Reversible cell morphology change renders tolerance to therapies in MCL cell lines. (A) Parental Mino-VEN-R cells (DMSO) and giant cells (pirtobrutinib, 10 μM; 5 days); H&E staining (left) and polyploidy analysis (right). Scale bar, 50 μm. (B) After treatment with pirtobrutinib (10 μM; 5 days), Mino-VEN-R cells developed into giant cells. Compared with proliferating parental cells, giant cells went into quiescence with increased protein and DNA quantity in each cell. (C) Apoptosis assay shows that giant cells are resistant to further treatment of pirtobrutinib. (D) Cell proliferation analysis shows halted proliferation of giant cells compared with parental cells in 2D culture. (E) Subcutaneous inoculated giant cells (105 cells) in NSG mice (n = 5) are tumorigenic with delayed progression compared with parental cells (105 cells; n = 8). (F) After pirtobrutinib removal, individual giant cells in 3D culture proliferated and regressed to original size after 11 days (R1). When they were retreated with pirtobrutinib (10 μM; >3 days), each cell developed into giant cells again (G2), and they reproliferated after pirtobrutinib removal for 19 days (R2). Scale bar, 100 μm. (G) Schematic illustration of the ontogeny of reversible pirtobrutinib-tolerant persister MCL cells. Unpaired t test. ∗∗∗P < .001; ∗∗∗∗P < .0001. Parental Mino-VEN-R (P; without pirtobrutinib) underwent morphology change to generate giant first (G1; 10 μM pirtobrutinib), reproliferated first (R1; pirtobrutinib removed), giant second (G2; 10 μM pirtobrutinib), and reproliferated second (R2; pirtobrutinib removed). DMSO, dimethyl sulfoxide; H&E, hematoxylin and eosin; ns, nonsignificant.
