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In pancreatic cancer, neural invasion is one of the most common paths of cancer dissemination. Classically, cancer cells actively invade nerves and cause local recurrence and pain. Three-dimensional (3D) neural migration assay has become a standard tool for scientists to study neural invasion by confronting the involved cell types. This protocol introduces Schwann cells, i.e., the most prevalent cell type in peripheral nerves, in a novel heterotypic, glia-cancer-neuron, 3D migration assay for assessing their relevance in the early pathogenesis of neural invasion. Particularly, this assay allows the monitoring of the early Schwann cell migratory activity.

Cell-to-extracellular matrix adhesion is regulated by a multitude of pathways initiated distally to the core cell-matrix adhesion machinery, such as via growth factor signaling. In contrast to these extrinsically sourced pathways, we now identify a regulatory pathway that is intrinsic to the core adhesion machinery, providing an internal regulatory feedback loop to fine tune adhesion levels. This autoinhibitory negative feedback loop is initiated by cell adhesion to vitronectin, leading to PAK4 activation, which in turn limits total cell-vitronectin adhesion strength. Specifically, we show that PAK4 is activated by cell attachment to vitronectin as mediated by PAK4 binding partner integrin αvβ5, and that active PAK4 induces accelerated integrin αvβ5 turnover within adhesion complexes. Accelerated integrin turnover is associated with additional PAK4-mediated effects, including inhibited integrin αvβ5 clustering, reduced integrin to F-actin connectivity and perturbed adhesion complex maturation. These specific outcomes are ultimately associated with reduced cell adhesion strength and increased cell motility. We thus demonstrate a novel mechanism deployed by cells to tune cell adhesion levels through the autoinhibitory regulation of integrin adhesion.