Extended Data Fig. 6: Cancer-to-neuron contacts in vitro.
From: Functional synapses between neurons and small cell lung cancer
a) Co-culture of murine cortical neurons (immunolabeled against MAP2) and SCLC cells (COR-L88, expressing DsRed) showing the appearance of dense VGLUT1-positive puncta onto SCLC cells contacted by neuronal terminals b-d) Different views of a 3D-reconstruction of 3D-STED for co-cultures immunolabeled against axonal marker SMI-312, mNeonGreen to mark SCLC cells, dendritic marker MAP2, and postsynaptic marker HOMER1, showing that that the contacts on cancer cells are predominantly axonal. Representative of 3 experiments. e) Co-culture of human iPSC-derived cortical neurons and SCLC cells (COR-L88, expressing tdTomato), immunostained for the pre- and post-synaptic markers BSN and HOMER1. Right panels show a single confocal stack (top) and 3D reconstruction (bottom) of an SCLC cell contacted by a GFP-positive axonal fiber (white arrowheads) exhibiting BSN and HOMER1 co-localizing puncta (yellow arrowheads) located outside and inside the SCLC cell surface. f) Confocal overview of SCLC cells (mNeonGreen + , shown in white) co-cultured with murine nodose ganglia. g) A detailed view of the boxed region from panel f, followed by individual magnified regions, which indicate the arrangements of VGluT1 (presynaptic, neuronal) and HOMER1 (postsynaptic, within SCLC cell) molecules. h) Line scans were drawn automatically across HOMER1 spots, starting in their intensity maxima, and moving towards the periphery. The signal drops, as expected; a similar drop is seen in the VGluT1 signal, confirming their close apposition (N = 4 independent experiments, n = 782 line scans. Colocalization tested using a two-sided Pearson correlation test. Error bars: standard error of the mean). i) Correlative intensity scatter plot of SCLC mNeonGreen signal vs HOMER1 signal (N = 4 independent experiments, N = 782) indicates that a substantial proportion of the HOMER1-marked spots are formed on SCLC cells, and therefore show a measurable mNeonGreen signal (62.02%).
