Fig. 5: Microdroplet size and volume fraction regulate invasion and migration mode.

a Schematic representation of the organo-hydrogels (OHGs) with varying microdroplet diameter and volume fraction. b Storage modulus of collagen-based OHG with distinct microdroplet size (n = 4 gels; average ± s.e.m.). The dashed line shows the predicted trend. c Normalised stress-relaxation curves of collagen-based OHG with distinct microdroplet size (n = 4 gels). d BODIPY (green) and F-actin (red) and nuclei (blue) staining of OVCAR8 cells after 7 d in collagen-based OHG of 70 or 25 μm diameter microdroplets (representative images from n = 13 spheroids). Arrowheads indicate cells at the invasive front. e Quantification of relative OVCAR8 spheroid area in collagen-based OHG of distinct emulsion percentage of volume fraction and microdroplet diameter after 7 d in culture (n = 13 spheroids). f BODIPY (green) and mRFP (red) staining of mRFP-OVCAR8 cells after 7 d invasion into adipose peritoneal tissue explants (representative images from n = 3 explants per donor). g Correlation between patient average adipocyte diameter and invaded OVCAR8 cells after 7 d, at 42 μm tissue depth, relative to number of cells at the tissue surface (n = 6 donors; average ± s.e.m.). The dashed line shows the predicted trend. h Quantification of interdroplet distance in OHG (n = 100 microdroplets). i Quantification of the percentage of invasive front OVCAR8 cells in contact with microdroplets in OHG (n = 121 cells in 3 gels). j Quantification of percentage of Ki67 + cells, immunofluorescence signal intensity of cytoplasmic pFAK and pMLC relative to collagen average, and nuclear:cytoplasmic ratio of YAP/TAZ in OVCAR8 cells embedded in OHG of 70 or 25 μm diameter microdroplets for 24 h (n = 55 cells). k F-actin (green) and DAPI (blue) staining of CAOV3 spheroids embedded for 7 d in collagen-based 25 μm microdroplet OHG in the presence or absence of TGFβ. Arrowheads indicate invading cells (representative images from n = 6 spheroids). l Quantification of CAOV3 spheroid area relative to non-TGFβ-treated control after 7 d in culture (n = 6 spheroids). m Schematic illustration of the cell force-dependent invasion of adipose tissue enabled by the anisotropic mechanics of OHGs and the generation of migration tracks at the ECM-adipocyte/microdroplet interface. Arrows indicate the direction of the force. For the data in (h, i, j and l), a two-sided unpaired t test was performed. Coefficient of determination and Pearson correlation (two-tailed test) were performed in (g) to determine the relationship between tissue adipocyte diameter and OVCAR8 invasion. Error bars in (b) represent the s.e.m. Scale bars, 50 μm (bottom panels in d, k), 100 μm (f), 200 μm (top panels in d, k). Source data are provided as a Source Data file.