ABSTRACT
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.
Subject(s)
Carcinoma, Pancreatic Ductal , Pancreatic Neoplasms , Proteomics , Animals , Humans , Mice , Calcium-Binding Proteins/metabolism , Calcium-Binding Proteins/genetics , Cancer-Associated Fibroblasts/metabolism , Cancer-Associated Fibroblasts/pathology , Carcinoma, Pancreatic Ductal/metabolism , Carcinoma, Pancreatic Ductal/pathology , Carcinoma, Pancreatic Ductal/genetics , Cell Adhesion Molecules , Cell Line, Tumor , Deoxycytidine/analogs & derivatives , Deoxycytidine/pharmacology , Disease Models, Animal , Fibrosis , Gemcitabine , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/genetics , Proteomics/methodsABSTRACT
Aberrant AKT activation occurs in a number of cancers, metabolic syndrome, and immune disorders, making it an important target for the treatment of many diseases. To monitor spatial and temporal AKT activity in a live setting, we generated an Akt-FRET biosensor mouse that allows longitudinal assessment of AKT activity using intravital imaging in conjunction with image stabilization and optical window technology. We demonstrate the sensitivity of the Akt-FRET biosensor mouse using various cancer models and verify its suitability to monitor response to drug targeting in spheroid and organotypic models. We also show that the dynamics of AKT activation can be monitored in real time in diverse tissues, including in individual islets of the pancreas, in the brown and white adipose tissue, and in the skeletal muscle. Thus, the Akt-FRET biosensor mouse provides an important tool to study AKT dynamics in live tissue contexts and has broad preclinical applications.
Subject(s)
Biosensing Techniques , Proto-Oncogene Proteins c-akt , Mice , Animals , Proto-Oncogene Proteins c-akt/metabolism , Fluorescence Resonance Energy Transfer/methods , Biosensing Techniques/methodsABSTRACT
The extracellular matrix (ECM) is a non-cellular network of macromolecules, which provides cells and tissues with structural support and biomechanical feedback to regulate cellular function, tissue tension, and homeostasis. Even subtle changes to ECM abundance, architecture, and organization can affect downstream biological pathways, thereby influencing normal cell and tissue function and also driving disease conditions. For example, in cancer, the ECM is well known to provide both biophysical and biochemical cues that influence cancer initiation, progression, and metastasis, highlighting the need to better understand cell-ECM interactions in cancer and other ECM-enriched diseases. Initial cell-derived matrix (CDM) models were used as an in vitro system to mimic and assess the physiologically relevant three-dimensional (3D) cell-ECM interactions. Here, we describe an expansion to these initial CDM models generated by fibroblasts to assess the effect of genetic or pharmacological intervention on fibroblast-mediated matrix production and organization. Additionally, we highlight current methodologies to quantify changes in the ultrastructure and isotropy of the resulting ECM and also provide protocols for assessing cancer cell interaction with CDMs. Understanding the nature and influence of these complex and heterogeneous processes can offer insights into the biomechanical and biochemical mechanisms, which drive cancer development and metastasis, and how we can target them to improve cancer outcomes. This protocol was validated in: Sci Adv (2021), DOI: 10.1126/sciadv.abh0363.
ABSTRACT
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.
ABSTRACT
Basal breast cancer is associated with younger age, early relapse, and a high mortality rate. Here, we use unbiased droplet-based single-cell RNA sequencing (RNA-seq) to elucidate the cellular basis of tumor progression during the specification of the basal breast cancer subtype from the luminal progenitor population in the MMTV-PyMT (mouse mammary tumor virus-polyoma middle tumor-antigen) mammary tumor model. We find that basal-like cancer cells resemble the alveolar lineage that is specified upon pregnancy and encompass the acquisition of an aberrant post-lactation developmental program of involution that triggers remodeling of the tumor microenvironment and metastatic dissemination. This involution mimicry is characterized by a highly interactive multicellular network, with involution cancer-associated fibroblasts playing a pivotal role in extracellular matrix remodeling and immunosuppression. Our results may partially explain the increased risk and poor prognosis of breast cancer associated with childbirth.
Subject(s)
Cancer-Associated Fibroblasts/metabolism , Carcinoma, Basal Cell/genetics , Mammary Glands, Animal/metabolism , Mammary Neoplasms, Animal/genetics , Transcriptome , Animals , Breast Neoplasms/genetics , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cancer-Associated Fibroblasts/pathology , Carcinoma, Basal Cell/metabolism , Carcinoma, Basal Cell/pathology , Cell Lineage/genetics , Chemokine CXCL12/genetics , Chemokine CXCL12/metabolism , Collagen Type I, alpha 1 Chain/genetics , Collagen Type I, alpha 1 Chain/metabolism , Extracellular Matrix/metabolism , Extracellular Matrix/pathology , Female , Gene Expression Regulation, Neoplastic , High-Throughput Nucleotide Sequencing , Humans , Mammary Glands, Animal/pathology , Mammary Glands, Animal/virology , Mammary Neoplasms, Animal/metabolism , Mammary Neoplasms, Animal/pathology , Mammary Tumor Virus, Mouse/growth & development , Mammary Tumor Virus, Mouse/pathogenicity , Matrix Metalloproteinase 3/genetics , Matrix Metalloproteinase 3/metabolism , Mice , Neoplasm Metastasis , Pregnancy , Single-Cell Analysis , Tumor Microenvironment/geneticsABSTRACT
Whereas genomic aberrations in the SLIT-ROBO pathway are frequent in pancreatic ductal adenocarcinoma (PDAC), their function in the pancreas is unclear. Here we report that in pancreatitis and PDAC mouse models, epithelial Robo2 expression is lost while Robo1 expression becomes most prominent in the stroma. Cell cultures of mice with loss of epithelial Robo2 (Pdx1Cre;Robo2F/F) show increased activation of Robo1+ myofibroblasts and induction of TGF-ß and Wnt pathways. During pancreatitis, Pdx1Cre;Robo2F/F mice present enhanced myofibroblast activation, collagen crosslinking, T-cell infiltration and tumorigenic immune markers. The TGF-ß inhibitor galunisertib suppresses these effects. In PDAC patients, ROBO2 expression is overall low while ROBO1 is variably expressed in epithelium and high in stroma. ROBO2low;ROBO1high patients present the poorest survival. In conclusion, Robo2 acts non-autonomously as a stroma suppressor gene by restraining myofibroblast activation and T-cell infiltration. ROBO1/2 expression in PDAC patients may guide therapy with TGF-ß inhibitors or other stroma /immune modulating agents.
Subject(s)
Pancreas/metabolism , Pancreas/pathology , Receptors, Immunologic/metabolism , Transforming Growth Factor beta/metabolism , Animals , Blotting, Western , Carcinoma, Pancreatic Ductal/genetics , Carcinoma, Pancreatic Ductal/metabolism , Cells, Cultured , Female , Flow Cytometry , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , In Situ Hybridization , In Vitro Techniques , Male , Mice , Nerve Tissue Proteins/genetics , Nerve Tissue Proteins/metabolism , Pancreatitis/genetics , Pancreatitis/metabolism , Receptors, Immunologic/genetics , Signal Transduction/genetics , Signal Transduction/physiology , Trans-Activators/genetics , Trans-Activators/metabolism , Roundabout ProteinsABSTRACT
Intravital microscopy can provide unique insights into the function of biological processes in a native context. However, physiological motion caused by peristalsis, respiration and the heartbeat can present a significant challenge, particularly for functional readouts such as fluorescence lifetime imaging (FLIM), which require longer acquisition times to obtain a quantitative readout. Here, we present and benchmark Galene, a versatile multi-platform software tool for image-based correction of sample motion blurring in both time resolved and conventional laser scanning fluorescence microscopy data in two and three dimensions. We show that Galene is able to resolve intravital FLIM-FRET images of intra-abdominal organs in murine models and NADH autofluorescence of human dermal tissue imaging subject to a wide range of physiological motions. Thus, Galene can enable FLIM imaging in situations where a stable imaging platform is not always possible and rescue previously discarded quantitative imaging data.
Subject(s)
Imaging, Three-Dimensional , Intravital Microscopy , Motion , Algorithms , Animals , Biosensing Techniques , Cell Adhesion , Computer Simulation , Fluorescence Resonance Energy Transfer , Guanosine Triphosphate/metabolism , Humans , Intestines/physiology , Mice , Microscopy, Fluorescence , Models, Biological , Neoplasm Metastasis , Neuropeptides/metabolism , Pancreatic Neoplasms/pathology , Skin/anatomy & histology , Software , rac1 GTP-Binding Protein/metabolism , src-Family Kinases/metabolismABSTRACT
Application of advanced intravital imaging facilitates dynamic monitoring of pathway activity upon therapeutic inhibition. Here, we assess resistance to therapeutic inhibition of the PI3K pathway within the hypoxic microenvironment of pancreatic ductal adenocarcinoma (PDAC) and identify a phenomenon whereby pronounced hypoxia-induced resistance is observed for three clinically relevant inhibitors. To address this clinical problem, we have mapped tumor hypoxia by both immunofluorescence and phosphorescence lifetime imaging of oxygen-sensitive nanoparticles and demonstrate that these hypoxic regions move transiently around the tumor. To overlay this microenvironmental information with drug response, we applied a FRET biosensor for Akt activity, which is a key effector of the PI3K pathway. Performing dual intravital imaging of drug response in different tumor compartments, we demonstrate an improved drug response to a combination therapy using the dual mTORC1/2 inhibitor AZD2014 with the hypoxia-activated pro-drug TH-302.
Subject(s)
Drug Resistance, Neoplasm , Phosphatidylinositol 3-Kinases/metabolism , Signal Transduction , Animals , Benzamides , Cell Line, Tumor , Drug Resistance, Neoplasm/drug effects , Drug Therapy, Combination , Female , Fluorescence Resonance Energy Transfer , Humans , Hypoxia , Intravital Microscopy/methods , Mice , Mice, Inbred BALB C , Morpholines/pharmacology , Morpholines/therapeutic use , Nanoparticles/chemistry , Nitroimidazoles/pharmacology , Nitroimidazoles/therapeutic use , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Phosphoramide Mustards/pharmacology , Phosphoramide Mustards/therapeutic use , Proto-Oncogene Proteins c-akt/metabolism , Pyrimidines , Signal Transduction/drug effects , Transplantation, Heterologous , Tumor MicroenvironmentABSTRACT
The small GTPase RhoA is involved in a variety of fundamental processes in normal tissue. Spatiotemporal control of RhoA is thought to govern mechanosensing, growth, and motility of cells, while its deregulation is associated with disease development. Here, we describe the generation of a RhoA-fluorescence resonance energy transfer (FRET) biosensor mouse and its utility for monitoring real-time activity of RhoA in a variety of native tissues in vivo. We assess changes in RhoA activity during mechanosensing of osteocytes within the bone and during neutrophil migration. We also demonstrate spatiotemporal order of RhoA activity within crypt cells of the small intestine and during different stages of mammary gestation. Subsequently, we reveal co-option of RhoA activity in both invasive breast and pancreatic cancers, and we assess drug targeting in these disease settings, illustrating the potential for utilizing this mouse to study RhoA activity in vivo in real time.
Subject(s)
Biosensing Techniques , Fluorescence Resonance Energy Transfer/methods , Intravital Microscopy/methods , Time-Lapse Imaging/methods , rho GTP-Binding Proteins/genetics , Animals , Antineoplastic Agents/pharmacology , Bone and Bones/cytology , Bone and Bones/metabolism , Cell Movement/drug effects , Dasatinib/pharmacology , Erlotinib Hydrochloride/pharmacology , Female , Fluorescence Resonance Energy Transfer/instrumentation , Gene Expression Regulation , Intestine, Small/metabolism , Intestine, Small/ultrastructure , Intravital Microscopy/instrumentation , Mammary Glands, Animal/blood supply , Mammary Glands, Animal/drug effects , Mammary Glands, Animal/ultrastructure , Mammary Neoplasms, Experimental/blood supply , Mammary Neoplasms, Experimental/drug therapy , Mammary Neoplasms, Experimental/genetics , Mammary Neoplasms, Experimental/ultrastructure , Mechanotransduction, Cellular , Mice , Mice, Transgenic , Neutrophils/metabolism , Neutrophils/ultrastructure , Osteocytes/metabolism , Osteocytes/ultrastructure , Pancreatic Neoplasms/blood supply , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/genetics , Pancreatic Neoplasms/ultrastructure , Time-Lapse Imaging/instrumentation , rho GTP-Binding Proteins/metabolism , rhoA GTP-Binding ProteinABSTRACT
The emerging standard of care for patients with inoperable pancreatic cancer is a combination of cytotoxic drugs gemcitabine and Abraxane, but patient response remains moderate. Pancreatic cancer development and metastasis occur in complex settings, with reciprocal feedback from microenvironmental cues influencing both disease progression and drug response. Little is known about how sequential dual targeting of tumor tissue tension and vasculature before chemotherapy can affect tumor response. We used intravital imaging to assess how transient manipulation of the tumor tissue, or "priming," using the pharmaceutical Rho kinase inhibitor Fasudil affects response to chemotherapy. Intravital Förster resonance energy transfer imaging of a cyclin-dependent kinase 1 biosensor to monitor the efficacy of cytotoxic drugs revealed that priming improves pancreatic cancer response to gemcitabine/Abraxane at both primary and secondary sites. Transient priming also sensitized cells to shear stress and impaired colonization efficiency and fibrotic niche remodeling within the liver, three important features of cancer spread. Last, we demonstrate a graded response to priming in stratified patient-derived tumors, indicating that fine-tuned tissue manipulation before chemotherapy may offer opportunities in both primary and metastatic targeting of pancreatic cancer.
Subject(s)
Disease Progression , Pancreatic Neoplasms/drug therapy , Pancreatic Neoplasms/pathology , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , rho-Associated Kinases/antagonists & inhibitors , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/analogs & derivatives , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/pharmacology , 1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine/therapeutic use , Actin Cytoskeleton/drug effects , Actin Cytoskeleton/metabolism , Albumin-Bound Paclitaxel/pharmacology , Albumin-Bound Paclitaxel/therapeutic use , Animals , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Biosensing Techniques , CDC2 Protein Kinase/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Collagen/metabolism , Deoxycytidine/analogs & derivatives , Deoxycytidine/pharmacology , Deoxycytidine/therapeutic use , Extracellular Matrix/metabolism , Humans , Liver/pathology , Mice , Neoplasm Invasiveness , Neoplasm Metastasis , Signal Transduction/drug effects , Treatment Outcome , rho-Associated Kinases/metabolism , src-Family Kinases/metabolism , GemcitabineABSTRACT
The mechanisms protecting from immunopathology during acute bacterial infections are incompletely known. We found that in response to apoptotic immune cells and live or dead Listeria monocytogenes scavenger receptor BI (SR-BI), an anti-atherogenic lipid exchange mediator, activated internalization mechanisms with characteristics of macropinocytosis and, assisted by Golgi fragmentation, initiated autophagic responses. This was supported by scavenger receptor-induced local increases in membrane cholesterol concentrations which generated lipid domains particularly in cell extensions and the Golgi. SR-BI was a key driver of beclin-1-dependent autophagy during acute bacterial infection of the liver and spleen. Autophagy regulated tissue infiltration of neutrophils, suppressed accumulation of Ly6C+ (inflammatory) macrophages, and prevented hepatocyte necrosis in the core of infectious foci. Perifocal levels of Ly6C+ macrophages and Ly6C- macrophages were unaffected, indicating predominant regulation of the focus core. SR-BI-triggered autophagy promoted co-elimination of apoptotic immune cells and dead bacteria but barely influenced bacterial sequestration and survival or inflammasome activation, thus exclusively counteracting damage inflicted by immune responses. Hence, SR-BI- and autophagy promote a surveillance pathway that partially responds to products of antimicrobial defenses and selectively prevents immunity-induced damage during acute infection. Our findings suggest that control of infection-associated immunopathology can be based on a unified defense operation.
Subject(s)
Autophagy/immunology , Macrophages/immunology , Membrane Microdomains/immunology , Pinocytosis/immunology , Scavenger Receptors, Class B/immunology , Animals , Autophagy/genetics , Beclin-1/genetics , Beclin-1/immunology , Golgi Apparatus/genetics , Golgi Apparatus/immunology , Listeria monocytogenes/immunology , Listeriosis/genetics , Listeriosis/immunology , Listeriosis/pathology , Liver Diseases/genetics , Liver Diseases/immunology , Liver Diseases/pathology , Macrophages/pathology , Membrane Microdomains/genetics , Mice , Mice, Knockout , Neutrophil Infiltration/genetics , Neutrophil Infiltration/immunology , Neutrophils/immunology , Neutrophils/pathology , Pinocytosis/genetics , Scavenger Receptors, Class B/genetics , Splenic Diseases/genetics , Splenic Diseases/immunology , Splenic Diseases/pathologyABSTRACT
Endothelial cells respond to a large range of stimuli including circulating lipoproteins, growth factors and changes in haemodynamic mechanical forces to regulate the activity of endothelial nitric oxide synthase (eNOS) and maintain blood pressure. While many signalling pathways have been mapped, the identities of membrane domains through which these signals are transmitted are less well characterized. Here, we manipulated bovine aortic endothelial cells (BAEC) with cholesterol and the oxysterol 7-ketocholesterol (7KC). Using a range of microscopy techniques including confocal, 2-photon, super-resolution and electron microscopy, we found that sterol enrichment had differential effects on eNOS and caveolin-1 (Cav1) colocalisation, membrane order of the plasma membrane, caveolae numbers and Cav1 clustering. We found a correlation between cholesterol-induced condensation of the plasma membrane and enhanced high density lipoprotein (HDL)-induced eNOS activity and phosphorylation suggesting that cholesterol domains, but not individual caveolae, mediate HDL stimulation of eNOS. Vascular endothelial growth factor (VEGF)-induced and shear stress-induced eNOS activity was relatively independent of membrane order and may be predominantly controlled by the number of caveolae on the cell surface. Taken together, our data suggest that signals that activate and phosphorylate eNOS are transmitted through distinct membrane domains in endothelial cells.
Subject(s)
Endothelial Cells/metabolism , Membrane Microdomains/physiology , Nitric Oxide Synthase Type III/metabolism , Animals , Aorta/cytology , Cattle , Caveolae/physiology , Caveolin 1/analysis , Cell Line , Cholesterol/pharmacology , Chromatography, High Pressure Liquid , Endothelial Cells/drug effects , Endothelial Cells/ultrastructure , Enzyme Activation/drug effects , Enzyme Activation/physiology , Humans , Image Processing, Computer-Assisted , Ketocholesterols/pharmacology , Lipoproteins, HDL/pharmacology , Membrane Microdomains/drug effects , Membrane Microdomains/ultrastructure , Microscopy, Electron , Rheology , Stress, Mechanical , Vascular Endothelial Growth Factor A/pharmacology , beta-Cyclodextrins/pharmacologyABSTRACT
E-cadherin-mediated cell-cell junctions play a prominent role in maintaining the epithelial architecture. The disruption or deregulation of these adhesions in cancer can lead to the collapse of tumor epithelia that precedes invasion and subsequent metastasis. Here we generated an E-cadherin-GFP mouse that enables intravital photobleaching and quantification of E-cadherin mobility in live tissue without affecting normal biology. We demonstrate the broad applications of this mouse by examining E-cadherin regulation in multiple tissues, including mammary, brain, liver, and kidney tissue, while specifically monitoring E-cadherin mobility during disease progression in the pancreas. We assess E-cadherin stability in native pancreatic tissue upon genetic manipulation involving Kras and p53 or in response to anti-invasive drug treatment and gain insights into the dynamic remodeling of E-cadherin during in situ cancer progression. FRAP in the E-cadherin-GFP mouse, therefore, promises to be a valuable tool to fundamentally expand our understanding of E-cadherin-mediated events in native microenvironments.
Subject(s)
Cadherins/metabolism , Green Fluorescent Proteins/metabolism , Neoplasms, Experimental/metabolism , Neoplasms, Experimental/pathology , Optical Imaging/methods , Tumor Microenvironment , Animals , Cadherins/genetics , Green Fluorescent Proteins/genetics , Mice , Mice, Transgenic , Neoplasms, Experimental/genetics , Organ Specificity , Proto-Oncogene Proteins p21(ras)/genetics , Proto-Oncogene Proteins p21(ras)/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolismABSTRACT
ROCK signaling causes epidermal hyper-proliferation by increasing ECM production, elevating dermal stiffness, and enhancing Fak-mediated mechano-transduction signaling. Elevated dermal stiffness in turn causes ROCK activation, establishing mechano-reciprocity, a positive feedback loop that can promote tumors. We have identified a negative feedback mechanism that limits excessive ROCK signaling during wound healing and is lost in squamous cell carcinomas (SCCs). Signal flux through ROCK was selectively tuned down by increased levels of 14-3-3ζ, which interacted with Mypt1, a ROCK signaling antagonist. In 14-3-3ζ(-/-) mice, unrestrained ROCK signaling at wound margins elevated ECM production and reduced ECM remodeling, increasing dermal stiffness and causing rapid wound healing. Conversely, 14-3-3ζ deficiency enhanced cutaneous SCC size. Significantly, inhibiting 14-3-3ζ with a novel pharmacological agent accelerated wound healing 2-fold. Patient samples of chronic non-healing wounds overexpressed 14-3-3ζ, while cutaneous SCCs had reduced 14-3-3ζ. These results reveal a novel 14-3-3ζ-dependent mechanism that negatively regulates mechano-reciprocity, suggesting new therapeutic opportunities.
Subject(s)
14-3-3 Proteins/metabolism , Cell Proliferation/physiology , Homeostasis/physiology , Signal Transduction/physiology , Wound Healing/physiology , rho-Associated Kinases/metabolism , Animals , Epidermis/metabolism , MiceABSTRACT
During pregnancy, the ETS transcription factor ELF5 establishes the milk-secreting alveolar cell lineage by driving a cell fate decision of the mammary luminal progenitor cell. In breast cancer, ELF5 is a key transcriptional determinant of tumor subtype and has been implicated in the development of insensitivity to anti-estrogen therapy. In the mouse mammary tumor virus-Polyoma Middle T (MMTV-PyMT) model of luminal breast cancer, induction of ELF5 levels increased leukocyte infiltration, angiogenesis, and blood vessel permeability in primary tumors and greatly increased the size and number of lung metastasis. Myeloid-derived suppressor cells, a group of immature neutrophils recently identified as mediators of vasculogenesis and metastasis, were recruited to the tumor in response to ELF5. Depletion of these cells using specific Ly6G antibodies prevented ELF5 from driving vasculogenesis and metastasis. Expression signatures in luminal A breast cancers indicated that increased myeloid cell invasion and inflammation were correlated with ELF5 expression, and increased ELF5 immunohistochemical staining predicted much shorter metastasis-free and overall survival of luminal A patients, defining a group who experienced unexpectedly early disease progression. Thus, in the MMTV-PyMT mouse mammary model, increased ELF5 levels drive metastasis by co-opting the innate immune system. As ELF5 has been previously implicated in the development of antiestrogen resistance, this finding implicates ELF5 as a defining factor in the acquisition of the key aspects of the lethal phenotype in luminal A breast cancer.
Subject(s)
Breast Neoplasms/metabolism , Lung Neoplasms/secondary , Lung/metabolism , Neoplasm Proteins/metabolism , Proto-Oncogene Proteins c-ets/metabolism , Animals , Breast Neoplasms/immunology , Breast Neoplasms/physiopathology , Breast Neoplasms/virology , Capillary Permeability , Cell Proliferation , DNA-Binding Proteins , Female , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , Hemorrhage/etiology , Hemorrhage/prevention & control , Humans , Leukocytes/immunology , Leukocytes/pathology , Lung/blood supply , Lung/immunology , Lung/pathology , Lung Neoplasms/blood supply , Lung Neoplasms/pathology , Lung Neoplasms/prevention & control , Lymphocyte Depletion , Mice, Transgenic , Myeloid Cells/immunology , Myeloid Cells/pathology , Neoplasm Proteins/genetics , Neovascularization, Pathologic/etiology , Neovascularization, Pathologic/prevention & control , Neutrophil Infiltration , Polyomavirus/pathogenicity , Proto-Oncogene Proteins c-ets/genetics , Recombinant Fusion Proteins/metabolism , Survival Analysis , Transcription Factors , Tumor BurdenABSTRACT
INTRODUCTION: Mammographic density (MD), after adjustment for a women's age and body mass index, is a strong and independent risk factor for breast cancer (BC). Although the BC risk attributable to increased MD is significant in healthy women, the biological basis of high mammographic density (HMD) causation and how it raises BC risk remain elusive. We assessed the histological and immunohistochemical differences between matched HMD and low mammographic density (LMD) breast tissues from healthy women to define which cell features may mediate the increased MD and MD-associated BC risk. METHODS: Tissues were obtained between 2008 and 2013 from 41 women undergoing prophylactic mastectomy because of their high BC risk profile. Tissue slices resected from the mastectomy specimens were X-rayed, then HMD and LMD regions were dissected based on radiological appearance. The histological composition, aromatase immunoreactivity, hormone receptor status and proliferation status were assessed, as were collagen amount and orientation, epithelial subsets and immune cell status. RESULTS: HMD tissue had a significantly greater proportion of stroma, collagen and epithelium, as well as less fat, than LMD tissue did. Second harmonic generation imaging demonstrated more organised stromal collagen in HMD tissues than in LMD tissues. There was significantly more aromatase immunoreactivity in both the stromal and glandular regions of HMD tissues than in those regions of LMD tissues, although no significant differences in levels of oestrogen receptor, progesterone receptor or Ki-67 expression were detected. The number of macrophages within the epithelium or stroma did not change; however, HMD stroma exhibited less CD206(+) alternatively activated macrophages. Epithelial cell maturation was not altered in HMD samples, and no evidence of epithelial-mesenchymal transition was seen; however, there was a significant increase in vimentin(+)/CD45(+) immune cells within the epithelial layer in HMD tissues. CONCLUSIONS: We confirmed increased proportions of stroma and epithelium, increased aromatase activity and no changes in hormone receptor or Ki-67 marker status in HMD tissue. The HMD region showed increased collagen deposition and organisation as well as decreased alternatively activated macrophages in the stroma. The HMD epithelium may be a site for local inflammation, as we observed a significant increase in CD45(+)/vimentin(+) immune cells in this area.
Subject(s)
Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Breast/metabolism , Collagen/metabolism , Epithelium/metabolism , Mammary Glands, Human/abnormalities , Stromal Cells/metabolism , Adult , Biomarkers, Tumor/metabolism , Breast/pathology , Breast Density , Breast Neoplasms/immunology , Epithelial-Mesenchymal Transition , Epithelium/pathology , Female , Humans , Immunohistochemistry , Immunophenotyping , Macrophages/immunology , Macrophages/metabolism , Macrophages/pathology , Mammography , Middle Aged , Phenotype , Risk FactorsABSTRACT
Compartmentalization is a functionally important property of the plasma membrane, yet the underlying principles that organize membrane proteins into distinct domains are not well understood. Using single molecule localization microscopy, we assessed the clustering of five model membrane proteins in the plasma membrane of HeLa cells. All five proteins formed discrete and distinct nano-scaled clusters. The extent of clustering of the five proteins, independent of their membrane anchors, increased significantly when the fluorescent protein mEOS2 was employed, suggesting that protein-protein interactions are a key driver for clustering. Further, actin depolymerization or reduction of membrane order had a greater, and in some instances opposing effects on the clustering of membrane proteins fused to mEOS2 compared to PS-CFP2-fusion proteins. The data propose that protein interactions can override the lateral organization imposed by membrane anchors to provide an exquisite regulation of the mosaic-like compartmentalization of the plasma membrane.
Subject(s)
Membrane Proteins/chemistry , Membrane Proteins/metabolism , Microscopy/methods , Actins/chemistry , Actins/metabolism , Cell Membrane/chemistry , Cell Membrane/drug effects , Cell Membrane/metabolism , Cluster Analysis , Humans , Membrane Lipids/chemistry , Membrane Lipids/metabolism , Protein Binding , Protein Interaction Domains and Motifs , Protein Multimerization , Protein TransportABSTRACT
BACKGROUND AND PURPOSE: Annexin A6 (AnxA6) is a calcium-dependent phospholipid-binding protein that can be recruited to the plasma membrane to function as a scaffolding protein to regulate signal complex formation, endo- and exocytic pathways as well as distribution of cellular cholesterol. Here, we have investigated how AnxA6 influences the membrane order. EXPERIMENTAL APPROACH: We used Laurdan and di-4-ANEPPDHQ staining in (i) artificial membranes; (ii) live cells to investigate membrane packing and ordered lipid phases; and (iii) a super-resolution imaging (photoactivated localization microscopy, PALM) and Ripley's K second-order point pattern analysis approach to assess how AnxA6 regulates plasma membrane order domains and protein clustering. KEY RESULTS: In artificial membranes, purified AnxA6 induced a global increase in membrane order. However, confocal microscopy using di-4-ANEPPDHQ in live cells showed that cells expressing AnxA6, which reduces plasma membrane cholesterol levels and modifies the actin cytoskeleton meshwork, displayed a decrease in membrane order (â¼15 and 30% in A431 and MEF cells respectively). PALM data from Lck10 and Src15 membrane raft/non-raft markers revealed that AnxA6 expression induced clustering of both raft and non-raft markers. Altered clustering of Lck10 and Src15 in cells expressing AnxA6 was also observed after cholesterol extraction with methyl-ß-cyclodextrin or actin cytoskeleton disruption with latrunculin B. CONCLUSIONS AND IMPLICATIONS: AnxA6-induced plasma membrane remodelling indicated that elevated AnxA6 expression decreased membrane order through the regulation of cellular cholesterol homeostasis and the actin cytoskeleton. This study provides the first evidence from live cells that support current models of annexins as membrane organizers.
Subject(s)
Annexin A6/metabolism , Cell Membrane/metabolism , Animals , Cell Line , Cell Line, Tumor , Cell Membrane/chemistry , Humans , Lipids/chemistry , Mice, Knockout , Microscopy, FluorescenceABSTRACT
Fluorescence microscopy is an important tool in all fields of biology to visualize structures and monitor dynamic processes and distributions. Contrary to conventional microscopy techniques such as confocal microscopy, which are limited by their spatial resolution, super-resolution techniques such as photoactivated localization microscopy (PALM) and stochastic optical reconstruction microscopy (STORM) have made it possible to observe and quantify structure and processes on the single molecule level. Here, we describe a method to image and quantify the molecular distribution of membrane-associated proteins in two and three dimensions with nanometer resolution.
Subject(s)
Imaging, Three-Dimensional/methods , Microscopy, Fluorescence/methods , Electroporation/instrumentation , Electroporation/methods , Humans , Jurkat Cells , Microscopy/methods , Microscopy, Fluorescence/instrumentation , Proteins/analysis , Transfection/instrumentation , Transfection/methodsABSTRACT
Optimising response to tyrosine kinase inhibitors in cancer remains an extensive field of research. Intravital imaging is an emerging tool, which can be used in drug discovery to facilitate and fine-tune maximum drug response in live tumors. A greater understanding of intratumoural delivery and pharmacodynamics of a drug can be obtained by imaging drug target-specific fluorescence resonance energy transfer (FRET) biosensors in real time. Here, we outline our recent work using a Src-FRET biosensor as a readout of Src activity to gauge optimal tyrosine kinase inhibition in response to dasatinib treatment regimens in vivo. By simultaneously monitoring both the inhibition of Src using FRET imaging, and the modulation of the surrounding extracellular matrix using second harmonic generation (SHG) imaging, we were able to show enhanced drug penetrance and delivery to live pancreatic tumors. We discuss the implications of this dual intravital imaging approach in the context of altered tumor-stromal interactions, while summarising how this approach could be applied to assess other combination strategies or tyrosine kinase inhibitors in a preclinical setting.