Figure 1.Elevated pressure reduced αROR1-CAR T cell-mediated cytotoxicity in A549 lung cancer cells. (A) A schematic showing the αROR1-CAR T cells in activating cytotoxicity in cancer cells. The structure of the αROR1-CAR is composed of an αROR1 ScFv, CD28 transmembrane domain, a 4-1BB co-stimulatory domain, and a CD3ζ signaling domain. (B) A549 cells maintained in elevated pressure (+100 mmHg) for at least 7 days (2 passages) were co-cultured without or with αROR1-CAR T cells at 1:10 ratio in a pressured incubator for 4 hours. A549 cells were gated according to size (Supplementary Figure 4) and apoptotic (Annexin V+ Propidium iodide -) and dead (Annexin V+ Propidium iodide+) cells were quantified by flow cytometry. (C–D) Quantification and statistical analysis of apoptosis and cell death by 3 biological repeats. P indicates P values and ns indicates no significance. (E) Cytotoxicity assay was performed as in (A) except using cells stably expressing fly luciferase (A549-Red-Fluc) as a convenient readout. Cell viability was measured directly through luciferase activity and the reads were converted to cytotoxicity as mentioned in Methods. (F–H) Cytokines (IL-2, IFN-γ, and TNF-α) in the medium after 16 hours of co-culture were measured by ELISA.
Figure 1 — Pressure increases PD-L1 expression in A549 lung adenocarcinoma cells and causes resistance to anti-ROR1 CAR T cell-mediated cytotoxicity | Aging
