Figure 4.

Patients spheroids are responsive to WNT signaling and WNT5A ligand is present in ascites of group A patients. A) Representative images of isolated patient spheroids used in WB in B-C. B) Left, Group A spheroids have PS-DVL that is reduced upon LGK974 treatment and increased after WNT3A and WNT5A stimuli. This is indicative of endogenous WNT secretion and responsiveness to exogenous WNT ligands similarly as Kuramochi cell line. Right, unchanged PS-DVL levels of group B spheroids indicate that these cells did not secrete endogenous WNTs nor responded to activation by exogenous WNT ligands. C-D) WNT5A ligand is present in malignant ascites in group A patients. Non-cellular parts of ascites were fractionized using size exclusion chromatography to remove major serum contaminants such as albumin and immunoglobulins (IgG). SDS-PAGE gels were immunoblotted with WNT5A antibody. The fraction containing the majority of WNT proteins is shown for each ascites. Recombinant WNT proteins are shown as controls and indicators of correct molecular weight (MW), unlike human IgG which has a similar MW which can be mistaken for WNTs if not depleted (false positive signal from secondary antibody). Group B ascites are marked by orange color. Representative image is shown. X - patients excluded from the study. WBs from all ascites were simultaneously processed as seen in Supplementary Fig. S5A, and quantified in S5B and D) Violin plot. Group A ascites contain higher levels of WNT5A protein than Group B ascites. P = 0.0186, Student´s t-test. 0.15 was set as a cut-off value for the analysis of the patient outcome. E) OS of patients with WNT5A high and WNT5A low/negative ascites. F) PFS of patients with WNT5A high and WNT5A low/negative ascites.