Fig 2: Tyrosine kinase-inactive DDR2 partially rescues lung metastasis of tumor cells in vivo. (A) Lysates from the indicated 4T1 cell lines (shDdr2, DDR2 depleted; rescue EV, empty vector control) were immunoblotted using the indicated antibodies. ß-actin is shown as a loading control. Molecular masses are shown in kDa. Blots are representative of three experiments. (B) Time to end-stage tumors (2 cm diameter) for the indicated orthotopically transplanted 4T1 tumor cells. Each symbol represents an individual mouse. Mean and s.e.m. are indicated. (C) Lung metastases of mice orthotopically transplanted with the indicated 4T1 tumor cells. Data are represented as lung metastases (Lung Mets) per lobe, averaged over three step-sections of at least 50 µm. Metastases were only counted if more than ten tumor cells were present. Each symbol represents an individual mouse. Mean and s.e.m. are indicated. A minimum of 5 animals (shSCR) and a maximum of 16 animals (shDdr2) were used. *P<0.05; ***P<0.001; ****P<0.0001; ns, not significant (one-way ANOVA with Tukey's post-hoc test).

Fig 3: Ddr2 expression is up-regulated and DDR2 activation increases in MLin cells after injury.(A) TSNE plot visualizing DDR2 signaling score generated from a list of genes shown to be influenced by Ddr2 activation. (B) snATAC sequencing visualization of Ddr2 and surrounding gene tracks showing the proportion of reads in Pdgfra-expressing MLin cells 1 week after BT. Arrow labels TSS. (C) Representative confocal images (63×) of uninjured tendon and injury site 1 week after BT in Ddr2-LacZ reporter mice with quantification of the number of ß-galactosidase+ cells per image (n = 3 mice per group, n = 2 to 3 images per mouse). The yellow box shows inset region. The region within white outlines is the Achilles tendon. Scale bars on 63× and tile images are 100 and 1000 µm, respectively. (D) Representative confocal images (63×) of uninjured tendon and injury site 1 week after BT in wild-type C57B6 mice with quantification of PDGFRa+ and DDR2+ cells per image (n = 3 mice per group, n = 3 images per mouse). Scale bars, 100 µm. (E) Representative confocal images (63×) of uninjured tendon and injury site 1 week after BT in Ddr2-LacZ mice with quantification of the percentage of ß-galactosidase+ cells with pDDR2+ staining per image (n = 3 mice per group, n = 3 images per mouse). Scale bars, 50 µm. (F) Representative confocal images (20×) of uninjured human tendon and mature human HO tissue stained with Hoechst, anti-PDGFRa, and anti-phosphorylated DDR2 (pDDR2). Arrows show PDGFRa+ pDDR2+ cells in peritenon adjacent to uninjured tendon and within and on mature HO tissue. Scale bars, 100 µm. All graphs show means ± SEM. All individual channel images are from the areas within yellow dashes.

Fig 4: Tyrosine kinase-independent DDR2 signaling in CAFs supports tumor lung colonization in vivo. (A) Isolated syngeneic (FVB/N) PyMT tumor cells (7.5×105) were co-injected with CAFs (FVB/N) of indicated genotypes (1×105) in the tail vein of FVB/N female mice. After 7 days, lungs were harvested, and metastases (Mets) were counted in lung slices stained with hemotoxylin and eosin, as described in Materials and Methods. For each experimental condition, four mice were analyzed. The mean±s.e.m. is indicated. (B) PyMT tumor cells (7.5×105) were co-injected with CAFs of the indicated genotypes (1×105), and lung slices were analyzed as described in A. For each condition, five mice were analyzed. Data are mean±s.e.m. *P<0.05; **P<0.01; ***P<0.001 (one-way ANOVA with Tukey's post-hoc test). (C) To determine whether CAFs were present in lung colonization sites after co-injection, we generated RFP-positive (RFP+) WT mouse CAFs and co-injected them with Bo1 FVB/N mouse mammary tumor cells at the same 7:1 ratio as used in A and B, using 7×105 tumor cells. After 48 h or 7 d, lungs were collected and processed as described in the Materials and Methods. Immunofluorescence was performed on formalin-fixed paraffin-embedded lung sections using anti-RFP antibodies. The total number of RFP+ cells was counted per field of view (FOV) obtained using a 10× objective. n=8 FOVs. The mean±s.e.m. is indicated. ****P<0.0001 (unpaired t-test). (D) Representative FOV of a 48-h sample from the experiment in C, obtained using a 60× objective. DAPI staining (blue) and anti-RFP immunofluorescence (red) are shown.

Fig 5: DDR2 deletion contributes to a disorganized ECM following injury.(A) SHG imaging of fibrillar collagen at the injury site of wild-type mice—mobilized or immobilized (1 and 3 weeks)—3 weeks after BT. Anisotropy quantification of collagen ECM (n = 3 mice per group, n = 3 to 5 images per mouse). (B) µCT imaging of mobilized or immobilized (1 and 3 weeks) injured hindlimb from wild-type mice 9 weeks after BT (n = 5 mice per group). Orange labels ectopic bone. (C) UMAP dimensional reduction and (D) violin plots showing relative expression of Col1a1, Col3a1, and Ddr2 from scRNA sequencing of cells harvested from the injury site 1 week after BT with and without limb immobilization (n = 1 mouse per group) [Col1a1: average Log(FC) = 0.63, Padj. = 2.88 × 10-100, Col3a1: average Log(FC) = 0.65, Padj. = 4.94 × 10-107, Ddr2: no significant difference]. (E) SHG imaging of fibrillar collagen at the injury site 1 week after BT in LC and Ddr2slie/slie mice (n = 3 mice per group, n = 4 to 5 images per mouse). (F) Representative confocal images (40×) of MLin cells plated on aligned collagen-coated fibrils from LC and Ddr2slie/slie mice and stained with anti-vinculin, phalloidin for F-actin, and Hoechst. Graphs showing number of FAs and SD of FA orientation (n = 10 images per group). (G) Traction force microscopy heatmaps of LC and Pdgfra-CreER+/-;Ddr2fl/fl MLin cells on 8-kPa type-I collagen–coated substrates. Graphs quantifying strain energy per cell (n = 12 to 15 cells per group) and fraction of cell footprint with low traction, defined as the fraction of region of interest (ROI) area with traction magnitude less than or equal to half of the mean traction magnitude within the ROI area (n = 12 to 15 cells per group; P = 0.0832 by Mann-Whitney U test). All scale bars, 100 µm. All graphs show means ± SEM.