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BACKGROUND & OBJECTIVES: Screening for pancreatic cancer is recommended for individuals with a strong family history, certain genetic syndromes, or a neoplastic cyst of the pancreas. However, limited data supports a survival benefit attributable to screening these higher-risk individuals. METHODS: All patients enrolled in screening at a High-Risk Pancreatic Cancer Clinic (HRC) from July 2013 to June 2020 were identified from a prospectively maintained institutional database and compared to patients evaluated at a Surgical Oncology Clinic (SOC) at the same institution during the same period. Clinical outcomes of patients selected for surgical resection, particularly clinicopathologic stage and overall survival, were compared. RESULTS: Among 826 HRC patients followed for a median (IQR) of 2.3 (0.8-4.2) years, 128 were selected for surgical resection and compared to 402 SOC patients selected for resection. Overall survival was significantly longer among HRC patients (median survival: not reached vs. 2.6 years, p < 0.001). Among 31 HRC and 217 SOC patients with a diagnosis of pancreatic ductal adenocarcinoma (PDAC), the majority of HRC patients were diagnosed with stage 0 disease (carcinoma in situ), while the majority of SOC patients were diagnosed with stage II disease (p < 0.001). Overall survival after resection of invasive PDAC was also significantly longer among HRC patients compared to SOC patients (median survival 5.5 vs. 1.6 years, p = 0.002). CONCLUSION: Patients at increased risk for PDAC and followed with guideline-based screening exhibited downstaging of disease and improved survival from PDAC in comparison to patients who were not screened.
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BACKGROUND: Sperm acrosomal SLLP1 binding (SAS1B) protein is found in oocytes, which is necessary for sperm-oocyte interaction, and also in uterine and pancreatic cancers. Anti-SAS1B antibody-drug conjugates (ADCs) arrested growth in these cancers. However, SAS1B expression in cancers and normal tissues has not been characterized. We hypothesized that SAS1B is expressed on the surface of other common solid cancer cells, but not on normal tissue cells, and might be selectively targeted therapeutically. METHODS: SAS1B expression in human normal and cancer tissues was determined by immunohistochemistry, and complementary DNA (cDNA) libraries were employed to PCR amplify human SAS1B and its transcripts. Monoclonal antibodies (mAbs) to human SAS1B were generated using mouse hybridomas. SAS1B deletion constructs were developed to map SAS1B's epitope, enabling the creation of a blocking peptide. Indirect immunofluorescence (IIF) of human transfected normal and cancer cells was performed to assess SAS1B expression. SAS1B intracellular versus surface expression in normal and tumor tissues was evaluated by flow cytometry after staining with anti-SAS1B mAb, with specificity confirmed with the blocking peptide. Human cancer lines were treated with increasing mAb and ADC concentrations. ATP was quantitated as a measure of cell viability. RESULTS: SAS1B expression was identified in a subset of human cancers and the cytoplasm of pancreatic islet cells. Two new SAS1B splice variants were deduced. Monoclonal antibodies were generated to SAS1B splice variant A. The epitope for mAbs SB2 and SB5 is between SAS1B amino acids 32-39. IIF demonstrated intracellular SAS1B expression in transfected kidney cells and on the cell surface of squamous cell lung carcinoma. Flow cytometry demonstrated intracellular SAS1B expression in all tumors and some normal cells. However, surface expression of SAS1B was identified only on cancer cells. SB2 ADC mediated dose-dependent cytotoxic killing of multiple human cancer lines. CONCLUSION: SAS1B is a novel cancer-oocyte antigen with cell surface expression restricted to cancer cells. In vitro, it is an effective target for antibody-mediated cancer cell lysis. These findings support further exploration of SAS1B as a potential therapeutic cancer target in multiple human cancers, either with ADC or as a chimeric antigen receptor-T (CAR-T) cell target.
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Imunoconjugados , Neoplasias , Masculino , Humanos , Camundongos , Animais , Imunoconjugados/farmacologia , Imunoconjugados/uso terapêutico , Sêmen , Oócitos/metabolismo , Neoplasias/tratamento farmacológico , Neoplasias/genética , Neoplasias/metabolismo , Anticorpos Monoclonais/farmacologia , Anticorpos Monoclonais/uso terapêutico , Epitopos , Peptídeos/metabolismoRESUMO
The tumor microenvironment in pancreatic ductal adenocarcinoma (PDAC) plays a key role in tumor progression and response to therapy. The dense PDAC stroma causes hypovascularity, which leads to hypoxia. Here, we showed that hypoxia drives long-lasting epithelial-mesenchymal transition (EMT) in PDAC primarily through a positive-feedback histone methylation-MAPK signaling axis. Transformed cells preferentially underwent EMT in hypoxic tumor regions in multiple model systems. Hypoxia drove a cell autonomous EMT in PDAC cells, which, unlike EMT in response to growth factors, could last for weeks. Furthermore, hypoxia reduced histone demethylase KDM2A activity, suppressed PP2 family phosphatase expression, and activated MAPKs to post-translationally stabilize histone methyltransferase NSD2, leading to an H3K36me2-dependent EMT in which hypoxia-inducible factors played only a supporting role. Hypoxia-driven EMT could be antagonized in vivo by combinations of MAPK inhibitors. Collectively, these results suggest that hypoxia promotes durable EMT in PDAC by inducing a histone methylation-MAPK axis that can be effectively targeted with multidrug therapies, providing a potential strategy for overcoming chemoresistance. SIGNIFICANCE: Integrated regulation of histone methylation and MAPK signaling by the low-oxygen environment of pancreatic cancer drives long-lasting EMT that promotes chemoresistance and shortens patient survival and that can be pharmacologically inhibited. See related commentary by Wirth and Schneider, p. 1739.
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Carcinoma Ductal Pancreático , Transição Epitelial-Mesenquimal , Histonas , Neoplasias Pancreáticas , Humanos , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Camundongos , Histonas/metabolismo , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/tratamento farmacológico , Animais , Metilação , Sistema de Sinalização das MAP Quinases , Linhagem Celular Tumoral , Microambiente Tumoral , Camundongos Nus , Ensaios Antitumorais Modelo de Xenoenxerto , Hipóxia Celular , Hipóxia Tumoral , Hipóxia/metabolismo , Proteínas F-Box , Histona Desmetilases com o Domínio JumonjiRESUMO
Due to low numbers of circulating tumor cells (CTCs) in liquid biopsies, there is much interest in enrichment of alternative circulating-like mesenchymal cancer cell subpopulations from in vitro tumor cultures for utilization within molecular profiling and drug screening. Viable cancer cells that are released into the media of drug-treated adherent cancer cell cultures exhibit anoikis resistance or anchorage-independent survival away from their extracellular matrix with nutrient sources and waste sinks, which serves as a pre-requisite for metastasis. The enrichment of these cell subpopulations from tumor cultures can potentially serve as an in vitro source of circulating-like cancer cells with greater potential for scale-up in comparison with CTCs. However, these live circulating-like cancer cell subpopulations exhibit size overlaps with necrotic and apoptotic cells in the culture media, which makes it challenging to selectively enrich them, while maintaining them in their suspended state. We present optimization of a flowthrough high frequency (1 MHz) positive dielectrophoresis (pDEP) device with sequential 3D field non-uniformities that enables enrichment of the live chemo-resistant circulating cancer cell subpopulation from an in vitro culture of metastatic patient-derived pancreatic tumor cells. Central to this strategy is the utilization of single-cell impedance cytometry with gates set by supervised machine learning, to optimize the frequency for pDEP, so that live circulating cells are selected based on multiple biophysical metrics, including membrane physiology, cytoplasmic conductivity and cell size, which is not possible using deterministic lateral displacement that is solely based on cell size. Using typical drug-treated samples with low levels of live circulating cells (<3%), we present pDEP enrichment of the target subpopulation to â¼44% levels within 20 minutes, while rejecting >90% of dead cells. This strategy of utilizing single-cell impedance cytometry to guide the optimization of dielectrophoresis has implications for other complex biological samples.
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Células Neoplásicas Circulantes , Neoplasias Pancreáticas , Humanos , Linhagem Celular Tumoral , Células Neoplásicas Circulantes/patologia , Neoplasias Pancreáticas/patologia , PâncreasRESUMO
Immune checkpoint blockade immunotherapy has radically changed patient outcomes in multiple cancer types. Pancreatic cancer is one of the notable exceptions, being protected from immunotherapy by a variety of mechanisms, including the presence of a dense stroma and immunosuppressive myeloid cells. Previous studies have demonstrated that CD40 stimulation can remodel the tumor microenvironment in a manner that promotes effector immune cell responses and can cooperate with immune checkpoint inhibition for durable tumor control mediated by T cells. Here we confirm the capability of this combination therapy to dramatically, and durably, control pancreatic cancer growth in an orthotopic model and that the immune memory to this cancer is primarily a function of CD4+ T cells. We extend this understanding by demonstrating that recruitment of recently primed T cells from the draining lymph nodes is not necessary for the observed control, suggesting that the pre-existing intra-tumoral cells respond to the combination therapy. Further, we find that the efficacy of CD40 stimulation is not dependent upon CD70, which is commonly induced on dendritic cells in response to CD40 agonism. Finally, we find that directly targeting the receptor for CD70, CD27, in combination with the TLR3 agonist polyIC, provides some protection despite failing to increase the frequency of interferon gamma-secreting T cells.
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Chemotherapy failure in pancreatic cancer patients is widely attributed to cancer cell reprogramming towards drug resistance by cancer associated fibroblasts (CAFs), which are the abundant cell type in the tumor microenvironment. Association of drug resistance to specific cancer cell phenotypes within multicellular tumors can advance isolation protocols for enabling cell-type specific gene expression markers to identify drug resistance. This requires the distinction of drug resistant cancer cells versus CAFs, which is challenging since permeabilization of CAF cells during drug treatment can cause non-specific uptake of cancer cell-specific stains. Cellular biophysical metrics, on the other hand, can provide multiparametric information to assess the gradual alteration of target cancer cells towards drug resistance, but these phenotypes need to be distinguished versus CAFs. Using pancreatic cancer cells and CAFs from a metastatic patient-derived tumor that exhibits cancer cell drug resistance under CAF co-culture, the biophysical metrics from multifrequency single-cell impedance cytometry are utilized for distinction of the subpopulation of viable cancer cells versus CAFs, before and after gemcitabine treatment. This is accomplished through supervised machine learning after training the model using key impedance metrics for cancer cells and CAFs from transwell co-cultures, so that an optimized classifier model can recognize each cell type and predict their respective proportions in multicellular tumor samples, before and after gemcitabine treatment, as validated by their confusion matrix and flow cytometry assays. In this manner, an aggregate of the distinguishing biophysical metrics of viable cancer cells after gemcitabine treatment in co-cultures with CAFs can be used in longitudinal studies, to classify and isolate the drug resistant subpopulation for identifying markers.
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Técnicas Biossensoriais , Neoplasias Pancreáticas , Humanos , Gencitabina , Impedância Elétrica , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Fibroblastos , Microambiente Tumoral , Linhagem Celular Tumoral , Neoplasias PancreáticasRESUMO
Unrestricted cell death can lead to an immunosuppressive tumor microenvironment, with dysregulated apoptotic signaling that causes resistance of pancreatic cancer cells to cytotoxic therapies. Hence, modulating cell death by distinguishing the progression of subpopulations under drug treatment from viable towards early apoptotic, late apoptotic, and necrotic states is of interest. While flow cytometry after fluorescent staining can monitor apoptosis with single-cell sensitivity, the background of non-viable cells within non-immortalized pancreatic tumors from xenografts can confound distinction of the intensity of each apoptotic state. Based on single-cell impedance cytometry of drug-treated pancreatic cancer cells that are obtained from tumor xenografts with differing levels of gemcitabine sensitivity, we identify the biophysical metrics that can distinguish and quantify cellular subpopulations at the early apoptotic versus late apoptotic and necrotic states, by using machine learning methods to train for the recognition of each phenotype. While supervised learning has previously been used for classification of datasets with known classes, our advancement is the utilization of optimal positive controls for each class, so that clustering by unsupervised learning and classification by supervised learning can occur on unknown datasets, without human interference or manual gating. In this manner, automated biophysical classification can be used to follow the progression of apoptotic states in each heterogeneous drug-treated sample, for developing drug treatments to modulate cancer cell death and advance longitudinal analysis to discern the emergence of drug resistant phenotypes.
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Neoplasias Pancreáticas , Apoptose , Impedância Elétrica , Citometria de Fluxo/métodos , Humanos , Aprendizado de Máquina , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Microambiente Tumoral , Neoplasias PancreáticasRESUMO
Pancreatic ductal adenocarcinoma (PDA) cells reprogram their transcriptional and metabolic programs to survive the nutrient-poor tumor microenvironment. Through in vivo CRISPR screening, we discovered islet-2 (ISL2) as a candidate tumor suppressor that modulates aggressive PDA growth. Notably, ISL2, a nuclear and chromatin-associated transcription factor, is epigenetically silenced in PDA tumors and high promoter DNA methylation or its reduced expression correlates with poor patient survival. The exogenous ISL2 expression or CRISPR-mediated upregulation of the endogenous loci reduces cell proliferation. Mechanistically, ISL2 regulates the expression of metabolic genes, and its depletion increases oxidative phosphorylation (OXPHOS). As such, ISL2-depleted human PDA cells are sensitive to the inhibitors of mitochondrial complex I in vitro and in vivo. Spatial transcriptomic analysis shows heterogeneous intratumoral ISL2 expression, which correlates with the expression of critical metabolic genes. These findings nominate ISL2 as a putative tumor suppressor whose inactivation leads to increased mitochondrial metabolism that may be exploitable therapeutically.
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Carcinoma Ductal Pancreático , Proteínas com Homeodomínio LIM , Proteínas do Tecido Nervoso , Neoplasias Pancreáticas , Fatores de Transcrição , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Epigênese Genética , Genes Supressores de Tumor , Humanos , Proteínas com Homeodomínio LIM/genética , Proteínas com Homeodomínio LIM/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neoplasias Pancreáticas/metabolismo , Fatores de Transcrição/metabolismo , Microambiente Tumoral/genéticaRESUMO
The ability to rapidly and sensitively predict drug response and toxicity using in vitro models of patient-derived tumors is essential for assessing chemotherapy efficacy. Currently, drug sensitivity assessment for solid tumors relies on imaging adherent cells or by flow cytometry of cells lifted from drug-treated cultures after fluorescent staining for apoptotic markers. Subcellular apoptotic bodies (ABs), including microvesicles that are secreted into the culture media under drug treatment can potentially serve as markers for drug sensitivity, without the need to lift cells under culture. However, their stratification to quantify cell disassembly is challenging due to their compositional diversity, with tailored labeling strategies currently needed for the recognition and cytometry of each AB type. It is shown that the high frequency impedance phase versus size distribution of ABs determined by high-throughput single-particle impedance cytometry of supernatants in the media of gemcitabine-treated pancreatic tumor cultures exhibits phenotypic resemblance to lifted apoptotic cells and enables shape-based stratification within distinct size ranges, which is not possible by flow cytometry. It is envisioned that this tool can be applied in conjunction with the appropriate pancreatic tumor microenvironment model to assess drug sensitivity and toxicity of patient-derived tumors, without the need to lift cells from cultures.
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Vesículas Extracelulares , Neoplasias Pancreáticas , Preparações Farmacêuticas , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Impedância Elétrica , Humanos , Neoplasias Pancreáticas/tratamento farmacológico , Microambiente TumoralRESUMO
Pancreatic ductal adenocarcinoma (PDAC) remains one of the most challenging cancers to treat. Due to the asymptomatic nature of the disease and lack of curative treatment modalities, the 5-y survival rate of PDAC patients is one of the lowest of any cancer type. The recurrent genetic alterations in PDAC are yet to be targeted. Therefore, identification of effective drug combinations is desperately needed. Here, we performed an in vivo CRISPR screen in an orthotopic patient-derived xenograft (PDX) model to identify gene targets whose inhibition creates synergistic tumor growth inhibition with gemcitabine (Gem), a first- or second-line chemotherapeutic agent for PDAC treatment. The approach revealed protein arginine methyltransferase gene 5 (PRMT5) as an effective druggable candidate whose inhibition creates synergistic vulnerability of PDAC cells to Gem. Genetic depletion and pharmacological inhibition indicate that loss of PRMT5 activity synergistically enhances Gem cytotoxicity due to the accumulation of excessive DNA damage. At the molecular level, we show that inhibition of PRMT5 results in RPA depletion and impaired homology-directed DNA repair (HDR) activity. The combination (Gem + PRMT5 inhibition) creates conditional lethality and synergistic reduction of PDAC tumors in vivo. The findings demonstrate that unbiased genetic screenings combined with a clinically relevant model system is a practical approach in identifying synthetic lethal drug combinations for cancer treatment.
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Antineoplásicos/farmacologia , Desoxicitidina/análogos & derivados , Neoplasias Pancreáticas/metabolismo , Proteína-Arginina N-Metiltransferases , Animais , Sistemas CRISPR-Cas/genética , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Desoxicitidina/farmacologia , Desenvolvimento de Medicamentos , Técnicas de Inativação de Genes , Humanos , Camundongos Nus , Proteína-Arginina N-Metiltransferases/antagonistas & inibidores , Proteína-Arginina N-Metiltransferases/genética , Proteína-Arginina N-Metiltransferases/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto , GencitabinaRESUMO
Mitochondria undergo fission and fusion to maintain homeostasis, and tumors exhibit the dysregulation of mitochondrial dynamics. We recently demonstrated that ectopic HRasG12V promotes mitochondrial fragmentation and tumor growth through Erk phosphorylation of the mitochondrial fission GTPase Dynamin-related protein 1 (Drp1). However, the role of Drp1 in the setting of endogenous oncogenic KRas remains unknown. Here, we show that Drp1 is required for KRas-driven anchorage-independent growth in fibroblasts and patient-derived pancreatic cancer cell lines, and it promotes glycolytic flux, in part through the regulation of hexokinase 2 (HK2). Furthermore, Drp1 deletion imparts a significant survival advantage in a model of KRas-driven pancreatic cancer, and tumors exhibit a strong selective pressure against complete Drp1 deletion. Rare tumors that arise in the absence of Drp1 have restored glycolysis but exhibit defective mitochondrial metabolism. This work demonstrates that Drp1 plays dual roles in KRas-driven tumor growth: supporting both glycolysis and mitochondrial function through independent mechanisms.
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Dinaminas/metabolismo , Dinaminas/fisiologia , Mitocôndrias/patologia , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Animais , Apoptose , Proliferação de Células , Dinaminas/genética , Regulação Neoplásica da Expressão Gênica , Glicólise , Humanos , Camundongos , Camundongos Knockout , Mitocôndrias/metabolismo , Dinâmica Mitocondrial , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas p21(ras)/genética , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Predicting the response and identifying additional targets that will improve the efficacy of chemotherapy is a major goal in cancer research. Through large-scale in vivo and in vitro CRISPR knockout screens in pancreatic ductal adenocarcinoma cells, we identified genes whose genetic deletion or pharmacologic inhibition synergistically increase the cytotoxicity of MEK signaling inhibitors. Furthermore, we show that CRISPR viability scores combined with basal gene expression levels could model global cellular responses to the drug treatment. We develop drug response evaluation by in vivo CRISPR screening (DREBIC) method and validated its efficacy using large-scale experimental data from independent experiments. Comparative analyses demonstrate that DREBIC predicts drug response in cancer cells from a wide range of tissues with high accuracy and identifies therapeutic vulnerabilities of cancer-causing mutations to MEK inhibitors in various cancer types.
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Antineoplásicos/farmacologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas/genética , Técnicas de Química Combinatória , Sistemas de Liberação de Medicamentos , Técnicas de Inativação de Genes , Testes Genéticos , Modelos Biológicos , Neoplasias Pancreáticas/genética , Animais , Pontos de Checagem do Ciclo Celular , Morte Celular , Linhagem Celular Tumoral , Sinergismo Farmacológico , Humanos , Camundongos Nus , Quinases de Proteína Quinase Ativadas por Mitógeno/antagonistas & inibidores , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Reprodutibilidade dos TestesRESUMO
Successful therapeutic options remain elusive for pancreatic cancer. The exquisite sensitivity and specificity of humoral and cellular immunity may provide therapeutic approaches if antigens specific for pancreatic cancer cells can be identified. Here we characterize SAS1B (ovastacin, ASTL, astacin-like), a cancer-oocyte antigen, as an attractive immunotoxin target expressed at the surface of human pancreatic cancer cells, with limited expression among normal tissues. Immunohistochemistry shows that most pancreatic cancers are SAS1Bpos (68%), while normal pancreatic ductal epithelium is SAS1Bneg. Pancreatic cancer cell lines developed from patient-derived xenograft models display SAS1B cell surface localization, in addition to cytoplasmic expression, suggesting utility for SAS1B in multiple immunotherapeutic approaches. When pancreatic cancer cells were treated with an anti-SAS1B antibody-drug conjugate, significant cell death was observed at 0.01-0.1 µg/mL, while SAS1Bneg human keratinocytes were resistant. Cytotoxicity was correlated with SAS1B cell surface expression; substantial killing was observed for tumors with low steady state SAS1B expression, suggesting a substantial proportion of SAS1Bpos tumors can be targeted in this manner. These results demonstrate SAS1B is a surface target in pancreatic cancer cells capable of binding monoclonal antibodies, internalization, and delivering cytotoxic drug payloads, supporting further development of SAS1B as a novel target for pancreatic cancer.
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Purpose: Patients with pancreatic ductal adenocarcinoma (PDAC) who undergo surgical resection and adjuvant chemotherapy have an expected survival of only 2 years due to disease recurrence, frequently in the liver. We investigated the role of liver macrophages in progression of PDAC micrometastases to identify adjuvant treatment strategies that could prolong survival.Experimental Design: A murine splenic injection model of hepatic micrometastatic PDAC was used with five patient-derived PDAC tumors. The impact of liver macrophages on tumor growth was assessed by (i) depleting mouse macrophages in nude mice with liposomal clodronate injection, and (ii) injecting tumor cells into nude versus NOD-scid-gamma mice. Immunohistochemistry and flow cytometry were used to measure CD47 ("don't eat me signal") expression on tumor cells and characterize macrophages in the tumor microenvironment. In vitro engulfment assays and mouse experiments were performed with CD47-blocking antibodies to assess macrophage engulfment of tumor cells, progression of micrometastases in the liver and mouse survival.Results:In vivo clodronate depletion experiments and NOD-scid-gamma mouse experiments demonstrated that liver macrophages suppress the progression of PDAC micrometastases. Five patient-derived PDAC cell lines expressed variable levels of CD47. In in vitro engulfment assays, CD47-blocking antibodies increased the efficiency of PDAC cell clearance by macrophages in a manner which correlated with CD47 receptor surface density. Treatment of mice with CD47-blocking antibodies resulted in increased time-to-progression of metastatic tumors and prolonged survival.Conclusions: These findings suggest that following surgical resection of PDAC, adjuvant immunotherapy with anti-CD47 antibody could lead to substantially improved outcomes for patients. Clin Cancer Res; 24(6); 1415-25. ©2017 AACR.
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Antígeno CD47/antagonistas & inibidores , Imunomodulação , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/metabolismo , Animais , Antígeno CD47/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Progressão da Doença , Humanos , Imuno-Histoquímica , Imunoterapia/métodos , Macrófagos/imunologia , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos NOD , Metástase Neoplásica , Estadiamento de Neoplasias , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/terapia , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PURPOSE: Most patients with pancreatic ductal adenocarcinoma (PDAC) die within 5 years following resection plus adjuvant gemcitabine (Gem) from outgrowth of occult metastases. We hypothesized that inhibition of the KRAS pathway with the MEK inhibitor trametinib would inhibit the outgrowth of occult liver metastases in a preclinical model. METHODS: Liver metastases harvested from two patients with PDAC (Tumors 608, 366) were implanted orthotopically in mice. Tumor cell lines were derived and transduced with lentiviruses encoding luciferase and injected into spleens of mice generating microscopic liver metastases. Growth kinetics of liver metastases were measured with bioluminescent imaging and time-to-progression (TTP), progression-free survival (PFS), and overall survival (OS) were determined. RESULTS: Trametinib (0.3 mg/kg BID) significantly prolonged OS versus control (Tumor 608: 114 vs. 43 days, p < 0.001; Tumor 366: not reached vs. 167 days, p = 0.0488). In vivo target validation demonstrated trametinib significantly reduced phosphorylated-ERK and expression of the ERK-responsive gene DUSP6. In a randomized, preclinical trial, mice were randomized to: (1) control, (2) adjuvant Gem (100 mg/kg IP, Q3 days) × 7 days followed by surveillance, or (3) adjuvant Gem followed by trametinib. Sequential Gem-trametinib significantly decreased metastatic cell outgrowth and increased TTP and PFS. CONCLUSIONS: Treatment of mice bearing micrometastases with trametinib significantly delayed tumor outgrowth by effectively inhibiting KRAS-MEK-ERK signaling. In a randomized, preclinical, murine trial adjuvant sequential Gem followed by trametinib inhibited occult metastatic cell outgrowth in the liver and increased PFS versus adjuvant Gem alone. An adjuvant trial of sequential Gem-trametinib is being planned in patients with resected PDAC.
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Carcinoma Ductal Pancreático/prevenção & controle , Neoplasias Hepáticas/prevenção & controle , Neoplasias Pancreáticas/prevenção & controle , Inibidores de Proteínas Quinases/farmacologia , Piridonas/farmacologia , Pirimidinonas/farmacologia , Animais , Carcinoma Ductal Pancreático/secundário , Humanos , Neoplasias Hepáticas/secundário , Camundongos , Neoplasias Pancreáticas/patologia , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Currently, prognostication for pancreatic ductal adenocarcinoma (PDAC) is based upon a coarse clinical staging system. Thus, more accurate prognostic tests are needed for PDAC patients to aid treatment decisions. METHODS AND FINDINGS: Affymetrix gene expression profiling was carried out on 15 human PDAC tumors and from the data we identified a 13-gene expression signature (risk score) that correlated with patient survival. The gene expression risk score was then independently validated using published gene expression data and survival data for an additional 101 patients with pancreatic cancer. Patients with high-risk scores had significantly higher risk of death compared to patients with low-risk scores (HR 2.27, pâ=â0.002). When the 13-gene score was combined with lymph node status the risk-score further discriminated the length of patient survival time (p<0.001). Patients with a high-risk score had poor survival independent of nodal status; however, nodal status increased predictability for survival in patients with a low-risk gene signature score (low-risk N1 vs. low-risk N0: HRâ=â2.0, pâ=â0.002). While AJCC stage correlated with patient survival (pâ=â0.03), the 13-gene score was superior at predicting survival. Of the 13 genes comprising the predictive model, four have been shown to be important in PDAC, six are unreported in PDAC but important in other cancers, and three are unreported in any cancer. CONCLUSIONS: We identified a 13-gene expression signature that predicts survival of PDAC patients and could prove useful for making treatment decisions. This risk score should be evaluated prospectively in clinical trials for prognostication and for predicting response to chemotherapy. Investigation of new genes identified in our model may lead to novel therapeutic targets.
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Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Genes Neoplásicos , Neoplasias Pancreáticas/genética , Adenocarcinoma/genética , Carcinoma Ductal Pancreático/genética , Humanos , Linfonodos/patologia , Prognóstico , Reprodutibilidade dos Testes , Transdução de Sinais/genética , Análise de Sobrevida , Regulação para Cima/genética , Neoplasias PancreáticasRESUMO
Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations and epidermal growth factor receptor (EGFR) family signaling are drivers of tumorigenesis in pancreatic ductal adenocarcinoma (PDAC). Previous studies have demonstrated that combinatorial treatment of PDAC xenografts with the mitogen-activated protein kinase-extracellular-signal-regulated kinase (ERK) kinase1/2 (MEK1/2) inhibitor trametinib and the dual EGFR/human epidermal growth factor receptor 2 (HER2) inhibitor lapatinib provided more effective inhibition than either treatment alone. In this study, we have used the therapeutic antibodies, panitumumab (specific for EGFR) and trastuzumab (specific for HER2), to probe the role of EGFR and HER2 signaling in the proliferation of patient-derived xenograft (PDX) tumors. We show that dual anti-EGFR and anti-HER2 therapy significantly augmented the growth inhibitory effects of the MEK1/2 inhibitor trametinib in three different PDX tumors. While significant growth inhibition was observed in both KRAS mutant xenograft groups receiving trametinib and dual antibody therapy (tumors 366 and 608), tumor regression was observed in the KRAS wild-type xenografts (tumor 738) treated in the same manner. Dual antibody therapy in conjunction with trametinib was equally or more effective at inhibiting tumor growth and with lower apparent toxicity than trametinib plus lapatinib. Together, these studies provide further support for a role for EGFR and HER2 in pancreatic cancer proliferation and underscore the importance of therapeutic intervention in both the KRAS-rapidly accelerated fibrosarcoma kinase (RAF)-MEK-ERK and EGFR-HER2 pathways to achieve maximal therapeutic efficacy in patients.
Assuntos
Anticorpos Monoclonais Humanizados/farmacologia , Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Neoplasias Pancreáticas/metabolismo , Piridonas/farmacologia , Pirimidinonas/farmacologia , Animais , Anticorpos Monoclonais/administração & dosagem , Anticorpos Monoclonais Humanizados/administração & dosagem , Antineoplásicos/administração & dosagem , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Quimioterapia Combinada , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/metabolismo , Humanos , Masculino , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patologia , Panitumumabe , Fosforilação , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Piridonas/administração & dosagem , Pirimidinonas/administração & dosagem , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Trastuzumab , Carga Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND: Relevant preclinical models that recapitulate the key features of human pancreatic ductal adenocarcinoma (PDAC) are needed in order to provide biologically tractable models to probe disease progression and therapeutic responses and ultimately improve patient outcomes for this disease. Here, we describe the establishment and clinical, pathological, molecular and genetic validation of a murine, orthotopic xenograft model of PDAC. METHODS: Human PDACs were resected and orthotopically implanted and propagated in immunocompromised mice. Patient survival was correlated with xenograft growth and metastatic rate in mice. Human and mouse tumor pathology were compared. Tumors were analyzed for genetic mutations, gene expression, receptor tyrosine kinase activation, and cytokine expression. RESULTS: Fifteen human PDACs were propagated orthotopically in mice. Xenograft-bearing mice developed peritoneal and liver metastases. Time to tumor growth and metastatic efficiency in mice each correlated with patient survival. Tumor architecture, nuclear grade and stromal content were similar in patient and xenografted tumors. Propagated tumors closely exhibited the genetic and molecular features known to characterize pancreatic cancer (e.g. high rate of KRAS, P53, SMAD4 mutation and EGFR activation). The correlation coefficient of gene expression between patient tumors and xenografts propagated through multiple generations was 93 to 99%. Analysis of gene expression demonstrated distinct differences between xenografts from fresh patient tumors versus commercially available PDAC cell lines. CONCLUSIONS: The orthotopic xenograft model derived from fresh human PDACs closely recapitulates the clinical, pathologic, genetic and molecular aspects of human disease. This model has resulted in the identification of rational therapeutic strategies to be tested in clinical trials and will permit additional therapeutic approaches and identification of biomarkers of response to therapy.
Assuntos
Adenocarcinoma/genética , Carcinoma Ductal Pancreático/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Neoplasias Pancreáticas/genética , Adenocarcinoma/metabolismo , Adenocarcinoma/patologia , Animais , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Linhagem Celular Tumoral , Análise por Conglomerados , Citocinas/metabolismo , Receptores ErbB/metabolismo , Humanos , Estimativa de Kaplan-Meier , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundário , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Mutação , Transplante de Neoplasias , Análise de Sequência com Séries de Oligonucleotídeos , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Neoplasias Peritoneais/genética , Neoplasias Peritoneais/metabolismo , Neoplasias Peritoneais/secundário , Proteína Smad4/genética , Transplante Heterólogo , Proteína Supressora de Tumor p53/genética , Proteínas ras/genéticaRESUMO
Mutations of the oncogene KRAS are important drivers of pancreatic cancer progression. Activation of epidermal growth factor receptor (EGFR) and human EGFR2 (HER2) is observed frequent in pancreatic adenocarcinomas. Because of co-activation of these two signaling pathways, we assessed the efficacy of inhibition of EGFR/HER2 receptors and the downstream KRAS effector, mitogen-activated protein kinase/extracellular-signal regulated kinase (ERK) kinase 1 and 2 (MEK1/2), on pancreatic cancer proliferation in vitro and in a murine orthotopic xenograft model. Treatment of established and patient-derived pancreatic cancer cell lines with the MEK1/2 inhibitor trametinib (GSK1120212) inhibited proliferation, and addition of the EGFR/HER2 inhibitor lapatinib enhanced the inhibition elicited by trametinib in three of eight cell lines. Importantly, in the orthotopic xenograft model, treatment with lapatinib and trametinib resulted in significantly enhanced inhibition of tumor growth relative to trametinib treatment alone in four of five patient-derived tumors tested and was, in all cases, significantly more effective in reducing the size of established tumors than treatment with lapatinib or trametinib alone. Acute treatment of established tumors with trametinib resulted in an increase in AKT2 phosphorylation that was blunted in mice treated with both trametinib and lapatinib. These data indicate that inhibition of the EGFR family receptor signaling may contribute to the effectiveness of MEK1/2 inhibition of tumor growth possibly through the inhibition of feedback activation of receptor tyrosine kinases in response to inhibition of the RAS-RAF-MEK-ERK pathway. These studies provide a rationale for assessing the co-inhibition of these pathways in the treatment of pancreatic cancer patients.
Assuntos
Receptores ErbB/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Piridonas/administração & dosagem , Pirimidinonas/administração & dosagem , Quinazolinas/administração & dosagem , Animais , Antineoplásicos/administração & dosagem , Linhagem Celular Tumoral , Receptores ErbB/antagonistas & inibidores , Humanos , Lapatinib , MAP Quinase Quinase Quinases/antagonistas & inibidores , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Transplante de Neoplasias , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/administração & dosagem , Receptor ErbB-2/antagonistas & inibidores , Transdução de Sinais/efeitos dos fármacosRESUMO
Current therapies for pancreatic ductal adenocarcinoma (PDA) target individual tumor cells. Focal adhesion kinase (FAK) is activated in PDA, and levels are inversely associated with survival. We investigated the effects of PF-562,271 (a small-molecule inhibitor of FAK/PYK2) on (i) in vitro migration, invasion, and proliferation; (ii) tumor proliferation, invasion, and metastasis in a murine model; and (iii) stromal cell composition in the PDA microenvironment. Migration assays were conducted to assess tumor and stromal cell migration in response to cellular factors, collagen, and the effects of PF-562,271. An orthotopic murine model was used to assess the effects of PF-562,271 on tumor growth, invasion, and metastasis. Proliferation assays measured PF-562,271 effects on in vitro growth. Immunohistochemistry was used to examine the effects of FAK inhibition on the cellular composition of the tumor microenvironment. FAK and PYK2 were activated and expressed in patient-derived PDA tumors, stromal components, and human PDA cell lines. PF-562,271 blocked phosphorylation of FAK (phospho-FAK or Y397) in a dose-dependent manner. PF-562,271 inhibited migration of tumor cells, cancer-associated fibroblasts, and macrophages. Treatment of mice with PF-562,271 resulted in reduced tumor growth, invasion, and metastases. PF-562,271 had no effect on tumor necrosis, angiogenesis, or apoptosis, but it did decrease tumor cell proliferation and resulted in fewer tumor-associated macrophages and fibroblasts than control or gemcitabine. These data support a role for FAK in PDA and suggest that inhibitors of FAK may contribute to efficacious treatment of patients with PDA.