Fig. 3

MDM analysis in multicellular TME model system. (A) Primary M0 MDMs were cultured in mono-culture MDM mono (M0) for 10 days, co-culture with established LNCaPs (Co-LN) or 22Rv1 (Co-22) cultures for 3 days, and tri-culture with autologous T cell cultures and established tumor cells (Tri-LN or Tri-22) for 24 h. (B) mRNA expression in MDMs was interrogated in MDM mono-, tumor co-culture, and tumor-MDM tri-culture conditions. Data represents fold change of NRQ expression as related to house-keeping genes RPLP0 and POLR2A over matched MDM mono-culture levels (dashed line). Statistical analysis performed by One-way ANOVA nonparametric with Friedman post-hoc analysis n = 6. (C) Data represent secreted protein levels (pg/mL) using a multi-analyte bead assay. Statistical analysis was performed using One-way ANOVA nonparametric with Friedman post-hoc analysis comparing the mean rank of each column with the rank of every other column; n = 3; (D) CD86 surface protein expression was measured by ‘in-chip’ immunofluorescent staining and subsequent confocal microscopy in microdevice wells; CD86 Brilliant Blue 515 (BB5151; green) and Hoechst (blue). Representative cross-section images with (bottom row) or without (top row) anti-CD86 antibody channel shown as two-channel overlay. (E) Mean Fluorescent Intensity Ratio of CD86 binding of CD86⁺ cells projected to the nuclear Hoechst fluorescence mean intensity of all cells, for entire microwells in XYZ for each condition, in replicate. Cells were identified using NIS-Elements spot detection. Ordinary one-way ANOVA with post-hoc analysis comparing the mean rank of each column with the rank of every other column; n = 3; Data represent mean ± SEM.