RET‐Dependent Axonal Sprouting from Spinal Afferent Neurons in a Pancreatic Cancer Model

Abstract

ObjectiveActivation of the RET receptor tyrosine kinase stimulates signaling pathways associated with cell proliferation, growth, differentiation, and survival. Changes in the expression of this receptor and its associated ligands have been linked to tumorigenesis in several tumor types, including pancreatic cancer. It has been established that tumor cells can migrate towards and invade cells of the nervous system, a process called perineural invasion that is associated with metastasis and reduced survival. Given that RET ligands play important roles in spinal afferent neuronal development and morphology, we hypothesized that the release of RET ligands by pancreatic cancer cells has a neurotrophic effect on spinal afferent neurons, drawing their axons towards the tumor and facilitating perineural invasion.MethodsThoraco‐lumbar (T10‐L2) dorsal root ganglion (DRG) neurons from male C57BL/6 mice were collected, dissociated, and cultured for 4 days in F12 media along with a 1:3 dilution of conditioned media from two pancreatic cancer cell lines (PanC1 and MiaPaCa2) or a cell‐free media control (F12 + 1:3 DMEM). In a separate set of experiments, DRG neurons were exposed to the same conditioned media along with the RET inhibitor, Selpercatinib (5 µM, diluted in DMSO). For the RET inhibitor experiments two control groups were used, the 1st contained F12 media + 1:3 DMEM + DMSO (vehicle controls, VC), and the 2nd group of controls consisted of F12 media + 1:3 DMEM + Selpercatinib (VC + Selp). Neurons were stained with calcein‐AM (1 µM) and visualized using a fluorescent microscope. Changes in DRG neuron morphology (neurite length and branching relative to distance from cell soma) were quantified using Sholl analysis.ResultsSholl plots revealed significant changes in DRG neuron morphology following treatment with either PanC1 or MiaPaCa2 conditioned media compared to DRG neurons treated with the RET inhibitor, or the different control groups. Where DRG neurons incubated with PanC1 conditioned media demonstrated an approximate 3 to 4‐fold increase in neurite length compared to VC and VC + Selp (229 ± 32.7 µm vs 74.7 ± 12.2 µm vs 50.2 ± 6.3 µm; respectively). These effects on neurite length were significantly diminished following treatment of the DRG neurons with PanC1 + Selp (116.6 ± 15.5 µm). Furthermore, treatment of DRG neurons with the PanC1 conditioned media led to a 2‐fold increase in the maximum branching of neurites (10.4 ± 2.1 intersections; n=36) compared to VCs (5.75 ± 1.3 intersections; n=35). Again, these effects were significantly diminished following treatment of DRG neurons with PanC1 conditioned media + Selp (3.75 ± 0.6 intersections; n=40) (Kruskal‐Wallis with Dunn’s post‐hoc multiple comparison test, p<0.001). Similar RET‐dependent effects were observed for DRG neurons treated with conditioned media from the MiaPaCa2 cell line.ConclusionThese data suggest that pancreatic cell lines release RET ligands that may attract axons of spinal afferent neurons towards them, which may facilitate perineural invasion.