RESUMEN
BACKGROUND & AIMS: Continuing recalcitrance to therapy cements pancreatic cancer (PC) as the most lethal malignancy, which is set to become the second leading cause of cancer death in our society. The study aim was to investigate the association between DNA damage response (DDR), replication stress, and novel therapeutic response in PC to develop a biomarker-driven therapeutic strategy targeting DDR and replication stress in PC. METHODS: We interrogated the transcriptome, genome, proteome, and functional characteristics of 61 novel PC patient-derived cell lines to define novel therapeutic strategies targeting DDR and replication stress. Validation was done in patient-derived xenografts and human PC organoids. RESULTS: Patient-derived cell lines faithfully recapitulate the epithelial component of pancreatic tumors, including previously described molecular subtypes. Biomarkers of DDR deficiency, including a novel signature of homologous recombination deficiency, cosegregates with response to platinum (P < .001) and PARP inhibitor therapy (P < .001) in vitro and in vivo. We generated a novel signature of replication stress that predicts response to ATR (P < .018) and WEE1 inhibitor (P < .029) treatment in both cell lines and human PC organoids. Replication stress was enriched in the squamous subtype of PC (P < .001) but was not associated with DDR deficiency. CONCLUSIONS: Replication stress and DDR deficiency are independent of each other, creating opportunities for therapy in DDR-proficient PC and after platinum therapy.
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Adenocarcinoma/patología , Daño del ADN/genética , Reparación del ADN/genética , Replicación del ADN/genética , Neoplasias Pancreáticas/patología , Adenocarcinoma/genética , Adenocarcinoma/terapia , Biomarcadores , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Humanos , Terapia Molecular Dirigida , Organoides , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/terapia , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
AZD0171 (INN: Falbikitug) is being developed as a humanized monoclonal antibody (mAb), immunoglobulin G subclass 1 (IgG1), which binds specifically to the immunosuppressive human cytokine leukemia inhibitory factor (LIF) and inhibits downstream signaling by blocking recruitment of glycoprotein 130 (gp130) to the LIF receptor (LIFR) subunit (gp190) and the phosphorylation of signal transducer and activator of transcription 3 (STAT3) and is intended to treat adult participants with advanced solid tumors. LIF is a pleiotropic cytokine (and a member of the IL-6 family of cytokines) involved in many physiological and pathological processes and is highly expressed in a subset of solid tumors, including non-small cell lung cancer (NSCLC), colon, ovarian, prostate, and pancreatic cancer. The aim of this work was to develop a mechanistic PK/PD model to investigate the effect of AZD0171 on tumor LIF levels, predict the level of downstream signaling complex (LIF:LIFR:gp130) inhibition, and examine the dose-response relationship to support dose selection for a Phase II clinical study. Modeling results show that tumor LIF is inhibited in a dose-dependent manner with >90% inhibition for 95% of patients at the Phase II clinical dose of 1500 mg Q2W.
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Solid tumors consist of neoplastic cells, non-malignant stromal cells, and migratory hematopoietic cells. Complex interactions between the cell types in this microenvironment regulate tumor growth, progression, metastasis, and angiogenesis. The cells and mediators of inflammation form a major part of the epithelial tumor microenvironment. In some cancers, inflammatory conditions precede development of malignancy; in others, oncogenic change drives a tumor-promoting inflammatory milieu. Whatever its origin, this "smoldering" inflammation aids proliferation and survival of malignant cells, stimulates angiogenesis and metastasis, subverts adaptive immunity, and alters response to hormones and chemotherapy. Cytokines are major mediators of communication between cells in the inflammatory tumor microenvironment. It is known that neoplastic cells often over-express proinflammatory mediators including proteases, eicosanoids, cytokines, and chemokines. Several cytokines such as macrophage migratory inhibitory factor (MIF), TNF-α, IL-6, IL-17, IL-12, IL-23, IL-10, and TGF-ß have been linked with both experimental and human cancers and can either promote or inhibit tumor development. MIF is a major cytokine in many cancers and there is evidence that the cytokine is produced by both malignant cells and infiltrating leukocytes. In this article we will discuss the role of cancer-associated inflammation and the particular role of MIF in malignant disease.
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Inflamación/inmunología , Neoplasias/inmunología , Animales , Transformación Celular Neoplásica/inmunología , Transformación Celular Neoplásica/metabolismo , Citocinas/inmunología , Citocinas/metabolismo , Humanos , Inflamación/metabolismo , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Transducción de Señal , Microambiente Tumoral/inmunologíaRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) has a very poor prognosis because of its high propensity to metastasize and its immunosuppressive microenvironment. Using a panel of pancreatic cancer cell lines, three-dimensional (3D) invasion systems, microarray gene signatures, microfluidic devices, mouse models, and intravital imaging, we demonstrate that ROCK-Myosin II activity in PDAC cells supports a transcriptional program conferring amoeboid invasive and immunosuppressive traits and in vivo metastatic abilities. Moreover, we find that immune checkpoint CD73 is highly expressed in amoeboid PDAC cells and drives their invasive, metastatic, and immunomodulatory traits. Mechanistically, CD73 activates RhoA-ROCK-Myosin II downstream of PI3K. Tissue microarrays of human PDAC biopsies combined with bioinformatic analysis reveal that rounded-amoeboid invasive cells with high CD73-ROCK-Myosin II activity and their immunosuppressive microenvironment confer poor prognosis to patients. We propose targeting amoeboid PDAC cells as a therapeutic strategy.
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Adenocarcinoma , Amoeba , Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animales , Humanos , Ratones , Adenocarcinoma/patología , Amoeba/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Línea Celular Tumoral , Movimiento Celular/fisiología , Proteínas del Citoesqueleto , Terapia de Inmunosupresión , Miosina Tipo II/metabolismo , Neoplasias Pancreáticas/patología , Microambiente TumoralRESUMEN
As cancer-associated factors, kallikrein-related peptidases (KLKs) are components of the tumour microenvironment, which represents a rich substrate repertoire, and considered attractive targets for the development of novel treatments. Standard-of-care therapy of pancreatic cancer shows unsatisfactory results, indicating the need for alternative therapeutic approaches. We aimed to investigate the expression of KLKs in pancreatic cancer and to inhibit the function of KLK6 in pancreatic cancer cells. KLK6, KLK7, KLK8, KLK10 and KLK11 were coexpressed and upregulated in tissues from pancreatic cancer patients compared to normal pancreas. Their high expression levels correlated with each other and were linked to shorter survival compared to low KLK levels. We then validated KLK6 mRNA and protein expression in patient-derived tissues and pancreatic cancer cells. Coexpression of KLK6 with KRT19, αSMA or CD68 was independent of tumour stage, while KLK6 was coexpressed with KRT19 and CD68 in the invasive tumour area. High KLK6 levels in tumour and CD68+ cells were linked to shorter survival. KLK6 inhibition reduced KLK6 mRNA expression, cell metabolic activity and KLK6 secretion and increased the secretion of other serine and aspartic lysosomal proteases. The association of high KLK levels and poor prognosis suggests that inhibiting KLKs may be a therapeutic strategy for precision medicine.
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In tumor engineering, 3D approaches are used to model components of the tumor microenvironment and to test new treatments. Pancreatic cancers are a cancer of substantial unmet need and survival rates are lower compared to any other cancer. Bioengineering techniques are increasingly applied to understand the unique biology of pancreatic tumors and to design patient-specific models. Here we summarize how extracellular and cellular elements of the pancreatic tumor microenvironment and their interactions have been studied in 3D cell cultures. We review selected clinical trials, assess the benefits of therapies interfering with the tumor microenvironment and address their limitations and future perspectives.
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Neoplasias Pancreáticas/patología , Esferoides Celulares/patología , Ingeniería de Tejidos/métodos , Animales , Técnicas de Cultivo de Célula , Modelos Animales de Enfermedad , Humanos , Microambiente TumoralRESUMEN
Emerging evidence suggests that cancer cell metabolism can be regulated by cancer-associated fibroblasts (CAFs), but the mechanisms are poorly defined. Here we show that CAFs regulate malignant cell metabolism through pathways under the control of FAK. In breast and pancreatic cancer patients we find that low FAK expression, specifically in the stromal compartment, predicts reduced overall survival. In mice, depletion of FAK in a subpopulation of CAFs regulates paracrine signals that increase malignant cell glycolysis and tumour growth. Proteomic and phosphoproteomic analysis in our mouse model identifies metabolic alterations which are reflected at the transcriptomic level in patients with low stromal FAK. Mechanistically we demonstrate that FAK-depletion in CAFs increases chemokine production, which via CCR1/CCR2 on cancer cells, activate protein kinase A, leading to enhanced malignant cell glycolysis. Our data uncover mechanisms whereby stromal fibroblasts regulate cancer cell metabolism independent of genetic mutations in cancer cells.
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Fibroblastos Asociados al Cáncer/enzimología , Proteína-Tirosina Quinasas de Adhesión Focal/metabolismo , Neoplasias/metabolismo , Neoplasias/patología , Animales , Neoplasias de la Mama/irrigación sanguínea , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular , Quimiocinas/metabolismo , Femenino , Glucólisis , Humanos , Masculino , Redes y Vías Metabólicas , Ratones Endogámicos C57BL , Neoplasias/irrigación sanguínea , Neoplasias Pancreáticas/irrigación sanguínea , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patología , Fosfoproteínas/metabolismo , Células del Estroma/metabolismo , Análisis de Supervivencia , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Tissue engineering technologies have produced controllable and reproducible three-dimensional (3D) models that mimic the architecture and complexity of native tissues. In particular cell biology-based research is driven by the development of cell culture platforms and techniques that allow the analysis of cells cultured in 3D. Here we summarize several easy-to-follow methods for the characterization of cells that have been encapsulated and grown in hydrogels to measure their cell viability, metabolic activity, and mechanical properties of cell-containing hydrogels. We also describe an enzymatic approach for the digestion of cell-containing hydrogels and cell recovery thereby maintaining high cell viability for subsequent analysis.
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Técnicas de Cultivo de Célula/métodos , Encapsulación Celular/métodos , Hidrogeles , Línea Celular Tumoral , Proliferación Celular , Supervivencia Celular , Medios de Cultivo/química , Citometría de Flujo/métodos , Humanos , Microscopía Confocal , Microscopía Fluorescente , Esferoides CelularesRESUMEN
B cells are salient features of pancreatic ductal adenocarcinoma (PDAC) tumors, yet their role in this disease remains controversial. Murine studies have indicated a protumoral role for B cells, whereas clinical data show tumor-infiltrating B cells are a positive prognostic factor, both in PDAC and other cancers. This disparity needs to be clarified in order to develop effective immunotherapies. In this study, we provide new evidence that reconcile human and mouse data and highlight the importance of using relevant preclinical tumor models when assessing B cell function. We compared B cell infiltration and activation in both a genetic model of murine PDAC (KPC mouse) and an injectable orthotopic model. A pronounced B cell infiltrate was only observed in KPC tumors and correlated with T cell infiltration, mirroring human disease. In contrast, orthotopic tumors exhibited a relative paucity of B cells. Accordingly, KPC-derived B cells displayed markers of B cell activation (germinal center entry, B cell memory, and plasma cell differentiation) accompanied by significant intratumoral immunoglobulin deposition, a feature markedly weaker in orthotopic tumors. Tumor immunoglobulins, however, did not appear to form immune complexes. Furthermore, in contrast to the current paradigm that tumor B cells are immunosuppressive, when assessed as a bulk population, intratumoral B cells upregulated several proinflammatory and immunostimulatory genes, a distinctly different phenotype to that of splenic-derived B cells; further highlighting the importance of studying tumor-infiltrating B cells over B cells from secondary lymphoid organs. In agreement with the current literature, genetic deletion of B cells (µMT mice) resulted in reduced orthotopic tumor growth, however, this was not recapitulated by treatment with B-cell-depleting anti-CD20 antibody and, more importantly, was not observed in anti-CD20-treated KPC mice. This suggests the result from B cell deficient mice might be caused by their altered immune system, rather than lack of B cells. Therefore, our data indicate B cells do not favor tumor progression. In conclusion, our analysis of relevant preclinical models shows B cells to be active members of the tumor microenvironment, producing immunostimulatory factors that might support the adaptive antitumor immune response, as suggested by human PDAC studies.
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Linfocitos B/inmunología , Carcinoma Ductal Pancreático/inmunología , Linfocitos Infiltrantes de Tumor/inmunología , Neoplasias Pancreáticas/inmunología , Microambiente Tumoral/inmunología , Animales , Antígenos CD20/inmunología , Modelos Animales de Enfermedad , Femenino , Activación de Linfocitos/inmunología , Masculino , Ratones , Ratones Endogámicos C57BL , Páncreas/citología , Páncreas/inmunología , Páncreas/patología , Neoplasias Pancreáticas/patología , Linfocitos T/inmunología , Neoplasias PancreáticasRESUMEN
Pancreatic ductal adenocarcinoma (PDAC) is resistant to most therapies including single-agent immunotherapy and has a dense desmoplastic stroma, and most patients present with advanced metastatic disease. We reveal that macrophages are the dominant leukocyte population both in human PDAC stroma and autochthonous models, with an important functional contribution to the squamous subtype of human PDAC. We targeted macrophages in a genetic PDAC model using AZD7507, a potent selective inhibitor of CSF1R. AZD7507 caused shrinkage of established tumors and increased mouse survival in this difficult-to-treat model. Malignant cell proliferation diminished, with increased cell death and an enhanced T cell immune response. Loss of macrophages rewired other features of the TME, with global changes in gene expression akin to switching PDAC subtypes. These changes were markedly different to those elicited when neutrophils were targeted via CXCR2. These results suggest targeting the myeloid cell axis may be particularly efficacious in PDAC, especially with CSF1R inhibitors.
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Carcinoma Ductal Pancreático/inmunología , Macrófagos/inmunología , Modelos Inmunológicos , Proteínas de Neoplasias/inmunología , Neoplasias Pancreáticas/inmunología , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/inmunología , Linfocitos T/inmunología , Adulto , Compuestos de Anilina/farmacología , Animales , Carcinoma Ductal Pancreático/tratamiento farmacológico , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Femenino , Compuestos Heterocíclicos con 2 Anillos/farmacología , Humanos , Inmunidad Celular/efectos de los fármacos , Inmunidad Celular/genética , Macrófagos/patología , Masculino , Ratones , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Neoplasias Pancreáticas/tratamiento farmacológico , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/antagonistas & inhibidores , Receptores de Factor Estimulante de Colonias de Granulocitos y Macrófagos/genética , Linfocitos T/patología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
CXCR2 has been suggested to have both tumor-promoting and tumor-suppressive properties. Here we show that CXCR2 signaling is upregulated in human pancreatic cancer, predominantly in neutrophil/myeloid-derived suppressor cells, but rarely in tumor cells. Genetic ablation or inhibition of CXCR2 abrogated metastasis, but only inhibition slowed tumorigenesis. Depletion of neutrophils/myeloid-derived suppressor cells also suppressed metastasis suggesting a key role for CXCR2 in establishing and maintaining the metastatic niche. Importantly, loss or inhibition of CXCR2 improved T cell entry, and combined inhibition of CXCR2 and PD1 in mice with established disease significantly extended survival. We show that CXCR2 signaling in the myeloid compartment can promote pancreatic tumorigenesis and is required for pancreatic cancer metastasis, making it an excellent therapeutic target.
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Anticuerpos Monoclonales/administración & dosificación , Carcinoma Ductal Pancreático/tratamiento farmacológico , Neoplasias Pancreáticas/tratamiento farmacológico , Receptores de Interleucina-8B/genética , Animales , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales Humanizados , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/patología , Línea Celular Tumoral , Desoxicitidina/administración & dosificación , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Inmunoterapia , Ratones , Metástasis de la Neoplasia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/patología , Pronóstico , Receptores de Interleucina-8B/antagonistas & inhibidores , Transducción de Señal , Bibliotecas de Moléculas Pequeñas/administración & dosificación , Bibliotecas de Moléculas Pequeñas/farmacología , Análisis de Supervivencia , Regulación hacia Arriba , Ensayos Antitumor por Modelo de Xenoinjerto , GemcitabinaRESUMEN
The majority of human pancreatic cancers have activating mutations in the KRAS proto-oncogene. These mutations result in increased activity of the NF-κB pathway and the subsequent constitutive production of proinflammatory cytokines. Here, we show that inhibitor of κB kinase 2 (Ikk2), a component of the canonical NF-κB signaling pathway, synergizes with basal Notch signaling to upregulate transcription of primary Notch target genes, resulting in suppression of antiinflammatory protein expression and promotion of pancreatic carcinogenesis in mice. We found that in the Kras(G12D)Pdx1-cre mouse model of pancreatic cancer, genetic deletion of Ikk2 in initiated pre-malignant epithelial cells substantially delayed pancreatic oncogenesis and resulted in downregulation of the classical Notch target genes Hes1 and Hey1. Tnf-α stimulated canonical NF-κB signaling and, in collaboration with basal Notch signals, induced optimal expression of Notch targets. Mechanistically, Tnf-α stimulation resulted in phosphorylation of histone H3 at the Hes1 promoter, and this signal was lost with Ikk2 deletion. Hes1 suppresses expression of Pparg, which encodes the antiinflammatory nuclear receptor Pparγ. Thus, crosstalk between Tnf-α/Ikk2 and Notch sustains the intrinsic inflammatory profile of transformed cells. These findings reveal what we believe to be a novel interaction between oncogenic inflammation and a major cell fate pathway and show how these pathways can cooperate to promote cancer progression.