MicroScale: miniaturizing cell arrays for macrogenetic screens

Selective RAF inhibitors, including vemurafenib, have unprecedented clinical activity in patients with BRAFmutant melanoma (Chapman et al., 2011). Although de novo resistance is observed in only a minority of patients, drug resistance invariably develops typically within the first year of treatment. Defining the molecular basis of drug resistance is thus critical; as such, efforts may facilitate the development of combination strategies that delay or prevent the emergence of resistant clones. In a recent report by Wood et al. (2012), the authors describe the development and validation of a novel, highthroughput platform for performing functional genomic screens using adherent human cancer cell lines. The platform, which the authors name Microarrays of Spatially Confined Adhesive Lentiviral Features (MicroSCALE), utilizes advanced surface engineering, lentiviral vector generation, and microarray technology to develop a miniaturized cell microarray that is high throughput and scalable. Specifically, the authors use polyacrylamide hydrogel-coated glass slides that are spotted with gelatin to spatially confine the growth of adherent human cancer cell lines (See Figure 1). The polyacrylamide hydrogel-coating functions as a cell adhesion-resistant substrate that restricts growth of the cancer cells to only those areas spotted with gelatin. Open reading frame (ORF) or shRNA lentiviral libraries are then printed directly on top of the gelatin cell adhesive material. This allows for restricted lentiviral-mediated delivery of the ORF or shRNAs to cells growing on a small, spatially confined location on the glass slide. As a standard microarray chip reader can be used to interrogate the MicroScale arrays, multiple readings can be performed using this approach allowing for a dynamic assessment of a perturbation such as drug exposure over multiple extended time points. Furthermore, as the cells are seeded on glass slides, the option of examining particular transduced spots for changes in cell phenotypes (for example morphology) may also be performed using high-content microscopy. The MicroSCALE arrays can thus substitute for multi-well plates to allow for more efficient gainor loss-of-function screens using adherent cancer cell lines. To validate the potential utility of this methodology, the authors screened for kinases that when expressed in BRAF-mutant melanoma cells attenuated RAF and/or MEK dependence. Consistent with prior studies, they find that oncogenic RAS (both HRAS and KRAS), MEK kinase kinases, including CRAF, MAP3K8 (which encodes COT) and MOS, and activating MEK1 mutants conferred resistance to RAF inhibition. Notably, expression of PDGFRb and IGF1R, two receptor tyrosine kinases that have been associated in prior studies with acquired resistance to vemurafenib did not score in the screen. The authors speculate that the failure of these genes to score in the assay may have been the result of an absence of ligand or other stromally derived or cell intrinsic factors. In addition to the previously defined resistance factors discussed above the MicroSCALE screen also identified multiple SRC family kinases and several nuclear factor jB (NF-jB) pathway members as genes which when overexpressed conferred resistance to RAF and MEK inhibitors. The latter finding is notable, as dysregulation of the NF-jB pathway was recently shown to modulate the sensitivity of lung cancer cells with activating mutations in EGFR to selective inhibitors of this kinase (Bivona et al., 2011). The authors proceed to