HCS is emerging as an integrated process in HTS to efficiently integrate morphological and phenotypic cellular response into the drug discovery and development process.25–32
Aligning a high-content assay with the correct imaging platform is critical for its development and execution in HTS. The choice of an imaging platform would impact the overall quality of image acquisition for the recognition and quantification of objects by the analysis protocols. Thus, imaging platform validation, if at all feasible and available, would help ease the decision making process by means of comparative studies on different platforms. This certainly was the case for our cell-based assay development process where three imaging platforms the INCA1000, the INCA2000, and the INCA3000, all three available in our laboratory, were evaluated for the purpose of imaging clusters in 384-well microtiter plates. We show that imaging of multiple fields of view or tiles with the INCA1000 (4 fields/tiles) and INCA3000 (9 fields/tiles) were not of sufficient content to capture the entire cluster population distributions as observed by whole-well imaging on the INCA2000 ().
One can argue that tile stitching can easily address the issues of multiple tiles per well resulting from the INCA1000 and the INCA3000 platforms, a process that has been successfully reported for a high-content wound healing siRNA screen involving twelve 96-well microtiter plates where the wound was imaged as 12 distinct tiles using an Applied Precision CellWorx (Cellomics) microscope equipped with a 10
magnifying objective and later stitched as one to observe the entire wound.33
Tile stitching, though feasible in the case of this low-throughput wound healing assay, is hardly achievable in practice and not easily amenable to deal with stitching data from large screening campaigns (up to 400,000 compounds) where as many as 3,711,744 tiles from the INCA3000 would require stitching into 412,416 images for further analysis. Further, the obtained tiles typically cover predetermined areas of the well and the area covered by the different fields may not overlap with the region of interest, and in the case of our assay, stitching separate tiles may not accurately reflect the subtle changes in the KP-transformed phenotype due mainly to our discovery of random and differential growth of the clusters in the wells ().
Our high-content assay using whole-well imaging as a platform provides an attractive alternative to multi-tile imaging and requiring stitching for applications involving cluster forming cells. The most significant advantages over (1) the multi-tile imaging and stitching33
and (2) the manual identification of spheroids and subsequent imaging by phase-contrast10
are the sensing time of 4
s per well, the “on the fly” 2D deconvolution option, and the rapid image analysis using this custom analysis method to quantify reversal as NEF. In addition, our assay recapitulated the observations made from brightfield imaging () for both vatalanib and imatinib on the reduction of cluster formation through inhibiting the PDGFRα kinase function (), exhibited robust and acceptable screening parameters with a calculated Z
′ value of 0.79 and an S/N ratio of 15. Due to the nature of the oncogenic transformation of the NIH-3T3 cells by expression of KP, in this case a fusion protein between PDGFRα and KDR proteins, our assay lived up to its expectations by identifying all those compounds affecting the PDGFRα signaling function, with the exception of SU4312, which was not identified as a reverse of the KP-transformed phenotype and it is believed to be due to its selectivity as a PDGFRβ antagonist.22
Secondary studies confirmed that SU4312 was inactive toward reversing the KP-transformed phenotype (), further emphasizing the sensitivity and selectivity of our optimized assay together with its ability to identify the EGFR inhibitors as inactives. The high-content nature of the assay provide a measure of reversal of the transformed phenotype together with assessment of compound cytotoxicity against the KP cells and seem to overcome the limitations of the low-content viability assays.8–10
Hence, we could identify noncytotoxic compounds reversing the KP-transformed phenotype such as imatinib, sunitinib, sorafenib, DMPQ, vatalanib, and nilotinib that would have been overlooked in a low-content cytotoxicity screen.
In conclusion, we have shown the utility of whole-well imaging and its usefulness in developing and executing cell-based assays; to our knowledge, this is the first example of such a cell-based assay allowing for screening of modulators or reversers of oncogenic-induced transformed phenotype against chemical libraries, and with a broader applicability to RNAi screening and systems biology to study signaling pathways and connectivity nodes in the oncogenically addicted and rewired cells.