RESUMO
Tolerance of mouse kidney allografts arises in grafts that develop regulatory tertiary lymphoid organs (rTLOs). Single-cell RNA-seq (scRNA-seq) data and adoptive transfer of alloreactive T cells after transplantation showed that cytotoxic CD8+ T cells are reprogrammed within the accepted graft to an exhausted/regulatory-like phenotype mediated by IFN-γ. Establishment of rTLOs was required because adoptive transfer of alloreactive T cells prior to transplantation results in kidney allograft rejection. Despite the presence of intragraft CD8+ cells with a regulatory phenotype, they were not essential for the induction and maintenance of kidney allograft tolerance since renal allotransplantation into CD8-KO recipients resulted in acceptance and not rejection. Analysis of scRNA-seq data from allograft kidneys and malignant tumors identified similar regulatory-like cell types within the T cell clusters and trajectory analysis showed that cytotoxic CD8+ T cells are reprogrammed into an exhausted/regulatory-like phenotype intratumorally. Induction of cytotoxic CD8+ T cell dysfunction of infiltrating cells appears to be a beneficial mechanistic pathway that protects the kidney allotransplant from rejection through a process we call "defensive tolerance." This pathway has implications for our understanding of allotransplant tolerance and tumor resistance to host immunity.
Assuntos
Transplante de Rim , Tolerância ao Transplante , Animais , Camundongos , Tolerância ao Transplante/imunologia , Linfócitos T CD8-Positivos/imunologia , Linfócitos T Citotóxicos/imunologia , Camundongos Knockout , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/patologia , Aloenxertos/imunologia , Interferon gama/imunologia , Interferon gama/genética , Interferon gama/metabolismoRESUMO
Neoadjuvant therapy is routinely used in pancreatic ductal adenocarcinoma (PDAC), but not all tumors respond to this treatment. Current clinical imaging techniques are not able to precisely evaluate and predict the response to neoadjuvant therapies over several weeks. A strong fibrotic reaction is a hallmark of a positive response, and during fibrogenesis, allysine residues are formed on collagen proteins by the action of lysyl oxidases. Here, we report the application of an allysine-targeted molecular MRI probe, MnL3, to provide an early, noninvasive assessment of treatment response in PDAC. Allysine increased 2- to 3-fold after one dose of neoadjuvant therapy with FOLFIRINOX in sensitive human PDAC xenografts in mice. Molecular MRI with MnL3 could specifically detect and quantify fibrogenesis in PDAC xenografts. Comparing the MnL3 signal before and 3 days after one dose of FOLFIRINOX predicted subsequent treatment response. The MnL3 tumor signal increased by 70% from day 0 to day 3 in mice that responded to subsequent doses of FOLFIRINOX, whereas no signal increase was observed in FOLFIRINOX-resistant tumors. This study indicates the promise of allysine-targeted molecular MRI as a noninvasive tool to predict chemotherapy outcomes. Significance: Allysine-targeted molecular MRI can quantify fibrogenesis in pancreatic tumors and predict response to chemotherapy, which could guide rapid clinical management decisions by differentiating responders from nonresponders after treatment initiation.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica , Carcinoma Ductal Pancreático , Irinotecano , Imageamento por Ressonância Magnética , Neoplasias Pancreáticas , Ensaios Antitumorais Modelo de Xenoenxerto , Animais , Humanos , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/diagnóstico por imagem , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/metabolismo , Camundongos , Irinotecano/farmacologia , Irinotecano/administração & dosagem , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/diagnóstico por imagem , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/metabolismo , Imageamento por Ressonância Magnética/métodos , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Fluoruracila/farmacologia , Fluoruracila/administração & dosagem , Oxaliplatina/farmacologia , Oxaliplatina/administração & dosagem , Linhagem Celular Tumoral , Leucovorina/administração & dosagem , Leucovorina/farmacologia , Leucovorina/uso terapêutico , Terapia Neoadjuvante , Feminino , LisinaRESUMO
PURPOSE: Targeting solid tumors with chimeric antigen receptor (CAR) T cells remains challenging due to heterogenous target antigen expression, antigen escape, and the immunosuppressive tumor microenvironment (TME). Pancreatic cancer is characterized by a thick stroma generated by cancer-associated fibroblasts (CAF), which may contribute to the limited efficacy of mesothelin-directed CAR T cells in early-phase clinical trials. To provide a more favorable TME for CAR T cells to target pancreatic ductal adenocarcinoma (PDAC), we generated T cells with an antimesothelin CAR and a secreted T-cell-engaging molecule (TEAM) that targets CAF through fibroblast activation protein (FAP) and engages T cells through CD3 (termed mesoFAP CAR-TEAM cells). EXPERIMENTAL DESIGN: Using a suite of in vitro, in vivo, and ex vivo patient-derived models containing cancer cells and CAF, we examined the ability of mesoFAP CAR-TEAM cells to target PDAC cells and CAF within the TME. We developed and used patient-derived ex vivo models, including patient-derived organoids with patient-matched CAF and patient-derived organotypic tumor spheroids. RESULTS: We demonstrated specific and significant binding of the TEAM to its respective antigens (CD3 and FAP) when released from mesothelin-targeting CAR T cells, leading to T-cell activation and cytotoxicity of the target cell. MesoFAP CAR-TEAM cells were superior in eliminating PDAC and CAF compared with T cells engineered to target either antigen alone in our ex vivo patient-derived models and in mouse models of PDAC with primary or metastatic liver tumors. CONCLUSIONS: CAR-TEAM cells enable modification of tumor stroma, leading to increased elimination of PDAC tumors. This approach represents a promising treatment option for pancreatic cancer.
Assuntos
Complexo CD3 , Endopeptidases , Proteínas Ligadas por GPI , Imunoterapia Adotiva , Mesotelina , Neoplasias Pancreáticas , Receptores de Antígenos Quiméricos , Microambiente Tumoral , Ensaios Antitumorais Modelo de Xenoenxerto , Humanos , Animais , Camundongos , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/terapia , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/metabolismo , Microambiente Tumoral/imunologia , Imunoterapia Adotiva/métodos , Receptores de Antígenos Quiméricos/imunologia , Receptores de Antígenos Quiméricos/metabolismo , Complexo CD3/imunologia , Complexo CD3/metabolismo , Proteínas Ligadas por GPI/imunologia , Proteínas Ligadas por GPI/metabolismo , Linhagem Celular Tumoral , Carcinoma Ductal Pancreático/imunologia , Carcinoma Ductal Pancreático/terapia , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/metabolismo , Linfócitos T/imunologia , Linfócitos T/metabolismo , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/imunologia , Proteínas de Membrana/imunologia , Proteínas de Membrana/metabolismo , Serina Endopeptidases/imunologia , Serina Endopeptidases/metabolismo , Adenocarcinoma/imunologia , Adenocarcinoma/terapia , Adenocarcinoma/patologiaRESUMO
Increased use of cross-sectional imaging has resulted in frequent detection of incidental cystic pancreatic lesions. Serous cystadenomas (SCAs) are benign cysts that do not require surgical intervention unless symptomatic. Unfortunately, up to half of SCAs do not have typical imaging findings ("atypical SCAs"), overlap with potentially malignant precursor lesions, and thus pose a diagnostic challenge. We tested whether the analysis of circulating extracellular vesicle (EV) biomarkers using a digital EV screening technology (DEST) could enhance the discrimination of cystic pancreatic lesions and avoid unnecessary surgical intervention in these atypical SCAs. Analysis of 25 different protein biomarkers in plasma EV from 68 patients identified a putative biomarker signature of Das-1, Vimentin, Chromogranin A, and CAIX with high discriminatory power (AUC of 0.99). Analysis of plasma EV for multiplexed markers may thus be helpful in clinical decision-making.
Assuntos
Cistadenoma Seroso , Cisto Pancreático , Neoplasias Pancreáticas , Humanos , Cistadenoma Seroso/diagnóstico , Cistadenoma Seroso/patologia , Cistadenoma Seroso/cirurgia , Cisto Pancreático/diagnóstico , Diagnóstico Diferencial , Neoplasias Pancreáticas/patologia , BiomarcadoresRESUMO
Tumor cell-derived extracellular vesicles (EVs) are being explored as circulating biomarkers, but it is unclear whether bulk measurements will allow early cancer detection. We hypothesized that a single-EV analysis (sEVA) technique could potentially improve diagnostic accuracy. Using pancreatic cancer (PDAC), we analyzed the composition of putative cancer markers in 11 model lines. In parental PDAC cells positive for KRASmut and/or P53mut proteins, only ~40% of EVs were also positive. In a blinded study involving 16 patients with surgically proven stage 1 PDAC, KRASmut and P53mut protein was detectable at much lower levels, generally in <0.1% of vesicles. These vesicles were detectable by the new sEVA approach in 15 of the 16 patients. Using a modeling approach, we estimate that the current PDAC detection limit is at ~0.1-cm3 tumor volume, below clinical imaging capabilities. These findings establish the potential for sEVA for early cancer detection.
Assuntos
Carcinoma Ductal Pancreático , Vesículas Extracelulares , Neoplasias Pancreáticas , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Carcinoma Ductal Pancreático/diagnóstico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , Neoplasias Pancreáticas/diagnóstico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Neoplasias PancreáticasRESUMO
BACKGROUND: Mutations in GNAS drive pancreatic tumorigenesis and frequently occur in intraductal papillary mucinous neoplasm (IPMN); however, their value as a therapeutic target is yet to be determined. This study aimed at evaluating the involvement of mutant GNAS in tumor aggressiveness in established pancreatic cancer. METHODS: CRISPR/Cas9-mediated GNAS R201H silencing was performed using human primary IPMN-associated pancreatic cancer cells. The role of oncogenic GNAS in tumor maintenance was evaluated by conducting cell culture and xenograft experiments, and western blotting and transcriptome analyses were performed to uncover GNAS-driven signatures. RESULTS: Xenografts of GNAS wild-type cells were characterized by a higher Ki-67 labeling index relative to GNAS-mutant cells. Phenotypic alterations in the GNAS wild-type tumors resulted in a significant reduction in mucin production accompanied by solid with massive stromal components. Transcriptional profiling suggested an apparent conflict of mutant GNAS with KRAS signaling. A significantly higher Notch intercellular domain (NICD) was observed in the nuclear fraction of GNAS wild-type cells. Meanwhile, inhibition of protein kinase A (PKA) induced NICD in GNAS-mutant IPMN cells, suggesting that NOTCH signaling is negatively regulated by the GNAS-PKA pathway. GNAS wild-type cells were characterized by a significant invasive property relative to GNAS-mutant cells, which was mediated through the NOTCH regulatory pathway. CONCLUSIONS: Oncogenic GNAS induces mucin production, not only via MUC2 but also via MUC5AC/B, which may enlarge cystic lesions in the pancreas. The mutation may also limit tumor aggressiveness by attenuating NOTCH signaling; therefore, such tumor-suppressing effects must be considered when therapeutically inhibiting the GNAS pathway.
Assuntos
Carcinoma Ductal Pancreático , Cromograninas , Subunidades alfa Gs de Proteínas de Ligação ao GTP , Neoplasias Pancreáticas , Proteínas Proto-Oncogênicas p21(ras) , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Cromograninas/genética , Subunidades alfa Gs de Proteínas de Ligação ao GTP/genética , Humanos , Mutação , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas Proto-Oncogênicas p21(ras)/antagonistas & inibidores , Proteínas Proto-Oncogênicas p21(ras)/genética , Proteínas Proto-Oncogênicas p21(ras)/metabolismoRESUMO
Neuroendocrine carcinoma (NEC) of the esophagogastric junction (EGJ) is a rare disease with no established treatments. Herein, we describe a case of recurrent squamous cell carcinoma (SCC) after achieving complete response to chemotherapy against NEC of the EGJ. A 67-year-old man was referred to our hospital because of epigastric discomfort. Computed tomography imaging and esophagogastroduodenoscopy revealed ulcerated tumors at the EGJ. Endoscopic biopsy revealed small tumor cells with a high nuclear/cytoplasmic ratio, suggesting small-cell NEC. Immunohistochemistry (IHC) analysis showed tumor cells with an MIB-1 index of 80%. The patient achieved complete response after 10 cycles of chemotherapy. Follow-up endoscopic examination revealed small red-colored mucosal lesions in the center of the cicatrized primary lesion. Re-biopsy detected cancer cells harboring large eosinophilic cytoplasm with keratinization and no evidence of NEC components. IHC of the cells were cytokeratin 5/6-positive and p53-negative. The tumor persisted without evidence of metastases after chemoradiotherapy, and total gastrectomy with lymph node dissection was performed. Pathological assessment of the resected specimens revealed SCC, without evidence of NEC. The patient survived without a recurrence for >3 years after the initial presentation. Somatic mutation profiles of the primary NEC and recurrent SCC were analyzed by targeted amplicon sequencing covering common cancer-related mutations. Both tumors possessed TP53 Q192X mutation, whereas SMAD4 S517T was found only in SCC, suggesting that both tumor components originated from a founder clone with a stop-gain mutation in TP53. The somatic mutation profile of the tumors indicated that that loss of heterozygosity (LOH) at the TP53 gene might have occurred during the differentiation of the founder clone into NEC, while a SMAD4 mutation might have contributed to SCC development, indicating branching and subclonal evolution from common founder clone to both NEC and SCC. The mutation assessments provided valuable information to better understand the clonal evolution of metachronous cancers.
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The development of molecular targeted drugs with radiation and chemotherapy is critically important for improving the outcomes of patients with hard-to-treat, potentially curable cancers. However, too many preclinical studies have not translated into successful radiation oncology trials. Major contributing factors to this insufficiency include poor reproducibility of preclinical data, inadequate preclinical modeling of intertumoral genomic heterogeneity that influences treatment sensitivity in the clinic, and a reliance on tumor growth delay instead of local control (TCD50) endpoints. There exists an urgent need to overcome these barriers to facilitate successful clinical translation of targeted radiosensitizers. To this end, we have used 3-dimensional (3D) cell culture assays to better model tumor behavior in vivo. Examples of successful prediction of in vivo effects with these 3D assays include radiosensitization of head and neck cancers by inhibiting epidermal growth factor receptor or focal adhesion kinase signaling, and radioresistance associated with oncogenic mutation of KRAS. To address the issue of tumor heterogeneity, we leveraged institutional resources that allow high-throughput 3D screening of radiation combinations with small-molecule inhibitors across genomically characterized cell lines from lung, head and neck, and pancreatic cancers. This high-throughput screen is expected to uncover genomic biomarkers that will inform the successful clinical translation of targeted agents from the National Cancer Institute Cancer Therapy Evaluation Program portfolio and other sources. Screening "hits" need to be subjected to refinement studies that include clonogenic assays, addition of disease-specific chemotherapeutics, target/biomarker validation, and integration of patient-derived tumor models. The chemoradiosensitizing activities of the most promising drugs should be confirmed in TCD50 assays in xenograft models with or without relevant biomarker and using clinically relevant radiation fractionation. We predict that appropriately validated and biomarker-directed targeted therapies will have a higher likelihood than past efforts of being successfully incorporated into the standard management of hard-to-treat tumors.
Assuntos
Terapia de Alvo Molecular , Biomarcadores Tumorais , Humanos , Neoplasias , Preparações Farmacêuticas , Radiossensibilizantes/uso terapêutico , Reprodutibilidade dos TestesRESUMO
PURPOSE: Pancreatic ductal adenocarcinoma (PDAC) lethality is multifactorial; although studies have identified transcriptional and genetic subsets of tumors with different prognostic significance, there is limited understanding of features associated with the minority of patients who have durable remission after surgical resection. In this study, we performed laser capture microdissection (LCM) of PDAC samples to define their cancer- and stroma-specific molecular subtypes and identify a prognostic gene expression signature for short-term and long-term survival. EXPERIMENTAL DESIGN: LCM and RNA sequencing (RNA-seq) analysis of cancer and adjacent stroma of 19 treatment-naïve PDAC tumors was performed. Gene expression signatures were tested for their robustness in a large independent validation set. An RNA-ISH assay with pooled probes for genes associated with disease-free survival (DFS) was developed to probe 111 PDAC tumor samples. RESULTS: Gene expression profiling identified four subtypes of cancer cells (C1-C4) and three subtypes of cancer-adjacent stroma (S1-S3). These stroma-specific subtypes were associated with DFS (P = 5.55E-07), with S1 associated with better prognoses when paired with C1 and C2. Thirteen genes were found to be predominantly expressed in cancer cells and corresponded with DFS in a validation using existing RNA-seq datasets. A second validation on an independent cohort of patients using RNA-ISH probes to six of these prognostic genes demonstrated significant association with overall survival (median 17 vs. 25 months; P < 0.02). CONCLUSIONS: Our results identified specific signatures from the epithelial and the stroma components of PDAC, which add clarity to the nature of PDAC molecular subtypes and may help predict survival.
Assuntos
Biomarcadores Tumorais/genética , Carcinoma Ductal Pancreático/genética , Pâncreas/patologia , Neoplasias Pancreáticas/genética , Idoso , Carcinoma Ductal Pancreático/mortalidade , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/cirurgia , Conjuntos de Dados como Assunto , Intervalo Livre de Doença , Feminino , Seguimentos , Regulação Neoplásica da Expressão Gênica , Humanos , Microdissecção e Captura a Laser , Masculino , Pessoa de Meia-Idade , Pâncreas/cirurgia , Pancreatectomia , Neoplasias Pancreáticas/mortalidade , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/cirurgia , Prognóstico , RNA-Seq , Medição de Risco/métodos , Medição de Risco/estatística & dados numéricos , Células Estromais/patologia , Microambiente Tumoral/genéticaRESUMO
Cancer patients with malignant involvement of tumor-draining lymph nodes (TDLNs) and distant metastases have the poorest prognosis. A drug delivery platform that targets the primary tumor, TDLNs, and metastatic niches simultaneously, remains to be developed. Here, we generated a novel monoclonal antibody (MHA112) against peripheral node addressin (PNAd), a family of glycoproteins expressed on high endothelial venules (HEVs), which are present constitutively in the lymph nodes (LNs) and formed ectopically in the tumor stroma. MHA112 was endocytosed by PNAd-expressing cells, where it passed through the lysosomes. MHA112 conjugated antineoplastic drug Paclitaxel (Taxol) (MHA112-Taxol) delivered Taxol effectively to the HEV-containing tumors, TDLNs, and metastatic lesions. MHA112-Taxol treatment significantly reduced primary tumor size as well as metastatic lesions in a number of mouse and human tumor xenografts tested. These data, for the first time, indicate that human metastatic lesions contain HEVs and provide a platform that permits simultaneous targeted delivery of antineoplastic drugs to the three key sites of primary tumor, TDLNs, and metastases.
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BACKGROUND AND AIMS: Advances in cross-sectional imaging have resulted in increased detection of intraductal papillary mucinous neoplasms (IPMNs), and their management remains controversial. At present, there is no reliable noninvasive method to distinguish between indolent and high risk IPMNs. We performed extracellular vesicle (EV) analysis to identify markers of malignancy in an attempt to better stratify these lesions. METHODS: Using a novel ultrasensitive digital extracellular vesicle screening technique (DEST), we measured putative biomarkers of malignancy (MUC1, MUC2, MUC4, MUC5AC, MUC6, Das-1, STMN1, TSP1, TSP2, EGFR, EpCAM, GPC1, WNT-2, EphA2, S100A4, PSCA, MUC13, ZEB1, PLEC1, HOOK1, PTPN6, and FBN1) in EV from patient-derived cell lines and then on circulating EV obtained from peripheral blood drawn from patients with IPMNs. We enrolled a total of 133 patients in two separate cohorts: a clinical discovery cohort (n = 86) and a validation cohort (n = 47). RESULTS: From 16 validated EV proteins in plasma samples collected from the discovery cohort, only MUC5AC showed significantly higher levels in high-grade lesions. Of the 11 patients with invasive IPMN (inv/HG), 9 had high MUC5AC expression in plasma EV of the 11 patients with high-grade dysplasia alone, only 1 had high MUC5AC expression (sensitivity of 82%, specificity of 100%). These findings were corroborated in a separate validation cohort. The addition of MUC5AC as a biomarker to imaging and high-riskstigmata allowed detection of all cases requiring surgery, whereas imaging and high-risk stigmata alone would have missed 5 of 14 cases (36%). CONCLUSIONS: MUC5AC in circulating EV can predict the presence of invasive carcinoma within IPMN. This approach has the potential to improve the management and follow-up of patients with IPMN including avoiding unnecessary surgery.
Assuntos
Biomarcadores Tumorais/sangue , Carcinoma Ductal Pancreático/diagnóstico , Vesículas Extracelulares/metabolismo , Neoplasias Intraductais Pancreáticas/diagnóstico , Neoplasias Pancreáticas/diagnóstico , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Biomarcadores Tumorais/metabolismo , Carcinoma Ductal Pancreático/sangue , Carcinoma Ductal Pancreático/patologia , Diagnóstico Diferencial , Feminino , Voluntários Saudáveis , Humanos , Biópsia Líquida/métodos , Masculino , Camundongos , Pessoa de Meia-Idade , Mucina-5AC/sangue , Mucina-5AC/metabolismo , Invasividade Neoplásica/patologia , Ductos Pancreáticos/diagnóstico por imagem , Ductos Pancreáticos/patologia , Neoplasias Intraductais Pancreáticas/sangue , Neoplasias Intraductais Pancreáticas/patologia , Neoplasias Pancreáticas/sangue , Neoplasias Pancreáticas/patologia , Estudo de Prova de Conceito , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is characterized by a highly desmoplastic reaction, warranting intense cancer-stroma communication. In this study, we interrogated the contribution of the BET family of chromatin adaptors to the cross-talk between PDAC cells and the tumor stroma. Short-term treatment of orthotopic xenograft tumors with CPI203, a small-molecule inhibitor of BET proteins, resulted in broad changes in the expression of genes encoding components of the extracellular matrix (matrisome) in both cancer and stromal cells. Remarkably, more than half of matrisome genes were expressed by cancer cells. In vitro cocultures of PDAC cells and cancer-associated fibroblasts (CAF) demonstrated that matrisome expression was regulated by BET-dependent cancer-CAF cross-talk. Disrupting this cross-talk in vivo resulted in diminished growth of orthotopic patient-derived xenograft tumors, reduced proliferation of cancer cells, and changes in collagen structure consistent with that of patients who experienced better survival. Examination of matrisome gene expression in publicly available data sets of 573 PDAC tumors identified a 65-gene signature that was able to distinguish long- and short-term PDAC survivors. Importantly, the expression of genes predictive of short-term survival was diminished in the cancer cells of orthotopic xenograft tumors of mice treated with CPI203. Taken together, these results demonstrate that inhibiting the activity BET proteins results in transcriptional and structural differences in the matrisome are associated with better patient survival. IMPLICATIONS: These studies highlight the biological relevance of the matrisome program in PDAC and suggest targeting of epigenetically driven tumor-stroma cross-talk as a potential therapeutic avenue.
Assuntos
Acetamidas/administração & dosagem , Azepinas/administração & dosagem , Fibroblastos Associados a Câncer/citologia , Carcinoma Ductal Pancreático/patologia , Proteínas da Matriz Extracelular/genética , Neoplasias Pancreáticas/patologia , Acetamidas/farmacologia , Animais , Azepinas/farmacologia , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Comunicação Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Colágeno/metabolismo , Proteínas da Matriz Extracelular/efeitos dos fármacos , Proteínas da Matriz Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Camundongos , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
BACKGROUND/AIM: FOLFIRINOX [fluorouracil (5-FU), irinotecan, oxaliplatin] and gemcitabine plus nab-paclitaxel are standard treatments for patients with pancreatic ductal adenocarcinoma (PDAC). Despite efficacy rates of less than 32%, evidence is lacking to guide the use of one drug over the other. Herein, we compared the sensitivity of patient-derived PDAC cell lines to each of these regimens. MATERIALS AND METHODS: Changes in the growth of 19 low-passage patient-derived PDAC cell lines were evaluated in response to treatment with FOLFIRINOX and gemcitabine plus paclitaxel (Gem-Pac). RESULTS: Six cell lines exhibited optimal sensitivity (high EMax and low GI50) to FOLFIRINOX and three cell lines exhibited optimal sensitivity to Gem-Pac. Several cell lines that were optimally sensitive to one drug regimen exhibited very poor response to the other. CONCLUSION: Further characterization of cancer cells exhibiting preferential sensitivity to each of these regimens may allow the identification of biomarkers to guide the selection of appropriate chemotherapy for a given patient.
Assuntos
Adenocarcinoma/tratamento farmacológico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Carcinoma Ductal Pancreático/tratamento farmacológico , Desoxicitidina/análogos & derivados , Neoplasias Pancreáticas/tratamento farmacológico , Adulto , Idoso , Idoso de 80 Anos ou mais , Linhagem Celular Tumoral , Desoxicitidina/uso terapêutico , Feminino , Fluoruracila/uso terapêutico , Humanos , Irinotecano/uso terapêutico , Leucovorina/uso terapêutico , Masculino , Pessoa de Meia-Idade , Oxaliplatina/uso terapêutico , Gencitabina , Neoplasias PancreáticasRESUMO
Pancreatic ductal adenocarcinoma (PDAC) lethality is due to metastatic dissemination. Characterization of rare, heterogeneous circulating tumor cells (CTCs) can provide insight into metastasis and guide development of novel therapies. Using the CTC-iChip to purify CTCs from PDAC patients for RNA-seq characterization, we identify three major correlated gene sets, with stemness genes LIN28B/KLF4, WNT5A, and LGALS3 enriched in each correlated gene set; only LIN28B CTC expression was prognostic. CRISPR knockout of LIN28B-an oncofetal RNA-binding protein exerting diverse effects via negative regulation of let-7 miRNAs and other RNA targets-in cell and animal models confers a less aggressive/metastatic phenotype. This correlates with de-repression of let-7 miRNAs and is mimicked by silencing of downstream let-7 target HMGA2 or chemical inhibition of LIN28B/let-7 binding. Molecular characterization of CTCs provides a unique opportunity to correlated gene set metastatic profiles, identify drivers of dissemination, and develop therapies targeting the "seeds" of metastasis.
Assuntos
Carcinoma Ductal Pancreático/genética , Proteína HMGA2/genética , MicroRNAs/genética , Células Neoplásicas Circulantes/metabolismo , Neoplasias Pancreáticas/genética , Proteínas de Ligação a RNA/genética , Adulto , Idoso , Animais , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Movimento Celular/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Proteína HMGA2/metabolismo , Humanos , Estimativa de Kaplan-Meier , Fator 4 Semelhante a Kruppel , Masculino , Camundongos Endogâmicos NOD , Camundongos Knockout , Camundongos SCID , Pessoa de Meia-Idade , Metástase Neoplásica , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/metabolismo , Proteínas de Ligação a RNA/metabolismoRESUMO
BACKGROUND: Patient-derived xenograft (PDX) mouse models of cancer have been recognized as better mouse models that recapitulate the characteristics of original malignancies including preserved tumor heterogeneity, lineage hierarchy, and tumor microenvironment. However, common challenges of PDX models are the significant time required for tumor expansion, reduced tumor take rates, and higher costs. Here, we describe a fast, simple, and cost-effective method of expanding PDX of pancreatic ductal adenocarcinoma (PDAC) in mice. METHODS: We used two established frozen PDAC PDX tissues (derived from two different patients) and implanted them subcutaneously into SCID mice. After tissues reached 10-20 mm in diameter, we performed survival surgery on each mouse to harvest 90-95% of subcutaneous PDX (incomplete resection), allowing the remaining 5-10% of PDX to continue growing in the same mouse. RESULTS: We expanded three consecutive passages (P1, P2, and P3) of PDX in the same mouse. Comparing the times required for in vivo expansion, P2 and P3 (expanded through incomplete resection) grew 26-60% faster than P1. Moreover, such expanded PDX tissues were successfully implanted orthotopically into mouse pancreases. Within 20 weeks using only 14 mice, we generated sufficient PDX tissue for future implantation of 200 mice. Our histology study confirmed that the morphologies of cancer cells and stromal structures were similar across all three passages of subcutaneous PDX and the orthotopic PDX and were reflective of the original patient tumors. CONCLUSIONS: Taking advantage of incomplete resection of tumors associated with high local recurrence, we established a fast method of PDAC PDX expansion in mice.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animais , Análise Custo-Benefício , Xenoenxertos , Humanos , Camundongos , Camundongos SCID , Recidiva Local de Neoplasia , Microambiente Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
INTRODUCTION: The inhibition of Hedgehog (Hh) signaling in pancreatic ductal adenocarcinoma (PDAC) reduces desmoplasia and promotes increased vascularity. In contrast to these findings, the Hh ligand Sonic Hedgehog (SHH) is a potent proangiogenic factor in non-tumor models. The aim of this study was to determine the molecular mechanisms by which SHH affects the tumor stroma and angiogenesis. METHODS: Mice bearing three different xenografted human PDAC (n = 5/group) were treated with neutralizing antibodies to SHH. After treatment for 7 days, tumors were evaluated and the expression of 38 pro- and antiangiogenic factors was assessed in the tumor cells and their stroma. The effect of SHH on the regulation of pro- and antiangiogenic factors in fibroblasts and its impact on endothelial cells was then further assessed in in vitro model systems. RESULTS: Inhibition of SHH affected tumor growth, stromal content, and vascularity. Its effect on the Hh signaling pathway was restricted to the stromal compartment of the three cancers. SHH-stimulated angiogenesis indirectly through the reduction of antiangiogenic THBS2 and TIMP2 in stromal cells. An additional direct effect of SHH on endothelial cells depended on the presence of VEGF. CONCLUSION: Inhibition of Hh signaling reduces tumor vascularity, suggesting that Hh plays a role in the maintenance or formation of the tumor vasculature. Whether the reduction in tumor growth and viability seen in the epithelium is a direct consequence of Hh pathway inhibition, or indirectly caused by its effect on the stroma and vasculature, remains to be evaluated.
Assuntos
Carcinoma Ductal Pancreático , Proteínas Hedgehog/metabolismo , Proteínas de Neoplasias/metabolismo , Neovascularização Patológica , Neoplasias Pancreáticas , Transdução de Sinais , Animais , Carcinoma Ductal Pancreático/irrigação sanguínea , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Feminino , Xenoenxertos , Humanos , Masculino , Camundongos , Camundongos Nus , Transplante de Neoplasias , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Neoplasias Pancreáticas/irrigação sanguínea , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologiaRESUMO
OBJECTIVES: Pancreatic ductal adenocarcinoma (PDAC) is characterized by early metastatic spread in more than 50% of patients. In this study, we sought to understand the migratory properties of (non)metastatic PDAC cells and determine whether the migration of cancer stem cell (CSC) populations accounts for the aggressive nature of this disease. METHODS: The migratory abilities of primary and metastatic PDAC cell lines were investigated using a microfluidic device and time-lapse photography. The velocity, time of delay of mobilization, and number of migratory cells were analyzed. Cancer stem cell subpopulations were isolated by fluorescence-activated cell sorting and their migratory properties compared with their non-CSC counterparts. RESULTS: Primary cancer cells exhibited higher velocities, greater number of migratory cells, and a shorter time of delay of mobilization in comparison to metastatic cell lines. Characterization of CSC populations revealed primary PDAC cell lines were composed of fewer CD133 and CD24CD44 CSC subpopulations than metastatic cells. Moreover, migratory analysis of CSC subpopulations revealed lower velocities, fewer migratory cells, and a greater time of delay of mobilization than non-CSC. CONCLUSIONS: Primary cancer cells demonstrate enhanced migratory abilities in comparison to metastatic PDAC cells. Those differences may result from lower CSC subpopulations in primary cells because CSC populations demonstrated impaired migratory abilities in contrast to non-CSC.
Assuntos
Carcinoma Ductal Pancreático/patologia , Movimento Celular , Células-Tronco Neoplásicas/patologia , Neoplasias Pancreáticas/patologia , Antígeno AC133/metabolismo , Biomarcadores Tumorais/metabolismo , Antígeno CD24/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Linhagem Celular Tumoral , Humanos , Receptores de Hialuronatos/metabolismo , Metástase Neoplásica , Células-Tronco Neoplásicas/metabolismo , Neoplasias Pancreáticas/metabolismoRESUMO
Transcriptional profiling has defined pancreatic ductal adenocarcinoma (PDAC) into distinct subtypes with the majority being classical epithelial (E) or quasi-mesenchymal (QM). Despite clear differences in clinical behavior, growing evidence indicates these subtypes exist on a continuum with features of both subtypes present and suggestive of interconverting cell states. Here, we investigated the impact of different therapies being evaluated in PDAC on the phenotypic spectrum of the E/QM state. We demonstrate using RNA-sequencing and RNA-in situ hybridization (RNA-ISH) that FOLFIRINOX combination chemotherapy induces a common shift of both E and QM PDAC toward a more QM state in cell lines and patient tumors. In contrast, Vitamin D, another drug under clinical investigation in PDAC, induces distinct transcriptional responses in each PDAC subtype, with augmentation of the baseline E and QM state. Importantly, this translates to functional changes that increase metastatic propensity in QM PDAC, but decrease dissemination in E PDAC in vivo models. These data exemplify the importance of both the initial E/QM subtype and the plasticity of E/QM states in PDAC in influencing response to therapy, which highlights their relevance in guiding clinical trials.
RESUMO
Single-cell technologies have described heterogeneity across tissues, but the spatial distribution and forces that drive single-cell phenotypes have not been well defined. Combining single-cell RNA and protein analytics in studying the role of stromal cancer-associated fibroblasts (CAFs) in modulating heterogeneity in pancreatic cancer (pancreatic ductal adenocarcinoma [PDAC]) model systems, we have identified significant single-cell population shifts toward invasive epithelial-to-mesenchymal transition (EMT) and proliferative (PRO) phenotypes linked with mitogen-activated protein kinase (MAPK) and signal transducer and activator of transcription 3 (STAT3) signaling. Using high-content digital imaging of RNA in situ hybridization in 195 PDAC tumors, we quantified these EMT and PRO subpopulations in 319,626 individual cancer cells that can be classified within the context of distinct tumor gland "units." Tumor gland typing provided an additional layer of intratumoral heterogeneity that was associated with differences in stromal abundance and clinical outcomes. This demonstrates the impact of the stroma in shaping tumor architecture by altering inherent patterns of tumor glands in human PDAC.