Fig 2: Tumor Cathepsin L mediates extracellular payload release of T-DXd in tumor microenvironment, contributing to therapeutic efficacy in HER2-low BC.a, b Au565 cells were assessed for T-DXd (a) or T-DM1 (b) cytotoxicity in vitro in the presence of CTSL inhibitor (3 µM Z-Phe-Phe-FMK). c Au565 cells overexpressing CTSL or lacking CTSL were assessed for T-DXd cytotoxicity in vitro. d Extracellular CTSL secretion by MDA-MB-231 lines was quantified with ELISA analysis of conditioned media. e Secreted proteins in conditioned media of indicated cell lines were concentrated and quantified for CTSL enzymatic activity in assay buffer. a–e Two-way ANOVA with Tukey’s multiple comparisons test (n = 3 per group). Nonlinear regression curve fit to calculate IC50 values. f Parental and CTSL-overexpressed MDA-MB-231 cells were implanted in SCID mice, and treated with T-DXd (5 mg/kg) or control PBS. g Control-KO and CTSL-KO MDA-MB-231 cells were implanted in SCID mice, and treated with T-DXd (10 mg/kg) or control PBS. f–h Mixed-effects analysis (REML) with Tukey’s multiple comparisons (n = 10). Arrows indicate time of treatments administered. h Tumor sizes (mm3) measured at end point for studies using MDA-MB-231 CTSL-KO lines. i Immunohistochemistry verifications of CTSL expression in parental or CTSL-modified MDA-MB-231 tumors. j LC-MS quantification of DXd in MDA-MB-231 parental (n = 15) versus CTSL-overexpressed tumors (n = 10), treated with T-DXd (10 mg/kg) for 5 days. k LC-MS quantification of DXd in MDA-MB-231 control-KO versus three different CTSL-KO pools, treated with T-DXd (10 mg/kg) for 3 days (n = 10 for all groups, except n = 6 for CTSL-KO #2). h, k One-way ANOVA with Tukey’s multiple comparisons. j Two-sided Mann–Whitney test. All data is presented as mean ± SEM with p values indicated.

Fig 3: Validation of the expression of PLOD2 and CTSL in keratocytes subjected to 12% strain. (A) Keratocytes were subjected to 12% strain for 48 hours. Protein expression level of PLOD2 and CTSL was assessed by Western blot. β-Actin served as a loading control for total protein. Representative image from one of three independent Western blot experiments conducted using samples from three different donors. (B, C) Densitometry analysis of the Western blot bands of PLOD2 and CTSL. n = 3. The densitometry of β-actin bands served as the normalization reference. Statistical analyses were conducted by an unpaired t-test. Data are presented as mean ± SD and normalized to the ctrl group. *P < 0.05, **P < 0.01.