Extended Data Fig. 5: Effect of culture media exchange and cancer cell co-culture on neuron viability and activity.

(a) Representative images of RealDRG neurons (stained with NeuO) after 72 h in standard sensory neuron media (Senso-MM) followed by different treatments: no media exchange, fresh media exchange, co-culture with MOC1 squamous cell carcinoma cells, or culture in MOC1-conditioned media for an additional 72 h. Initial seeding density: 0.15 × 10⁵ cells/well in poly-l-ornithine and iMatrix-511 coated 8-well ibidi plates. (b) Bar plot showing the viability of NeuO+ neurons, normalized to the “No media change” control. Neuronal viability was reduced only in the co-culture group. Data are represented as mean ± s.e.m., based on 6 biologically independent samples for each group. (c) Neurite analysis pipeline demonstration showing the methodology for quantifying neuronal morphology. Representative multichannel fluorescence images demonstrate the automated analysis approach using the Neurite Outgrowth Module in Gen5 Cytation 7 software, with neurons identified by neurofilament heavy chain (NFH) staining and DAPI nuclear labelling. (d) Representative monochrome immunofluorescence images of neurofilament heavy chain (NFH) staining showing neuronal morphology under different experimental conditions used for neurite metric quantification. Images were acquired using an Agilent BioTek Cytation 5 cell imaging multimode reader at 20x magnification as 12 × 12 image montages from the centre of each well. Upper row: Fresh media, no media change, B16 melanoma-conditioned media, and co-culture with B16 melanoma cells. Lower row: Fresh media, no media change, MIA PaCa-2-conditioned media, and co-culture with MIA PaCa-2 cells. Scale bars = 100 μm. (e) Quantification of average neurite length showing a trending decrease in B16 melanoma co-culture conditions (left, p = 0.054) and no significant difference in MIA PaCa-2 (PDAC) co-culture (right, p = 0.606). Data represent mean ± standard deviation from a minimum of 10 regions of interest per condition. Analysis performed using automated neurite outgrowth module with short ending branches <5 μm excluded. (f) Quantification of neurite thickness (calculated as NFH+ area divided by neurite length) showing a significant decrease in both B16 melanoma (left, p = 0.006) and MIA PaCa-2 (right, p = 0.014) co-culture conditions compared to controls. Data represent mean ± standard deviation from a minimum of 10 regions of interest per condition, with duplicate wells per condition. (g) Basal spontaneous firing rate of iPSC-derived sensory neurons assessed by multi-microelectrode array (MEA). After 10 days in normal culture, mature, firing neurons were cultured for 6 days under control conditions, stressed conditions (no media exchange), in MOC1-conditioned media, or co-cultured with MOC1 cells. Spontaneous firing rates were recorded at baseline (day 10), 24 h (day 11), and 72 h (day 13) after applying experimental conditions. Data are expressed as mean ± s.e.m. derived from two biologically independent samples for each group. Statistical significance was assessed using one-way ANOVA (p = 0.678 at baseline, p = 0.241 at 24 h, p = 0.302 at 72 h). (h) Mean firing rate of iPSC-derived sensory neurons co-cultured with MIA PaCa-2 cells over 5 days, measured by MEA. RealDRGs were plated at 20 K cells/well (n = 3 biologically independent samples, left panel) and 50 K cells/well (n = 2 biologically independent samples, right panel) on 24-well CytoView MEA plates, with media changes every 48 h. After 10 days in normal culture establishing stable spontaneous activity, mature neurons were cultured for 6 days under control conditions, stressed conditions (no media exchange), in MIA PaCa-2-conditioned media, or in MIA PaCa-2 co-culture. Spontaneous functional activity was recorded for a minimum of 10 min every 24 h from day 10 through day 15. Data are presented as mean ± s.e.m.