Fig. 2

Analysis of T cells in multicellular TME model system. (A) Primary activated T cells were cultured in mono-culture (Mono/T cell Mono), or co-culture with established LNCaPs (Co-LN) or 22Rv1 (Co-22) tumor cells, or in tri-culture with established autologous M0 MDM cultures and tumor cells (Tri-LN or Tri-22) for 24 h. Additionally, M0 MDM were also cultured in mono-culture (M0 Mono) for 10 days, and tumor cells were mono-cultured for 4 days (LN Mono or 22 Mono, respectively). (B-E) T cell migration analysis was performed by confocal microscopy (B) condensed z-layers represent T cells stained with DNA Hoechst (blue), CD4 BB515 (green), and CD8 PE (red); (C-E) quantification of images with spot detection. Data represents fold change in T cell migration distance projected over matched T cell mono-culture condition for (C) total T cell population and (D) CD4 or CD8 T cell subsets, individually. (E) Violin plots show ratio of CD4 to CD8 absolute migration distances; (C–E) n = 6; Data represent mean ± SEM. (F) mRNA expression in T cells was interrogated in T cell mono-, tumor co-culture, and tumor-MDM tri-culture conditions. Data expressed as normalized relative quantity (NRQ) as related to house-keeping genes RPLP0 and POLR2A; n = 5. (G,H) Cell culture supernatant was isolated from five unique cellular conditions (T cell Mono and Co- and Tri-cultures for both tumor models) and analyzed for secreted protein expression by multi-analyte bead assay. Data expressed as protein concentration (pg/mL). (G) n = 5, (H) n = 3; One-way ANOVA nonparametric with Friedman post-hoc analysis. Data represent mean ± SEM.