Bio agar for cyto inclusion

Samples undergoing immunofluorescence were either FFPE tumor specimens or growing agnospheres or colosphere mCRC729. The latter were harvested, fixed 10 min with PFA 4% at 4 °C, washed in PBS, suspended in bio-agar for cyto-inclusion (Bio-Optica) at 42 °C, and processed for inclusion in paraffin. All staining were performed as previously described^{67 (link)}. Images were acquired using a LEICA SPEII confocal microscope, equipped with a 40× oil objective and a 1.5× zoom for a final magnification of 600×. Optical single sections were acquired with a scanning mode format of 1024 × 1024 pixels. Fluorochrome unmixing was performed by acquisition of automated-sequential collection of multichannel images, to reduce spectral crosstalk between channels. For immunohistochemical staining, an additional peroxidase blocking was performed in H₂O₂ 3% methanol 50% incubated 20 min in the dark. For primary and secondary antibody concentrations, see reagents. Secondary antibodies were HRP-conjugated (Dako), and DAB substrate chromogen kit (Dako) was used for detection. Nuclei were counterstained with hematoxylin. Images were acquired through LASV4.2 software and are representative of at least three independent immunostainings.

In situ proximity ligation assay (PLA) assays were performed using the Duolink™ In Situ Red Starter Kit Mouse/Rabbit (Sigma) according to the manufacturer's instructions. Briefly, m‐colospheres were harvested, fixed 10 min with PFA 4% at 4 °C, washed in PBS, suspended in bio‐agar for cyto‐inclusion (Bio‐Optica) at 42 °C, paraffin‐embedded and sectioned as previously described [19 (link)]. Sections were incubated with primary anti‐EGFR, anti‐ERBB2, and anti‐ERBB3 (Table S2) at 4 °C overnight followed by species‐specific secondary antibodies conjugated with oligonucleotides (PLA probes). Negative control was performed by the sole EGFR primary antibody. After ligation and amplification, the signal from each pair of PLA probes in close proximity (< 40 nm) was visualized as an individual red spot and analyzed by LEICA SPEII confocal microscope and lasv software. PLA Multicolor Reagent Pack PLA kits DUO96000 and In Situ Red Starter Kit Mouse/Rabbit DUO92101 were purchased from Sigma‐Aldrich, Duolink™.