DDR2-null mice have decreased ovarian tumor burden

Immunohistochemical evaluation of advanced stage, human ovarian cancer specimens demonstrate that high expression of DDR2 in tumors is associated with poor survival [22]. Since DDR2 is predominantly expressed by mesenchymal cells, we asked whether presence of DDR2 in ovarian cancer-associated stromal cells impacted ovarian tumor burden in mouse models. To do so, we utilized a previously published intraperitoneal tumor model by introducing three mouse ovarian tumor cell lines into the intraperitoneal cavity of adult female Ddr2−/− or control WT C57BL/6 mice (i.e., a syngeneic mouse tumor model). The three ovarian cancer cell lines were: (1) ID8TB−/− (mouse ovarian surface epithelium cell line [23]), (2) BPPNM (fallopian tube epithelial-derived cell line [24]), and (3) KPCA (fallopian tube epithelial-derived cell line [24]). ID8TB−/− and BPPNM tumor cell lines expressed DDR2, while KPCA did not (Supplementary Fig. S1A). In all three experimental settings, ubiquitous Ddr2−/− recipient mice developed significantly less tumor burden than the WT mice, and the ID8TB−/− model in DDR2 KO mice had increased survival and decreased ascites compared to the DDR2 WT mice (Fig. 1A–D, Supplementary Fig. S1B). These data indicated that the presence of DDR2 in stromal cells or the host, in general, impacted ovarian cancer burden, regardless of tumor cell DDR2 expression status.

Fig. 1: Deletion of host DDR2 leads to decrease in tumor burden.

A DDR2-positive ID8 Trp53−/−Brca2−/− (ID8TB−/−) mouse ovarian surface epithelium cell (MOSEC) tumor line was intraperitoneally injected into C57BL/6J Ddr2 WT mice (8 mice; n = 8) or Ddr2 KO mice (8 mice; n = 8). B Kaplan–Meier survival curves for C57BL/6J Ddr2 WT (5 mice; n = 5) or Ddr2 KO (5 mice; n = 5) mice injected intraperitoneally with ID8TB−/− tumor line and monitored till disease endpoint. C DDR2-positive BPPNM (p53−/−R172HBrca1−/−Pten−/−Nf1−/−MycOE) mouse fallopian tube epithelial (FTE) tumor cell line was intraperitoneally injected into C57BL/6J Ddr2 WT mice (6 mice; n = 6) or Ddr2 KO mice (6 mice; n = 6). (D) DDR2-negative KPCA (p53−/−R172HCcne1OEAkt2OEKRASG12V) mouse FTE tumor cell line was intraperitoneally injected into C57BL/6J Ddr2 WT mice (4 mice; n = 4) or Ddr2 KO mice (4 mice; n = 4). In panels A, C, and D, Student’s t-test was used for statistics ****p < 0.0001, ***p < 0.001. In panel B, Logrank (Mantel Cox) test was used for analysis.

Arginase-1 mRNA expression is decreased in tumors from Ddr2−/− mice

To determine how tumor-associated stromal expression of DDR2 affected tumor burden, we performed targeted mRNA expression profiling of ID8TB−/− tumors dissected from WT and Ddr2−/− mice using the Nanostring nCounter Tumor Signaling 360 panel which includes 760 genes and 20 internal reference genes. Volcano plot analysis revealed that arginase-1 (Arg1) mRNA level, in particular, was dramatically decreased in tumors from Ddr2−/− hosts (Fig. 2A, Supplementary Fig. S2A, Supplementary Table 2). Quantitative PCR on mRNA isolated from omental ID8TB−/− tumor nodules, different than those used for Nanostring analysis, confirmed that Arg1 mRNA was indeed decreased in ID8TB−/− and BPPNM tumor nodules from Ddr2−/− hosts (Fig. 2B). Related Arg2 mRNA level was also decreased in tumor nodules (Fig. 2C). Arginase enzyme activity in whole tumor extracts and serum from tumor-bearing mice was also significantly decreased in Ddr2−/− mice (Fig. 2D, E). Finally, when fixed tumor slices were immunostained for arginase-1 protein, tumors from Ddr2−/− hosts had decreased arginase-1 expression (Fig. 2F).

Fig. 2: Arginase-1 is downregulated in tumor nodules from DDR2 KO mice.

A Volcano plot showing differentially expresses genes in RNA from ID8TB−/− tumor nodules from Ddr2 WT mice (5 mice; n = 5) or Ddr2−/− mice (5 mice; n = 5). B Quantitative PCR assay showing Arg1 gene expression using RNA from ID8TB−/− (6 mice; n = 6) and BPPNM (3 mice; n = 3) tumor nodules from Ddr2 WT mice or Ddr2−/− mice. C Quantitative PCR assay showing Arg2 gene expression using RNA from ID8TB−/− and BPPNM tumor nodules from Ddr2 WT mice (3 mice; n = 3) or Ddr2−/− mice (3 mice; n = 3). D Arginase activity assay on tumor nodules from ID8TB−/− tumor-bearing Ddr2 WT mice (3 mice; n = 3) or Ddr2−/− mice (3 mice; n = 3). E Arginase activity assay on serum from ID8TB−/− tumor-bearing Ddr2 WT mice (6 mice; n = 6) or Ddr2−/− mice (6 mice; n = 6). F Immunohistochemistry images for Arginase-1 expression on ID8TB−/− tumor nodules from Ddr2 WT mice (6 mice; n = 6) or Ddr2−/− mice (6 mice; n = 6). Scale bars = 50 μm (left). One tumor slice per mouse (total of 6 tumor slices per group) was used and the entire tumor slice was analyzed using Halo. Graph on right shows percent of area positive for Arg1. In all panels, Student’s t-test was used for statistics ****p < 0.0001, ***p < 0.001, **p < 0.01.

The action of DDR2 in CAFs controls arginase activity

To determine which cell(s) in the host tumor stromal compartment expressed DDR2, we first interrogated published human and BPPNM mouse ovarian cancer single-cell RNA sequencing datasets [24]. In both, DDR2 was found to be primarily expressed in CAFs but was also present in some tumor cell clusters (Fig. 3A, B, Supplementary Fig. S3A and B, Supplementary Tables 3 and 4). Notably, in both samples, none of the identified immune cell clusters expressed DDR2 mRNA.

Fig. 3: DDR2 regulates arginase activity in omental cancer-associated fibroblasts (CAFs).

A UMAP plot showing DDR2 expression in various cell type clusters from human ovarian cancer samples from PMID: 34238352. B UMAP plot showing Ddr2 expression in various cell type clusters from BPPNM mouse tumors from PMID: 33158843. C Western blot of DDR2-expressing (shSCRM), DDR2-depleted (shDdr2) and DDR2 rescue (shDdr2 + DDR2 rescue) CAFs with the indicated antibodies. Two separate short-hairpin RNA targeting DDR2 (#1 and #2) were used. D Arginase activity assay of DDR2-expressing (shSCRM), DDR2-depleted (shDdr2) and DDR2 rescue (shDdr2 + DDR2 rescue) CAFs (n = 3). E Western blot of CAFs from ID8 Trp53−/− Brca2−/− tumor-bearing DDR2 WT and Ddr2−/− mice as well as DDR2-null CAFs with constitutive overexpression of arginase-1 (Ddr2−/−Arg1OE) or control vector (Ddr2−/−EV) with the indicated antibodies. F Arginase activity assay on CAFs from ID8 Trp53−/− Brca2−/− tumors from DDR2-expressing (WT) and DDR2-null (Ddr2−/−) mice as well as DDR2-null CAFs with constitutive overexpression of arginase-1 (Ddr2−/−Arg1OE) or control vector (Ddr2−/−EV) (n = 3). In all panels, Student’s t-test was used for statistics ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.

Based on these results, we examined three distinct validated human omental CAF cell lines for DDR2 expression [25], and all expressed DDR2 (Fig. 3C and Supplementary Fig. S3C). When DDR2 expression was shRNA-depleted in all CAF cell lines, to varying degrees (Fig. 3C and Supplementary Fig. S3C), Arg1 mRNA expression decreased (Supplementary Fig. S3D) as did cellular arginase activity (Fig. 3D and Supplementary Fig. S3E). Importantly, these changes in Arg1 expression and activity were rescued, to levels approximating that in WT CAFs by expressing a RNAi-resistant isoform of DDR2 in hCAF68 cells depleted of Ddr2 (Fig. 3C, D).

To determine if DDR2 regulates Arg1 expression and arginase activity in omental CAFs in vivo, we made use of CAFs from the mouse ID8TB−/− syngeneic ovarian tumor model. CAFs from WT and Ddr2−/− tumor-bearing mice were isolated as previously published [26]. Following negative selection to deplete immune cells with an anti-CD45 antibody and epithelial cells with an anti-EpCAM antibody, remaining stromal cells were immortalized using SV40 large T virus. Similar to human omental CAF cell lines, mouse CAFs from ID8TB−/− tumors in Ddr2−/− mice had decreased Arg1 mRNA levels (Supplementary Fig. S3F), Arg1 expression, and arginase activity (Fig. 3E, F) which was increased upon constitutive Arg1 overexpression in Ddr2−/− CAFs (Fig. 3E, F).

Given the findings that Arg1 is expressed in DDR2+ CAFs, we used another in vivo approach to identify whether other cell populations expressed Arg1. We performed single-cell mRNA sequencing (scRNAseq) on ID8TB−/− mouse tumors dissected from WT mice (Fig. 4A, Supplementary Table 5). Using established tumor, CAF, and immune markers [24] (Supplementary Fig. S4A), we identified two tumor, three CAF and four immune cell clusters (Fig. 4A). Violin plot analysis of the various cell clusters present revealed that DDR2 was expressed in the three PDGFRΑ+ CAF clusters and one tumor cell subpopulation (Fig. 4A, B). Arg1 mRNA expression was present in two of the three Ddr2+ CAF clusters as well as in two immune cell clusters and one tumor cell cluster (Fig. 4B).

Fig. 4: Identification of a subpopulation of DDR2-expressing CAFs that express arginase-1 in vivo.

A UMAP plot showing showing cell clusters for ID8TB−/− tumors in WT mice. B Violin plots showing expression of Ddr2, Arg1 and Pdgfra in cell clusters for ID8TB−/− tumors in WT mice. C Analysis of multiplex immunohistochemistry on ID8TB−/− tumor slices showing the percent of double positive Arg1+ and Pdgfra+ cells as a proportion of all Pdgfra+ cells in tumor slices (n = 7 mice). Entire tumor slice was analyzed in Halo. D Analysis of multiplex immunohistochemistry on ID8TB−/− tumor slices showing the percent of Pdgfra+ cells in tumor slices (n = 7 mice). Entire tumor slice was analyzed in Halo. E Representative images of multiplex immunohistochemistry ID8TB−/− tumor slices from WT and Ddr2−/− mice stained for Pdgfra, Arg1 and hematoxylin (n = 7 mice). Scale bar = 50 μm. In all panels, Student’s t-test was used for statistics ***p < 0.001, **p < 0.01, ns = p > 0.05.

Next, we performed multiplex immunohistochemistry analysis on ID8TB−/− tumors from WT and Ddr2−/− mice for expression of Arg1 and various tumor stromal cell type markers (CAF – PDGFRα; macrophage – F4/80). Arg1 expression was present in 30% of cells expressing the CAF marker protein PDGFRα (Fig. 4C–E). In tumors from Ddr2−/− mice, the proportion of Arg1-positive CAFs (%Arg1+ and PDGFRα+/ PDGFRα+) was significantly decreased compared to WT mice (Fig. 4C). This was not a result of overall decreased CAF populations in tumors from Ddr2−/− mice as the proportion of PDGFRα+ CAFs were similar between tumors from WT and Ddr2−/− mice (Fig. 4D). The proportion of Arg1-positive macrophages (%Arg1+ and F4-80+/ F4-80+) and Arg1-positive tumor cells (%Arg1 and CK8+/CK8+) were similar between tumors from WT and Ddr2−/− mice (Supplementary Fig. S4B–G). Flow analysis of single-cell suspensions from ID8TB−/− tumors in WT hosts revealed that 17.4% of total cells were CAFs (e.g., PDGFRα positive) (Supplementary Fig. S4H, Supplementary Table 6) while 20% of cells isolated from tumors were CD45+ immune cells (Supplementary Fig. S4H).

Taken together, these accumulated cell line and in vivo data indicate that Arg1 is expressed in Ddr2+ CAFs, and DDR2 regulates arginase-1 protein levels and arginase activity. Moreover, in this mouse ovarian tumor model, CAF-derived Arg1, as opposed to immune cell- or tumor cell-derived Arg1, was likely a significant contributor to overall arginase activity in ovarian cancer.

Ovarian tumor omental CAFs with high DDR2 and ARG1 expression promote in vivo omental colonization

Omental colonization can be part of tumor progression in ovarian cancer. To determine if DDR2-regulated arginase-1 activity in CAFs impacted tumor cell colonization in vivo, we co-injected WT mice intraperitoneally with syngeneic luciferase-positive KPCA tumor cells (low DDR2 expression and low arginase activity) (Supplementary Fig. S1A, Supplementary Fig. S5A) +/− various luciferase-negative mouse omental CAF cell lines from WT or Ddr2−/− mice. After 5 days, mice were sacrificed, omentum digested, and luciferase assay performed which reflected the amount of KPCA tumor cells that had colonized the omentum (Supplementary Fig. S5B). When KPCA cells were co-injected with WT mouse CAFs, there was a significant increase in omental colonization by KPCA cells (Fig. 5). Compared to WT CAFs, when Ddr2−/− CAFs were used, there was significantly less tumor cell colonization (Fig. 5). Mice co-injected with Ddr2−/− Arg1OE CAFs had increased omental colonization compared to those co-injected with Ddr2−/− CAFs. This data suggested that in vivo DDR2 and Arg1 expressing CAFs might impact early steps of omental colonization or the proliferation of tumor cells after attachment.

Fig. 5: Omental CAFs with high DDR2 and/or arginase-1 expression promote in vivo omental colonization.

A Normalized luminescence counts of omental tissue from KPCA tumor cells alone (lane 1), ID8TB−/− WT mouse CAFs alone (lane 2), WT mice injected with KPCA and WT mouse CAFs (lane 3), KPCA and Ddr2−/− mouse CAFs (lane 4), and KPCA and Ddr2−/− mouse CAFs with Arg1 constitutive overexpression (lane 5) (n = 5 mice). In this experiment, luciferase-positive KPCA tumor cells were used and CAFs are negative for luciferase. Student’s t-test was used for statistics ****p < 0.0001, ***p < 0.001, **p < 0.01.

SNAIL protein was detected at the promoter region of arginase-1 gene

The action of DDR2 in CAFs appeared to regulate Arg1 expression at the transcriptional level (Fig. 2A, B). We have previously shown that SNAIL (SNAI1), an EMT inducing transcription factor that promotes tumor cell migration and invasion [27], is regulated by the action of DDR2 in tumors, post-transcriptionally [28]. SNAI1 can act as both a transcriptional repressor and activator [29,30,31]. We confirmed that SNAIL protein level was indeed decreased in Ddr2-depleted CAFs (Fig. 6A). To determine if SNAIL protein could impact Arg1 transcription, we performed chromatin immunoprecipitation (ChIP) experiments to determine if SNAIL was present at the promoter region of the endogenous Arg1 gene. In human ovarian tumor CAFs, SNAIL protein was detected at the promoter region of the human Arg1 gene, while in Ddr2-depleted CAFs, there was less SNAIL detected (Fig. 6B, Supplementary Fig. S6A). To confirm this finding, we constitutively overexpressed SNAIL1 in DDR2-depleted CAFs (shDDR2 SNAIL OE) (Fig. 6C) and performed quantitative PCR for Arg1. Arg1 mRNA levels were increased in shDDR2 SNAIL OE CAFs compared to DDR2-depleted CAFs (shDDR2). In control experiments, we confirmed nuclear localization of SNAIL in shDDR2 SNAIL OE CAFs (Supplementary Fig. S6B) This suggested that DDR2-regulated SNAIL1 expression impacts Arg1 transcription in ovarian CAFs.

Fig. 6: DDR2’s regulation of arginase-1 is dependent on SNAI1 transcriptional activity.

A Western blot of DDR2-expressing (shSCRM) and DDR2-depleted (shDdr2) CAFs with the indicated antibodies. B Chromatin immunoprecipitation-qPCR (ChIP-qPCR) of Arg1 expression using SNAIL or IgG control antibodies on shSCRM and shDdr2 CAFs. E-cadherin expression was used as a positive control. ChIP-qPCR of E-cadherin expression using SNAIL or IgG control antibodies on shSCRM and shDdr2 CAFs (n = 3 replicates). C Quantitative PCR assay showing DDR2, SNAIL and Arg1 gene expression using RNA from shSCRM, shDDR2 and DDR2-depleted SNAIL-overexpressing CAFs (shDDR2 SNAIL OE). In all panels, ****p < 0.0001, ***p < 0.001, **p < 0.01, ns = p > 0.05.

DDR2-dependent arginase activity in CAFs is important for ovarian tumor collagen protein production and secretion

Arg1 is a central cytosolic enzyme controlling cellular L-Arginine metabolism. Arg1 cleaves L-Arg to generate urea and L-Ornithine. L-Ornithine is subsequently metabolized to generate L-Proline and polyamines [32,33,34] (Fig. 7A). To determine if DDR2 signaling impacted L-Arginine metabolite production in ovarian tumor CAFs, we generated a series of human omental CAF cells: (1) DDR2-expressing WT control (shSCRM), (2) DDR2-depleted (shDdr2), (3) DDR2-depleted and constitutively overexpressing Arg1 (shDdr2 Arg1OE) and (4) a transfection control empty vector (shDdr2 EV) (Supplementary Fig. S7A, B). In Ddr2-depleted CAFs, both intracellular and secreted L-Arginine levels, as determined by a standard biochemical assay, were increased (Fig. 7B). Compared to WT CAFs, L-Ornithine levels were significantly decreased in Ddr2-depleted CAFs (Fig. 7C).

Fig. 7: DDR2-dependent arginase activity in CAFs promotes increased collagen synthesis.

A Schematic showing arginine metabolism by arginase into L-ornithine and other downstream products. B Arginine assay showing levels of intracellular and extracellular arginine in DDR2-expressing (shSCRM) and DDR2-depleted (shDdr2) CAFs (n = 3). C Ornithine assay showing levels of intracellular ornithine in DDR2-expressing (shSCRM) and DDR2-depleted (shDdr2) CAFs (n = 3). D Representative images of modified Masson’s trichrome stain for collagen (blue) from ID8 Trp53−/− Brca2−/− tumors from Ddr2 WT and Ddr2 KO mice (n = 6 mice). Scale bar = 50 μm. One tumor slice per mouse was used (6 total tumor slices per group). Percent area of collagen was quantified using the entire tumor slice in Halo. E Representative images of collagen immunofluorescence stain in DDR2-expressing (shSCRM) and DDR2-depleted (shDdr2) CAFs (n = 3 replicates, 12 images per group). F Proline assay showing levels of intracellular proline in DDR2-expressing (shSCRM) and DDR2-depleted (shDdr2) CAFs (n = 3). G Hydroxyproline assay measurement of DDR2-expressing (shSCRM), DDR2-depleted (shDdr2), DDR2-depeleted and constitutive arginase-1 overexpressing (shDdr2 Arg1OE) and DDR2-depleted empty vector control (shDdr2 EV) (n = 3 replicates). H Procollagen1a1 assay measurement of conditioned media from shSCRM, shDdr2, shDdr2 Arg1OE and shDdr2 EV CAFs (n = 3 replicates). In all panels, Student’s t-test was used for statistics ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns = p > 0.05.

In breast tumor CAFs, the action of DDR2 has been shown to contribute to the production of collagens, by affecting mRNA synthesis [13]. However, whether DDR2 signals could also regulate the production of collagen proteins, and if so, how has not been addressed. When ovarian tumor nodules from WT and Ddr2−/− mice were stained for fibrillar collagens with trichrome blue, the amount of detected fibrillar collagen in tumors from Ddr2−/− mice was significantly decreased (Fig. 7D). In addition, cultured DDR2-depleted human omental CAFs expressed decreased collagen1α1 protein as detected by immunofluorescence (Fig. 7E). We next determined the L-Proline content in human omental CAF cell lines using a standard biochemical assay. Ddr2-depleted CAFs had decreased L-Proline content (Fig. 7F). In collagen proteins, much of the proline exists in its hydroxylated form, hydroxyproline [35]. In Ddr2-depleted CAFs, there was also decreased cellular hydroxyproline (Fig. 7G). Constitutive Arg1 overexpression in Ddr2-depleted cells rescued hydroxyproline levels to that present in control WT CAFs (Fig. 7G).

While hydroxyproline is a sensitive marker for collagen level in cells, it is not a direct measure of collagen synthesis since hydroxyproline residues may be elevated due to collagen synthesis and degradation. Newly synthesized triple helical procollagen is secreted into the extracellular space where additional cleavage and crosslinking occurs to form mature collagen fibers [36]. To determine if DDR2 signals affected collagen protein synthesis and secretion, we measured secreted procollagen 1α1 levels in the culture media from various CAFs. Ddr2-depleted CAFs secreted less procollagen 1α1 compared to WT CAFs (Fig. 7H). Constitutive Arg1 overexpression in Ddr2-depleted CAFs (shDDR2 Arg1OE) increased the amount of procollagen 1α1 secreted to levels produced by control WT CAFs (Fig. 7H). In other control experiments, siRNA-mediated depletion of Arg1 in WT CAFs resulted in decreased procollagen 1α1 secretion (Supplementary Fig. S7C, D).

To confirm that L-arginine was able to be converted to collagen in both WT and Ddr2-depleted CAFs, we performed arginine metabolic tracing experiments. Human omental CAFs (+/− DDR2) were loaded with 13C C6-labeled L-arginine and after 72 h intracellular collagen peptides were isolated following cell lysis and subjected to mass spectrometry. This type of experiment does not distinguish the levels of labeled collagen between different cells. As expected based on prior literature [37] in both WT and Ddr2-depleted CAFs we identified 21 collagen peptides from 11 distinct collagens that contained 13C C5-labeled proline or hydroxyproline (Supplementary Table 7) in both DDR2-depleted and WT control.

To determine if fibrillar collagen increases DDR2 expression in CAFs, we cultured DDR2-expressing and DDR2-depleted CAFs on plastic or polymerized collagen and checked DDR2 expression after 24 h. We observed a modest increase in DDR2 protein levels in CAFs cultured on polymerized collagen compared to CAFs cultured on plastic (Supplementary Fig. S7E).

In sum, these accumulated data indicated that DDR2-dependent regulation of arginase activity in omental CAFs contribute to collagen protein synthesis and secretion. This could explain, in part, why Ddr2−/− ovarian cancer tumor nodules contain less ECM fibrillar collagens, and possibly as a result, have decreased tumor burden in vivo. Additionally, collagen may increase DDR2 levels in CAFs, suggesting the possibility of a feedback loop between collagen and DDR2.

DDR2-dependent arginase polyamine production in CAFs contributes to tumor cell invasion

Ovarian tumor progression is dependent upon tumor cell attachment to and invasion through the basement membrane with subsequent interaction with CAFs. Given our findings that DDR2 inactivated CAFs have lower L-Ornithine levels and this can lead to a decrease in polyamines, we asked whether DDR2-regulated arginase activity in CAFs impacted polyamine production. To do this, we biochemically determined the total polyamine level in CAFs cells (+/− DDR2) and their secreted media. Ddr2-depleted CAFs had decreased intracellular and extracellular polyamine levels compared to WT control (Fig. 8A). We next performed mass spectrometry on conditioned media produced by human CAFs (+/− DDR2). Ddr2-depleted (shDDR2) CAFs produced decreased levels of spermidine and putrescine compared to WT control (shSCRM) (Fig. 8B).

Fig. 8: Polyamine-mediated and DDR2-dependent arginase activity in CAFs promotes tumor invasion.

A Total polyamine assay showing levels of intracellular and extracellular polyamines in DDR2-expressing (shSCRM) and DDR2-depleted (shDdr2) CAFs and CAF conditioned media, respectively (n = 3 replicates). B Heatmap showing fold change expression of each polyamine and intermediate from mass spectrometry intensity data (n = 3 replicates). C Matrigel invasion assay of Tyknu tumor cells using conditioned media from WT control (shSCRM), DDR2-depleted (shDdr2), DDR2-depeleted and constitutive arginase-1 overexpressing (shDdr2 Arg1OE) and DDR2-depleted empty vector control (shDdr2 EV) as chemoattractant (n = 10 images analyzed from 3 replicates per condition). D Matrigel invasion assay of Tyknu tumor cells using conditioned media from siCTRL, siArg1-1 and siArg1-2 CAFs (n = 12 images analyzed from 3 replicates per condition). E Matrigel invasion assay of Tyknu tumor cells using conditioned media from CAFs treated with PBS solvent control (Vehicle), 10 μM or 100 μM CB1158 (n = 12 images analyzed from 3 replicates per condition). F Matrigel invasion assay of Tyknu tumor cells using CAF conditioned media from WT control (shSCRM), DDR2-depleted (shDdr2), DDR2-depleted CM supplemented with 10 μM spermidine (shDdr2 + spmd), DDR2-depleted CM supplemented with 10 μM putrescine (shDdr2 + put) and solvent control (shDdr2 + PBS). (n = 12 images analyzed from 3 replicates per condition). In all panels, Student’s t-test was used for statistics ****p < 0.0001, ***p < 0.001, **p < 0.01.

Media secreted by Ddr2-depleted human ovarian tumor CAFs leads to decreased ovarian tumor cell invasion and migration compared to media secreted by Ddr2-expressing CAFs [25]. We confirmed this result in Matrigel invasion assays in Boyden chambers using two human ovarian tumor cell lines (Tyknu; OVCAR8) and CAF conditioned media (CM) added to the lower well (Fig. 8C, and Supplementary Fig. S8A, B). CM from shDdr2 Arg1OE CAFs rescued this defect (Fig. 8C). CM from Arg1-depleted CAFs did not support ovarian tumor cell invasion through Matrigel (Fig. 8D, and Supplementary Fig. S7D, S8C and S8D). We also performed Matrigel invasion assays using CM from WT CAFs pretreated with the arginase inhibitor, CB1158 [38]. CB1158 was removed from CM prior to invasion assay using a 10 kDa molecular cutoff filter. We observed a dose-response inhibition of tumor cell invasion through Matrigel when conditioned media from CAFs treated with an arginase inhibitor was added (Fig. 8E, and Supplementary Fig. S8E and S8F).

Polyamines are polycationic molecules that can contribute to cellular proliferation and invasion [39, 40]. To determine if DDR2-dependent (Arg1-dependent) polyamine production specifically could contribute to ovarian tumor cell invasion, we added exogenous spermidine or putrescine to CM from Ddr2-depleted CAFs and repeated the Boyden-chamber Matrigel invasion assays. Both polyamines rescued the tumor cell invasion defect of CM from Ddr2-depleted CAFs (Fig. 8F, and Supplementary Fig. S8G and S8H).

In sum, these data indicated that the presence of DDR2 in omental ovarian tumor CAFs controlled polyamine production, likely through DDR2-regulated Arginase-1 production. Moreover, polyamine production by CAFs could support ovarian tumor cell invasion through Matrigel.

High stromal ARG1 expression in ovarian cancer correlate with poor overall survival

We have previously shown that high stromal expression of DDR2 protein in human ovarian tumors correlates with worse overall survival [22]. Given that DDR2-regulated arginase activity in CAFs affected ovarian cancer tumor collagen production, we asked whether stromal arginase-1 expression correlated with ovarian cancer patient survival. We quantified stromal arginase-1 expression in a human ovarian cancer tumor microarray by immunohistochemistry and correlated stromal arginase-1 protein expression with survival outcomes. Patients with high stromal DDR2 and high stromal arginase-1 expression had median overall survival of 23 months whereas patients with low stromal DDR2 and low stromal arginase-1 had a median overall survival of 171 months (Supplementary Fig. S9A). We then performed a multivariate analysis for DDR2 and arginase-1 controlling for known clinical factors that influence survival. We identified that advanced stage or high stromal arginase-1 were associated with poor survival in ovarian cancer patients (Supplementary Fig. S9B).