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BACKGROUND & AIMS: RNF43 is an E3 ubiquitin ligase that is recurrently mutated in pancreatic ductal adenocarcinoma (PDAC) and precursor cystic neoplasms of the pancreas. The impact of RNF43 mutations on PDAC is poorly understood and autochthonous models have not been characterized sufficiently. In this study, we describe a genetically engineered mouse model (GEMM) of PDAC with conditional expression of oncogenic Kras and deletion of the catalytic domain of Rnf43 in exocrine cells. METHODS: We generated Ptf1a-Cre;LSL-KrasG12D;Rnf43flox/flox (KRC) and Ptf1a-Cre; LSL-KrasG12D (KC) mice and animal survival was assessed. KRC mice were sacrificed at 2 months, 4 months, and at moribund status followed by analysis of pancreata by single-cell RNA sequencing. Comparative analyses between moribund KRC and a moribund Kras/Tp53-driven PDAC GEMM (KPC) was performed. Cell lines were isolated from KRC and KC tumors and interrogated by cytokine array analyses, ATAC sequencing, and in vitro drug assays. KRC GEMMs were also treated with an anti-CTLA4 neutralizing antibody with treatment response measured by magnetic response imaging. RESULTS: We demonstrate that KRC mice display a marked increase in incidence of high-grade cystic lesions of the pancreas and PDAC compared with KC. Importantly, KRC mice have a significantly decreased survival compared with KC mice. Using single-cell RNA sequencing, we demonstrated that KRC tumor progression is accompanied by a decrease in macrophages, as well as an increase in T and B lymphocytes, with evidence of increased immune checkpoint molecule expression and affinity maturation, respectively. This was in stark contrast to the tumor immune microenvironment observed in the KPC PDAC GEMM. Furthermore, expression of the chemokine CXCL5 was found to be specifically decreased in KRC cancer cells by means of epigenetic regulation and emerged as a putative candidate for mediating the unique KRC immune landscape. CONCLUSIONS: The KRC GEMM establishes RNF43 as a bona fide tumor suppressor gene in PDAC. This GEMM features a markedly different immune microenvironment compared with previously reported PDAC GEMMs and puts forth a rationale for an immunotherapy approach in this subset of PDAC cases.
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Adenocarcinoma , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Ubiquitina-Proteína Ligasas , Adenocarcinoma/genética , Animales , Carcinoma Ductal Pancreático/patología , Modelos Animales de Enfermedad , Epigénesis Genética , Humanos , Ratones , Neoplasias Pancreáticas/patología , Proteínas Proto-Oncogénicas p21(ras)/genética , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Microambiente Tumoral , Ubiquitina-Proteína Ligasas/genética , Neoplasias PancreáticasRESUMEN
The central nervous system (CNS) contains a complex network of blood vessels that promote normal tissue development and physiology. Abnormal control of blood vessel morphogenesis and maturation is linked to the pathogenesis of various neurodevelopmental diseases. The CNS-specific genes that regulate blood vessel morphogenesis in development and disease remain largely unknown. Here, we have characterized functions for the gene encoding prion protein 2 (Prnd) in CNS blood vessel development and physiology. Prnd encodes the glycosylphosphatidylinositol (GPI)-linked protein doppel, which is expressed on the surface of angiogenic vascular endothelial cells, but is absent in quiescent endothelial cells of the adult CNS. During CNS vascular development, doppel interacts with receptor tyrosine kinases and activates cytoplasmic signaling pathways involved in endothelial cell survival, metabolism and migration. Analysis of mice genetically null for Prnd revealed impaired CNS blood vessel morphogenesis and associated endothelial cell sprouting defects. Prnd-/- mice also displayed defects in endothelial barrier integrity. Collectively, these data reveal novel mechanisms underlying doppel control of angiogenesis in the developing CNS, and may provide new insights about dysfunctional pathways that cause vascular-related CNS disorders.
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Barrera Hematoencefálica/metabolismo , Células Endoteliales/metabolismo , Neovascularización Patológica/metabolismo , Neovascularización Fisiológica/fisiología , Proteínas Priónicas/metabolismo , Animales , Sistema Nervioso Central/metabolismo , Citoplasma/metabolismo , Proteínas Ligadas a GPI/metabolismo , Ratones , Morfogénesis/fisiología , Proteínas Tirosina Quinasas/metabolismo , Transducción de Señal/fisiologíaRESUMEN
BACKGROUND: Limited accessibility of the tumour precludes longitudinal characterisation for therapy guidance in pancreatic ductal adenocarcinoma (PDAC). METHODS: We utilised dielectrophoresis-field flow fractionation (DEP-FFF) to isolate circulating tumour cells (CTCs) in 272 blood draws from 74 PDAC patients (41 localised, 33 metastatic) to non-invasively monitor disease progression. RESULTS: Analysis using multiplex imaging flow cytometry revealed four distinct sub-populations of CTCs: epithelial (E-CTC), mesenchymal (M-CTC), partial epithelial-mesenchymal transition (pEMT-CTC) and stem cell-like (SC-CTC). Overall, CTC detection rate was 76.8% (209/272 draws) and total CTC counts did not correlate with any clinicopathological variables. However, the proportion of pEMT-CTCs (prop-pEMT) was correlated with advanced disease, worse progression-free and overall survival in all patients, and earlier recurrence after resection. CONCLUSION: Our results underscore the importance of immunophenotyping and quantifying specific CTC sub-populations in PDAC.
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Carcinoma Ductal Pancreático/patología , Transición Epitelial-Mesenquimal/fisiología , Células Neoplásicas Circulantes/patología , Neoplasias Pancreáticas/patología , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores Farmacológicos/análisis , Biomarcadores Farmacológicos/sangre , Biomarcadores de Tumor/análisis , Biomarcadores de Tumor/sangre , Carcinoma Ductal Pancreático/sangre , Carcinoma Ductal Pancreático/diagnóstico , Células Cultivadas , Progresión de la Enfermedad , Monitoreo de Drogas/métodos , Femenino , Humanos , Inmunofenotipificación , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Estadificación de Neoplasias , Células Neoplásicas Circulantes/clasificación , Neoplasias Pancreáticas/sangre , Neoplasias Pancreáticas/diagnóstico , Fenotipo , Valor Predictivo de las Pruebas , PronósticoRESUMEN
BACKGROUND AND AIMS: EUS-guided FNA is recommended as a first-line procedure for the histopathologic diagnosis of pancreatic cancer. Molecular analysis of EUS-FNA samples might be used as an auxiliary tool to strengthen the diagnosis. The current study aimed to evaluate the diagnostic performances of K-ras testing using droplet digital polymerase chain reaction (ddPCR) and a novel single-nucleotide variant (SNV) assay performed on pancreatic EUS-FNA samples. METHODS: EUS-FNA specimens from 31 patients with pancreatic masses (22 pancreatic ductal adenocarcinomas, 7 chronic pancreatitis, and 2 pancreatic neuroendocrine tumors) were included in the study. K-ras testing was initially performed by ddPCR. In addition, mutational status was evaluated using an SNV assay by NanoString technology, using digital enumeration of unique barcoded probes to detect 97 SNVs from 24 genes of clinical significance. RESULTS: The overall specificity and sensitivity of cytologic examination were 100% and 63%, respectively. K-ras mutation testing was performed using ddPCR, and the sensitivity increased to 87% with specificity 90%. The SNV assay detected at least 1 variant in 90% of pancreatic ductal adenocarcinoma samples; the test was able to detect 2 K-ras codon 61 mutations in 2 cases of pancreatic ductal adenocarcinoma, which were missed by ddPCR. The overall diagnostic accuracy of the cytologic examination alone was 74%, and it increased to 91% when the results of both molecular tests were considered for the cases with negative and inconclusive results. CONCLUSIONS: The current study illustrated that integration of K-ras analysis with cytologic evaluation, especially in inconclusive cases, can enhance the diagnostic accuracy of EUS-FNA for pancreatic lesions.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Carcinoma Ductal Pancreático/diagnóstico , Carcinoma Ductal Pancreático/genética , Biopsia por Aspiración con Aguja Fina Guiada por Ultrasonido Endoscópico , Endosonografía , Humanos , Nucleótidos , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/genética , Reacción en Cadena de la Polimerasa , Sensibilidad y EspecificidadRESUMEN
BACKGROUND & AIMS: We aimed to investigate the clinical utility of circulating tumor cell DNA (ctDNA) and exosome DNA (exoDNA) in pancreatic cancer. METHODS: We collected liquid biopsy samples from 194 patients undergoing treatment for localized or metastatic pancreatic adenocarcinoma from April 7, 2015, through October 13, 2017 (425 blood samples collected before [baseline] and during therapy). Additional liquid biopsy samples were collected from 37 disease control individuals. Droplet digital polymerase chain reaction was used to determine KRAS mutant allele fraction (MAF) from ctDNA and exoDNA purified from plasma. For the longitudinal analysis, we analyzed exoDNA and ctDNA in 123 serial blood samples from 34 patients. We performed analysis including Cox regression, Fisher exact test, and Bayesian inference to associate KRAS MAFs in exoDNA and ctDNA with prognostic and predictive outcomes. RESULTS: In the 34 patients with potentially resectable tumors, an increase in exoDNA level after neoadjuvant therapy was significantly associated with disease progression (P = .003), whereas ctDNA did not show correlations with outcomes. Concordance rates of KRAS mutations present in surgically resected tissue and detected in liquid biopsy samples were greater than 95%. On univariate analysis, patients with metastases and detectable ctDNA at baseline status had significantly shorter times of progression-free survival (PFS) (hazard ratio [HR] for death, 1.8; 95% CI, 1.1-3.0; P = .019), and overall survival (OS) (HR, 2.8; 95% CI, 1.4-5.7; P = .0045) compared with patients without detectable ctDNA. On multivariate analysis, MAFs ≥5% in exoDNA were a significant predictor of PFS (HR, 2.28; 95% CI, 1.18-4.40; P = .014) and OS (HR, 3.46; 95% CI, 1.40-8.50; P = .007). A multianalyte approach showed detection of both ctDNA and exoDNA MAFs ≥5% at baseline status to be a significant predictor of OS (HR, 7.73, 95% CI, 2.61-22.91, P = .00002) on multivariate analysis. In the longitudinal analysis, an MAF peak above 1% in exoDNA was significantly associated with radiologic progression (P = .0003). CONCLUSIONS: In a prospective cohort of pancreatic cancer patients, we show how longitudinal monitoring using liquid biopsy samples through exoDNA and ctDNA provides both predictive and prognostic information relevant to therapeutic stratification.
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Adenocarcinoma/genética , Biomarcadores de Tumor/genética , ADN Tumoral Circulante/genética , Exosomas/genética , Mutación , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogénicas p21(ras)/genética , Adenocarcinoma/sangre , Adenocarcinoma/secundario , Adenocarcinoma/terapia , Biomarcadores de Tumor/sangre , ADN Tumoral Circulante/sangre , Análisis Mutacional de ADN , Progresión de la Enfermedad , Exosomas/patología , Humanos , Biopsia Líquida , Terapia Neoadyuvante , Pancreatectomía , Neoplasias Pancreáticas/sangre , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/terapia , Reacción en Cadena de la Polimerasa , Valor Predictivo de las Pruebas , Estudios Prospectivos , Proteínas Proto-Oncogénicas p21(ras)/sangre , Factores de Riesgo , Factores de Tiempo , Resultado del TratamientoRESUMEN
Longitudinal monitoring of patients with advanced cancers is crucial to evaluate both disease burden and treatment response. Current liquid biopsy approaches mostly rely on the detection of DNA-based biomarkers. However, plasma RNA analysis can unleash tremendous opportunities for tumor state interrogation and molecular subtyping. Through the application of deep learning algorithms to the deconvolved transcriptomes of RNA within plasma extracellular vesicles (evRNA), we successfully predicted consensus molecular subtypes in patients with metastatic colorectal cancer. Analysis of plasma evRNA also enabled monitoring of changes in transcriptomic subtype under treatment selection pressure and identification of molecular pathways associated with recurrence. This approach also revealed expressed gene fusions and neoepitopes from evRNA. These results demonstrate the feasibility of using transcriptomic-based liquid biopsy platforms for precision oncology approaches, spanning from the longitudinal monitoring of tumor subtype changes to the identification of expressed fusions and neoantigens as cancer-specific therapeutic targets, sans the need for tissue-based sampling. SIGNIFICANCE: The development of an approach to interrogate molecular subtypes, cancer-associated pathways, and differentially expressed genes through RNA sequencing of plasma extracellular vesicles lays the foundation for liquid biopsy-based longitudinal monitoring of patient tumor transcriptomes.
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Biomarcadores de Tumor , Vesículas Extracelulares , Perfilación de la Expresión Génica , Transcriptoma , Humanos , Vesículas Extracelulares/genética , Vesículas Extracelulares/metabolismo , Perfilación de la Expresión Génica/métodos , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/sangre , Biopsia Líquida/métodos , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/sangre , Neoplasias Colorrectales/patología , Regulación Neoplásica de la Expresión Génica , Neoplasias/genética , Neoplasias/sangre , Neoplasias/patologíaRESUMEN
KRAS inhibitors demonstrate clinical efficacy in pancreatic ductal adenocarcinoma (PDAC); however, resistance is common. Among patients with KRASG12C-mutant PDAC treated with adagrasib or sotorasib, mutations in PIK3CA and KRAS, and amplifications of KRASG12C, MYC, MET, EGFR, and CDK6 emerged at acquired resistance. In PDAC cell lines and organoid models treated with the KRASG12D inhibitor MRTX1133, epithelial-to-mesenchymal transition and PI3K-AKT-mTOR signaling associate with resistance to therapy. MRTX1133 treatment of the KrasLSL-G12D/+;Trp53LSL-R172H/+;p48-Cre (KPC) mouse model yielded deep tumor regressions, but drug resistance ultimately emerged, accompanied by amplifications of Kras, Yap1, Myc, and Cdk6/Abcb1a/b, and co-evolution of drug-resistant transcriptional programs. Moreover, in KPC and PDX models, mesenchymal and basal-like cell states displayed increased response to KRAS inhibition compared to the classical state. Combination treatment with KRASG12D inhibition and chemotherapy significantly improved tumor control in PDAC mouse models. Collectively, these data elucidate co-evolving resistance mechanisms to KRAS inhibition and support multiple combination therapy strategies.
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PURPOSE: Cancer of unknown primary (CUP) accounts for 2-5% of all cancer diagnoses, wherein standard investigations fail to reveal the original tumor site. Basket trials allocate targeted therapeutics based on actionable somatic mutations, independent of tumor entity. These trials, however, mostly rely on variants identified in tissue biopsies. Since liquid biopsies (LB) represent the overall tumor genomic landscape, they may provide an ideal diagnostic source in CUP patients. To identify the most informative liquid biopsy compartment, we compared the utility of genomic variant analysis for therapy stratification in two LB compartments (circulating cell-free (cf) and extracellular vesicle (ev) DNA). METHODS: CfDNA and evDNA from 23 CUP patients were analyzed using a targeted gene panel covering 151 genes. Identified genetic variants were interpreted regarding diagnostic and therapeutic relevance using the MetaKB knowledgebase. RESULTS: LB revealed a total of 22 somatic mutations in evDNA and/or cfDNA in 11/23 patients. Out of the 22 identified somatic variants, 14 are classified as Tier I druggable somatic variants. Comparison of variants identified in evDNA and cfDNA revealed an overlap of 58% of somatic variants in both LB compartments, whereas over 40% of variants were only found in one or the other compartment. CONCLUSION: We observed substantial overlap between somatic variants identified in evDNA and cfDNA of CUP patients. Nonetheless, interrogation of both LB compartments can potentially increase the rate of druggable alterations, stressing the significance of liquid biopsies for possible primary-independent basket and umbrella trial inclusion.
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Ácidos Nucleicos Libres de Células , Neoplasias Primarias Desconocidas , Humanos , Neoplasias Primarias Desconocidas/tratamiento farmacológico , Neoplasias Primarias Desconocidas/genética , ADN de Neoplasias/genética , Biopsia Líquida , MutaciónRESUMEN
Intraductal papillary mucinous neoplasms (IPMN) of the pancreas are bona fide precursor lesions of pancreatic ductal adenocarcinoma (PDAC). The most common subtype of IPMNs harbors a gastric foveolar-type epithelium, and these low-grade mucinous neoplasms are harbingers of IPMNs with high-grade dysplasia and cancer. The molecular underpinning of gastric differentiation in IPMNs is unknown, although identifying drivers of this indolent phenotype might enable opportunities for intercepting progression to high-grade IPMN and cancer. We conducted spatial transcriptomics on a cohort of IPMNs, followed by orthogonal and cross-species validation studies, which established the transcription factor NKX6-2 as a key determinant of gastric cell identity in low-grade IPMNs. Loss of NKX6-2 expression is a consistent feature of IPMN progression, while reexpression of Nkx6-2 in murine IPMN lines recapitulates the aforementioned gastric transcriptional program and glandular morphology. Our study identifies NKX6-2 as a previously unknown transcription factor driving indolent gastric differentiation in IPMN pathogenesis. SIGNIFICANCE: Identification of the molecular features driving IPMN development and differentiation is critical to prevent cancer progression and enhance risk stratification. We used spatial profiling to characterize the epithelium and microenvironment of IPMN, which revealed a previously unknown link between NKX6-2 and gastric differentiation, the latter associated with indolent biological potential. See related commentary by Ben-Shmuel and Scherz-Shouval, p. 1768. This article is highlighted in the In This Issue feature, p. 1749.
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Carcinoma Ductal Pancreático , Neoplasias Quísticas, Mucinosas y Serosas , Neoplasias Intraductales Pancreáticas , Neoplasias Pancreáticas , Animales , Ratones , Carcinoma Ductal Pancreático/patología , Diferenciación Celular/genética , Páncreas/patología , Neoplasias Intraductales Pancreáticas/genética , Neoplasias Pancreáticas/patología , Transcriptoma , Microambiente TumoralRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is associated with mutations in Kras, a known oncogenic driver of PDAC; and the KRAS G12D mutation is present in nearly half of PDAC patients. Recently, a non-covalent small molecule inhibitor (MRTX1133) was identified with specificity to the Kras G12D mutant protein. Here we explore the impact of Kras G12D inhibition by MRTX1133 on advanced PDAC and its influence on the tumor microenvironment. Employing different orthotopic xenograft and syngeneic tumor models, eight different PDXs, and two different autochthonous genetic models, we demonstrate that MRTX1133 reverses early PDAC growth, increases intratumoral CD8 + effector T cells, decreases myeloid infiltration, and reprograms cancer associated fibroblasts. Autochthonous genetic mouse models treated with MRTX1133 leads to regression of both established PanINs and advanced PDAC. Regression of advanced PDAC requires CD8 + T cells and immune checkpoint blockade therapy (iCBT) synergizes with MRTX1133 to eradicate PDAC and prolong overall survival. Mechanistically, inhibition of mutant Kras in advanced PDAC and human patient derived organoids (PDOs) induces Fas expression in cancer cells and facilitates CD8 + T cell mediated death. These results demonstrate the efficacy of MRTX1133 in different mouse models of PDAC associated with reprogramming of stromal fibroblasts and a dependency on CD8 + T cell mediated tumor clearance. Collectively, this study provides a rationale for a synergistic combination of MRTX1133 with iCBT in clinical trials.
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Intraductal papillary mucinous neoplasms (IPMN) are cystic precursor lesions to pancreatic ductal adenocarcinoma (PDAC). IPMNs undergo multistep progression from low-grade (LG) to high-grade (HG) dysplasia, culminating in invasive neoplasia. While patterns of IPMN progression have been analyzed using multiregion sequencing for somatic mutations, there is no integrated assessment of molecular events, including copy-number alterations (CNA) and transcriptional changes that accompany IPMN progression. We performed laser capture microdissection on surgically resected IPMNs of varying grades of histologic dysplasia obtained from 23 patients, followed by whole-exome and whole-transcriptome sequencing. Overall, HG IPMNs displayed a significantly greater aneuploidy score than LG lesions, with chromosome 1q amplification being associated with HG progression and with cases that harbored co-occurring PDAC. Furthermore, the combined assessment of single-nucleotide variants (SNV) and CNAs identified both linear and branched evolutionary trajectories, underscoring the heterogeneity in the progression of LG lesions to HG and PDAC. At the transcriptome level, upregulation of MYC-regulated targets and downregulation of transcripts associated with the MHC class I antigen presentation machinery as well as pathways related to glycosylation were a common feature of progression to HG. In addition, the established PDAC transcriptional subtypes (basal-like and classical) were readily apparent within IPMNs. Taken together, this work emphasizes the role of 1q copy-number amplification as a putative biomarker of high-risk IPMNs, underscores the importance of immune evasion even in noninvasive precursor lesions, and reinforces that evolutionary pathways in IPMNs are heterogenous, comprised of both SNV and CNA-driven events. SIGNIFICANCE: Integrated molecular analysis of genomic and transcriptomic alterations in the multistep progression of IPMNs, which are bona fide precursors of pancreatic cancer, identifies features associated with progression of low-risk lesions to high-risk lesions and cancer, which might enable patient stratification and cancer interception strategies.
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Carcinoma Ductal Pancreático , Neoplasias Quísticas, Mucinosas y Serosas , Neoplasias Intraductales Pancreáticas , Neoplasias Pancreáticas , Humanos , Proyectos Piloto , Neoplasias Intraductales Pancreáticas/genética , Neoplasias Pancreáticas/genética , Carcinoma Ductal Pancreático/genéticaRESUMEN
The KRASG12D mutation is present in nearly half of pancreatic adenocarcinomas (PDAC). We investigated the effects of inhibiting the KRASG12D mutant protein with MRTX1133, a non-covalent small molecule inhibitor of KRASG12D, on early and advanced PDAC and its influence on the tumor microenvironment. Employing 16 different models of KRASG12D-driven PDAC, we demonstrate that MRTX1133 reverses early PDAC growth, increases intratumoral CD8+ effector T cells, decreases myeloid infiltration, and reprograms cancer-associated fibroblasts. MRTX1133 leads to regression of both established PanINs and advanced PDAC. Regression of advanced PDAC requires CD8+ T cells and immune checkpoint blockade (ICB) synergizes with MRTX1133 to eradicate PDAC and prolong overall survival. Mechanistically, inhibition of KRASG12D in advanced PDAC and human patient derived organoids induces FAS expression in cancer cells and facilitates CD8+ T cell-mediated death. Collectively, this study provides a rationale for a synergistic combination of MRTX1133 with ICB in clinical trials.
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Linfocitos T CD8-positivos , Neoplasias Pancreáticas , Proteínas Proto-Oncogénicas p21(ras) , Humanos , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Proteínas Proto-Oncogénicas p21(ras)/antagonistas & inhibidores , Proteínas Proto-Oncogénicas p21(ras)/genética , Microambiente TumoralRESUMEN
Background: The development of diverse spatial profiling technologies has provided an unprecedented insight into molecular mechanisms driving cancer pathogenesis. Here, we conducted the first integrated cross-species assessment of spatial transcriptomics and spatial metabolomics alterations associated with progression of intraductal papillary mucinous neoplasms (IPMN), bona fide cystic precursors of pancreatic ductal adenocarcinoma (PDAC). Methods: Matrix Assisted Laster Desorption/Ionization (MALDI) mass spectrometry (MS)-based spatial imaging and Visium spatial transcriptomics (ST) (10X Genomics) was performed on human resected IPMN tissues (N= 23) as well as pancreata from a mutant Kras;Gnas mouse model of IPMN. Findings were further compared with lipidomic analyses of cystic fluid from 89 patients with histologically confirmed IPMNs, as well as single-cell and bulk transcriptomic data of PDAC and normal tissues. Results: MALDI-MS analyses of IPMN tissues revealed long-chain hydroxylated sulfatides, particularly the C24:0(OH) and C24:1(OH) species, to be selectively enriched in the IPMN and PDAC neoplastic epithelium. Integrated ST analyses confirmed that the cognate transcripts engaged in sulfatide biosynthesis, including UGT8, Gal3St1 , and FA2H , were co-localized with areas of sulfatide enrichment. Lipidomic analyses of cystic fluid identified several sulfatide species, including the C24:0(OH) and C24:1(OH) species, to be significantly elevated in patients with IPMN/PDAC compared to those with low-grade IPMN. Targeting of sulfatide metabolism via the selective galactosylceramide synthase inhibitor, UGT8-IN-1, resulted in ceramide-induced lethal mitophagy and subsequent cancer cell death in vitro , and attenuated tumor growth of mutant Kras;Gnas allografts. Transcript levels of UGT8 and FA2H were also selectively enriched in PDAC transcriptomic datasets compared to non-cancerous areas, and elevated tumoral UGT8 was prognostic for poor overall survival. Conclusion: Enhanced sulfatide metabolism is an early metabolic alteration in cystic pre-cancerous lesions of the pancreas that persists through invasive neoplasia. Targeting sulfatide biosynthesis might represent an actionable vulnerability for cancer interception.
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Heterogeneity is a hallmark of cancer. The advent of single-cell technologies has helped uncover heterogeneity in a high-throughput manner in different cancers across varied contexts. Here we apply single-cell sequencing technologies to reveal inherent heterogeneity in assumptively monoclonal pancreatic cancer (PDAC) cell lines and patient-derived organoids (PDOs). Our findings reveal a high degree of both genomic and transcriptomic polyclonality in monolayer PDAC cell lines, custodial variation induced by growing apparently identical cell lines in different laboratories, and transcriptomic shifts in transitioning from 2D to 3D spheroid growth models. Our findings also call into question the validity of widely available immortalized, non-transformed pancreatic lines as contemporaneous "control" lines in experiments. We confirm these findings using a variety of independent assays, including but not limited to whole exome sequencing, single-cell copy number variation sequencing (scCNVseq), single-nuclei assay for transposase-accessible chromatin with sequencing, fluorescence in-situ hybridization, and single-cell RNA sequencing (scRNAseq). We map scRNA expression data to unique genomic clones identified by orthogonally-gathered scCNVseq data of these same PDAC cell lines. Further, while PDOs are known to reflect the cognate in vivo biology of the parental tumor, we identify transcriptomic shifts during ex vivo passage that might hamper their predictive abilities over time. The impact of these findings on rigor and reproducibility of experimental data generated using established preclinical PDAC models between and across laboratories is uncertain, but a matter of concern.
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Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Variaciones en el Número de Copia de ADN/genética , Humanos , Páncreas/metabolismo , Neoplasias Pancreáticas/patología , Reproducibilidad de los Resultados , Neoplasias PancreáticasRESUMEN
Single-cell RNA sequencing studies have suggested that total mRNA content correlates with tumor phenotypes. Technical and analytical challenges, however, have so far impeded at-scale pan-cancer examination of total mRNA content. Here we present a method to quantify tumor-specific total mRNA expression (TmS) from bulk sequencing data, taking into account tumor transcript proportion, purity and ploidy, which are estimated through transcriptomic/genomic deconvolution. We estimate and validate TmS in 6,590 patient tumors across 15 cancer types, identifying significant inter-tumor variability. Across cancers, high TmS is associated with increased risk of disease progression and death. TmS is influenced by cancer-specific patterns of gene alteration and intra-tumor genetic heterogeneity as well as by pan-cancer trends in metabolic dysregulation. Taken together, our results indicate that measuring cell-type-specific total mRNA expression in tumor cells predicts tumor phenotypes and clinical outcomes.
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Neoplasias , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Heterogeneidad Genética , Genómica , ARN Mensajero/genética , Progresión de la EnfermedadRESUMEN
PURPOSE: Precision medicine approaches in pancreatic ductal adenocarcinoma (PDAC) are imperative for improving disease outcomes. With molecular subtypes of PDAC gaining relevance in the context of therapeutic stratification, the ability to characterize heterogeneity of cancer-specific gene expression patterns is of great interest. In addition, understanding patterns of immune evasion within PDAC is of importance as novel immunotherapeutic strategies are developed. EXPERIMENTAL DESIGN: Single-cell RNA sequencing (scRNA-seq) is readily applicable to limited biopsies from human primary and metastatic PDAC and identifies most cancers as being an admixture of previously described epithelial transcriptomic subtypes. RESULTS: Integrative analyses of our data provide an in-depth characterization of the heterogeneity within the tumor microenvironment, including cancer-associated fibroblast subclasses, and predicts for a multitude of ligand-receptor interactions, revealing potential targets for immunotherapy approaches. CONCLUSIONS: Our analysis demonstrates that the use of de novo biopsies from patients with PDAC paired with scRNA-seq may facilitate therapeutic prediction from limited biopsy samples.
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Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Transcriptoma , Biopsia , Humanos , Microambiente Tumoral , Secuenciación del ExomaRESUMEN
PURPOSE: Most patients with pancreatic ductal adenocarcinoma (PDAC) present with surgically unresectable cancer. As a result, endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) is the most common biospecimen source available for diagnosis in treatment-naïve patients. Unfortunately, these limited samples are often not considered adequate for genomic analysis, precluding the opportunity for enrollment on precision medicine trials. EXPERIMENTAL DESIGN: Applying an epithelial cell adhesion molecule (EpCAM)-enrichment strategy, we show the feasibility of using real-world EUS-FNA for in-depth, molecular-barcoded, whole-exome sequencing (WES) and somatic copy-number alteration (SCNA) analysis in 23 patients with PDAC. RESULTS: Potentially actionable mutations were identified in >20% of patients. Further, an increased mutational burden and higher aneuploidy in WES data were associated with an adverse prognosis. To identify predictive biomarkers for first-line chemotherapy, we developed an SCNA-based complexity score that was associated with response to platinum-based regimens in this cohort. CONCLUSIONS: Collectively, these results emphasize the feasibility of real-world cytology samples for in-depth genomic characterization of PDAC and show the prognostic potential of SCNA for PDAC diagnosis.
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Carcinoma Ductal Pancreático/genética , Neoplasias Pancreáticas/genética , Anciano , Anciano de 80 o más Años , Carcinoma Ductal Pancreático/mortalidad , Carcinoma Ductal Pancreático/patología , Carcinoma Ductal Pancreático/terapia , Variaciones en el Número de Copia de ADN , Análisis Mutacional de ADN/métodos , Biopsia por Aspiración con Aguja Fina Guiada por Ultrasonido Endoscópico , Estudios de Factibilidad , Femenino , Heterogeneidad Genética , Genómica , Humanos , Masculino , Persona de Mediana Edad , Mutación , Páncreas/diagnóstico por imagen , Páncreas/patología , Neoplasias Pancreáticas/mortalidad , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/terapia , Proyectos Piloto , Pronóstico , Supervivencia sin Progresión , Secuenciación del ExomaRESUMEN
Primary cancers disseminate both single circulating tumor cells (CTCs) and CTC "clusters," the latter of which have been shown to demonstrate greater metastatic propensity and adverse impact on prognosis. Many devices developed to isolate single CTCs also capture CTC clusters, but there is translational potential for a platform specifically designed to isolate CTC clusters. Herein, we introduce our microfluidic device for isolating CTC clusters ("Microfluidic Isolation of CTC Clusters" or MICC), which is equipped with â¼10 000 trap chambers that isolate tumor cell clusters based on their large sizes and dynamic force balance against a pillar obstacle in the trap chamber. Whole blood is injected, followed by a wash step to remove blood cells and a final backflush to release intact clusters for downstream analysis. Using clusters from tumor cell-line and confocal microscopy, we verified the ability of the MICC platform to specifically capture tumor cell clusters in the trap chambers. Our flow rate optimization experiments identified 25 µl/min for blood injection, 100 µl/min as wash flow rate, and 300 µl/min as the release flow rate - indicating that 1 ml of whole blood can be processed in less than an hour. Under these optimal flow conditions, we assessed the MICC platform's capture and release performance using blood samples spiked with different concentrations of clusters, revealing a capture efficiency of 66%-87% and release efficiency of 76%-90%. The results from our study suggest that the MICC platform has the potential to isolate CTC clusters from cancer patient blood, enabling it for clinical applications in cancer management.
RESUMEN
PURPOSE: Early detection of pancreatic ductal adenocarcinoma (PDAC) remains elusive. Precursor lesions of PDAC, specifically intraductal papillary mucinous neoplasms (IPMNs), represent a bona fide pathway to invasive neoplasia, although the molecular correlates of progression remain to be fully elucidated. Single-cell transcriptomics provides a unique avenue for dissecting both the epithelial and microenvironmental heterogeneities that accompany multistep progression from noninvasive IPMNs to PDAC. EXPERIMENTAL DESIGN: Single-cell RNA sequencing was performed through droplet-based sequencing on 5,403 cells from 2 low-grade IPMNs (LGD-IPMNs), 2 high-grade IPMNs (HGD-IPMN), and 2 PDACs (all surgically resected). RESULTS: Analysis of single-cell transcriptomes revealed heterogeneous alterations within the epithelium and the tumor microenvironment during the progression of noninvasive dysplasia to invasive cancer. Although HGD-IPMNs expressed many core signaling pathways described in PDAC, LGD-IPMNs harbored subsets of single cells with a transcriptomic profile that overlapped with invasive cancer. Notably, a proinflammatory immune component was readily seen in low-grade IPMNs, composed of cytotoxic T cells, activated T-helper cells, and dendritic cells, which was progressively depleted during neoplastic progression, accompanied by infiltration of myeloid-derived suppressor cells. Finally, stromal myofibroblast populations were heterogeneous and acquired a previously described tumor-promoting and immune-evading phenotype during invasive carcinogenesis. CONCLUSIONS: This study demonstrates the ability to perform high-resolution profiling of the transcriptomic changes that occur during multistep progression of cystic PDAC precursors to cancer. Notably, single-cell analysis provides an unparalleled insight into both the epithelial and microenvironmental heterogeneities that accompany early cancer pathogenesis and might be a useful substrate to identify targets for cancer interception.See related commentary by Hernandez-Barco et al., p. 2027.
Asunto(s)
Adenocarcinoma Mucinoso/genética , Carcinoma Ductal Pancreático/genética , Neoplasias Pancreáticas/genética , Progresión de la Enfermedad , Humanos , Fenotipo , Microambiente TumoralRESUMEN
Even though pancreatic ductal adenocarcinoma (PDAC) is associated with fibrotic stroma, the molecular pathways regulating the formation of cancer associated fibroblasts (CAFs) are not well elucidated. An epigenomic analysis of patient-derived and de-novo generated CAFs demonstrated widespread loss of cytosine methylation that was associated with overexpression of various inflammatory transcripts including CXCR4. Co-culture of neoplastic cells with CAFs led to increased invasiveness that was abrogated by inhibition of CXCR4. Metabolite tracing revealed that lactate produced by neoplastic cells leads to increased production of alpha-ketoglutarate (aKG) within mesenchymal stem cells (MSCs). In turn, aKG mediated activation of the demethylase TET enzyme led to decreased cytosine methylation and increased hydroxymethylation during de novo differentiation of MSCs to CAF. Co-injection of neoplastic cells with TET-deficient MSCs inhibited tumor growth in vivo. Thus, in PDAC, a tumor-mediated lactate flux is associated with widespread epigenomic reprogramming that is seen during CAF formation.