiprotein complexes but also require dimerization. Nevertheless, details matter as aromatic amino acids at the deletion junction of some BRAFΔß3-αC oncoproteins, e.g., BRAFΔLNVTAP>F, increase their stability and dimerization propensity while conferring resistance to monomer-favoring RAFi such as dabrafenib or HSP 90/CDC37 inhibition. In contrast, dimer-favoring inhibitors such as naporafenib inhibit all BRAFΔß3-αC mutants in cell lines and patient-derived organoids, suggesting that tumors driven by such oncoproteins are vulnerable to these compounds.
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Proteínas HSP90 de Choque Térmico , Proteínas Proto-Oncogénicas B-raf , Humanos , Dimerización , Proteínas Proto-Oncogénicas B-raf/genética , AminoácidosRESUMEN
Kinetics of hematopoietic recovery driven by different types of human stem and progenitor cells after transplantation are not fully understood. Short-term repopulating cells (STRCs) dominate early hematopoiesis after transplantation. STRCs are highly enriched in adult mobilized peripheral blood compared with cord blood, but the length of their contribution to hematopoiesis remains unclear. To understand posttransplantation durability and lineage contribution of STRCs, we compared repopulation kinetics of mobilized peripheral blood (high STRC content) with cord blood transplants (low STRC content) in long-lived NOD.Cg-Prkdc(scid)Il2rg(tm1Wjl)/SzJ (IL2RG(-/-)) mice. This comparison demonstrates that quantitative contribution of human STRCs to hematopoiesis is restricted to the first 5 months after transplantation. The ratio of STRCs to long-term repopulating cells dramatically changes during ontogeny. This model enables to precisely determine early and late engraftment kinetics of defined human repopulating cell types and to preclinically assess the engraftment kinetics of engineered stem cell transplants.
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Trasplante de Células Madre de Sangre del Cordón Umbilical , Supervivencia de Injerto/fisiología , Hematopoyesis/fisiología , Movilización de Célula Madre Hematopoyética , Trasplante de Células Madre Hematopoyéticas , Animales , División Celular/fisiología , Linaje de la Célula/fisiología , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Receptores de Interleucina-2/genética , Recuperación de la Función/fisiología , Trasplante HeterólogoRESUMEN
Introduction: Pancreatic ductal adenocarcinoma (PDAC) is largely refractory to cancer immunotherapy with PD-1 immune checkpoint blockade (ICB). Oncolytic virotherapy has been shown to synergize with ICB. In this work, we investigated the combination of anti-PD-1 and oncolytic measles vaccine in an immunocompetent transplantable PDAC mouse model. Methods: We characterized tumor-infiltrating T cells by immunohistochemistry, flow cytometry and T cell receptor sequencing. Further, we performed gene expression profiling of tumor samples at baseline, after treatment, and when tumors progressed. Moreover, we analyzed systemic anti-tumor and anti-viral immunity. Results: Combination treatment significantly prolonged survival compared to monotherapies. Tumor-infiltrating immune cells were increased after virotherapy. Gene expression profiling revealed a unique, but transient signature of immune activation after combination treatment. However, systemic anti-tumor immunity was induced by virotherapy and remained detectable even when tumors progressed. Anti-PD-1 treatment did not impact anti-viral immunity. Discussion: Our results indicate that combined virotherapy and ICB induces anti-tumor immunity and reshapes the tumor immune environment. However, further refinement of this approach may be required to develop its full potential and achieve durable efficacy.
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Carcinoma Ductal Pancreático , Viroterapia Oncolítica , Neoplasias Pancreáticas , Ratones , Animales , Neoplasias Pancreáticas/patología , Carcinoma Ductal Pancreático/genética , Inmunoterapia/métodos , Viroterapia Oncolítica/métodos , Neoplasias PancreáticasRESUMEN
Protein-coding and non-coding genes like miRNAs tightly control hematopoietic differentiation programs. Although miRNAs are frequently located within introns of protein-coding genes, the molecular interplay between intronic miRNAs and their host genes is unclear. By genomic integration site mapping of gamma-retroviral vectors in genetically corrected peripheral blood from gene therapy patients, we identified the EVL/MIR342 gene locus as a hotspot for therapeutic vector insertions indicating its accessibility and expression in human hematopoietic stem and progenitor cells. We therefore asked if and how EVL and its intronic miRNA-342 regulate hematopoiesis. Here we demonstrate that overexpression (OE) of Evl in murine primary Lin- Sca1+ cKit+ cells drives lymphopoiesis whereas miR-342 OE increases myeloid colony formation in vitro and in vivo, going along with a profound upregulation of canonical pathways essential for B-cell development or myelopoietic functions upon Evl or miR-342 OE, respectively. Strikingly, miR-342 counteracts its host gene by targeting lymphoid signaling pathways, resulting in reduced pre-B-cell output. Moreover, EVL overexpression is associated with lymphoid leukemia in patients. In summary, our data show that one common gene locus regulates distinct hematopoietic differentiation programs depending on the gene product expressed, and that the balance between both may determine hematopoietic cell fate decision.
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Moléculas de Adhesión Celular/metabolismo , Diferenciación Celular , Hematopoyesis , Células Madre Hematopoyéticas/citología , MicroARNs/genética , Animales , Moléculas de Adhesión Celular/genética , Células Madre Hematopoyéticas/metabolismo , Humanos , Intrones , RatonesRESUMEN
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.
RESUMEN
BACKGROUND: The development of secondary resistance (SR) in metastatic colorectal cancer (mCRC) treated with anti-epidermal growth factor receptor (anti-EGFR) antibodies is not fully understood at the molecular level. Here we tested in vivo selection of anti-EGFR SR tumors in CRC patient-derived xenograft (PDX) models as a strategy for a molecular dissection of SR mechanisms. METHODS: We analyzed 21 KRAS, NRAS, BRAF, and PI3K wildtype CRC patient-derived xenograft (PDX) models for their anti-EGFR sensitivity. Furthermore, 31 anti-EGFR SR tumors were generated via chronic in vivo treatment with cetuximab. A multi-omics approach was employed to address molecular primary and secondary resistance mechanisms. Gene set enrichment analyses were used to uncover SR pathways. Targeted therapy of SR PDX models was applied to validate selected SR pathways. RESULTS: In vivo anti-EGFR SR could be established with high efficiency. Chronic anti-EGFR treatment of CRC PDX tumors induced parallel evolution of multiple resistant lesions with independent molecular SR mechanisms. Mutations in driver genes explained SR development in a subgroup of CRC PDX models, only. Transcriptional reprogramming inducing anti-EGFR SR was discovered as a common mechanism in CRC PDX models frequently leading to RAS signaling pathway activation. We identified cAMP and STAT3 signaling activation, as well as paracrine and autocrine signaling via growth factors as novel anti-EGFR secondary resistance mechanisms. Secondary resistant xenograft tumors could successfully be treated by addressing identified transcriptional changes by tailored targeted therapies. CONCLUSIONS: Our study demonstrates that SR PDX tumors provide a unique platform to study molecular SR mechanisms and allow testing of multiple treatments for efficient targeting of SR mechanisms, not possible in the patient. Importantly, it suggests that the development of anti-EGFR tolerant cells via transcriptional reprogramming as a cause of anti-EGFR SR in CRC is likely more prevalent than previously anticipated. It emphasizes the need for analyses of SR tumor tissues at a multi-omics level for a comprehensive molecular understanding of anti-EGFR SR in CRC.
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Biomarcadores de Tumor , Reprogramación Celular/genética , Neoplasias Colorrectales/etiología , Resistencia a Antineoplásicos/genética , Transcripción Genética , Alelos , Animales , Línea Celular , Evolución Clonal , Neoplasias Colorrectales/tratamiento farmacológico , Neoplasias Colorrectales/metabolismo , Neoplasias Colorrectales/patología , Biología Computacional , Variaciones en el Número de Copia de ADN , Modelos Animales de Enfermedad , Receptores ErbB/antagonistas & inhibidores , Receptores ErbB/metabolismo , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Ratones , Terapia Molecular Dirigida , Mutación , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Secuenciación del Exoma , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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.
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Antineoplásicos/farmacología , Neoplasias Colorrectales/metabolismo , Quinasas Ciclina-Dependientes/metabolismo , Ciclinas/metabolismo , Proteolisis , Antineoplásicos/química , Apoptosis/efectos de los fármacos , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Autorrenovación de las Células/efectos de los fármacos , Daño del ADN , Femenino , Ensayos Analíticos de Alto Rendimiento , Humanos , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis/efectos de los fármacos , Proteómica , Esferoides Celulares/efectos de los fármacos , Esferoides Celulares/metabolismo , Esferoides Celulares/patología , Proteína p53 Supresora de Tumor/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación/efectos de los fármacosRESUMEN
The oncogene yes-associated protein (YAP) controls liver tumor initiation and progression via cell extrinsic functions by creating a tumor-supporting environment in conjunction with cell autonomous mechanisms. However, how YAP controls organization of the microenvironment and in particular the vascular niche, which contributes to liver disease and hepatocarcinogenesis, is poorly understood. To investigate heterotypic cell communication, we dissected murine and human liver endothelial cell (EC) populations into liver sinusoidal endothelial cells (LSEC) and continuous endothelial cells (CEC) through histomorphological and molecular characterization. In YAPS127A-induced tumorigenesis, a gradual replacement of LSECs by CECs was associated with dynamic changes in the expression of genes involved in paracrine communication. The formation of new communication hubs connecting CECs and LSECs included the hepatocyte growth factor (Hgf)/c-Met signaling pathway. In hepatocytes and tumor cells, YAP/TEA domain transcription factor 4 (TEAD4)-dependent transcriptional induction of osteopontin (Opn) stimulated c-Met expression in EC with CEC phenotype, which sensitized these cells to the promigratory effects of LSEC-derived Hgf. In human hepatocellular carcinoma, the presence of a migration-associated tip-cell signature correlated with poor clinical outcome and the loss of LSEC marker gene expression. The occurrence of c-MET-expressing CECs in human liver cancer samples was confirmed at the single-cell level. In summary, YAP-dependent changes of the liver vascular niche comprise the formation of heterologous communication hubs in which tumor cell-derived factors modify the cross-talk between LSECs and CECs via the HGF/c-MET axis. SIGNIFICANCE: YAP-dependent changes of the liver vascular niche comprise the formation of heterologous communication hubs in which tumor cell-derived factors modify the cross-talk between EC subpopulations. GRAPHICAL ABSTRACT: http://cancerres.aacrjournals.org/content/canres/80/24/5502/F1.large.jpg.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Carcinogénesis/metabolismo , Carcinoma Hepatocelular/metabolismo , Comunicación Celular/genética , Proteínas de Ciclo Celular/metabolismo , Células Endoteliales/metabolismo , Factor de Crecimiento de Hepatocito/metabolismo , Neoplasias Hepáticas/metabolismo , Proteínas Proto-Oncogénicas c-met/metabolismo , Transducción de Señal/genética , Factores de Transcripción/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Carcinoma Hepatocelular/patología , Proteínas de Ciclo Celular/genética , Células Hep G2 , Humanos , Neoplasias Hepáticas/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Señalizadoras YAPRESUMEN
Patient-derived 3D cell culture systems are currently advancing cancer research since they potentiate the molecular analysis of tissue-like properties and drug response under well-defined conditions. However, our understanding of the relationship between the heterogeneity of morphological phenotypes and the underlying transcriptome is still limited. To address this issue, we here introduce "pheno-seq" to directly link visual features of 3D cell culture systems with profiling their transcriptome. As prototypic applications breast and colorectal cancer (CRC) spheroids were analyzed by pheno-seq. We identified characteristic gene expression signatures of epithelial-to-mesenchymal transition that are associated with invasive growth behavior of clonal breast cancer spheroids. Furthermore, we linked long-term proliferative capacity in a patient-derived model of CRC to a lowly abundant PROX1-positive cancer stem cell subtype. We anticipate that the ability to integrate transcriptome analysis and morphological patho-phenotypes of cancer cells will provide novel insight on the molecular origins of intratumor heterogeneity.