Fig. 2: Adhesion-mediated self-assembly of hybrid tumoroids.

A Representative confocal microscopy maximal z-projection of a DSLB synthetic cell in the process of integrating into a Panc-1 tumoroid. Forming 3D aggregates were fixed 8 hours after co-incubation and stained for membrane (wheat germ agglutinin) and actin (phalloidin) to resolve adhesive interfaces between synthetic and natural cells. The inset shows a magnified area from the synthetic cell contact site. Scale bar is 20 µm. B Representative transmission electron microscopy image of a DSLB (magenta) integrated into a Panc-1 tumoroid showing cellular protrusion (cyan) around the synthetic cell. Scale bar is 500 nm. C Correlation of synthetic cell serum opsonization with synthetic cell integration into Panc-1 tumoroids. Exponential fit is shown in magenta. Results shown as mean ±SD from at least n = 14 tumoroids per condition. D Bright field live cell imaging of a Panc-1 tumoroid with integrated DSLBs. An exemplary synthetic cell is segmented to derive deformation and roundness over time. Scale bar 50 µm. E, F Quantification of the roundness of four synthetic cells from sequences in D over 17 hours of observation (magenta). Two control synthetic cells sitting outside of the tumoroid (gray) shown as a control. P-value calculated by one-way ANOVA. Shown as individual data points for each droplet (n = 17) and the mean value as a black line. G Schematic illustration of the recombinant protein coupling to DSLB-based synthetic cells to induce receptor-ligand interactions with natural cells in tumoroids. H Representative confocal microscopy maximal z-projections of DSLBs integrated into Panc-1 tumoroids without biofunctionalization (top panel) or with conjugated recombinant CD40L ectodomains (bottom panel). CD40 receptor localization in Panc-1 cells was detected by anti-CD40 staining after permeabilization. Scale bar is 10 µm. A, B and H show one representative example from one experiment each in which 10 tumoroids were imaged.