Journal: Cancer Communications

Article Title: Targeting autophagy overcomes cancer‐intrinsic resistance to CAR‐T immunotherapy in B‐cell malignancies

doi: 10.1002/cac2.12525

Figure Lengend Snippet: Autophagy protects B‐cell malignancies from CAR‐T cell‐mediated cytotoxicity in vitro. (A) Western blotting showing the expression levels of p62 and LC3B proteins 72 hours after the addition of vehicle (as control), rapamycin, SAR405 and autophinib in Nalm6 and Raji cell lines. GAPDH was used as a loading control. (B‐C) Representative images of LC3B immunofluorescent staining of Nalm6 and Raji cells treated with vehicle (as control), rapamycin, SAR405 and autophinib. Number of LC3B puncta per cell was compared between the three groups ( n = 6). Values are shown as the mean ± SD. Differences among groups were calculated with one‐way ANOVA tests. (D) Cytotoxic analysis of vehicle, rapamycin, SAR405 and autophinib pre‐treated Nalm6 and Raji cell lines when co‐cultured with CD19 CAR‐T cells at different E:T ratios (0:1, 1:4, 1:2, 1:1) at 37 °C for 24 hours ( n = 3). Values are shown as the mean ± SD. Differences among groups were calculated with two‐way ANOVA tests. (E) Western blotting showing the expression levels of three autophagy‐related (ATG3, BECN1 and RB1CC1) proteins in the sgControl and indicated gene‐KO groups in Nalm6 and Raji cell lines. GAPDH was used as a loading control. (F) Western blotting showing the expression levels of LC3B proteins in the sgControl and indicated gene‐KO groups in Nalm6 and Raji cell lines. GAPDH was used as a loading control. (G‐H) Representative images of LC3B immunofluorescent staining of Nalm6 and Raji cells with sgControl and indicated gene‐KO. Number of LC3B puncta per cell was compared between the groups ( n = 6). Values are shown as the mean ± SD. Differences among groups were calculated with one‐way ANOVA tests. (I‐J) Flow cytometry showing the level of CD19 expression in negative control, sgControl and indicated gene‐KO Nalm6 and Raji cell lines. (K) Cytotoxic analysis of sgControl and indicated gene KO Nalm6 and Raji cells co‐cultured with CD19 CAR‐T cells (E:T ratio = 1:4) at 37 °C for 24 hours ( n = 3). Values are shown as the mean ± SD. Statistical differences are calculated with one‐way ANOVA with tests. * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0,001; ns: not significant. Abbreviations: ANOVA analysis of variance; CAR‐T chimeric antigen receptor T; E:T effector: target; KO knockout; ns: not significant; SD standard deviation; sgRNA single‐guide RNA.

Article Snippet: The CD3 (Proteintech, 60181‐1‐Ig, 1:200, Rosemont, USA), LC3B (Cell Signaling, #43566, 1:6,000, Boston, IL, USA), PD‐1 (Cell Signaling, #86163, 1:200, Boston, IL, USA), CD69 (Proteintech, 10803‐1‐AP, 1:100, Rosemont, USA), dsDNA (Santa Cruz Biotechnology, sc‐58749, 1:100, Dallas, USA) antibodies were used for immunofluorescence staining.

Techniques: In Vitro, Western Blot, Expressing, Control, Staining, Cell Culture, Flow Cytometry, Negative Control, Knock-Out, Standard Deviation