RESUMO
Pancreatic cancer is accompanied by a fibrotic reaction that alters interactions between tumor cells and the stroma to promote tumor progression. Consequently, strategies to target the tumor stroma might be used to treat patients with pancreatic cancer. We review recently developed approaches for reshaping the pancreatic tumor stroma and discuss how these might improve patient outcomes. We also describe relationships between the pancreatic tumor extracellular matrix, the vasculature, the immune system, and metabolism, and discuss the implications for the development of stromal compartment-specific therapies.
Assuntos
Antineoplásicos/uso terapêutico , Matriz Extracelular/efeitos dos fármacos , Neoplasias Pancreáticas/tratamento farmacológico , Células Estromais/efeitos dos fármacos , Microambiente Tumoral , Animais , Biomarcadores Tumorais/metabolismo , Comunicação Celular/efeitos dos fármacos , Metabolismo Energético/efeitos dos fármacos , Matriz Extracelular/imunologia , Matriz Extracelular/metabolismo , Matriz Extracelular/patologia , Humanos , Terapia de Alvo Molecular , Neoplasias Pancreáticas/imunologia , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Transdução de Sinais/efeitos dos fármacos , Células Estromais/imunologia , Células Estromais/metabolismo , Células Estromais/patologiaRESUMO
OBJECTIVE: Extensive molecular heterogeneity of pancreatic ductal adenocarcinoma (PDA), few effective therapies and high mortality make this disease a prime model for advancing development of tailored therapies. The p16-cyclin D-cyclin-dependent kinase 4/6-retinoblastoma (RB) protein (CDK4) pathway, regulator of cell proliferation, is deregulated in PDA. Our aim was to develop a novel personalised treatment strategy for PDA based on targeting CDK4. DESIGN: Sensitivity to potent CDK4/6 inhibitor PD-0332991 (palbociclib) was correlated to protein and genomic data in 19 primary patient-derived PDA lines to identify biomarkers of response. In vivo efficacy of PD-0332991 and combination therapies was determined in subcutaneous, intrasplenic and orthotopic tumour models derived from genome-sequenced patient specimens and genetically engineered model. Mechanistically, monotherapy and combination therapy were investigated in the context of tumour cell and extracellular matrix (ECM) signalling. Prognostic relevance of companion biomarker, RB protein, was evaluated and validated in independent PDA patient cohorts (>500 specimens). RESULTS: Subtype-specific in vivo efficacy of PD-0332991-based therapy was for the first time observed at multiple stages of PDA progression: primary tumour growth, recurrence (second-line therapy) and metastatic setting and may potentially be guided by a simple biomarker (RB protein). PD-0332991 significantly disrupted surrounding ECM organisation, leading to increased quiescence, apoptosis, improved chemosensitivity, decreased invasion, metastatic spread and PDA progression in vivo. RB protein is prevalent in primary operable and metastatic PDA and may present a promising predictive biomarker to guide this therapeutic approach. CONCLUSION: This study demonstrates the promise of CDK4 inhibition in PDA over standard therapy when applied in a molecular subtype-specific context.
Assuntos
Carcinoma Ductal Pancreático/tratamento farmacológico , Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Neoplasias Pancreáticas/tratamento farmacológico , Animais , Antineoplásicos/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Biomarcadores Tumorais/metabolismo , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Modelos Animais de Doenças , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Terapia de Alvo Molecular/métodos , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Fosforilação , Piperazinas/uso terapêutico , Prognóstico , Piridinas/uso terapêutico , Proteína do Retinoblastoma/metabolismo , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Basic in vitro systems can be used to model and assess complex diseases, such as cancer. Recent advances in this field include the incorporation of multiple cell types and extracellular matrix proteins into three-dimensional (3D) models to recapitulate the structure, organization and functionality of live tissue in situ. Cells within such a 3D environment behave very differently from cells on two-dimensional (2D) substrates, as cell-matrix interactions trigger signalling pathways and cellular responses in 3D, which may not be observed in 2D. Thus, the use of 3D systems can be advantageous for the assessment of disease progression over 2D set-ups alone. Here, we highlight the current advantages and challenges of employing 3D systems in the study of cancer and provide an overview to guide the appropriate use of distinct models in cancer research.
Assuntos
Comunicação Celular , Modelos Biológicos , Neoplasias/patologia , Microambiente Tumoral , Animais , Técnicas de Cocultura , Humanos , Transdução de SinaisRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is characterized by increasing fibrosis, which can enhance tumor progression and spread. Here, we undertook an unbiased temporal assessment of the matrisome of the highly metastatic KPC (Pdx1-Cre, LSL-KrasG12D/+, LSL-Trp53R172H/+) and poorly metastatic KPflC (Pdx1-Cre, LSL-KrasG12D/+, Trp53fl/+) genetically engineered mouse models of pancreatic cancer using mass spectrometry proteomics. Our assessment at early-, mid-, and late-stage disease reveals an increased abundance of nidogen-2 (NID2) in the KPC model compared to KPflC, with further validation showing that NID2 is primarily expressed by cancer-associated fibroblasts (CAFs). Using biomechanical assessments, second harmonic generation imaging, and birefringence analysis, we show that NID2 reduction by CRISPR interference (CRISPRi) in CAFs reduces stiffness and matrix remodeling in three-dimensional models, leading to impaired cancer cell invasion. Intravital imaging revealed improved vascular patency in live NID2-depleted tumors, with enhanced response to gemcitabine/Abraxane. In orthotopic models, NID2 CRISPRi tumors had less liver metastasis and increased survival, highlighting NID2 as a potential PDAC cotarget.
Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Proteômica , Animais , Humanos , Camundongos , Proteínas de Ligação ao Cálcio/metabolismo , Proteínas de Ligação ao Cálcio/genética , Fibroblastos Associados a Câncer/metabolismo , Fibroblastos Associados a Câncer/patologia , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/genética , Moléculas de Adesão Celular , Linhagem Celular Tumoral , Desoxicitidina/análogos & derivados , Desoxicitidina/farmacologia , Modelos Animais de Doenças , Fibrose , Gencitabina , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/genética , Proteômica/métodosRESUMO
The basement membrane (BM) around tumor lobes forms a barrier to prevent cancer cells from invading the surrounding tissue. Although myoepithelial cells are key producers of the healthy mammary epithelium BM, they are nearly absent in mammary tumors. To study the origin and dynamics of the BM, we developed and imaged a laminin beta1-Dendra2 mouse model. We show that the turnover of laminin beta1 is faster in the BMs that surround the tumor lobes than in the BMs that surround the healthy epithelium. Moreover, we find that epithelial cancer cells and tumor-infiltrating endothelial cells synthesize laminin beta1 and that this production is temporarily and locally heterogeneous, leading to local discontinuity of the BM laminin beta1. Collectively, our data draw a new paradigm for tumor BM turnover in which the disassembly happens at a constant rate, and a local misbalance of compensating production leads to reduction or even complete disappearance of the BM.
Assuntos
Neoplasias da Mama , Laminina , Animais , Feminino , Humanos , Camundongos , Membrana Basal , Neoplasias da Mama/patologia , Células Endoteliais , Células Epiteliais , Modelos Animais de DoençasRESUMO
Although initially successful, treatments with chemotherapy often fail because of the recurrence of chemoresistant metastases. Since these tumors develop after treatment, resistance is generally thought to occur in response to chemotherapy. However, alternative mechanisms of intrinsic chemoresistance in the chemotherapy-naïve setting may exist but remain poorly understood. Here, we study drug-naïve murine breast cancer brain metastases (BCBMs) to identify how cancer cells growing in a secondary site can acquire intrinsic chemoresistance without cytotoxic agent exposure. We demonstrate that drug-naïve murine breast cancer cells that form cancer lesions in the brain undergo vascular mimicry and concomitantly express the adenosine 5'-triphosphate-binding cassette transporter breast cancer resistance protein (BCRP), a common marker of brain endothelial cells. We reveal that expression of BCRP by the BCBM tumor cells protects them against doxorubicin and topotecan. We conclude that BCRP overexpression can cause intrinsic chemoresistance in cancer cells growing in metastatic sites without prior chemotherapy exposure.
Assuntos
Antineoplásicos , Neoplasias Encefálicas , Neoplasias da Mama , Animais , Feminino , Camundongos , Antineoplásicos/farmacologia , Antineoplásicos/uso terapêutico , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Resistencia a Medicamentos Antineoplásicos/genética , Células Endoteliais/metabolismo , Proteínas de Neoplasias/metabolismoRESUMO
Although treatment with taxanes does not always lead to clinical benefit, all patients are at risk of their detrimental side effects such as peripheral neuropathy. Understanding the in vivo mode of action of taxanes can help design improved treatment regimens. Here, we demonstrate that in vivo, taxanes directly trigger T cells to selectively kill cancer cells in a non-canonical, T cell receptor-independent manner. Mechanistically, taxanes induce T cells to release cytotoxic extracellular vesicles, which lead to apoptosis specifically in tumor cells while leaving healthy epithelial cells intact. We exploit these findings to develop an effective therapeutic approach, based on transfer of T cells pre-treated with taxanes ex vivo, thereby avoiding toxicity of systemic treatment. Our study reveals a different in vivo mode of action of one of the most commonly used chemotherapies, and opens avenues to harness T cell-dependent anti-tumor effects of taxanes while avoiding systemic toxicity.
Assuntos
Vesículas Extracelulares , Neoplasias , Humanos , Linfócitos T , Taxoides/farmacologia , Apoptose , Células Epiteliais , Neoplasias/tratamento farmacológicoRESUMO
The lysyl oxidase family represents a promising target in stromal targeting of solid tumors due to the importance of this family in crosslinking and stabilizing fibrillar collagens and its known role in tumor desmoplasia. Using small-molecule drug-design approaches, we generated and validated PXS-5505, a first-in-class highly selective and potent pan-lysyl oxidase inhibitor. We demonstrate in vitro and in vivo that pan-lysyl oxidase inhibition decreases chemotherapy-induced pancreatic tumor desmoplasia and stiffness, reduces cancer cell invasion and metastasis, improves tumor perfusion and enhances the efficacy of chemotherapy in the autochthonous genetically engineered KPC model, while also demonstrating antifibrotic effects in human patient-derived xenograft models of pancreatic cancer. PXS-5505 is orally bioavailable, safe and effective at inhibiting lysyl oxidase activity in tissues. Our findings present the rationale for progression of a pan-lysyl oxidase inhibitor aimed at eliciting a reduction in stromal matrix to potentiate chemotherapy in pancreatic ductal adenocarcinoma.
Assuntos
Pancreatopatias , Neoplasias Pancreáticas , Humanos , Gencitabina , Proteína-Lisina 6-Oxidase , Neoplasias Pancreáticas/tratamento farmacológicoRESUMO
Glioma is the most common form of malignant primary brain tumours in adults. Their highly invasive nature makes the disease incurable to date, emphasizing the importance of better understanding the mechanisms driving glioma invasion. Glial fibrillary acidic protein (GFAP) is an intermediate filament protein that is characteristic for astrocyte- and neural stem cell-derived gliomas. Glioma malignancy is associated with changes in GFAP alternative splicing, as the canonical isoform GFAPα is downregulated in higher-grade tumours, leading to increased dominance of the GFAPδ isoform in the network. In this study, we used intravital imaging and an ex vivo brain slice invasion model. We show that the GFAPδ and GFAPα isoforms differentially regulate the tumour dynamics of glioma cells. Depletion of either isoform increases the migratory capacity of glioma cells. Remarkably, GFAPδ-depleted cells migrate randomly through the brain tissue, whereas GFAPα-depleted cells show a directionally persistent invasion into the brain parenchyma. This study shows that distinct compositions of the GFAPnetwork lead to specific migratory dynamics and behaviours of gliomas.
Assuntos
Neoplasias Encefálicas/patologia , Encéfalo/patologia , Movimento Celular , Proteína Glial Fibrilar Ácida/metabolismo , Glioma/patologia , Animais , Neoplasias Encefálicas/metabolismo , Linhagem Celular Tumoral , Feminino , Glioma/metabolismo , Microscopia Intravital , Masculino , Camundongos Endogâmicos C57BL , Invasividade Neoplásica , Isoformas de ProteínasRESUMO
(1) Background: an increasing number of breast cancer patients develop lethal brain metastases (BM). The complete removal of these tumors by surgery becomes complicated when cells infiltrate into the brain parenchyma. However, little is known about the nature of these invading cells in breast cancer brain metastasis (BCBM). (2) Methods: we use intravital microscopy through a cranial window to study the behavior of invading cells in a mouse model of BCBM. (3) Results: we demonstrate that BCBM cells that escape from the metastatic mass and infiltrate into brain parenchyma undergo epithelial-to-mesenchymal transition (EMT). Moreover, cells undergoing EMT revert to an epithelial state when growing tumor masses in the brain. Lastly, through multiplex immunohistochemistry, we confirm the presence of these infiltrative cells in EMT in patient samples. (4) Conclusions: together, our data identify the critical role of EMT in the invasive behavior of BCBM, which warrants further consideration to target those cells when treating BCBM.
RESUMO
An increasing number of breast cancer patients develop brain metastases (BM). Standard-of-care treatments are largely inefficient, and breast cancer brain metastasis (BCBM) patients are considered untreatable. Immunotherapies are not successfully employed in BCBM, in part because breast cancer is a "cold" tumor and also because the brain tissue has a unique immune landscape. Here, we generate and characterize immunocompetent models of BCBM derived from PyMT and Neu mammary tumors to test how harnessing the pro-senescence properties of doxorubicin can be used to prime the specific immune BCBM microenvironment. We reveal that BCBM senescent cells, induced by doxorubicin, trigger the recruitment of PD1-expressing T cells to the brain. Importantly, we demonstrate that induction of senescence with doxorubicin improves the efficacy of immunotherapy with anti-PD1 in BCBM in a CD8 T cell-dependent manner, thereby providing an optimized strategy to introduce immune-based treatments in this lethal disease. In addition, our BCBM models can be used for pre-clinical testing of other therapeutic strategies in the future.
Assuntos
Neoplasias Encefálicas , Neoplasias da Mama , Humanos , Feminino , Neoplasias da Mama/tratamento farmacológico , Neoplasias Encefálicas/tratamento farmacológico , Doxorrubicina/farmacologia , Imunoterapia , Microambiente TumoralRESUMO
PURPOSE: Extensive work in preclinical models has shown that microenvironmental cells influence many aspects of cancer cell behavior, including metastatic potential and their sensitivity to therapeutics. In the human setting, this behavior is mainly correlated with the presence of immune cells. Here, in addition to T cells, B cells, macrophages, and mast cells, we identified the relevance of nonimmune cell types for breast cancer survival and therapy benefit, including fibroblasts, myoepithelial cells, muscle cells, endothelial cells, and seven distinct epithelial cell types. EXPERIMENTAL DESIGN: Using single-cell sequencing data, we generated reference profiles for all these cell types. We used these reference profiles in deconvolution algorithms to optimally detangle the cellular composition of more than 3,500 primary breast tumors of patients that were enrolled in the SCAN-B and MATADOR clinical trials, and for which bulk mRNA sequencing data were available. RESULTS: This large data set enables us to identify and subsequently validate the cellular composition of microenvironments that distinguish differential survival and treatment benefit for different treatment regimens in patients with primary breast cancer. In addition to immune cells, we have identified that survival and therapy benefit are characterized by various contributions of distinct epithelial cell types. CONCLUSIONS: From our study, we conclude that differential survival and therapy benefit of patients with breast cancer are characterized by distinct microenvironments that include specific populations of immune and epithelial cells.
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Neoplasias da Mama , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/terapia , Microambiente Celular , Células Endoteliais/patologia , Feminino , Humanos , Microambiente Tumoral/genéticaRESUMO
Double strand breaks (DSBs) are highly toxic to a cell, a property that is exploited in radiation therapy. A critical component for the damage induction is cellular oxygen, making hypoxic tumor areas refractory to the efficacy of radiation treatment. During a fractionated radiation regimen, these hypoxic areas can be re-oxygenated. Nonetheless, hypoxia still constitutes a negative prognostic factor for the patient's outcome. We hypothesized that this might be attributed to specific hypoxia-induced cellular traits that are maintained upon reoxygenation. Here, we show that reoxygenation of hypoxic non-transformed RPE-1 cells fully restored induction of DSBs but the cells remain radioresistant as a consequence of hypoxia-induced quiescence. With the use of the cell cycle indicators (FUCCI), cell cycle-specific radiation sensitivity, the cell cycle phase duration with live cell imaging, and single cell tracing were assessed. We observed that RPE-1 cells experience a longer G1 phase under hypoxia and retain a large fraction of cells that are non-cycling. Expression of HPV oncoprotein E7 prevents hypoxia-induced quiescence and abolishes the radioprotective effect. In line with this, HPV-negative cancer cell lines retain radioresistance, while HPV-positive cancer cell lines are radiosensitized upon reoxygenation. Quiescence induction in hypoxia and its HPV-driven prevention was observed in 3D multicellular spheroids. Collectively, we identify a new hypoxia-dependent radioprotective phenotype due to hypoxia-induced quiescence that accounts for a global decrease in radiosensitivity that can be retained upon reoxygenation and is absent in cells expressing oncoprotein E7.
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Hipóxia Celular/fisiologia , Radiação Ionizante , Linhagem Celular Tumoral , HumanosRESUMO
The concept that extracellular vesicles (EVs) from the diet can be absorbed by the intestinal tract of the consuming organism, be bioavailable in various organs, and in-turn exert phenotypic changes is highly debatable. Here, we isolate EVs from both raw and commercial bovine milk and characterize them by electron microscopy, nanoparticle tracking analysis, western blotting, quantitative proteomics and small RNA sequencing analysis. Orally administered bovine milk-derived EVs survive the harsh degrading conditions of the gut, in mice, and is subsequently detected in multiple organs. Milk-derived EVs orally administered to mice implanted with colorectal and breast cancer cells reduce the primary tumor burden. Intriguingly, despite the reduction in primary tumor growth, milk-derived EVs accelerate metastasis in breast and pancreatic cancer mouse models. Proteomic and biochemical analysis reveal the induction of senescence and epithelial-to-mesenchymal transition in cancer cells upon treatment with milk-derived EVs. Timing of EV administration is critical as oral administration after resection of the primary tumor reverses the pro-metastatic effects of milk-derived EVs in breast cancer models. Taken together, our study provides context-based and opposing roles of milk-derived EVs as metastasis inducers and suppressors.
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Vesículas Extracelulares , Leite/citologia , Neoplasias Experimentais/patologia , Administração Oral , Animais , Disponibilidade Biológica , Neoplasias da Mama/patologia , Neoplasias da Mama/terapia , Bovinos , Linhagem Celular Tumoral , Proliferação de Células , Transição Epitelial-Mesenquimal , Vesículas Extracelulares/química , Vesículas Extracelulares/genética , Feminino , Humanos , Neoplasias Hepáticas Experimentais/patologia , Neoplasias Hepáticas Experimentais/secundário , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Camundongos Endogâmicos BALB C , Neoplasias Experimentais/terapia , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/terapia , Distribuição Tecidual , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is a highly metastatic, chemoresistant malignancy and is characterized by a dense, desmoplastic stroma that modulates PDAC progression. Here, we visualized transient manipulation of focal adhesion kinase (FAK), which integrates bidirectional cell-environment signaling, using intravital fluorescence lifetime imaging microscopy of the FAK-based Förster resonance energy transfer biosensor in mouse and patient-derived PDAC models. Parallel real-time quantification of the FUCCI cell cycle reporter guided us to improve PDAC response to standard-of-care chemotherapy at primary and secondary sites. Critically, micropatterned pillar plates and stiffness-tunable matrices were used to pinpoint the contribution of environmental cues to chemosensitization, while fluid flowinduced shear stress assessment, patient-derived matrices, and personalized in vivo models allowed us to deconstruct how FAK inhibition can reduce PDAC spread. Last, stratification of PDAC patient samples via Merlin status revealed a patient subset with poor prognosis that are likely to respond to FAK priming before chemotherapy.
RESUMO
Metastasis is a highly dynamic process during which cancer and microenvironmental cells undergo a cascade of events required for efficient dissemination throughout the body. During the metastatic cascade, tumor cells can change their state and behavior, a phenomenon commonly defined as cellular plasticity. To monitor cellular plasticity during metastasis, high-resolution intravital microscopy (IVM) techniques have been developed and allow us to visualize individual cells by repeated imaging in animal models. In this review, we summarize the latest technological advancements in the field of IVM and how they have been applied to monitor metastatic events. In particular, we highlight how longitudinal imaging in native tissues can provide new insights into the plastic physiological and developmental processes that are hijacked by cancer cells during metastasis.
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Plasticidade Celular , Microscopia Intravital/métodos , Metástase Neoplásica/patologia , Animais , Humanos , Neoplasias/diagnóstico por imagem , Microambiente TumoralRESUMO
Introduction: Pancreatic cancer (PC) is responsible for significant worldwide cancer-associated mortality and has one of the lowest five-year survival rate post-diagnosis of all epithelial cancers. A major contributor to this dismal outcome is the extensive stromal reaction that occurs during PC progression. As such, targeting key components of the pancreatic tumor stroma in combination with standard-of-care chemotherapy has been a recent focus in both the pre-clinical and clinical settings.Areas Covered: In this commentary, we highlight how perlecan was identified as a new potential target for this disease.Expert Opinion: Perlecan is deposited by cancer-associated fibroblasts (CAFs) in the pancreatic tumor stroma, and work from our laboratory group recently demonstrated that depleting perlecan reduces metastatic spread, while also improving chemotherapy efficacy in pancreatic tumors harboring a gain-of-function p53 mutation. We also discuss potential strategies to therapeutically target perlecan which could be tested in pre-clinical models prior to translation into the clinic.
Assuntos
Fibroblastos Associados a Câncer , Neoplasias Pancreáticas , Proteoglicanas de Heparan Sulfato , Humanos , Células EstromaisRESUMO
Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease; the identification of novel targets and development of effective treatment strategies are urgently needed to improve patient outcomes. Remodeling of the pancreatic stroma occurs during PDAC development, which drives disease progression and impairs responses to therapy. The actomyosin regulatory ROCK1 and ROCK2 kinases govern cell motility and contractility, and have been suggested to be potential targets for cancer therapy, particularly to reduce the metastatic spread of tumor cells. However, ROCK inhibitors are not currently used for cancer patient treatment, largely due to the overwhelming challenge faced in the development of anti-metastatic drugs, and a lack of clarity as to the cancer types most likely to benefit from ROCK inhibitor therapy. In 2 recent publications, we discovered that ROCK1 and ROCK2 expression were increased in PDAC, and that increased ROCK activity was associated with reduced survival and PDAC progression by enabling extracellular matrix (ECM) remodeling and invasive growth of pancreatic cancer cells. We also used intravital imaging to optimize ROCK inhibition using the pharmacological ROCK inhibitor fasudil (HA-1077), and demonstrated that short-term ROCK targeting, or 'priming', improved chemotherapy efficacy, disrupted cancer cell collective movement, and impaired metastasis. This body of work strongly indicates that the use of ROCK inhibitors in pancreatic cancer therapy as 'priming' agents warrants further consideration, and provides insights as to how transient mechanical manipulation, or fine-tuning the ECM, rather than chronic stromal ablation might be beneficial for improving chemotherapeutic efficacy in the treatment of this deadly disease.
Assuntos
Terapia de Alvo Molecular/métodos , Neoplasias Pancreáticas/tratamento farmacológico , Quinases Associadas a rho/metabolismo , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/uso terapêutico , Animais , Progressão da Doença , Humanos , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/metabolismo , Quinases Associadas a rho/antagonistas & inibidoresRESUMO
BACKGROUND: The use of in vitro cell cultures is a powerful tool for obtaining key insights into the behaviour and response of cells to interventions in normal and disease situations. Unlike in vivo settings, in vitro experiments allow a fine-tuned control of a range of microenvironmental elements independently within an isolated setting. The recent expansion in the use of three-dimensional (3D) in vitro assays has created a number of representative tools to study cell behaviour in a more physiologically 3D relevant microenvironment. Complex 3D in vitro models that can recapitulate human tissue biology are essential for understanding the pathophysiology of disease. AIM: The development of the 3D coculture collagen contraction and invasion assay, the "organotypic assay," has been widely adopted as a powerful approach to bridge the gap between standard two-dimensional tissue culture and in vivo mouse models. In the cancer setting, these assays can then be used to dissect how stromal cells, such as cancer-associated fibroblasts (CAFs), drive extracellular matrix (ECM) remodelling to alter cancer cell behaviour and response to intervention. However, to date, many of the published organotypic protocols are low-throughput, time-consuming (up to several weeks), and work-intensive with often limited scalability. Our aim was to develop a fast, high-throughput, scalable 3D organotypic assay for use in oncology screening and drug development. METHODS AND RESULTS: Here, we describe a modified 96-well organotypic assay, the "Mini-Organo," which can be easily completed within 5 days. We demonstrate its application in a wide range of mouse and human cancer biology approaches including evaluation of stromal cell 3D ECM remodelling, 3D cancer cell invasion, and the assessment of efficacy of potential anticancer therapeutic targets. Furthermore, the organotypic assay described is highly amenable to customisation using different cell types under diverse experimental conditions. CONCLUSIONS: The Mini-Organo high-throughput 3D organotypic assay allows the rapid screening of potential cancer therapeutics in human and mouse models in a time-efficient manner.