RESUMO
OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) has limited therapeutic options, particularly with immune checkpoint inhibitors. Highly chemoresistant 'stem-like' cells, known as cancer stem cells (CSCs), are implicated in PDAC aggressiveness. Thus, comprehending how this subset of cells evades the immune system is crucial for advancing novel therapies. DESIGN: We used the KPC mouse model (LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre) and primary tumour cell lines to investigate putative CSC populations. Transcriptomic analyses were conducted to pinpoint new genes involved in immune evasion. Overexpressing and knockout cell lines were established with lentiviral vectors. Subsequent in vitro coculture assays, in vivo mouse and zebrafish tumorigenesis studies, and in silico database approaches were performed. RESULTS: Using the KPC mouse model, we functionally confirmed a population of cells marked by EpCAM, Sca-1 and CD133 as authentic CSCs and investigated their transcriptional profile. Immune evasion signatures/genes, notably the gene peptidoglycan recognition protein 1 (PGLYRP1), were significantly overexpressed in these CSCs. Modulating PGLYRP1 impacted CSC immune evasion, affecting their resistance to macrophage-mediated and T-cell-mediated killing and their tumourigenesis in immunocompetent mice. Mechanistically, tumour necrosis factor alpha (TNFα)-regulated PGLYRP1 expression interferes with the immune tumour microenvironment (TME) landscape, promoting myeloid cell-derived immunosuppression and activated T-cell death. Importantly, these findings were not only replicated in human models, but clinically, secreted PGLYRP1 levels were significantly elevated in patients with PDAC. CONCLUSIONS: This study establishes PGLYRP1 as a novel CSC-associated marker crucial for immune evasion, particularly against macrophage phagocytosis and T-cell killing, presenting it as a promising target for PDAC immunotherapy.
Assuntos
Carcinoma Ductal Pancreático , Células-Tronco Neoplásicas , Neoplasias Pancreáticas , Animais , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Camundongos , Células-Tronco Neoplásicas/imunologia , Células-Tronco Neoplásicas/metabolismo , Carcinoma Ductal Pancreático/imunologia , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Humanos , Linhagem Celular Tumoral , Evasão Tumoral/imunologia , Modelos Animais de Doenças , Evasão da Resposta Imune , Microambiente Tumoral/imunologiaRESUMO
OBJECTIVE: The lysyl oxidase-like protein 2 (LOXL2) contributes to tumour progression and metastasis in different tumour entities, but its role in pancreatic ductal adenocarcinoma (PDAC) has not been evaluated in immunocompetent in vivo PDAC models. DESIGN: Towards this end, we used PDAC patient data sets, patient-derived xenograft in vivo and in vitro models, and four conditional genetically-engineered mouse models (GEMMS) to dissect the role of LOXL2 in PDAC. For GEMM-based studies, K-Ras +/LSL-G12D;Trp53 LSL-R172H;Pdx1-Cre mice (KPC) and the K-Ras +/LSL-G12D;Pdx1-Cre mice (KC) were crossed with Loxl2 allele floxed mice (Loxl2Exon2 fl/fl) or conditional Loxl2 overexpressing mice (R26Loxl2 KI/KI) to generate KPCL2KO or KCL2KO and KPCL2KI or KCL2KI mice, which were used to study overall survival; tumour incidence, burden and differentiation; metastases; epithelial to mesenchymal transition (EMT); stemness and extracellular collagen matrix (ECM) organisation. RESULTS: Using these PDAC mouse models, we show that while Loxl2 ablation had little effect on primary tumour development and growth, its loss significantly decreased metastasis and increased overall survival. We attribute this effect to non-cell autonomous factors, primarily ECM remodelling. Loxl2 overexpression, on the other hand, promoted primary and metastatic tumour growth and decreased overall survival, which could be linked to increased EMT and stemness. We also identified tumour-associated macrophage-secreted oncostatin M (OSM) as an inducer of LOXL2 expression, and show that targeting macrophages in vivo affects Osm and Loxl2 expression and collagen fibre alignment. CONCLUSION: Taken together, our findings establish novel pathophysiological roles and functions for LOXL2 in PDAC, which could be potentially exploited to treat metastatic disease.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Humanos , Camundongos , Animais , Transição Epitelial-Mesenquimal/genética , Neoplasias Pancreáticas/patologia , Carcinoma Ductal Pancreático/patologia , Modelos Animais de Doenças , Macrófagos/metabolismo , Aminoácido Oxirredutases/genética , Neoplasias PancreáticasRESUMO
The authors recently reported on the potential of targeting SRC kinase signaling in pancreatic cancer stem cells [...].
RESUMO
The proto-oncogene nonreceptor tyrosine-protein kinase SRC is a member of the SRC family of tyrosine kinases (SFKs), and its activation and overexpression have been shown to play a protumorigenic role in multiple solid cancers, including pancreatic ductal adenocarcinoma (PDAC). PDAC is currently the seventh-leading cause of cancer-related death worldwide, and, by 2030, it is predicted to become the second-leading cause of cancer-related death in the United States. PDAC is characterized by its high lethality (5-year survival of rate of <10%), invasiveness, and chemoresistance, all of which have been shown to be due to the presence of pancreatic cancer stem cells (PaCSCs) within the tumor. Due to the demonstrated overexpression of SRC in PDAC, we set out to determine if SRC kinases are important for PaCSC biology using pharmacological inhibitors of SRC kinases (dasatinib or PP2). Treatment of primary PDAC cultures established from patient-derived xenografts with dasatinib or PP2 reduced the clonogenic, self-renewal, and tumor-initiating capacity of PaCSCs, which we attribute to the downregulation of key signaling factors such as p-FAK, p-ERK1-2, and p-AKT. Therefore, this study not only validates that SRC kinases are relevant and biologically important for PaCSCs but also suggests that inhibitors of SRC kinases may represent a possible future treatment option for PDAC patients, although further studies are still needed.
RESUMO
OBJECTIVE: Pancreatic ductal adenocarcinoma (PDAC) is a disease of unmet medical need. While immunotherapy with chimeric antigen receptor T (CAR-T) cells has shown much promise in haematological malignancies, their efficacy for solid tumours is challenged by the lack of tumour-specific antigens required to avoid on-target, off-tumour effects. Switchable CAR-T cells whereby activity of the CAR-T cell is controlled by dosage of a tumour antigen-specific recombinant Fab-based 'switch' to afford a fully tunable response may overcome this translational barrier. DESIGN: In this present study, we have used conventional and switchable CAR-T cells to target the antigen HER2, which is upregulated on tumour cells, but also present at low levels on normal human tissue. We used patient-derived xenograft models derived from patients with stage IV PDAC that mimic the most aggressive features of PDAC, including severe liver and lung metastases. RESULTS: Switchable CAR-T cells followed by administration of the switch directed against human epidermal growth factor receptor 2 (HER2)-induced complete remission in difficult-to-treat, patient-derived advanced pancreatic tumour models. Switchable HER2 CAR-T cells were as effective as conventional HER2 CAR-T cells in vivo testing a range of different CAR-T cell doses. CONCLUSION: These results suggest that a switchable CAR-T system is efficacious against aggressive and disseminated tumours derived from patients with advanced PDAC while affording the potential safety of a control switch.
Assuntos
Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/terapia , Imunoterapia Adotiva/métodos , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/terapia , Animais , Antígenos de Neoplasias/genética , Biópsia por Agulha , Carcinoma Ductal Pancreático/imunologia , Ensaio de Imunoadsorção Enzimática , Citometria de Fluxo , Regulação Neoplásica da Expressão Gênica , Humanos , Imuno-Histoquímica , Imunoterapia/métodos , Invasividade Neoplásica/patologia , Metástase Neoplásica , Estadiamento de Neoplasias , Neoplasias Pancreáticas/imunologia , Receptor ErbB-2/genética , Estatísticas não Paramétricas , Resultado do Tratamento , Células Tumorais Cultivadas , Ensaios Antitumorais Modelo de Xenoenxerto/métodosRESUMO
Cancer stem cells (CSCs) are thought to drive tumor growth, metastasis and chemoresistance. Although surface markers such as CD133 and CD44 have been successfully used to isolate CSCs, their expression is not exclusively linked to the CSC phenotype and is prone to environmental alteration. We identified cells with an autofluorescent subcellular compartment that exclusively showed CSC features across different human tumor types. Primary tumor-derived autofluorescent cells did not overlap with side-population (SP) cells, were enriched in sphere culture and during chemotherapy, strongly expressed pluripotency-associated genes, were highly metastatic and showed long-term in vivo tumorigenicity, even at the single-cell level. Autofluorescence was due to riboflavin accumulation in membrane-bounded cytoplasmic structures bearing ATP-dependent ABCG2 transporters. In summary, we identified and characterized an intrinsic autofluorescent phenotype in CSCs of diverse epithelial cancers and used this marker to isolate and characterize these cells.
Assuntos
Biomarcadores Tumorais/metabolismo , Separação Celular/métodos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Imagem Óptica/métodos , Riboflavina/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Autofagia , Proteína 12 Relacionada à Autofagia , Carcinoma Hepatocelular/patologia , Carcinoma Pulmonar de Células não Pequenas/patologia , Carcinoma Ductal Pancreático/patologia , Neoplasias Colorretais/patologia , Feminino , Humanos , Neoplasias Hepáticas/patologia , Neoplasias Pulmonares/patologia , Camundongos , Camundongos Nus , Proteínas Associadas aos Microtúbulos/biossíntese , Proteínas de Neoplasias/metabolismo , Neoplasias Pancreáticas/patologia , Proteínas Modificadoras Pequenas Relacionadas à Ubiquitina/biossíntese , Células Tumorais CultivadasRESUMO
Nanomedicine nowadays offers novel solutions in cancer therapy by introducing multimodal treatments in one single formulation. In addition, nanoparticles act as nanocarriers changing the solubility, biodistribution and efficiency of the therapeutic molecules, thus generating more efficient treatments and reducing their side effects. To apply these novel therapeutic approaches, efforts are focused on the multi-functionalization of the nanoparticles and will open up new avenues to advanced combinational therapies. Pancreatic ductal adenocarcinoma (PDAC) is a cancer with unmet medical needs. Abundant expression of the anti-phagocytosis signal CD47 has also been observed on pancreatic cancer cells, in particular a subset of cancer stem cells (CSCs) responsible for resistance to standard therapy and metastatic potential. CD47 receptor is found on pancreatic cancer and highly expressed on CSCs, but not on normal pancreas. Inhibiting CD47 using monoclonal antibodies has been shown as an effective strategy to treat PDAC in vivo. However, CD47 inhibition effectively slowed tumor growth only in combination with Gemcitabine or Abraxane. In this work, we present the generation of multifunctionalized iron oxide magnetic nanoparticles (MNPs) that include the anti-CD47 antibody and the chemotherapeutic drug Gemcitabine in a single formulation. We demonstrate the in vitro efficacy of the formulation against CD47-positive pancreatic cancer cells. This article is part of a Special Issue entitled "Recent Advances in Bionanomaterials" Guest Editor: Dr. Marie-Louise Saboungi and Dr. Samuel D. Bader.
Assuntos
Anticorpos Monoclonais/farmacologia , Antineoplásicos/farmacologia , Antígeno CD47/metabolismo , Carcinoma Ductal Pancreático/tratamento farmacológico , Desoxicitidina/análogos & derivados , Portadores de Fármacos , Magnetismo/métodos , Nanopartículas de Magnetita , Nanomedicina/métodos , Células-Tronco Neoplásicas/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Anticorpos Monoclonais/química , Anticorpos Monoclonais/metabolismo , Antineoplásicos/química , Antineoplásicos/metabolismo , Apoptose/efeitos dos fármacos , Antígeno CD47/imunologia , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Sobrevivência Celular/efeitos dos fármacos , Desoxicitidina/química , Desoxicitidina/metabolismo , Desoxicitidina/farmacologia , Composição de Medicamentos , Humanos , Nanopartículas de Magnetita/química , Células-Tronco Neoplásicas/patologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Propriedades de Superfície , Células Tumorais Cultivadas , GencitabinaRESUMO
Noonan syndrome (NS) is an autosomal dominant genetic disorder characterized by short stature, craniofacial dysmorphism, and congenital heart defects. NS also is associated with a risk for developing myeloproliferative disorders (MPD), including juvenile myelomonocytic leukemia (JMML). Mutations responsible for NS occur in at least 11 different loci including KRAS. Here we describe a mouse model for NS induced by K-Ras(V14I), a recurrent KRAS mutation in NS patients. K-Ras(V14I)-mutant mice displayed multiple NS-associated developmental defects such as growth delay, craniofacial dysmorphia, cardiac defects, and hematologic abnormalities including a severe form of MPD that resembles human JMML. Homozygous animals had perinatal lethality whose penetrance varied with genetic background. Exposure of pregnant mothers to a MEK inhibitor rescued perinatal lethality and prevented craniofacial dysmorphia and cardiac defects. However, Mek inhibition was not sufficient to correct these defects when mice were treated after weaning. Interestingly, Mek inhibition did not correct the neoplastic MPD characteristic of these mutant mice, regardless of the timing at which the mice were treated, thus suggesting that MPD is driven by additional signaling pathways. These genetically engineered K-Ras(V14I)-mutant mice offer an experimental tool for studying the molecular mechanisms underlying the clinical manifestations of NS. Perhaps more importantly, they should be useful as a preclinical model to test new therapies aimed at preventing or ameliorating those deficits associated with this syndrome.
Assuntos
Modelos Animais de Doenças , Genes ras , Camundongos Mutantes , Mutação de Sentido Incorreto , Síndrome de Noonan/genética , Mutação Puntual , Proteínas Proto-Oncogênicas p21(ras)/genética , Anormalidades Múltiplas/embriologia , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/prevenção & controle , Alelos , Substituição de Aminoácidos , Animais , Tamanho Corporal/genética , Linhagem da Célula , Cruzamentos Genéticos , Nanismo/genética , Epistasia Genética , Face/anormalidades , Feminino , Genes Dominantes , Genótipo , Cardiopatias Congênitas/genética , Hematopoese/genética , Leucemia Mielomonocítica Juvenil/genética , MAP Quinase Quinase Quinases/antagonistas & inibidores , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes/genética , Transtornos Mieloproliferativos/genética , Síndromes Neoplásicas Hereditárias/embriologia , Síndromes Neoplásicas Hereditárias/genética , Fenótipo , Gravidez , Efeitos Tardios da Exposição Pré-Natal , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas p21(ras)/fisiologia , Quimera por Radiação , Transdução de Sinais/efeitos dos fármacosRESUMO
Cancer cells adapt cellular metabolism to cope with their high proliferation rate. Instead of primarily using oxidative phosphorylation (OXPHOS), cancer cells use less efficient glycolysis for the production of ATP and building blocks (Warburg effect). However, tumours are not uniform, but rather functionally heterogeneous and harbour a subset of cancer cells with stemness features. Such cancer cells have the ability to repopulate the entire tumour and thus have been termed cancer stem cells (CSCs) or tumour-initiating cells (TICs). As opposed to differentiated bulk tumour cells relying on glycolysis, CSCs show a distinct metabolic phenotype that, depending on the cancer type, can be highly glycolytic or OXPHOS dependent. In either case, mitochondrial function is critical and takes centre stage in CSC functionality. Remaining controversies in this young and emerging research field may be related to CSC isolation techniques and/or the use of less suitable model systems. Still, the apparent dependence of CSCs on mitochondrial function, regardless of their primary metabolic phenotype, represents a previously unrecognised Achilles heel amendable for therapeutic intervention. Elimination of highly chemoresistant CSCs as the root of many cancers via inhibition of mitochondrial function bears the potential to prevent relapse from disease and thus improve patients' long-term outcome.
Assuntos
Células-Tronco Neoplásicas/metabolismo , Animais , Diferenciação Celular/fisiologia , Proliferação de Células/fisiologia , Glicólise , Humanos , Neoplasias/metabolismo , Neoplasias/patologia , Células-Tronco Neoplásicas/patologia , Fosforilação OxidativaRESUMO
Pancreatic cancer stem cells (CSCs) have been first described in 2007 and since then have emerged as an intriguing entity of cancer cells with distinct functional features including self-renewal and exclusive in vivo tumorigenicity. The heterogeneous pancreatic CSC pool has been implicated in tumor propagation as well as metastatic spread. Clinically, the most important feature of CSCs is their strong resistance to standard chemotherapy, which results in fast disease relapse, even with today's more advanced chemotherapeutic regimens. Therefore, novel therapeutic strategies to most efficiently target pancreatic CSCs are being developed and their careful clinical translation should provide new avenues to eradicate this deadly disease.
Assuntos
Resistencia a Medicamentos Antineoplásicos/genética , Células-Tronco Neoplásicas , Neoplasias Pancreáticas/genética , Animais , Proliferação de Células , Humanos , Metástase Neoplásica , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/patologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
OBJECTIVES: The tumour stroma/microenvironment not only provides structural support for tumour development, but more importantly it provides cues to cancer stem cells (CSCs) that regulate their self-renewal and metastatic potential. This is certainly true for pancreatic ductal adenocarcinomas (PDAC), where tumour-associated fibroblasts, pancreatic stellate cells and immune cells create an abundant paracrine niche for CSCs via microenvironment-secreted factors. Thus understanding the role that tumour stroma cells play in PDAC development and CSC biology is of utmost importance. DESIGN: Microarray analyses, tumour microarray immunohistochemical assays, in vitro co-culture experiments, recombinant protein treatment approaches and in vivo intervention studies were performed to understand the role that the immunomodulatory cationic antimicrobial peptide 18/LL-37 (hCAP-18/LL-37) plays in PDAC biology. RESULTS: We found that hCAP-18/LL-37 was strongly expressed in the stroma of advanced primary and secondary PDAC tumours and is secreted by immune cells of the stroma (eg, tumour-associated macrophages) in response to tumour growth factor-ß1 and particularly CSC-secreted Nodal/ActivinA. Treatment of pancreatic CSCs with recombinant LL-37 increased pluripotency-associated gene expression, self-renewal, invasion and tumourigenicity via formyl peptide receptor 2 (FPR2)- and P2X purinoceptor 7 receptor (P2X7R)-dependent mechanisms, which could be reversed by inhibiting these receptors. Importantly, in a genetically engineered mouse model of K-Ras-driven pancreatic tumourigenesis, we also showed that tumour formation was inhibited by either reconstituting these mice with bone marrow from cathelicidin-related antimicrobial peptide (ie, murine homologue of hCAP-18/LL-37) knockout mice or by pharmacologically inhibiting FPR2 and P2X7R. CONCLUSIONS: Thus, hCAP-18/LL-37 represents a previously unrecognised PDAC microenvironment factor that plays a critical role in pancreatic CSC-mediated tumourigenesis.
Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Células-Tronco Neoplásicas/metabolismo , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Microambiente Tumoral , Ativinas/metabolismo , Animais , Peptídeos Catiônicos Antimicrobianos/farmacologia , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , Carcinogênese/metabolismo , Carcinoma Ductal Pancreático/genética , Autorrenovação Celular/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Camundongos Nus , Invasividade Neoplásica , Células-Tronco Neoplásicas/efeitos dos fármacos , Neoplasias Pancreáticas/genética , Análise Serial de Proteínas , Antagonistas do Receptor Purinérgico P2X/farmacologia , Receptores de Formil Peptídeo/antagonistas & inibidores , Receptores de Formil Peptídeo/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Transdução de Sinais/efeitos dos fármacos , Análise Serial de Tecidos , Fator de Crescimento Transformador beta1/farmacologia , CatelicidinasRESUMO
OBJECTIVE: Cancer stem cells (CSCs) represent the root of many solid cancers including pancreatic ductal adenocarcinoma, are highly chemoresistant and represent the cellular source for disease relapse. However the mechanisms involved in these processes still need to be fully elucidated. Understanding the mechanisms implicated in chemoresistance and metastasis of pancreatic cancer is critical to improving patient outcomes. DESIGN: Micro-RNA (miRNA) expression analyses were performed to identify functionally defining epigenetic signatures in pancreatic CSC-enriched sphere-derived cells and gemcitabine-resistant pancreatic CSCs. RESULTS: We found the miR-17-92 cluster to be downregulated in chemoresistant CSCs versus non-CSCs and demonstrate its crucial relevance for CSC biology. In particular, overexpression of miR-17-92 reduced CSC self-renewal capacity, in vivo tumourigenicity and chemoresistance by targeting multiple NODAL/ACTIVIN/TGF-ß1 signalling cascade members as well as directly inhibiting the downstream targets p21, p57 and TBX3. Overexpression of miR-17-92 translated into increased CSC proliferation and their eventual exhaustion via downregulation of p21 and p57. Finally, the translational impact of our findings could be confirmed in preclinical models for pancreatic cancer. CONCLUSIONS: Our findings therefore identify the miR-17-92 cluster as a functionally determining family of miRNAs in CSCs, and highlight the putative potential of developing modulators of this cluster to overcome drug resistance in pancreatic CSCs.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Carcinoma Ductal Pancreático/metabolismo , Desoxicitidina/análogos & derivados , Resistencia a Medicamentos Antineoplásicos/genética , MicroRNAs/metabolismo , Células-Tronco Neoplásicas/metabolismo , Neoplasias Pancreáticas/metabolismo , Ativinas/metabolismo , Animais , Antimetabólitos Antineoplásicos/uso terapêutico , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Autorrenovação Celular , Transformação Celular Neoplásica , Inibidor de Quinase Dependente de Ciclina p21/genética , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Inibidor de Quinase Dependente de Ciclina p57/metabolismo , Desoxicitidina/farmacologia , Desoxicitidina/uso terapêutico , Regulação para Baixo , Epigênese Genética , Feminino , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Camundongos , Camundongos Nus , MicroRNAs/antagonistas & inibidores , MicroRNAs/genética , Células-Tronco Neoplásicas/efeitos dos fármacos , Proteína Nodal/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , RNA Longo não Codificante , Transdução de Sinais , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Transcriptoma , Fator de Crescimento Transformador beta1/metabolismo , GencitabinaRESUMO
BACKGROUND & AIMS: Although smoking is a leading risk factor for pancreatic ductal adenocarcinoma (PDAC), little is known about the mechanisms by which smoking promotes initiation or progression of PDAC. METHODS: We studied the effects of nicotine administration on pancreatic cancer development in Kras(+/LSLG12Vgeo);Elas-tTA/tetO-Cre (Ela-KRAS) mice, Kras(+/LSLG12D);Trp53+/LSLR172H;Pdx-1-Cre (KPC) mice (which express constitutively active forms of KRAS), and C57/B6 mice. Mice were given nicotine for up to 86 weeks to produce blood levels comparable with those of intermediate smokers. Pancreatic tissues were collected and analyzed by immunohistochemistry and reverse transcriptase polymerase chain reaction; cells were isolated and assayed for colony and sphere formation and gene expression. The effects of nicotine were also evaluated in primary pancreatic acinar cells isolated from wild-type, nAChR7a(-/-), Trp53(-/-), and Gata6(-/-);Trp53(-/-) mice. We also analyzed primary PDAC cells that overexpressed GATA6 from lentiviral expression vectors. RESULTS: Administration of nicotine accelerated transformation of pancreatic cells and tumor formation in Ela-KRAS and KPC mice. Nicotine induced dedifferentiation of acinar cells by activating AKT-ERK-MYC signaling; this led to inhibition of Gata6 promoter activity, loss of GATA6 protein, and subsequent loss of acinar differentiation and hyperactivation of oncogenic KRAS. Nicotine also promoted aggressiveness of established tumors as well as the epithelial-mesenchymal transition, increasing numbers of circulating cancer cells and their dissemination to the liver, compared with mice not exposed to nicotine. Nicotine induced pancreatic cells to acquire gene expression patterns and functional characteristics of cancer stem cells. These effects were markedly attenuated in K-Ras(+/LSL-G12D);Trp53(+/LSLR172H);Pdx-1-Cre mice given metformin. Metformin prevented nicotine-induced pancreatic carcinogenesis and tumor growth by up-regulating GATA6 and promoting differentiation toward an acinar cell program. CONCLUSIONS: In mice, nicotine promotes pancreatic carcinogenesis and tumor development via down-regulation of Gata6 to induce acinar cell dedifferentiation.
Assuntos
Células Acinares/efeitos dos fármacos , Carcinoma Ductal Pancreático/induzido quimicamente , Desdiferenciação Celular/efeitos dos fármacos , Fator de Transcrição GATA6/metabolismo , Nicotina/toxicidade , Agonistas Nicotínicos/toxicidade , Pâncreas/efeitos dos fármacos , Neoplasias Pancreáticas/induzido quimicamente , Proteínas Proto-Oncogênicas p21(ras)/metabolismo , Células Acinares/metabolismo , Células Acinares/patologia , Animais , Carcinoma Ductal Pancreático/genética , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/prevenção & controle , Carcinoma Ductal Pancreático/secundário , Linhagem Celular Tumoral , Transformação Celular Neoplásica/induzido quimicamente , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Fator de Transcrição GATA6/deficiência , Fator de Transcrição GATA6/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/secundário , Metformina/farmacologia , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Nus , Mutação , Células Neoplásicas Circulantes/efeitos dos fármacos , Células Neoplásicas Circulantes/metabolismo , Células Neoplásicas Circulantes/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Pâncreas/metabolismo , Pâncreas/patologia , Neoplasias Pancreáticas/genética , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/prevenção & controle , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Proto-Oncogênicas p21(ras)/deficiência , Proteínas Proto-Oncogênicas p21(ras)/genética , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Transfecção , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Receptor Nicotínico de Acetilcolina alfa7/genéticaRESUMO
It has been demonstrated that even localized tumors without clinically apparent metastasis give rise to circulating tumor cells (CTCs). A growing number of technically diverse platforms are being developed for detecting/isolating CTCs in the circulating blood. Despite the technical challenges of isolating rare CTCs from blood, recent studies have already shown the predictive value of CTCs enumeration. Thus, it is becoming increasingly accepted that CTC numbers are linked to patients' outcome and may also be used to monitor treatment response and disease relapse, respectively. Further CTCs provide a non-invasive source for tumor material, 'liquid biopsy', which is particularly important for patients, where no biopsy material can be obtained or where serial biopsies of the tumor, e.g., following treatment, are practically impossible. On the other hand the molecular and biological characterization of CTCs has still remained at a rather experimental stage. Future studies are necessary to define CTC heterogeneity to establish the crucial role of circulating cancer stem cells for driving metastasis, which represent a distinct subpopulation of CTCs that bear metastasis-initiating capabilities based on their stemness properties and invasiveness and thus are critical for the patients' clinical outcome. As compared to non-tumorigenic/metastatic bulk CTCs, circulating cancer stem cells may not only be capable of evading from the primary tumor, but also escape from immune surveillance, survive in the circulating blood and subsequently form metastases in distant organs. Thus, circulating cancer stem cells represent a subset of exclusively tumorigenic cancer stem cells characterized by their invasive characteristics and are potential therapeutic targets for preventing disease progression. To date, only a few original reports and reviews have been published focusing on circulating cancer stem cells. This review discusses the potential importance of isolating and characterizing these circulating cancer stem cells, but also highlights current technological limitations.
RESUMO
The advent of liquid biopsies has brought significant changes to the diagnosis and monitoring of non-small cell lung cancer (NSCLC), presenting both promise and challenges. Molecularly targeted drugs, capable of enhancing survival rates, are now available to around a quarter of NSCLC patients. However, to ensure their effectiveness, precision diagnosis is essential. Circulating tumor DNA (ctDNA) analysis as the most advanced liquid biopsy modality to date offers a non-invasive method for tracking genomic changes in NSCLC. The potential of ctDNA is particularly rooted in its ability to furnish comprehensive (epi-)genetic insights into the tumor, thereby aiding personalized treatment strategies. One of the key advantages of ctDNA-based liquid biopsies in NSCLC is their ability to capture tumor heterogeneity. This capability ensures a more precise depiction of the tumor's (epi-)genomic landscape compared to conventional tissue biopsies. Consequently, it facilitates the identification of (epi-)genetic alterations, enabling informed treatment decisions, disease progression monitoring, and early detection of resistance-causing mutations for timely therapeutic interventions. Here we review the current state-of-the-art in ctDNA-based liquid biopsy technologies for NSCLC, exploring their potential to revolutionize clinical practice. Key advancements in ctDNA detection methods, including PCR-based assays, next-generation sequencing (NGS), and digital PCR (dPCR), are discussed, along with their respective strengths and limitations. Additionally, the clinical utility of ctDNA analysis in guiding treatment decisions, monitoring treatment response, detecting minimal residual disease, and identifying emerging resistance mechanisms is examined. Liquid biopsy analysis bears the potential of transforming NSCLC management by enabling non-invasive monitoring of Minimal Residual Disease and providing early indicators for response to targeted treatments including immunotherapy. Furthermore, considerations regarding sample collection, processing, and data interpretation are highlighted as crucial factors influencing the reliability and reproducibility of ctDNA-based assays. Addressing these challenges will be essential for the widespread adoption of ctDNA-based liquid biopsies in routine clinical practice, ultimately paving the way toward personalized medicine and improved outcomes for patients with NSCLC.
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Pancreatic ductal adenocarcinoma (PDAC) is characterized by a dense and stiff extracellular matrix (ECM) associated with tumor progression and therapy resistance. To further the understanding of how stiffening of the tumor microenvironment (TME) contributes to aggressiveness, a three-dimensional (3D) self-assembling hydrogel disease model is developed based on peptide amphiphiles (PAs, PA-E3Y) designed to tailor stiffness. The model displays nanofibrous architectures reminiscent of native TME and enables the study of the invasive behavior of PDAC cells. Enhanced tuneability of stiffness is demonstrated by interacting thermally annealed aqueous solutions of PA-E3Y (PA-E3Yh) with divalent cations to create hydrogels with mechanical properties and ultrastructure similar to native tumor ECM. It is shown that stiffening of PA-E3Yh hydrogels to levels found in PDAC induces ECM deposition, promotes epithelial-to-mesenchymal transition (EMT), enriches CD133+/CXCR4+ cancer stem cells (CSCs), and subsequently enhances drug resistance. The findings reveal how a stiff 3D environment renders PDAC cells more aggressive and therefore more faithfully recapitulates in vivo tumors.
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Resistencia a Medicamentos Antineoplásicos , Transição Epitelial-Mesenquimal , Matriz Extracelular , Hidrogéis , Células-Tronco Neoplásicas , Neoplasias Pancreáticas , Microambiente Tumoral , Humanos , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/tratamento farmacológico , Hidrogéis/química , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Matriz Extracelular/metabolismo , Microambiente Tumoral/efeitos dos fármacos , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/metabolismo , Peptídeos/química , Peptídeos/farmacologia , Fenótipo , Receptores CXCR4/metabolismoRESUMO
Pancreatic ductal adenocarcinoma (PDAC) poses significant clinical challenges, often presenting as unresectable with limited biopsy options. Here, we show that circulating tumor cells (CTCs) offer a promising alternative, serving as a "liquid biopsy" that enables the generation of in vitro 3D models and highly aggressive in vivo models for functional and molecular studies in advanced PDAC. Within the retrieved CTC pool (median 65 CTCs/5 mL), we identify a subset (median content 8.9%) of CXCR4+ CTCs displaying heightened stemness and metabolic traits, reminiscent of circulating cancer stem cells. Through comprehensive analysis, we elucidate the importance of CTC-derived models for identifying potential targets and guiding treatment strategies. Screening of stemness-targeting compounds identified stearoyl-coenzyme A desaturase (SCD1) as a promising target for advanced PDAC. These results underscore the pivotal role of CTC-derived models in uncovering therapeutic avenues and ultimately advancing personalized care in PDAC.
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It is now well established that human pancreatic ductal adenocarcinoma (PDAC) contains a subset of cells with self-renewal capabilities and subsequent exclusive in vivo tumorigenic capacity as assessed by limiting dilution tumorigenic transplantation assays into immunodeficient mice. These cells are considered pancreatic cancer stem cells (CSCs) and are able to form tumors indistinguishable from parental ones. Furthermore they display strong chemotherapy resistance and are implicated in tumor relapses and metastatic spread. Important next steps for advancing the field of pancreatic CSC research include the identification and characterization of CSCs in the unperturbed in vivo setting. This has been achieved just recently for other solid tumors such as glioblastoma using clonal analysis after lineage tracing in mice [1]. In vivo imaging of CSCs during tumor development should not only provide new insights into the in vivo features of CSCs, but also help to further unravel the influence of the stroma on CSC biology. Comprehensive studies of the tumor heterogeneity with respect to the coexistence of different clones potentially generated by distinct population of CSCs that are evolving by stochastic cell fate decisions may actually unite the CSC concept and the model of clonal evolution for pancreatic cancer. Eventually, the design of specific therapies against CSCs should open new alleys to improve survival of patients with PDAC. Combined therapies targeting CSCs and their progenies as well as the supportive stroma may represent the most promising approach for the future treatment of patients with PDAC.
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Adenocarcinoma/patologia , Células-Tronco Neoplásicas/citologia , Pâncreas/citologia , Neoplasias Pancreáticas/patologia , Adenocarcinoma/tratamento farmacológico , Animais , Antígenos CD/metabolismo , Antineoplásicos/administração & dosagem , Antineoplásicos/uso terapêutico , Biomarcadores , Humanos , Camundongos , Células-Tronco Neoplásicas/classificação , Células-Tronco Neoplásicas/fisiologia , Neoplasias Pancreáticas/tratamento farmacológicoRESUMO
OBJECTIVE: Cord blood-derived human endothelial colony-forming cells (ECFCs) bear a high proliferative capacity and potently enhance tissue neovascularization in vivo. Here, we investigated whether the leading mechanism for the functional improvement relates to their physical vascular incorporation or perivascular paracrine effects and whether the effects can be further enhanced by dual-cell-based therapy, including mesenchymal stem cells (MSCs). METHODS AND RESULTS: ECFCs or MSCs were lentivirally transduced with thymidine kinase suicide gene driven by the endothelial-specific vascular endothelial growth factor 2 (kinase insert domain receptor) promoter and evaluated in a hindlimb ischemia model. ECFCs and MSCs enhanced neovascularization after ischemic events to a similar extent. Dual therapy using ECFCs and MSCs further enhanced neovascularization. Mechanistically, 3 weeks after induction of ischemia followed by cell therapy, ganciclovir-mediated elimination of kinase insert domain receptor(+) cells completely reversed the therapeutic effect of ECFCs but not that of MSCs. Histological analysis revealed that ganciclovir effectively eliminated ECFCs incorporated into the vasculature. CONCLUSIONS: Endothelial-specific suicide gene technology demonstrates distinct mechanisms for ECFCs and MSCs, with complete abolishment of ECFC-mediated effects, whereas MSC-mediated effects remained unaffected. These data strengthen the notion that a dual-cell-based therapy represents a promising approach for vascular regeneration of ischemic tissue.
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Terapia Baseada em Transplante de Células e Tecidos/métodos , Endotélio Vascular/citologia , Membro Posterior/irrigação sanguínea , Isquemia/terapia , Células-Tronco Mesenquimais/citologia , Neovascularização Fisiológica/fisiologia , Células-Tronco/citologia , Animais , Proliferação de Células , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/fisiologia , Feminino , Ganciclovir/farmacologia , Humanos , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/fisiologia , Camundongos , Camundongos Nus , Modelos Animais , Fenótipo , Recuperação de Função Fisiológica/fisiologia , Células-Tronco/efeitos dos fármacos , Células-Tronco/fisiologiaRESUMO
Emerging evidence suggests that stem cells play a crucial role not only in the generation and maintenance of different tissues, but also in the development and progression of malignancies. For the many solid cancers, it has now been shown that they harbor a distinct subpopulation of cancer cells that bear stem cell features and therefore, these cells are termed cancer stem cells (CSC) or tumor-propagating cells. CSC are exclusively tumorigenic and essential drivers for tumor progression and metastasis. Moreover, it has been shown that pancreatic ductal adenocarcinoma does not only contain one homogeneous population of CSC rather than diverse subpopulations that may have evolved during tumor progression. One of these populations is called migrating CSC and can be characterized by CXCR4 co-expression. Only these cells are capable of evading the primary tumor and traveling to distant sites such as the liver as the preferred site of metastatic spread. Clinically even more important, however, is the observation that CSC are highly resistant to chemo- and radiotherapy resulting in their relative enrichment during treatment and rapid relapse of disease. Many laboratories are now working on the further in-depth characterization of these cells, which may eventually allow for the identification of their Achilles heal and lead to novel treatment modalities for fighting this deadly disease.