Publications by authors named "Srivatsan Raghavan"

22 Publications

  • Page 1 of 1

FGFR2 Extracellular Domain In-Frame Deletions are Therapeutically Targetable Genomic Alterations that Function as Oncogenic Drivers in Cholangiocarcinoma.

Cancer Discov 2021 Apr 29. Epub 2021 Apr 29.

Department of Medical Oncology, Dana-Farber Cancer Institute.

We conducted next generation DNA sequencing on 335 biliary tract cancers and characterized the genomic landscape by anatomic site within the biliary tree. In addition to frequent FGFR2 fusions among patients with intrahepatic cholangiocarcinoma (IHCC), we identified FGFR2 extracellular domain in-frame deletions (EIDs) in 5 of 178 (2.8%) patients with IHCC, including two patients with FGFR2 p.H167_N173del. Expression of this FGFR2 EID in NIH3T3 cells resulted in constitutive FGFR2 activation, oncogenic transformation, and sensitivity to FGFR inhibitors. Three patients with FGFR2 EIDs were treated with Debio 1347, an oral FGFR-1/2/3 inhibitor, and all showed partial responses. One patient developed an acquired L618F FGFR2 kinase domain mutation at disease progression and experienced a further partial response for 17 months to an irreversible FGFR2 inhibitor, futibatinib. Together, these findings reveal FGFR2 EIDs as an alternative mechanism of FGFR2 activation in IHCC that predict sensitivity to FGFR inhibitors in the clinic.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/2159-8290.CD-20-1669DOI Listing
April 2021

Altered RNA Splicing by Mutant p53 Activates Oncogenic RAS Signaling in Pancreatic Cancer.

Cancer Cell 2020 08 18;38(2):198-211.e8. Epub 2020 Jun 18.

The Kinghorn Cancer Centre, and the Cancer Research Program, Garvan Institute of Medical Research, Darlinghurst, Sydney, NSW, Australia; Department of Surgery, Bankstown Hospital, Eldridge Road, Bankstown, Sydney, NSW, Australia; South Western Sydney Clinical School, Faculty of Medicine, University of New South Wales, Liverpool, NSW, Australia.

Pancreatic ductal adenocarcinoma (PDAC) is driven by co-existing mutations in KRAS and TP53. However, how these mutations collaborate to promote this cancer is unknown. Here, we uncover sequence-specific changes in RNA splicing enforced by mutant p53 which enhance KRAS activity. Mutant p53 increases expression of splicing regulator hnRNPK to promote inclusion of cytosine-rich exons within GTPase-activating proteins (GAPs), negative regulators of RAS family members. Mutant p53-enforced GAP isoforms lose cell membrane association, leading to heightened KRAS activity. Preventing cytosine-rich exon inclusion in mutant KRAS/p53 PDACs decreases tumor growth. Moreover, mutant p53 PDACs are sensitized to inhibition of splicing via spliceosome inhibitors. These data provide insight into co-enrichment of KRAS and p53 mutations and therapeutics targeting this mechanism in PDAC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.ccell.2020.05.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8028848PMC
August 2020

Discovery of a selective inhibitor of doublecortin like kinase 1.

Nat Chem Biol 2020 06 6;16(6):635-643. Epub 2020 Apr 6.

Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA, USA.

Doublecortin like kinase 1 (DCLK1) is an understudied kinase that is upregulated in a wide range of cancers, including pancreatic ductal adenocarcinoma (PDAC). However, little is known about its potential as a therapeutic target. We used chemoproteomic profiling and structure-based design to develop a selective, in vivo-compatible chemical probe of the DCLK1 kinase domain, DCLK1-IN-1. We demonstrate activity of DCLK1-IN-1 against clinically relevant patient-derived PDAC organoid models and use a combination of RNA-sequencing, proteomics and phosphoproteomics analysis to reveal that DCLK1 inhibition modulates proteins and pathways associated with cell motility in this context. DCLK1-IN-1 will serve as a versatile tool to investigate DCLK1 biology and establish its role in cancer.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41589-020-0506-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7246176PMC
June 2020

Non-viral delivery of CRISPR/Cas9 complex using CRISPR-GPS nanocomplexes.

Nanoscale 2019 Nov 31;11(44):21317-21323. Epub 2019 Oct 31.

Marble Center for Cancer Nanomedicine, Institute for Medical Engineering & Science, Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology, Cambridge, MA 02139, USA and Broad Institute of Massachusetts Institute of Technology and Harvard, Cambridge, MA 02139, USA and Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115, USA and Howard Hughes Medical Institute, Cambridge, MA 02139, USA.

There is a critical need for the development of safe and efficient delivery technologies for CRISPR/Cas9 to advance translation of genome editing to the clinic. Non-viral methods that are simple, efficient, and completely based on biologically-derived materials could offer such potential. Here we report a simple and modular tandem peptide-based nanocomplex system with cell-targeting capacity that efficiently combines guide RNA (sgRNA) with Cas9 protein, and facilitates internalization of sgRNA/Cas9 ribonucleoprotein complexes to yield robust genome editing across multiple cell lines.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1039/c9nr01786kDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7709491PMC
November 2019

Synthetic Lethal Interaction of SHOC2 Depletion with MEK Inhibition in RAS-Driven Cancers.

Cell Rep 2019 10;29(1):118-134.e8

Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA; Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, MA 02215, USA; Harvard Medical School, 25 Shattuck Street, Boston, MA 02115, USA; Department of Medicine, Brigham and Women's Hospital and Harvard Medical School, 75 Francis Street, Boston, MA 02115, MA. Electronic address:

The mitogen-activated protein kinase (MAPK) pathway is a critical effector of oncogenic RAS signaling, and MAPK pathway inhibition may be an effective combination treatment strategy. We performed genome-scale loss-of-function CRISPR-Cas9 screens in the presence of a MEK1/2 inhibitor (MEKi) in KRAS-mutant pancreatic and lung cancer cell lines and identified genes that cooperate with MEK inhibition. While we observed heterogeneity in genetic modifiers of MEKi sensitivity across cell lines, several recurrent classes of synthetic lethal vulnerabilities emerged at the pathway level. Multiple members of receptor tyrosine kinase (RTK)-RAS-MAPK pathways scored as sensitizers to MEKi. In particular, we demonstrate that knockout, suppression, or degradation of SHOC2, a positive regulator of MAPK signaling, specifically cooperated with MEK inhibition to impair proliferation in RAS-driven cancer cells. The depletion of SHOC2 disrupted survival pathways triggered by feedback RTK signaling in response to MEK inhibition. Thus, these findings nominate SHOC2 as a potential target for combination therapy.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.celrep.2019.08.090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6918830PMC
October 2019

TAS-120 Overcomes Resistance to ATP-Competitive FGFR Inhibitors in Patients with FGFR2 Fusion-Positive Intrahepatic Cholangiocarcinoma.

Cancer Discov 2019 08 20;9(8):1064-1079. Epub 2019 May 20.

Gastrointestinal Oncology Service, Department of Medicine, Memorial Sloan Kettering Cancer Center, New York, New York.

ATP-competitive fibroblast growth factor receptor (FGFR) kinase inhibitors, including BGJ398 and Debio 1347, show antitumor activity in patients with intrahepatic cholangiocarcinoma (ICC) harboring activating gene fusions. Unfortunately, acquired resistance develops and is often associated with the emergence of secondary kinase domain mutations. Here, we report that the irreversible pan-FGFR inhibitor TAS-120 demonstrated efficacy in 4 patients with 2 fusion-positive ICC who developed resistance to BGJ398 or Debio 1347. Examination of serial biopsies, circulating tumor DNA (ctDNA), and patient-derived ICC cells revealed that TAS-120 was active against multiple FGFR2 mutations conferring resistance to BGJ398 or Debio 1347. Functional assessment and modeling the clonal outgrowth of individual resistance mutations from polyclonal cell pools mirrored the resistance profiles observed clinically for each inhibitor. Our findings suggest that strategic sequencing of FGFR inhibitors, guided by serial biopsy and ctDNA analysis, may prolong the duration of benefit from FGFR inhibition in patients with fusion-positive ICC. SIGNIFICANCE: ATP-competitive FGFR inhibitors (BGJ398, Debio 1347) show efficacy in -altered ICC; however, acquired kinase domain mutations cause drug resistance and tumor progression. We demonstrate that the irreversible FGFR inhibitor TAS-120 provides clinical benefit in patients with resistance to BGJ398 or Debio 1347 and overcomes several FGFR2 mutations in ICC models..
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/2159-8290.CD-19-0182DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6677584PMC
August 2019

WRN helicase is a synthetic lethal target in microsatellite unstable cancers.

Nature 2019 04 10;568(7753):551-556. Epub 2019 Apr 10.

Broad Institute of Harvard and MIT, Cambridge, MA, USA.

Synthetic lethality-an interaction between two genetic events through which the co-occurrence of these two genetic events leads to cell death, but each event alone does not-can be exploited for cancer therapeutics. DNA repair processes represent attractive synthetic lethal targets, because many cancers exhibit an impairment of a DNA repair pathway, which can lead to dependence on specific repair proteins. The success of poly(ADP-ribose) polymerase 1 (PARP-1) inhibitors in cancers with deficiencies in homologous recombination highlights the potential of this approach. Hypothesizing that other DNA repair defects would give rise to synthetic lethal relationships, we queried dependencies in cancers with microsatellite instability (MSI), which results from deficient DNA mismatch repair. Here we analysed data from large-scale silencing screens using CRISPR-Cas9-mediated knockout and RNA interference, and found that the RecQ DNA helicase WRN was selectively essential in MSI models in vitro and in vivo, yet dispensable in models of cancers that are microsatellite stable. Depletion of WRN induced double-stranded DNA breaks and promoted apoptosis and cell cycle arrest selectively in MSI models. MSI cancer models required the helicase activity of WRN, but not its exonuclease activity. These findings show that WRN is a synthetic lethal vulnerability and promising drug target for MSI cancers.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41586-019-1102-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6580861PMC
April 2019

iRGD-guided Tumor-penetrating Nanocomplexes for Therapeutic siRNA Delivery to Pancreatic Cancer.

Mol Cancer Ther 2018 11 10;17(11):2377-2388. Epub 2018 Aug 10.

Koch Institute for Integrative Cancer Research, Massachusetts Institute of Technology (MIT), Cambridge, Massachusetts.

Pancreatic cancer is one of the leading causes of cancer-related death, with 5-year survival of 8.5%. The lack of significant progress in improving therapy reflects our inability to overcome the desmoplastic stromal barrier in pancreatic ductal adenocarcinoma (PDAC) as well as a paucity of new approaches targeting its genetic underpinnings. RNA interference holds promise in targeting key mutations driving PDAC; however, a nucleic acid delivery vehicle that homes to PDAC and breaches the stroma does not yet exist. Noting that the cyclic peptide iRGD mediates tumor targeting and penetration through interactions with αβ integrins and neuropilin-1, we hypothesized that "tandem" peptides combining a cell-penetrating peptide and iRGD can encapsulate siRNA to form tumor-penetrating nanocomplexes (TPN) capable of delivering siRNA to PDAC. The use of directly conjugated iRGD is justified by receptor expression patterns in human PDAC biopsies. In this work, we optimize iRGD TPNs with polyethylene glycol (PEG)-peptide conjugates for systemic delivery to sites of disease. We show that TPNs effectively knockdown siRNA targets in PDAC cell lines and in an immunocompetent genetically engineered mouse model of PDAC. Furthermore, we validate their tumor-penetrating ability in three-dimensional organoids and autochthonous tumors. In murine therapeutic trials, TPNs delivering anti- siRNA significantly delay tumor growth. Thus, iRGD TPNs hold promise in treating PDAC by not only overcoming physical barriers to therapy, but by leveraging the stroma to achieve knockdown of the gold-standard genetic target. Moreover, the modular construction of this delivery platform allows for facile adaptation to future genetic target candidates in pancreatic cancer. .
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/1535-7163.MCT-17-1090DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6298224PMC
November 2018

Real-time Genomic Characterization of Advanced Pancreatic Cancer to Enable Precision Medicine.

Cancer Discov 2018 09 14;8(9):1096-1111. Epub 2018 Jun 14.

Dana-Farber Cancer Institute, Boston, Massachusetts.

Clinically relevant subtypes exist for pancreatic ductal adenocarcinoma (PDAC), but molecular characterization is not yet standard in clinical care. We implemented a biopsy protocol to perform time-sensitive whole-exome sequencing and RNA sequencing for patients with advanced PDAC. Therapeutically relevant genomic alterations were identified in 48% (34/71) and pathogenic/likely pathogenic germline alterations in 18% (13/71) of patients. Overall, 30% (21/71) of enrolled patients experienced a change in clinical management as a result of genomic data. Twenty-six patients had germline and/or somatic alterations in DNA-damage repair genes, and 5 additional patients had mutational signatures of homologous recombination deficiency but no identified causal genomic alteration. Two patients had oncogenic in-frame deletions, and we report the first clinical evidence that this alteration confers sensitivity to MAPK pathway inhibition. Moreover, we identified tumor/stroma gene expression signatures with clinical relevance. Collectively, these data demonstrate the feasibility and value of real-time genomic characterization of advanced PDAC. Molecular analyses of metastatic PDAC tumors are challenging due to the heterogeneous cellular composition of biopsy specimens and rapid progression of the disease. Using an integrated multidisciplinary biopsy program, we demonstrate that real-time genomic characterization of advanced PDAC can identify clinically relevant alterations that inform management of this difficult disease. .
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/2159-8290.CD-18-0275DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6192263PMC
September 2018

Publisher Correction: Investigating the relationship between Aerosol Optical Depth and Precipitation over Southeast Asia with Relative Humidity as an influencing factor.

Sci Rep 2018 May 10;8(1):7600. Epub 2018 May 10.

Department of Civil and Environmental Engineering, National University of Singapore, Singapore, Singapore.

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-018-25179-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5945860PMC
May 2018

Investigating the relationship between Aerosol Optical Depth and Precipitation over Southeast Asia with Relative Humidity as an influencing factor.

Sci Rep 2017 10 17;7(1):13395. Epub 2017 Oct 17.

Department of Civil and Environmental Engineering, National University of Singapore, Singapore, Singapore.

Atmospheric aerosols influence precipitation by changing the earth's energy budget and cloud properties. A number of studies have reported correlations between aerosol properties and precipitation data. Despite previous research, it is still hard to quantify the overall effects that aerosols have on precipitation as multiple influencing factors such as relative humidity (RH) can distort the observed relationship between aerosols and precipitation. Thus, in this study, both satellite-retrieved and reanalysis data were used to investigate the relationship between aerosols and precipitation in the Southeast Asia region from 2001 to 2015, with RH considered as a possible influencing factor. Different analyses in the study indicate that a positive correlation was present between Aerosol Optical Depth (AOD) and precipitation over northern Southeast Asia region during the autumn and the winter seasons, while a negative correlation was identified over the Maritime Continent during the autumn season. Subsequently, a partial correlation analysis revealed that while RH influences the long-term negative correlations between AOD and precipitation, it did not significantly affect the positive correlations seen in the winter season. The result of this study provides additional evidence with respect to the critical role of RH as an influencing factor in AOD-precipitation relationship over Southeast Asia.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-017-10858-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5645411PMC
October 2017

KEAP1 loss modulates sensitivity to kinase targeted therapy in lung cancer.

Elife 2017 02 1;6. Epub 2017 Feb 1.

Department of Medical Oncology, Dana-Farber Cancer Institute, Boston, United States.

Inhibitors that target the receptor tyrosine kinase (RTK)/Ras/mitogen-activated protein kinase (MAPK) pathway have led to clinical responses in lung and other cancers, but some patients fail to respond and in those that do resistance inevitably occurs (Balak et al., 2006; Kosaka et al., 2006; Rudin et al., 2013; Wagle et al., 2011). To understand intrinsic and acquired resistance to inhibition of MAPK signaling, we performed CRISPR-Cas9 gene deletion screens in the setting of BRAF, MEK, EGFR, and ALK inhibition. Loss of , a negative regulator of NFE2L2/NRF2, modulated the response to BRAF, MEK, EGFR, and ALK inhibition in BRAF-, NRAS-, KRAS-, EGFR-, and ALK-mutant lung cancer cells. Treatment with inhibitors targeting the RTK/MAPK pathway increased reactive oxygen species (ROS) in cells with intact KEAP1, and loss of KEAP1 abrogated this increase. In addition, loss of KEAP1 altered cell metabolism to allow cells to proliferate in the absence of MAPK signaling. These observations suggest that alterations in the KEAP1/NRF2 pathway may promote survival in the presence of multiple inhibitors targeting the RTK/Ras/MAPK pathway.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.7554/eLife.18970DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5305212PMC
February 2017

Metastatic pancreatic neuroendocrine tumor with ectopic adrenocorticotropic hormone production.

Proc (Bayl Univ Med Cent) 2015 Jan;28(1):46-9

Department of Medicine (Vaduganathan, Nagarur, Lauter, Padmanabhan, Raghavan, Pallais, Fenves) and the Department of Pathology (Kerr), Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts.

We describe a 71-year-old man who presented with abdominal pain, lower-extremity edema, recent unintentional weight loss, hypertension, hyperglycemia, hypokalemia, and metabolic alkalosis. Serum cortisol levels remained elevated after overnight high-dose dexamethasone suppression. Magnetic resonance imaging revealed a small mass in the head of the pancreas with scattered liver metastases. Both endoscopic ultrasound-guided pancreatic biopsy and liver biopsy revealed a well-differentiated neuroendocrine tumor. These lesions did not show significant uptake on octreotide scan. Medical management and hepatic artery chemoembolization were attempted. Ultimately, the patient underwent bilateral adrenalectomy, but died within 4 months of symptom onset secondary to postoperative complications.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4264709PMC
http://dx.doi.org/10.1080/08998280.2015.11929184DOI Listing
January 2015

Manipulation of 3D Cluster Size and Geometry by Release from 2D Micropatterns.

Cell Mol Bioeng 2012 Sep;5(3):299-306

Department of Bioengineering, University of Pennsylvania, 510 Skirkanich Hall, 210 South 33rd Street, Philadelphia, PA 19104, USA.

A novel method to control three-dimensional cell cluster size and geometry using two-dimensional patterning techniques is described. Cells were first cultured on two-dimensional micropatterned collagen using conventional soft lithography techniques. Collagenase was used to degrade the micropatterned collagen and release cells from the micropatterns, forming clusters of cells which were then resuspended in a three-dimensional collagen matrix. This method facilitated the formation of uniformly sized clusters within a single sample. By systematically varying the geometry of the two-dimensional micropatterned islands, final cluster size and cell number in three dimensions could be controlled. Using this technique, we showed that proliferation of cells within collagen gels depended on the size of clusters, suggesting an important role for multicellular structure on biological function. Furthermore, by utilizing more complex two-dimensional patterns, non-spherical structures could be produced. This technique demonstrates a simple way to exploit two-dimensional micro-patterning in order to create complex and structured multicellular clusters in a three-dimensional environment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/s12195-012-0236-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3665517PMC
September 2012

Decreased cell adhesion promotes angiogenesis in a Pyk2-dependent manner.

Exp Cell Res 2011 Aug 18;317(13):1860-71. Epub 2011 May 18.

Department of Bioengineering, University of Pennsylvania, 210 South 33rd Street, Philadelphia, PA 19104, USA.

Angiogenesis is regulated by both soluble growth factors and cellular interactions with the extracellular matrix (ECM). While cell adhesion via integrins has been shown to be required for angiogenesis, the effects of quantitative changes in cell adhesion and spreading against the ECM remain less clear. Here, we show that angiogenic sprouting in natural and engineered three-dimensional matrices exhibited a biphasic response, with peak sprouting when adhesion to the matrix was limited to intermediate levels. Examining changes in global gene expression to determine a genetic basis for this response, we demonstrate a vascular endothelial growth factor (VEGF)-induced upregulation of genes associated with vascular invasion and remodeling when cell adhesion was limited, whereas cells on highly adhesive surfaces upregulated genes associated with proliferation. To explore a mechanistic basis for this effect, we turned to focal adhesion kinase (FAK), a central player in adhesion signaling previously implicated in angiogenesis, and its homologue, proline-rich tyrosine kinase 2 (Pyk2). While FAK signaling had some impact, our results suggested that Pyk2 can regulate both gene expression and endothelial sprouting through its enhanced activation by VEGF in limited adhesion contexts. We also demonstrate decreased sprouting of tissue explants from Pyk2-null mice as compared to wild type mice as further confirmation of the role of Pyk2 in angiogenic sprouting. These results suggest a surprising finding that limited cell adhesion can enhance endothelial responsiveness to VEGF and demonstrate a novel role for Pyk2 in the adhesive regulation of angiogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.yexcr.2011.05.006DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3123418PMC
August 2011

Micropatterned dynamically adhesive substrates for cell migration.

Langmuir 2010 Nov 1;26(22):17733-8. Epub 2010 Oct 1.

Department of Biomedical Engineering, Johns Hopkins School of Medicine, Baltimore, Maryland 21205, United States.

We present a novel approach to examine cell migration using dynamically adhesive substrates consisting of three spatially and functionally distinct regions: the first is permanently nonadhesive to cells, the second is permanently adhesive, and the final region is electrochemically switched from nonadhesive to adhesive. We applied a double microcontact printing approach to pattern gold surfaces with carboxylic acid-terminated self-assembled monolayers (SAMs) that permit initial cell adhesion, with methyl-terminated SAMs that permit adsorption of a nonadhesive, and with tri(ethylene glycol)-terminated SAMs that can be electrochemically "switched" to permit cell migration from a prespecified pattern onto a new pattern. Using these substrates, we investigated the migration of epithelial cells from monolayers onto narrow, branching tracks of extracellular matrix in order to characterize how lead cells influence the direction of movement of followers. Time-lapse imaging revealed that, on average, five cells consistently chose one branch before other cells entered the second branch, providing evidence to suggest that intercellular communication plays an important role in guiding the cohesive movement of epithelial sheets. This platform may be of use in furthering our understanding of the mechanisms underlying cellular decision-making during migration in both individual and multicellular contexts.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1021/la102955mDOI Listing
November 2010

Decoupling diffusional from dimensional control of signaling in 3D culture reveals a role for myosin in tubulogenesis.

J Cell Sci 2010 Sep 3;123(Pt 17):2877-83. Epub 2010 Aug 3.

Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

We present a novel microfabricated platform to culture cells within arrays of micrometer-scale three-dimensional (3D) extracellular matrix scaffolds (microgels). These microscale cultures eliminate diffusion barriers that are intrinsic to traditional 3D culture systems (macrogels) and enable uniform cytokine stimulation of the entire culture population, as well as allow immunolabeling, imaging and population-based biochemical assays across the relatively coplanar microgels. Examining early signaling associated with hepatocyte growth factor (HGF)-mediated scattering and tubulogenesis of MDCK cells revealed that 3D culture modulates cellular responses both through dimensionality and altered stimulation rates. Comparing responses in 2D culture, microgels and macrogels demonstrated that HGF-induced ERK signaling was driven by the dynamics of stimulation and not by whether cells were in a 2D or 3D environment, and that this ERK signaling was equally important for HGF-induced cell scattering on 2D substrates and tubulogenesis in 3D. By contrast, we discovered a specific HGF-induced increase in myosin expression leading to sustained downregulation of myosin activity that occurred only within 3D contexts and was required for 3D tubulogenesis but not 2D scattering. Interestingly, although absent in cells on collagen-coated plates, downregulation of myosin activity also occurred for cells on collagen gels, but was transient and mediated by a combination of myosin dephosphorylation and enhanced myosin expression. Furthermore, upregulating myosin activity via siRNA targeted to a myosin phosphatase did not attenuate scattering in 2D but did inhibit tubulogenesis in 3D. Together, these results demonstrate that cellular responses to soluble cues in 3D culture are regulated by both rates of stimulation and by matrix dimensionality, and highlight the importance of decoupling these effects to identify early signals relevant to cellular function in 3D environments.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/jcs.055079DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2923566PMC
September 2010

Geometrically controlled endothelial tubulogenesis in micropatterned gels.

Tissue Eng Part A 2010 Jul;16(7):2255-63

Department of Biomedical Engineering, Johns Hopkins School of Medicine, Baltimore, Maryland, USA.

We present a novel approach to control endothelial tubulogenesis by spatially patterning cells within micromolded collagen gels. Endothelial cells cultured within microscale channels that were filled with collagen gel organized into tubes with lumens within 24-48 h of seeding. These tubes extended up to 1 cm in length, and exhibited cell-cell junction formation characteristic of early stage capillary vessels. Tube diameter could be controlled by varying collagen concentrations or channel width. The geometry of the microfabricated template also could be used to guide the development of branches during tube formation, allowing for the generation of more complex capillary architectures. Time-lapse imaging of tube formation revealed a highly dynamic process involving coalescence of endothelial cells, reorganization and alignment of collagen fibers into a central core, and arrangement of cells into cords. This platform may be of use to generate geometrically defined vascular networks for tissue engineering applications as well as a means to better understand the process of endothelial tubulogenesis.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/ten.TEA.2009.0584DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2947948PMC
July 2010

Cell polarity triggered by cell-cell adhesion via E-cadherin.

J Cell Sci 2009 Apr 3;122(Pt 7):905-11. Epub 2009 Mar 3.

Department of Bioengineering, University of Pennsylvania, Philadelphia, PA 19104, USA.

Cell polarity is orchestrated by numerous extracellular cues, and guides events such as chemotaxis, mitosis and wound healing. In scrape-wound assays of cell monolayers, wound-edge cells orient their centrosomes towards the wound, a process that appears to depend on the formation of new cell-extracellular-matrix adhesions as cells spread into the wound. In direct contrast to scrape-wounded cells, isolated cells without cell-cell contacts failed to polarize, suggesting that asymmetry of cell-cell adhesions resulting from monolayer disruption might contribute to polarization. By using micropatterned substrates to engineer such asymmetries in kidney epithelial cells, we found that cell-cell contact induced displacement of the nucleus towards the contact, and also caused centrosomal reorientation and lamellipodial ruffling to the distal side of the nucleus. Upon release from micropatterned constraints, cells exhibited directed migration away from the cell-cell contact. Disrupting E-cadherin engagement randomized nuclear position and lamellipodial ruffling in patterned cultures, and abrogated scrape-wound-induced cell reorientation, but not migration rate. Polarity that was induced by cell-cell contact required an intact actin cytoskeleton and Cdc42 activity, but not RhoA or Rac signaling. Together, these findings demonstrate a novel role for cell-cell adhesion in polarization, and have implications for wound healing and developmental patterning.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1242/jcs.028183DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2720926PMC
April 2009

An inhibitory role for FAK in regulating proliferation: a link between limited adhesion and RhoA-ROCK signaling.

J Cell Biol 2006 Jul;174(2):277-88

Department of Biomedical Engineering, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA.

Focal adhesion kinase (FAK) transduces cell adhesion to the extracellular matrix into proliferative signals. We show that FAK overexpression induced proliferation in endothelial cells, which are normally growth arrested by limited adhesion. Interestingly, displacement of FAK from adhesions by using a FAK-/- cell line or by expressing the C-terminal fragment FRNK also caused an escape of adhesion-regulated growth arrest, suggesting dual positive and negative roles for FAK in growth regulation. Expressing kinase-dead FAK-Y397F in FAK-/- cells prevented uncontrolled growth, demonstrating the antiproliferative function of inactive FAK. Unlike FAK overexpression-induced growth, loss of growth control in FAK-/- or FRNK-expressing cells increased RhoA activity, cytoskeletal tension, and focal adhesion formation. ROCK inhibition rescued adhesion-dependent growth control in these cells, and expression of constitutively active RhoA or ROCK dysregulated growth. These findings demonstrate the ability of FAK to suppress and promote growth, and underscore the importance of multiple mechanisms, even from one molecule, to control cell proliferation.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1083/jcb.200510062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2064187PMC
July 2006

Simple approach to micropattern cells on common culture substrates by tuning substrate wettability.

Tissue Eng 2004 May-Jun;10(5-6):865-72

Department of Biomedical Engineering, Johns Hopkins University, Baltimore, Maryland 21205-2196, USA.

The ability to spatially control cell adhesion and multicellular organization is critical to many biomedical and tissue-engineering applications. This work describes a straightforward method to micropattern cells onto glass, silicone rubber, and polystyrene using commercially available reagents. An elastomeric polydimethylsiloxane stamp is used to contact-transfer extracellular matrix protein onto a surface followed by blocking cell adhesion in the surrounding regions by the physisorption of Pluronic surfactants. Using self-assembled monolayers of alkanethiols on gold as model surfaces to control surface wettability, we found that protein printing was most effective at intermediate to highly wetting surfaces whereas Pluronic adsorption occurred at intermediate to low wetting surfaces. Within a regimen of intermediate wettability both techniques were applied in conjunction to restrict cell adhesion to specified patterns. Adjusting the wettability of common tissue culture substrates to the same intermediate range again allowed the micropatterning of cells, suggesting that this approach is likely to be generally applicable to many types of materials. This technique therefore may allow for wider adoption of cell patterning.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/1076327041348365DOI Listing
January 2005