Fig. 4

Efficacy of anti-CAIX CAR-T cells on RCC patient derived organotypic tumor spheroids (PDOTS). (A) Schematic of workflow. ccRCC patient tumor specimens were collected, digested and filtered into S1, S2, S3 fractions, in which S2 fraction was used to generate ccRCC PDOTS. On Day −1, PDOTS mixed with collagen were injected into the central channel of the microfluidic device. Quality control (QC) was performed on Day 0 to profile ccRCC TME and determine the viability of PDOTS. The CAR-T cells were added to the side channels and co-incubated with PDOTS for 6 days. And the downstream analysis was performed. (B) Representative images of PDOTS on Day 0. IF was performed to show biomarkers on PDOTS. In the left panel, PDOTS were stained with Hoechst, EpCAM and CD45. In the middle panel, PDOTS were stained with Hoechst, Calcein, CD8, and EpCAM. In the right panel, PDOTS were stained with Hoechst, EpCAM, PI, and CAIX. Scale bars shown in the images represent 20 μm. (C) CAR-T migration was evaluated on Day 6 on PDOTS of a primary ccRCC sample (51321216). By using the ZsGreen fluorescence of CAR-T cells, the signal of middle channel was quantified. Scale bars shown in the images represent 200 μm. (D) CAR-T migration was quantified on PDOTS of a primary ccRCC sample (51321216). G9 (pink), G36 (orange), G250 (green) are shown in the plot. (E) Heatmap of log2 fold change of selected cytokine and chemokine in the supernatant. Cytokine profiling was performed via Luminex using supernatant collected on Day 6 from PDOTS (51321216 RCC sample) and CAR-T co-cultures. Selected cytokines and chemokines are shown here, including IP-10, IFN-γ, GM-CSF, IL-2, TNFβ, IL-15, IFN-α2, and IL-17α. (F) Bar plots of IP-10, and IFN-γ secretion of PDOTS (51,321,216) co-culturing with G9 (pink), G36 (orange), and G250 (green) were shown in the plot. All data with error bars are presented as mean ± SD. P values are defined by unpaired two-tailed t-tests (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; and ∗∗∗∗p < 0.0001)