![]() ![]() Control (Scramble shRNA-expressing, Scr) 3D acini could be distinguished from non-shRNA-expressing acini by the presence ( Fig. We developed a high-throughput, arrayed, live imaging-based screening approach to determine the 3D phenotype of ARFome component depletion on multi-day morphogenesis. We used these PC3 acini to examine ARFome contribution to 3D morphogenesis as (i) they have high levels of all ARF GTPases, (ii) they, upon intraprostatic xenograft, provide a model for metastatic tumorigenesis, and (iii) we have shown that they can be used to identify ARF GTPase modules that regulate 3D invasion, in vivo metastasis, and predict patient survival ( Nacke et al., 2021). When PC3 cells were plated on a thin coat of ECM as a suspension of single cells in low percentage ECM-containing medium, they formed heterogenous multicellular structures polarized around a central lumen, which we termed acini ( Freckmann et al., 2022 Nacke et al., 2021). PC3 cells also expressed almost all components of the ARFome ( Fig. S1, A–G), particularly in 3D compared to 2D culture ( Fig. Examination of ARF GTPase expression across nine prostate cancer cell lines indicated that metastatic PC3 cancer cells showed high expression levels of all ARF GTPases compared to normal prostate cells (RWPE-1, PRECLH Fig. ![]() 1 B), and generated a highly validated library targeting all ARFs, GEFs, GAPs, and 72 known interactors ( Fig. We engineered a lentiviral system that co-encodes an shRNA and membrane-targeted mVenus (mem:Venus) fluorescent protein to transduced cells ( Fig. We interrogated the functional contribution of ARF GTPases, their GEFs, GAPs, and known interactors and effectors, which we term the “ARFome,” to cancer cell morphogenesis ( Fig. Our analysis defines a unique function for the ARF3 GTPase in controlling how cells collectively organize during invasion and metastasis. In vivo, ARF3 levels acted as a rheostat for metastasis from intraprostatic tumor transplants and ARF3/N-cadherin expression can be used to identify prostate cancer patients with metastatic, poor-outcome disease. Functionally, the ability of ARF3 to control invasion modality is dependent on association and subsequent control of turnover of N-cadherin. This revealed that ARF3 GTPase regulates the modality of invasion, acting as a switch between leader cell-led chains of invasion or collective sheet movement. Through a functional genomic screen of three-dimensional (3D) prostate cancer cell behavior, we explore the contribution of ARF GTPases, GEFs, GAPs, and interactors to collective invasion. Unraveling their function is complicated by the overlapping association of ARFs with guanine nucleotide exchange factors (GEFs), GTPase-activating proteins (GAPs), and numerous interactors. ARF GTPases are central regulators of membrane trafficking that control local membrane identity and remodeling facilitating vesicle formation. ![]()
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