Figure 5.

EGFR CAR‐T cells activate multiple signalling pathways in TNBC cells. (a) MDA‐MB‐231 cells were mixed with CTL T or EGFR CAR‐T cells at a ratio of 2:1 for 3 days. T cells in suspension were then removed, and the adherent tumor cells were collected, followed by RNA‐seq analysis. Up‐ and down‐regulated genes of CAR‐T cells upon incubation in MDA‐MB‐231 cells (FPKM > 0.5, FC > 1.5) are shown in the pie chart. RNA from three biological replicates was pooled for RNA‐seq. (b, c) Heat map (b) and box plot (c) representation of the expression levels (FPKM, log₂) of the up‐ and down‐regulated genes described in a. P‐values are shown at the top (c). (d, e) GO analysis of up‐ (d) and down‐regulated (e) genes described in a. (f, g) The expression (FPKM, log₂) of representative up‐ (f) and down‐regulated (g) genes of MDA‐MB‐231 cells in response to EGFR CAR‐T cell co‐incubation as detected by RNA‐seq is shown in the heat map. (h, i) UCSC Genome Browser views of representative up‐ (h) and down‐regulated (i) genes detected by RNA‐seq. (j, k) Cells described in a were subjected to RNA extraction and RT‐qPCR analysis to examine the expression of representative up‐ (j) and down‐regulated (k) genes of MDA‐MB‐231 cells. Data were obtained from three replicates and are presented as mean ± s.e.m. (**P < 0.01, ***P < 0.001). (l) MDA‐MB‐231 cells treated with CTL T or EGFR CAR‐T cells were subjected to IB analysis with the indicated antibodies. Molecular weight is indicated on the right. Experiments were repeated three times, and representative blots are shown. 231, MDA‐MB‐231; CAR‐T, chimeric antigen receptor‐modified T cells; chr, chromosome; CTL T, control T cells; E2F1, E2F transcription factor 1; EGFR, epidermal growth factor receptor; Fas, factor‐associated suicide; FC, fold change; FPKM, fragments per kilobase per million; GO, gene ontology; GZMB, granzyme B; IB, immunoblotting; IFNγ, interferon γ; IRF1, interferon regulatory factor 1; MHC, major histocompatibility complex; PARP, poly (ADP‐ribose) polymerase; pJAK1, Janus kinase‐1 phosphorylation; pRb, retinoblastoma protein phosphorylation; pSTAT1, signal transducer and activator of transcription 1 phosphorylation.