A combinational approach of high throughput 3D screening and “Cloudbuster 2.0” analysis reveals a novel interplay between cell migratory signaling pathways.

Session

Poster Session

Authors

Sabine Knipp (1, 2), Arndt Rohwedder (1), Anke Brüning-Richardson (2)

Affiliations

1. Johannes Kepler University Linz, Zentrum für Medizinische Forschung, Linz
2. University of Huddersfield, School of Applied Sciences, Huddersfield

Keywords

Cell migration; three-dimensional spheroids; three-dimensional imaging; point cloud quantification; image analysis software; open source;

Abstract text

We are first to report a so far unknown interplay between migratory signaling pathways of RhoGTPase activating proteins (ARHGAPs) and a member of the SH3 and multiple ankyrin repeat domains protein family (SHANK2). Data resulted from a high throughput analysis with an improved version of the python based open source application “Cloudbuster” (Rohwedder et al. 2022) of three dimensional (3D) glioblastoma cell spheroids growing in a collagen matrix.

The role of ARHGAPs as regulators of cell migration via activation of small GTPases is well established. SHANK proteins are so far mainly studied for their function as scaffolding proteins in the post-synaptic density of neuronal cells and are linked to multiple neurodevelopmental disorders including autism spectrum disorders. However, more recent studies highlight their ability to affect cell migration, e.g. via regulation of integrin mediated cell-matrix adhesion through interaction with small GTPases (Lilja et al. 2017). We recently developed the python based open source application “Cloudbuster” to allow the analysis of data generated in a 3D setting to obtain information on cell migratory vectors and spheroid shape changes over time, e.g. cell extension formation or cell number and distance covered by migratory cells. Our “Cloudbuster 2.0” version is now augmented with a shape recognition function for best fit of differentially growing spheroids, and an AI assisted descriptive statistics including a “most interesting” (= most diverging) parameter hit list. Both versions are suitable for high throughput automated analysis with an economical usage of computer resources.

We used a simple approach of different combinations of double and single knockdowns of ARHGAPs and SHANK2 respectively and analysed the effects on cell invasive behaviour of glioblastoma (GBM) cell lines grown as spheroids in a 3D collagen matrix. We next performed a high throughput analysis of the 3D spheroid invasion assay using the improved application version “Cloudbuster 2.0”. Strikingly, individual double knockdown combinations of different ARHGAPs with SHANK2 can either reinforce or reverse the observed single knockdown effects.

Our approach of combining high throughput screening in 3D and automated analysis with the improved “Cloudbuster 2.0” therefore allowed us to elucidate a so far uncharted interplay between known regulators of cell migration, the ARHGAPs and SHANK scaffolding proteins. This will open up new avenues to study migratory and invasive behaviour in development and disease, especially cancer cell migration.

References

Rohwedder, A. et al. (2022), Interface Focus 12: 20220016;

Lilja J, et al. (2017) Nat Cell Biol;19:292-305;