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. 2022 Apr 28;12:6919. doi: 10.1038/s41598-022-10905-6

Figure 1.

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 h. A549 cells were gated according to size (Fig. S4) 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 h of co-culture were measured by ELISA.