Extended Data Fig. 5 |. Mutational and biochemical analysis of DDR1-ECD in vitro and in vivo.

(a) Diagram of full-length (FL) DDR1 (top) and tumour curves of either E0771 Ddr1-WT or KO tumour cells carrying various DDR1 expression vectors: empty vector (EV), FL, deletion of the kinase domain (ΔKD), and extracellular domain (ECD) only. All p values were compared to KO + EV group. TM: transmembrane domain. WT: n = 9 tumours, KO+EV: n = 10 tumours, KO+FL: n = 10 tumours, KO+ ΔKD: n = 6 tumours, KO+ECD: n = 5 tumours. (b) Crystal structure of mouse DDR1 collagen-binding domain, generated by Jmol software (http://www.jmol.org/). Amino acid residues targeted in the mutational analysis are shown. (c) Immunoblots of Flag-tagged mouse WT DDR1-ECD and point mutants ectopically expressed in M-Wnt tumour cells, with GAPDH as the loading control. Images are representatives from three independent experiments. (d) Immunoblots of Flag-tagged mouse WT DDR1-ECD and point mutants ectopically expressed in AT-3 tumour cells, with GAPDH as the loading control. Images are representatives from three independent experiments. (e–f) Growth curves of M-Wnt (e) and AT-3 (f) Ddr1-KO tumours with ectopically expressed mouse WT DDR1-ECD or collagen-binding point mutants. The numbers in parenthesis indicate outgrowing tumours (larger than 100 mm³) versus total injected. (g, h) Immunoblots of full-length DDR1 in cells and soluble ECD in conditioned medium from various mouse (g) and triple-negative human breast cancer cell lines plus ER-positive MCF7 (h). Images are representatives from three independent experiments. (i) Coomassie staining of recombinant Fc-ECD under non-reducing and reducing conditions. (j) Rescue of Ddr1-KO E0771 tumour growth in immunocompetent hosts by recombinant Fc-ECD versus PBS vehicle (n = 6 tumours/group). (k) Diagram of the Transwell assay for CD8⁺ T cell migration. Primary CD8⁺ T cells were loaded in the upper chamber that had been pre-seeded with decellularized ECM derived from tumour cells. The lower chamber contained medium with or without CCL21. (l) CD8⁺ T cells in vitro migration activity was abrogated by decellularized ECM from AT-3 tumour cells in a DDR1-dependent manner. Value of migrated CD8⁺ T cell number without ECM and CCL12 is set at “1” (lanes 1 and 2: n = 3; lanes 3 and 4: n = 7), n refers to technical repeats. Values represent mean ± SEM. p value as indicated, two-tailed Student’s t-test for all tests except for tumour volumes, which were done by two-way ANOVA.