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1.
Sci Adv ; 10(27): eadl1197, 2024 Jul 05.
Article in English | MEDLINE | ID: mdl-38959305

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 , Fibrosis , Pancreatic Neoplasms , Proteomics , Animals , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/genetics , Proteomics/methods , Mice , Humans , Carcinoma, Pancreatic Ductal/metabolism , Carcinoma, Pancreatic Ductal/pathology , Carcinoma, Pancreatic Ductal/genetics , Cancer-Associated Fibroblasts/metabolism , Cancer-Associated Fibroblasts/pathology , Disease Models, Animal , Cell Line, Tumor , Calcium-Binding Proteins/metabolism , Calcium-Binding Proteins/genetics , Gemcitabine , Deoxycytidine/analogs & derivatives , Deoxycytidine/pharmacology , Cell Adhesion Molecules
2.
Nat Biotechnol ; 42(1): 72-86, 2024 Jan.
Article in English | MEDLINE | ID: mdl-37024678

ABSTRACT

Transfer RNAs (tRNAs) play a central role in protein translation. Studying them has been difficult in part because a simple method to simultaneously quantify their abundance and chemical modifications is lacking. Here we introduce Nano-tRNAseq, a nanopore-based approach to sequence native tRNA populations that provides quantitative estimates of both tRNA abundances and modification dynamics in a single experiment. We show that default nanopore sequencing settings discard the vast majority of tRNA reads, leading to poor sequencing yields and biased representations of tRNA abundances based on their transcript length. Re-processing of raw nanopore current intensity signals leads to a 12-fold increase in the number of recovered tRNA reads and enables recapitulation of accurate tRNA abundances. We then apply Nano-tRNAseq to Saccharomyces cerevisiae tRNA populations, revealing crosstalks and interdependencies between different tRNA modification types within the same molecule and changes in tRNA populations in response to oxidative stress.


Subject(s)
Nanopore Sequencing , Nanopores , RNA , RNA, Transfer/chemistry , Sequence Analysis, RNA/methods
3.
Nat Cancer ; 4(9): 1326-1344, 2023 09.
Article in English | MEDLINE | ID: mdl-37640930

ABSTRACT

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.


Subject(s)
Pancreatic Diseases , Pancreatic Neoplasms , Humans , Gemcitabine , Protein-Lysine 6-Oxidase , Pancreatic Neoplasms/drug therapy
4.
Nucleic Acids Res ; 51(11): 5301-5324, 2023 06 23.
Article in English | MEDLINE | ID: mdl-36882085

ABSTRACT

The existence of naturally occurring ribosome heterogeneity is now a well-acknowledged phenomenon. However, whether this heterogeneity leads to functionally diverse 'specialized ribosomes' is still a controversial topic. Here, we explore the biological function of RPL3L (uL3L), a ribosomal protein (RP) paralogue of RPL3 (uL3) that is exclusively expressed in skeletal muscle and heart tissues, by generating a viable homozygous Rpl3l knockout mouse strain. We identify a rescue mechanism in which, upon RPL3L depletion, RPL3 becomes up-regulated, yielding RPL3-containing ribosomes instead of RPL3L-containing ribosomes that are typically found in cardiomyocytes. Using both ribosome profiling (Ribo-seq) and a novel orthogonal approach consisting of ribosome pulldown coupled to nanopore sequencing (Nano-TRAP), we find that RPL3L modulates neither translational efficiency nor ribosome affinity towards a specific subset of transcripts. In contrast, we show that depletion of RPL3L leads to increased ribosome-mitochondria interactions in cardiomyocytes, which is accompanied by a significant increase in ATP levels, potentially as a result of fine-tuning of mitochondrial activity. Our results demonstrate that the existence of tissue-specific RP paralogues does not necessarily lead to enhanced translation of specific transcripts or modulation of translational output. Instead, we reveal a complex cellular scenario in which RPL3L modulates the expression of RPL3, which in turn affects ribosomal subcellular localization and, ultimately, mitochondrial activity.


Ribosomes are macromolecular machines responsible for protein synthesis in all living beings. Recent studies have shown that ribosomes can be heterogeneous in their structure, possibly leading to a specialized function. Here, we focus on RPL3L, a ribosomal protein expressed exclusively in striated muscles. We find that the deletion of the Rpl3l gene in a mouse model triggers a compensation mechanism, in which the missing RPL3L protein is replaced by its paralogue, RPL3. Furthermore, we find that RPL3-containing ribosomes establish closer interactions with mitochondria, cellular organelles responsible for energy production, leading to higher energy production when compared with RPL3L-containing ribosomes. Finally, we show that the RPL3­RPL3L compensation mechanism is also triggered in heart disease conditions, such as hypertrophy and myocardial infarction.


Subject(s)
Heart , Mitochondria , Ribosomal Proteins , Ribosomes , Animals , Mice , Mitochondria/metabolism , Muscle, Skeletal/metabolism , Protein Biosynthesis , Ribosomal Proteins/genetics , Ribosomal Proteins/metabolism , Ribosomes/genetics , Ribosomes/metabolism
6.
Nat Commun ; 13(1): 4587, 2022 08 06.
Article in English | MEDLINE | ID: mdl-35933466

ABSTRACT

The tumour stroma, and in particular the extracellular matrix (ECM), is a salient feature of solid tumours that plays a crucial role in shaping their progression. Many desmoplastic tumours including breast cancer involve the significant accumulation of type I collagen. However, recently it has become clear that the precise distribution and organisation of matrix molecules such as collagen I is equally as important in the tumour as their abundance. Cancer-associated fibroblasts (CAFs) coexist within breast cancer tissues and play both pro- and anti-tumourigenic roles through remodelling the ECM. Here, using temporal proteomic profiling of decellularized tumours, we interrogate the evolving matrisome during breast cancer progression. We identify 4 key matrisomal clusters, and pinpoint collagen type XII as a critical component that regulates collagen type I organisation. Through combining our proteomics with single-cell transcriptomics, and genetic manipulation models, we show how CAF-secreted collagen XII alters collagen I organisation to create a pro-invasive microenvironment supporting metastatic dissemination. Finally, we show in patient cohorts that collagen XII may represent an indicator of breast cancer patients at high risk of metastatic relapse.


Subject(s)
Breast Neoplasms , Collagen Type XII/metabolism , Neoplasm Metastasis , Tumor Microenvironment , Breast Neoplasms/pathology , Collagen , Collagen Type I , Extracellular Matrix/pathology , Female , Humans , Neoplasm Metastasis/pathology , Neoplasm Recurrence, Local/pathology , Proteomics
7.
J Chromatogr A ; 1665: 462803, 2022 Feb 22.
Article in English | MEDLINE | ID: mdl-35042139

ABSTRACT

Over 170 post-transcriptional RNA modifications have been described and are common in all kingdoms of life. These modifications range from methylation to complex chemical structures, with methylation being the most abundant. RNA modifications play a key role in RNA folding and function and their dysregulation in humans has been linked to several diseases such as cancer, metabolic diseases or neurological disorder. Nowadays, liquid chromatography-tandem mass spectrometry is considered the gold standard method for the identification and quantification of these modifications due to its sensitivity and accuracy. However, the analysis of modified ribonucleosides by mass spectrometry is complex due to the presence of positional isomers. In this scenario, optimal separation of these compounds by highly sensitive liquid chromatography combined with the generation of high-information spectra is critical to unequivocally identify them, especially in high-complex mixtures. Here we present an analytical method that comprises a new type of mixed-mode nano-flow liquid chromatography column combined with high- and low-collision energy data-independent mass spectrometric acquisition for the identification and quantitation of modified ribonucleosides. The method produces content-rich spectra and combines targeted and screening capabilities thus enabling the identification of a variety of modified nucleosides in biological matrices by single-shot liquid chromatographic analysis coupled to mass spectrometry.


Subject(s)
Ribonucleosides , Ribonucleotides , Chromatography, High Pressure Liquid , Chromatography, Liquid , Humans , Mass Spectrometry
8.
Sci Adv ; 7(40): eabh0363, 2021 Oct.
Article in English | MEDLINE | ID: mdl-34586840

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.

9.
Nat Commun ; 12(1): 3950, 2021 06 24.
Article in English | MEDLINE | ID: mdl-34168137

ABSTRACT

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.


Subject(s)
Extracellular Vesicles , Milk/cytology , Neoplasms, Experimental/pathology , Administration, Oral , Animals , Biological Availability , Breast Neoplasms/pathology , Breast Neoplasms/therapy , Cattle , Cell Line, Tumor , Cell Proliferation , Epithelial-Mesenchymal Transition , Extracellular Vesicles/chemistry , Extracellular Vesicles/genetics , Female , Humans , Liver Neoplasms, Experimental/pathology , Liver Neoplasms, Experimental/secondary , Lung Neoplasms/pathology , Lung Neoplasms/secondary , Mice, Inbred BALB C , Neoplasms, Experimental/therapy , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/therapy , Tissue Distribution , Xenograft Model Antitumor Assays
10.
Nat Biotechnol ; 39(10): 1278-1291, 2021 10.
Article in English | MEDLINE | ID: mdl-33986546

ABSTRACT

Nanopore RNA sequencing shows promise as a method for discriminating and identifying different RNA modifications in native RNA. Expanding on the ability of nanopore sequencing to detect N6-methyladenosine, we show that other modifications, in particular pseudouridine (Ψ) and 2'-O-methylation (Nm), also result in characteristic base-calling 'error' signatures in the nanopore data. Focusing on Ψ modification sites, we detected known and uncovered previously unreported Ψ sites in mRNAs, non-coding RNAs and rRNAs, including a Pus4-dependent Ψ modification in yeast mitochondrial rRNA. To explore the dynamics of pseudouridylation, we treated yeast cells with oxidative, cold and heat stresses and detected heat-sensitive Ψ-modified sites in small nuclear RNAs, small nucleolar RNAs and mRNAs. Finally, we developed a software, nanoRMS, that estimates per-site modification stoichiometries by identifying single-molecule reads with altered current intensity and trace profiles. This work demonstrates that Nm and Ψ RNA modifications can be detected in cellular RNAs and that their modification stoichiometry can be quantified by nanopore sequencing of native RNA.


Subject(s)
Nanopore Sequencing/methods , Pseudouridine/metabolism , RNA/metabolism , Sequence Analysis, RNA/methods , Algorithms , Gene Expression Profiling , Intramolecular Transferases/metabolism , Mitochondria/genetics , Pseudouridine/genetics , RNA/genetics , RNA Processing, Post-Transcriptional/genetics , RNA, Fungal/genetics , RNA, Fungal/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Ribosomal/genetics , RNA, Ribosomal/metabolism , Saccharomyces cerevisiae/genetics , Software , Stress, Physiological/genetics
11.
Genome Res ; 30(9): 1345-1353, 2020 09.
Article in English | MEDLINE | ID: mdl-32907883

ABSTRACT

Nanopore sequencing enables direct measurement of RNA molecules without conversion to cDNA, thus opening the gates to a new era for RNA biology. However, the lack of molecular barcoding of direct RNA nanopore sequencing data sets severely affects the applicability of this technology to biological samples, where RNA availability is often limited. Here, we provide the first experimental protocol and associated algorithm to barcode and demultiplex direct RNA nanopore sequencing data sets. Specifically, we present a novel and robust approach to accurately classify raw nanopore signal data by transforming current intensities into images or arrays of pixels, followed by classification using a deep learning algorithm. We demonstrate the power of this strategy by developing the first experimental protocol for barcoding and demultiplexing direct RNA sequencing libraries. Our method, DeePlexiCon, can classify 93% of reads with 95.1% accuracy or 60% of reads with 99.9% accuracy. The availability of an efficient and simple multiplexing strategy for native RNA sequencing will improve the cost-effectiveness of this technology, as well as facilitate the analysis of lower-input biological samples. Overall, our work exemplifies the power, simplicity, and robustness of signal-to-image conversion for nanopore data analysis using deep learning.


Subject(s)
Deep Learning , Nanopore Sequencing/methods , Sequence Analysis, RNA/methods , Algorithms
12.
Genome Biol ; 21(1): 97, 2020 05 07.
Article in English | MEDLINE | ID: mdl-32375858

ABSTRACT

BACKGROUND: RNA modifications play central roles in cellular fate and differentiation. However, the machinery responsible for placing, removing, and recognizing more than 170 RNA modifications remains largely uncharacterized and poorly annotated, and we currently lack integrative studies that identify which RNA modification-related proteins (RMPs) may be dysregulated in each cancer type. RESULTS: Here, we perform a comprehensive annotation and evolutionary analysis of human RMPs, as well as an integrative analysis of their expression patterns across 32 tissues, 10 species, and 13,358 paired tumor-normal human samples. Our analysis reveals an unanticipated heterogeneity of RMP expression patterns across mammalian tissues, with a vast proportion of duplicated enzymes displaying testis-specific expression, suggesting a key role for RNA modifications in sperm formation and possibly intergenerational inheritance. We uncover many RMPs that are dysregulated in various types of cancer, and whose expression levels are predictive of cancer progression. Surprisingly, we find that several commonly studied RNA modification enzymes such as METTL3 or FTO are not significantly upregulated in most cancer types, whereas several less-characterized RMPs, such as LAGE3 and HENMT1, are dysregulated in many cancers. CONCLUSIONS: Our analyses reveal an unanticipated heterogeneity in the expression patterns of RMPs across mammalian tissues and uncover a large proportion of dysregulated RMPs in multiple cancer types. We provide novel targets for future cancer research studies targeting the human epitranscriptome, as well as foundations to understand cell type-specific behaviors that are orchestrated by RNA modifications.


Subject(s)
Neoplasms/genetics , RNA Processing, Post-Transcriptional , Animals , Carrier Proteins/metabolism , Epididymis/metabolism , Evolution, Molecular , Humans , Male , Meiosis/genetics , Methyltransferases/metabolism , Mice , Molecular Sequence Annotation , Neoplasms/metabolism , Organ Specificity , Spermatogenesis/genetics
13.
Oncogene ; 39(8): 1821-1829, 2020 02.
Article in English | MEDLINE | ID: mdl-31735913

ABSTRACT

Pancreatic ductal adenocarcinoma (PDAC) remains one of the deadliest malignancies. It is phenotypically heterogeneous with a highly unstable genome and provides few common therapeutic targets. We found that MCL1, Cofilin1 (CFL1) and SRC mRNA were highly expressed by a wide range of these cancers, suggesting that a strategy of dual MCL-1 and SRC inhibition might be efficacious for many patients. Immunohistochemistry revealed that MCL-1 protein was present at high levels in 94.7% of patients in a cohort of PDACs from Australian Pancreatic Genome Initiative (APGI). High MCL1 and Cofilin1 mRNA expression was also strongly predictive of poor outcome in the TCGA dataset and in the APGI cohort. In culture, MCL-1 antagonism reduced the level of the cytoskeletal remodeling protein Cofilin1 and phosphorylated SRC on the active Y416 residue, suggestive of reduced invasive capacity. The MCL-1 antagonist S63845 synergized with the SRC kinase inhibitor dasatinib to reduce cell viability and invasiveness through 3D-organotypic matrices. In preclinical murine models, this combination reduced primary tumor growth and liver metastasis of pancreatic cancer xenografts. These data suggest that MCL-1 antagonism, while reducing cell viability, may have an additional benefit in increasing the antimetastatic efficacy of dasatinib for the treatment of PDAC.


Subject(s)
Adenocarcinoma/pathology , Dasatinib/pharmacology , Myeloid Cell Leukemia Sequence 1 Protein/antagonists & inhibitors , Pancreatic Neoplasms/pathology , Cell Line, Tumor , Drug Synergism , Humans , Neoplasm Invasiveness
14.
Nat Commun ; 10(1): 4079, 2019 09 09.
Article in English | MEDLINE | ID: mdl-31501426

ABSTRACT

The epitranscriptomics field has undergone an enormous expansion in the last few years; however, a major limitation is the lack of generic methods to map RNA modifications transcriptome-wide. Here, we show that using direct RNA sequencing, N6-methyladenosine (m6A) RNA modifications can be detected with high accuracy, in the form of systematic errors and decreased base-calling qualities. Specifically, we find that our algorithm, trained with m6A-modified and unmodified synthetic sequences, can predict m6A RNA modifications with ~90% accuracy. We then extend our findings to yeast data sets, finding that our method can identify m6A RNA modifications in vivo with an accuracy of 87%. Moreover, we further validate our method by showing that these 'errors' are typically not observed in yeast ime4-knockout strains, which lack m6A modifications. Our results open avenues to investigate the biological roles of RNA modifications in their native RNA context.


Subject(s)
Adenosine/analogs & derivatives , RNA/genetics , RNA/metabolism , Adenosine/metabolism , Base Sequence , Electricity , Saccharomyces cerevisiae/genetics , Sequence Analysis, RNA , Support Vector Machine
15.
Nat Commun ; 10(1): 3637, 2019 08 12.
Article in English | MEDLINE | ID: mdl-31406163

ABSTRACT

Heterogeneous subtypes of cancer-associated fibroblasts (CAFs) coexist within pancreatic cancer tissues and can both promote and restrain disease progression. Here, we interrogate how cancer cells harboring distinct alterations in p53 manipulate CAFs. We reveal the existence of a p53-driven hierarchy, where cancer cells with a gain-of-function (GOF) mutant p53 educate a dominant population of CAFs that establish a pro-metastatic environment for GOF and null p53 cancer cells alike. We also demonstrate that CAFs educated by null p53 cancer cells may be reprogrammed by either GOF mutant p53 cells or their CAFs. We identify perlecan as a key component of this pro-metastatic environment. Using intravital imaging, we observe that these dominant CAFs delay cancer cell response to chemotherapy. Lastly, we reveal that depleting perlecan in the stroma combined with chemotherapy prolongs mouse survival, supporting it as a potential target for anti-stromal therapies in pancreatic cancer.


Subject(s)
Cancer-Associated Fibroblasts/pathology , Drug Resistance, Neoplasm/genetics , Heparan Sulfate Proteoglycans/metabolism , Pancreatic Neoplasms/pathology , Tumor Suppressor Protein p53/metabolism , Animals , Cell Line, Tumor , Cell Movement , Cell Proliferation , Gene Expression Regulation, Neoplastic/genetics , Mice , Mice, Inbred BALB C , Neoplasm Invasiveness/pathology , Pancreas/pathology , Pancreatic Neoplasms/genetics , Signal Transduction/physiology , Tumor Suppressor Protein p53/genetics
16.
F1000Res ; 72018.
Article in English | MEDLINE | ID: mdl-30135716

ABSTRACT

Tumour metastasis is a dynamic and systemic process. It is no longer seen as a tumour cell-autonomous program but as a multifaceted and complex series of events, which is influenced by the intrinsic cellular mutational burden of cancer cells and the numerous bidirectional interactions between malignant and non-malignant cells and fine-tuned by the various extrinsic cues of the extracellular matrix. In cancer biology, metastasis as a process is one of the most technically challenging aspects of cancer biology to study. As a result, new platforms and technologies are continually being developed to better understand this process. In this review, we discuss some of the recent advances in metastasis and how the information gleaned is re-shaping our understanding of metastatic dissemination.


Subject(s)
Neoplasm Metastasis , Animals , Cell Movement , Humans , Neoplasm Invasiveness , Neoplasms/blood , Neoplasms/pathology , Neoplastic Cells, Circulating/pathology
17.
J Cell Sci ; 131(5)2018 03 06.
Article in English | MEDLINE | ID: mdl-29511095

ABSTRACT

Molecular mobility, localisation and spatiotemporal activity are at the core of cell biological processes and deregulation of these dynamic events can underpin disease development and progression. Recent advances in intravital imaging techniques in mice are providing new avenues to study real-time molecular behaviour in intact tissues within a live organism and to gain exciting insights into the intricate regulation of live cell biology at the microscale level. The monitoring of fluorescently labelled proteins and agents can be combined with autofluorescent properties of the microenvironment to provide a comprehensive snapshot of in vivo cell biology. In this Review, we summarise recent intravital microscopy approaches in mice, in processes ranging from normal development and homeostasis to disease progression and treatment in cancer, where we emphasise the utility of intravital imaging to observe dynamic and transient events in vivo We also highlight the recent integration of advanced subcellular imaging techniques into the intravital imaging pipeline, which can provide in-depth biological information beyond the single-cell level. We conclude with an outlook of ongoing developments in intravital microscopy towards imaging in humans, as well as provide an overview of the challenges the intravital imaging community currently faces and outline potential ways for overcoming these hurdles.


Subject(s)
Intravital Microscopy/trends , Molecular Imaging/trends , Neoplasms/genetics , Tumor Microenvironment/genetics , Disease Progression , Humans , Neoplasms/ultrastructure
18.
Cell Rep ; 21(1): 274-288, 2017 Oct 03.
Article in English | MEDLINE | ID: mdl-28978480

ABSTRACT

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 Protein
19.
Sci Transl Med ; 9(384)2017 04 05.
Article in English | MEDLINE | ID: mdl-28381539

ABSTRACT

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 , Gemcitabine
20.
Oncotarget ; 8(16): 26066-26078, 2017 Apr 18.
Article in English | MEDLINE | ID: mdl-28199967

ABSTRACT

Lysyl Oxidase-like 2 (LOXL2), a member of the lysyl oxidase family of amine oxidases is known to be important in normal tissue development and homeostasis, as well as the onset and progression of solid tumors. Here we tested the anti-tumor properties of two generations of novel small molecule LOXL2 inhibitor in the MDA-MB-231 human model of breast cancer. We confirmed a functional role for LOXL2 activity in the progression of primary breast cancer. Inhibition of LOXL2 activity inhibited the growth of primary tumors and reduced primary tumor angiogenesis. Dual inhibition of LOXL2 and LOX showed a greater effect and also led to a lower overall metastatic burden in the lung and liver. Our data provides the first evidence to support a role for LOXL2 specific small molecule inhibitors as a potential therapy in breast cancer.


Subject(s)
Amino Acid Oxidoreductases/antagonists & inhibitors , Antineoplastic Agents/pharmacology , Breast Neoplasms/metabolism , Enzyme Inhibitors/pharmacology , Amino Acid Oxidoreductases/genetics , Aminopropionitrile/pharmacology , Animals , Apoptosis , Breast Neoplasms/drug therapy , Breast Neoplasms/genetics , Breast Neoplasms/pathology , Cancer-Associated Fibroblasts/drug effects , Cancer-Associated Fibroblasts/metabolism , Cell Line, Tumor , Cell Movement/drug effects , Cell Proliferation/drug effects , Disease Models, Animal , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Female , Gene Knockdown Techniques , Gene Silencing , Humans , Mice , Neoplasm Metastasis , Neovascularization, Pathologic , Xenograft Model Antitumor Assays
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