Combining shRNA Screening With 3D Multiparametric Image Analysis, Emma Shanks, CRUK Beatson Institute, Glasgow

Emma Shanks

The Beatson Institute Screening Facility combines HCS with multiparametric phenotypic image analysis to support translational cancer research. Functional genomics tools including small interfering RNA (siRNA), short hairpin RNA (shRNA) and clustered regularly interspaced short palindromic repeats (CRISPR)-targeting RNA (crRNA) are used in both pooled and arrayed approaches to support target identification and validation in multiple types of cancer. Image-analysis capabilities support identification of a range of cellular phenotypes in 2D (adherent or suspension) and 3D (matrix and spheroid) environments, and tissue sections.

To explore the role of ADP-ribosylation factor (ARF) GTPases, regulators (i.e. GTPase-activating proteins [GAPs] and guanine nucleotide exchange factors [GEFs]) and effectors in the regulation of prostate spheroid polarity, Dr David Bryant and Dr Shanks and their respective teams collaboratively  developed an shRNA 3D spheroid imaging assay to quantify phenotypic alterations in response to functional genomic manipulations. Using a bespoke 40-well plate of 216 shRNAs containing ARF GTPases, regulators and effectors, HEK293FT cells are transfected, and PC3 target cells seeded and then infected. On day 9, cells are split onto Matrigel. One plate is fixed and stained after 3 days then imaged using the Operetta system. For the second plate, spheroid formation is imaged over 6 days prior to fixing, staining and imaging.

3D image analysis examines standard (i.e. area, roundness, width, length) and STAR (smooth, small spikes, long protrusions, spread) morphological properties. These four defined phenotypes are quantified in infected versus non-infected populations to determine if the difference between them is significant, and to look for trends in family members.

For Research Use Only. Not for Use in Diagnostic Procedures.