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, 58 (3), 1042-53

Platelet-derived Growth factor-D and Rho GTPases Regulate Recruitment of Cancer-Associated Fibroblasts in Cholangiocarcinoma


Platelet-derived Growth factor-D and Rho GTPases Regulate Recruitment of Cancer-Associated Fibroblasts in Cholangiocarcinoma

Massimiliano Cadamuro et al. Hepatology.


Cholangiocarcinoma (CCA) is characterized by an abundant stromal reaction. Cancer-associated fibroblasts (CAFs) are pivotal in tumor growth and invasiveness and represent a potential therapeutic target. To understand the mechanisms leading to CAF recruitment in CCA, we studied (1) expression of epithelial-mesenchymal transition (EMT) in surgical CCA specimens and CCA cells, (2) lineage tracking of an enhanced green fluorescent protein (EGFP)-expressing human male CCA cell line (EGI-1) after xenotransplantation into severe-combined-immunodeficient mice, (3) expression of platelet-derived growth factors (PDGFs) and their receptors in vivo and in vitro, (4) secretion of PDGFs by CCA cells, (5) the role of PDGF-D in fibroblast recruitment in vitro, and (6) downstream effectors of PDGF-D signaling. CCA cells expressed several EMT biomarkers, but not alpha smooth muscle actin (α-SMA). Xenotransplanted CCA masses were surrounded and infiltrated by α-SMA-expressing CAFs, which were negative for EGFP and the human Y-probe, but positive for the murine Y-probe. CCA cells were strongly immunoreactive for PDGF-A and -D, whereas CAFs expressed PDGF receptor (PDGFR)β. PDGF-D, a PDGFRβ agonist, was exclusively secreted by cultured CCA cells. Fibroblast migration was potently induced by PDGF-D and CCA conditioned medium and was significantly inhibited by PDGFRβ blockade with Imatinib and by silencing PDGF-D expression in CCA cells. In fibroblasts, PDGF-D activated the Rac1 and Cdc42 Rho GTPases and c-Jun N-terminal kinase (JNK). Selective inhibition of Rho GTPases (particularly Rac1) and of JNK strongly reduced PDGF-D-induced fibroblast migration.

Conclusion: CCA cells express several mesenchymal markers, but do not transdifferentiate into CAFs. Instead, CCA cells recruit CAFs by secreting PDGF-D, which stimulates fibroblast migration through PDGFRβ and Rho GTPase and JNK activation. Targeting tumor or stroma interactions with inhibitors of the PDGF-D pathway may offer a novel therapeutic approach.


Figure 1
Figure 1. Bioluminescence imaging and histological assessment of EGI-1 cells after xenotransplantation into SCID mice
High correspondence between the bioluminescence signal in the liver (A) and the macroscopic detection of liver tumors at autopsy (arrow, B) after xenotransplantation of EGI-1 cells into SCID mice by intraportal injection. Before transplantation, EGI-1 cells were transduced with lentiviral vectors encoding the firefly luciferase gene. Mice were sacrificed once the bioluminescence signal intensity in the liver reached a value >105 p/sec/cm2/sr. Histological analysis of liver metastases showed that EGI-1 cells laid embedded in a rich stroma (H&E staining, C). By dual immunofluorescence for EGFP (green) and α-SMA (red), we showed that CAF were strictly adjacent to EGI-1-derived tumors, but coincident labeling between EGFP and α-SMA was never observed (D). In liver tumors formed by EGI-1 cells, FISH showed that α-SMA-positive CAF (green, E, F) co-expressed mouse (red, white arrow, E), but not human (red, F) Y-Chr, which was instead expressed by tumoral EGI-1 cells (F). High specificity of both Y-probes was confirmed in preliminary experiments. Original magnification: C-F, 200x, insets in E,F, 400x.
Figure 2
Figure 2. Expression of PDGF ligands and receptors in human CCA samples
Neoplastic bile ducts (K7, green, A-E) were strongly positive for PDGF-A (red, A), and PDGF-D (red, D), weakly positive for PDGF-B (red, B), and negative for PDGF-C (red, C). In addition, neoplastic bile ducts expressed the PDGFRα, though extra-CCA PDGFRα staining was also found in some scattered surrounding cells, including CAF (red, E). On the other hand, CAF (α-SMA, green, F) were strongly decorated by PDGFRβ, which was negative in CCA cholangiocytes (red, F). Coincident staining appears in yellow. Original magnification: A-F, 200x, insets in D-F, 400x.
Figure 3
Figure 3. Proliferation (A) and migration (B) of human fibroblasts stimulated by conditioned media from CCA cholangiocytes
Proliferation of human fibroblasts was evaluated by MTS assay and expressed as absorbance at 490nm (A), whilst recruitment was evaluated by Matrigel-coated transwell chambers and expressed as number of transwell-invaded nuclei (B). A. Conditioned media obtained from EGI-1, TFK-1, and CCA1 cholangiocytes induced only a slight proliferative response with respect to starved fibroblasts and to control cholangiocytes (ctrl) (black columns: EGI-1, 132.33±5.05; TFK-1, 144.33±2.94; CCA1, 140.33±7.09, vs control, 123.67±6.19 and starved, 114.33±6.15; n=6 experiments). This effect, although small, was significantly reduced following treatment of cultured cells with Imatinib Mesylate 1µM (A) (gray columns: EGI-1, 119.83±7.22; TFK-1, 135.33±6.31; CCA1, 129.17±10.17; control, 117.83±6.74; n=6 experiments). B. Conditioned media from the same cell lines induced a potent chemotactic response on human fibroblasts with respect to ctrl (black columns: EGI-1, 45.9±4.01; TFK-1, 44.17±3.59; CCA1, 43.82±3.99 vs control, 11.76±9.83; n=3 experiments), an effect significantly reduced by Imatinib Mesylate 1µM (gray columns: EGI-1, 35.08±3.33; TFK-1, 37.83±1.55; CCA1, 36.82±0.41, control, 9.16±7.62; n=3 experiments). A similar significant reduction in fibroblast invasion was achieved using conditioned media from EGI-1 cells treated with PDGF-D siRNA, as compared with EGI-1 scramble (siRNA1, 37.45±2.08; siRNA2, 35.45±4.70 vs EGI-1 scramble, 44.98±2.30; n=3 experiments). *p<0.05 treated vs control; **p<0.01 treated vs control; #p<0.05 treated vs IM; ##p<0.01 treated vs IM; ^^p<0.01 treated vs starv; §p<0.05 siRNA vs scramble. IM, Imatinib Mesylate.
Figure 4
Figure 4. Activation of ERK1/2 and JNK in human fibroblasts stimulated with rhPDGF-D. A
By WB, stimulation of human fibroblasts with increasing doses of rhPDGF-D resulted in a mild increase of p-ERK1/2/ERK1/2 which reached significance only at the highest doses (black columns, 0.1ng/ml, 0.47±0.05; 1ng/ml, 0.57±0.08; 10ng/ml, 0.67±0.03; 100ng/ml, 0.94±0.09 vs untreated, 0.40±0.14; n=5 experiments), and was inhibited after addition of Imatinib 1µM (gray columns, untreated, 0.33±0.29; 0.1ng/ml, 0.30±0.26; 1ng/ml, 0.29±0.25; 10ng/ml, 0.51±0.09; 100ng/ml, 0.58±0.04). B. In contrast with ERK1/2, JNK was activated since the lowest doses of rhPDGF-D (black columns, 0.1ng/ml, 0.66±0.21; 1ng/ml, 0.93±0.17; 10ng/ml, 1.07±0.34; 100ng/ml, 0.91±0.33 vs untreated, n.d.; n=4 experiments), an effect that was significantly reduced by Imatinib 1µM (gray columns, untreated, 0.27±0.20; 0.1ng/ml, 0.18±0.23; 1ng/ml, 0.32±0.27; 10ng/ml, 0.27±0.26; 100ng/ml, 0.36±0.25). *p<0.05 treated vs untreated; **p<0.01 treated vs untreated; #p<0.05 treated vs Imatinib 1µM; ##p<0.01 treated vs Imatinib 1µM. IM, Imatinib Mesylate. The columns of bands in the Western blots below are respective of each of the five conditions displayed in the graph.
Figure 5
Figure 5. Dose-response activation of RhoA, Rac1 and Cdc42 in human fibroblasts stimulated with rhPDGF-D
Human fibroblasts were stimulated for 1min with increasing doses of rhPDGF-D (0.1, 1, 10, 100ng/ml) to assess a dose-response effect. Levels of activation are expressed as normalization on untreated cells. A linear dose-dependent increase was observed for Rac1 (B, black columns, 0.1ng/ml, 2.41±0.86; 1ng/ml, 3.14±1.70; 10 ng/ml, 3.39±1.40; 100ng/ml, 3.93±1.24), and for Cdc42 (C, black columns, 0.1ng/ml, 1.73±0.28; 1ng/ml, 1.77±0.22; 10 ng/ml, 2.26±0.61; 100ng/ml, 2.56±0.49) that was significant from the lowest dose. In contrast, activation of RhoA was observed only at the highest doses (A, black columns, 0.1ng/ml, 1.29±0.32; 1ng/ml, 1.39±0.49; 10 ng/ml, 2.08±0.50; 100ng/ml, 2.32±0.47). Imatinib 1µM blunted the activating effects of rhPDGF-D in all cases (gray columns, RhoA, 0.1ng/ml, 1.10±0.28; 1ng/ml, 0.97±0.25; 10 ng/ml, 1.02±0.51; 100ng/ml, 1.08±0.28. Rac1, 0.1ng/ml, 1.28±0.37; 1ng/ml, 1.49±0.48; 10 ng/ml, 1.30±0.26; 100ng/ml, 1.43±0.66. Cdc42, 0.1ng/ml, 1.11±0.07; 1ng/ml, 1.04±0.37; 10 ng/ml, 1.31±0.11; 100ng/ml, 1.05±0.16) (n=4 experiments). *p<0.05 treated vs untreated; **p<0.01 treated vs untreated; #p<0.05 treated vs Imatinib 1µM; ##p<0.01 treated vs Imatinib 1µM. IM, Imatinib Mesylate.
Figure 6
Figure 6. Effects of RhoA, Rac1, Cdc42 and JNK inhibitors on migration of human fibroblasts stimulated by rhPDGF-D
Human fibroblasts treated with rhPDGF-D 100ng/ml showed a significant reduction in migration after treatment with chemical inhibitors of small GTPases. A. Treatment with Y-27632 10µM (RhoA inhibitor), NSC23766 75nM (Rac1 inhibitor), CASIN 5µm (Cdc42 inhibitor), and SP600125 10µM (JNK inhibitor) induced a significant reduction in migration of different degrees (43.79±3.75 for RhoA antagonism, 22.53±3.81 for Rac1 antagonism, 33.78±3.15 for Cdc42 antagonism, and 20.09±3.08 for JNK antagonism vs 52.01±2.21 for PDGF-D treatment, p<0.01 in all cases). Combined treatment with the three inhibitors of small GTPases (mix) completely abolished the PDGF-D-stimulated fibroblast migration (8.78±3.97 vs untreated, 7.52±3.42, p=n.s.) (n=4 experiments). It is worth noting the spindle shaped morphology of fibroblasts induced by PDGF-D (B) is lost following treatment with NSC23766 (C). **p<0.01 PDGF-D treated vs untreated; ##p<0.01 PDGF-D treated vs inhibitors. Mix, Y-27632 10µM + NSC23766 75nM + CASIN 5µm.

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