Background Papillary renal cell carcinoma, accounting for 15% of renal cell carcinoma, is a heterogeneous disease consisting of different types of renal cancer, including tumors with indolent, multifocal presentation and solitary tumors with an aggressive, highly lethal phenotype. Little is known about the genetic basis of sporadic papillary renal cell carcinoma; no effective forms of therapy for advanced disease exist. Methods We performed comprehensive molecular characterization utilizing whole-exome sequencing, copy number, mRNA, microRNA, methylation and proteomic analyses of 161 primary papillary renal cell carcinomas. Results Type 1 and Type 2 papillary renal cell carcinomas were found to be different types of renal cancer characterized by specific genetic alterations, with Type 2 further classified into three individual subgroups based on molecular differences that influenced patient survival. MET alterations were associated with Type 1 tumors, whereas Type 2 tumors were characterized by CDKN2A silencing, SETD2 mutations, TFE3 fusions, and increased expression of the NRF2-ARE pathway. A CpG island methylator phenotype (CIMP) was found in a distinct subset of Type 2 papillary renal cell carcinoma characterized by poor survival and mutation of the fumarate hydratase (FH) gene. Conclusions Type 1 and Type 2 papillary renal cell carcinomas are clinically and biologically distinct. Alterations in the MET pathway are associated with Type 1 and activation of the NRF2-ARE pathway with Type 2; CDKN2A loss and CIMP in Type 2 convey a poor prognosis. Furthermore, Type 2 papillary renal cell carcinoma consists of at least 3 subtypes based upon molecular and phenotypic features.

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Past studies using micropatterned substrates coated with adhesive islands of extracellular matrix revealed that capillary endothelial cells can be geometrically switched between growth and apoptosis. Endothelial cells cultured on single islands larger than 1500 microm2 spread and progressed through the cell cycle, whereas cells restricted to areas less than 500 microm2 failed to extend and underwent apoptosis. The present study addressed whether island geometries that constrained cell spreading to intermediate degrees, neither supporting cell growth nor inducing apoptosis, cause cells to differentiate. Endothelial cells cultured on substrates micropatterned with 10-microm-wide lines of fibronectin formed extensive cell-cell contacts and spread to approximately 1000 microm2. Within 72 h, cells shut off both growth and apoptosis programs and underwent differentiation, resulting in the formation of capillary tube-like structures containing a central lumen. Accumulation of extracellular matrix tendrils containing fibronectin and laminin beneath cells and reorganization of platelet endothelial cell adhesion molecule-positive cell-cell junctions along the lengths of the tubes preceded the formation of these structures. Cells cultured on wider (30-microm) lines also formed cell-cell contacts and aligned their actin cytoskeleton, but these cells spread to larger areas (2200 microm2), proliferated, and did not form tubes. Use of micropatterned substrates revealed that altering the geometry of cell spreading can switch endothelial cells among the three major genetic programs that govern angiogenesis-growth, apoptosis and differentiation. The system presented here provides a well-defined adhesive environment in which to further investigate the steps involved in angiogenesis.

Extracellular matrix controls capillary endothelial cell sensitivity to soluble mitogens by binding integrin receptors and thereby activating a chemical signaling response that rapidly integrates with growth factor-induced signaling mechanisms. Here we report that in addition to integrins, growth factor receptors and multiple molecules that transduce signals conveyed by both types of receptors are immobilized on the cytoskeleton (CSK) and spatially integrated within the focal adhesion complex (FAC) at the site of integrin binding. FACs were rapidly induced in round cells and physically isolated from the remainder of the CSK after detergent-extraction using magnetic microbeads coated with fibronectin or a synthetic RGD-containing peptide. Immunofluorescence microscopy revealed that multiple signaling molecules (e.g., pp60csrc, ppl25FAKphosphatidylinositol-3-kinase, phospholipase C-'y, and Na+/H+ antiporter) involved in both integrin and growth factor receptor signaling pathways became associated with the CSK framework of the FAC within 15 min after binding to beads coated with integrin ligands. Recruitment of tyrosine kinases to the FAC was also accompanied by a local increase in tyrosine phosphorylation, as indicated by enhanced phosphotyrosine staining at the site of integrin binding. In contrast, neither recruitment of signaling molecules nor increased phosphotyrosine staining was observed when cells bound to beads coated with a control ligand (acetylated low density lipoprotein) that ligates transmembrane scavenger receptors, but does not induce FAC formation. Western blot analysis confirmed that FACs isolated using RGD-beads were enriched for pp6Ocsrc, pp125FAK, phospholipase G-y, and the Na+ /H+ antiporter when compared with intact CSK or basal cell surface preparations that retained lipid bilayer. Isolated FACs were also greatly enriched for the high affinity fibroblast growth factor receptor flg. Most importantly, isolated FACs continued to exhibit multiple chemical signaling activities in vitro, including protein tyrosine kinase activities (pp60csrc and pp125FAK) as well as the ability to undergo multiple sequential steps in the inositol lipid synthesis cascade. These data suggest that many of the chemical signaling events that are induced by integrins and growth factor receptors in capillary cells may effectively function in a "solid-state" on insoluble CSK scaffolds within the FAC and that the FAC may represent a major site for signal integration between these two regulatory pathways. Future investigations into the biochemical and biophysical basis of signal transduction may be facilitated by this method, which results in isolation of FACs that retain the CSK framework as well as multiple associated chemical signaling activities.

This paper describes a method based on experimentally simple techniques-microcontact printing and micromolding in capillaries-to prepare tissue culture substrates in which both the topology and molecular structure of the interface can be controlled. The method combines optically transparent contoured surfaces with self-assembled monolayers (SAMs) of alkanethiolates on gold to control interfacial characteristics; these tailored interfaces, in turn, control the adsorption of proteins and the attachment of cells. This report describes a simple and general method to fabricate optically transparent surfaces contoured into grooves of defined size and shape and to use self-assembled monolayers (SAMs) of alkanethiolates on gold to control cell attachment to these substrates. We have used SAMs extensively to control the adsorption of proteins and the attachment of mammalian cells to planar surfaces (refs. 1-5; for pioneering work by other groups, see refs. 6-10). By patterning the formation of SAMs using microcontact printing (11, 12)-an experimentally simple and nonphotolithographic technique-into regions that promote or resist the adsorption of protein, the attachment of cells to surfaces could be confined to rows 10-100 ,um wide (M.M., L.E.D., J. Tien, D.E.I., and G.M.W., unpublished results), or to islands, for the attachment of single cells (14). The present work extends this methodology to include control over the topography of surfaces used for cell culture; the method employs an elastomeric stamp having micrometer-scale patterns of relief to mold a thin film of polyurethane and SAMs to control the properties of these contoured surfaces.A number of groups have used contoured surfaces to study the effects of topography on cell alignment, migration, and metabolism (15)(16)(17)(18)(19)(20); this work has demonstrated the importance of substrate topography in controlling the behavior of cells. The procedures used to fabricate the substrates used in these studies have three limitations: (i) The molecular properties of the surfaces are not well-controlled (nor can these properties be tailored easily). (ii) The substrates (silicon) are optically opaque, and attached cells cannot be visualized using conventional light microscopy. (iii) The preparation of the substrates requires photolithographic techniques that are not routinely available in biological laboratories. The methodology described in this report uses more flexible and convenient techniques for microfabrication-microcontact printing (11, 12) and micromolding in capillary channels (21)-to create substrates contoured into grooves and plateaus. The methodology is general in that it allows surfaces having a variety of topologies to be fabricated easily, and it permits control at the molecular scale over the interfacial properties of the substrates.SAMs of alkanethiolates on gold are prepared by immersing a substrate coated with a thin film of gold in an ethanolic solution of a long-chain alkanethiol [HS(CH2)nX, 10 < n < 25]. The sulfur atoms coordinate to th...

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A methylcellulose suspension system that prevents cell-surface contact with the substrate was used to study the role of cell adhesion in the regulation of proliferation. The nonadhesive conditions established by suspension culture cause BALB/c 3T3 (A31) cells to enter a Go state of growth arrest within 48 hr as defined by an inhibition of DNA synthesis and a suppression of c-myc and histone mRNA expression. The adhesion of these suspension-arrested cells rapidly induces c-fos, c-myc, and actin gene expression. This stimulation did not depend on the presence of serum since the adhesion of suspension-arrested cells, in the absence of serum, also induced the expression of c-fos and c-myc mRNAs. In addition, adhesion onto fibronectin increased the number of cells able to respond to epidermal growth factor and insulin and progress into S phase. These results indicate that adhesion of suspensionarrested cells activates the Go/Gj transition independent of growth factors.The proliferation of nontransformed fibroblasts in culture is dependent on adhesion to a solid surface. This property is considered a criterion for normal cell growth and is termed anchorage dependence (1). The work of Penman and coworkers (2-4) has revealed that most macromolecular metabolic processes in normal cells are also dependent on adhesion. Suspension of both 3T3 and 3T6 cells in methylcellulose resulted in an inhibition of RNA production and protein synthesis. The recovery of these processes occurs in a coordinate fashion and is dependent on cell adhesion and extensive spreading (5). The stringent regulation of these metabolic processes during suspension and reattachment is lost as cells become increasingly transformed (6, 7).Adhesion has not been extensively studied as a growth regulatory mechanism. Earlier investigations established a direct correlation between the degree of cell spreading or change in cell shape and growth rate, as measured by DNA synthesis (8)(9)(10)(11)(12)(13). Most of our knowledge of the growth cycle, however, has arisen from studies on the action of soluble growth factors on cells arrested by serum deprivation or by contact inhibition. Activation of such cells initiates the transition from Go into G1 and involves a set of second messenger responses that ultimately alter gene expression (14, 15). The specific mechanisms responsible for integrating the effects of growth factors and cell adhesion during cell growth are unknown. Cellular interactions with particular extracellular matrix (ECM) proteins have been suggested as potential components in processes that regulate gene expression associated with growth and differentiation (16,17). In fact, recent studies have revealed that binding of growth factors during growth activation alters the organization of the cytoskeleton within the adhesion plaque (18).Studies with the growth control systems of serum deprivation and density inhibition cannot adequately address questions relating to adhesion since both systems are dependent on substrate interactions. We have ther...

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