Differential effects of sorafenib on liver versus tumor fibrosis mediated by stromal-derived factor 1 alpha/C-X-C receptor type 4 axis and myeloid differentiation antigen-positive myeloid cell infiltration in mice

HepatologyVolume 59, Issue 4 p. 1435-1447 Hepatobiliary MalignanciesFree Access Differential effects of sorafenib on liver versus tumor fibrosis mediated by stromal-derived factor 1 alpha/C-X-C receptor type 4 axis and myeloid differentiation antigen–positive myeloid cell infiltration in mice Yunching Chen, Yunching Chen Steele Laboratory for Tumor Biology, Department of Radiation Oncology, Boston, MASearch for more papers by this authorYuhui Huang, Yuhui Huang Steele Laboratory for Tumor Biology, Department of Radiation Oncology, Boston, MASearch for more papers by this authorThomas Reiberger, Thomas Reiberger Steele Laboratory for Tumor Biology, Department of Radiation Oncology, Boston, MASearch for more papers by this authorAnnique M. Duyverman, Annique M. Duyverman Steele Laboratory for Tumor Biology, Department of Radiation Oncology, Boston, MASearch for more papers by this authorPeigen Huang, Peigen Huang Steele Laboratory for Tumor Biology, Department of Radiation Oncology, Boston, MASearch for more papers by this authorRekha Samuel, Rekha Samuel Steele Laboratory for Tumor Biology, Department of Radiation Oncology, Boston, MASearch for more papers by this authorLotte Hiddingh, Lotte Hiddingh Steele Laboratory for Tumor Biology, Department of Radiation Oncology, Boston, MASearch for more papers by this authorSylvie Roberge, Sylvie Roberge Steele Laboratory for Tumor Biology, Department of Radiation Oncology, Boston, MASearch for more papers by this authorChristina Koppel, Christina Koppel Steele Laboratory for Tumor Biology, Department of Radiation Oncology, Boston, MASearch for more papers by this authorGregory Y. Lauwers, Gregory Y. Lauwers Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MASearch for more papers by this authorAndrew X. Zhu, Andrew X. Zhu Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MASearch for more papers by this authorRakesh K. Jain, Rakesh K. Jain Steele Laboratory for Tumor Biology, Department of Radiation Oncology, Boston, MASearch for more papers by this authorDan G. Duda, Corresponding Author Dan G. Duda Steele Laboratory for Tumor Biology, Department of Radiation Oncology, Boston, MAAddress reprint requests to: Dan G. Duda, D.M.D., Ph.D., Steele Laboratory for Tumor Biology, Massachusetts General Hospital, Cox-734, 100 Blossom Street, Boston, MA 02114. E-mail: [email protected]; fax: 617-726-1962.Search for more papers by this author Yunching Chen, Yunching Chen Steele Laboratory for Tumor Biology, Department of Radiation Oncology, Boston, MASearch for more papers by this authorYuhui Huang, Yuhui Huang Steele Laboratory for Tumor Biology, Department of Radiation Oncology, Boston, MASearch for more papers by this authorThomas Reiberger, Thomas Reiberger Steele Laboratory for Tumor Biology, Department of Radiation Oncology, Boston, MASearch for more papers by this authorAnnique M. Duyverman, Annique M. Duyverman Steele Laboratory for Tumor Biology, Department of Radiation Oncology, Boston, MASearch for more papers by this authorPeigen Huang, Peigen Huang Steele Laboratory for Tumor Biology, Department of Radiation Oncology, Boston, MASearch for more papers by this authorRekha Samuel, Rekha Samuel Steele Laboratory for Tumor Biology, Department of Radiation Oncology, Boston, MASearch for more papers by this authorLotte Hiddingh, Lotte Hiddingh Steele Laboratory for Tumor Biology, Department of Radiation Oncology, Boston, MASearch for more papers by this authorSylvie Roberge, Sylvie Roberge Steele Laboratory for Tumor Biology, Department of Radiation Oncology, Boston, MASearch for more papers by this authorChristina Koppel, Christina Koppel Steele Laboratory for Tumor Biology, Department of Radiation Oncology, Boston, MASearch for more papers by this authorGregory Y. Lauwers, Gregory Y. Lauwers Department of Pathology, Massachusetts General Hospital, Harvard Medical School, Boston, MASearch for more papers by this authorAndrew X. Zhu, Andrew X. Zhu Department of Medicine, Massachusetts General Hospital, Harvard Medical School, Boston, MASearch for more papers by this authorRakesh K. Jain, Rakesh K. Jain Steele Laboratory for Tumor Biology, Department of Radiation Oncology, Boston, MASearch for more papers by this authorDan G. Duda, Corresponding Author Dan G. Duda Steele Laboratory for Tumor Biology, Department of Radiation Oncology, Boston, MAAddress reprint requests to: Dan G. Duda, D.M.D., Ph.D., Steele Laboratory for Tumor Biology, Massachusetts General Hospital, Cox-734, 100 Blossom Street, Boston, MA 02114. E-mail: [email protected]; fax: 617-726-1962.Search for more papers by this author First published: 14 November 2013 https://doi.org/10.1002/hep.26790Citations: 144 Potential conflict of interest: Stock Ownership – R.K.J., SynDevRx, Enlight, XTuit; Employment or Leadership Position – none; Consulting – R.K.J., Enlight, Noxxon, Zyngenia, D.G.D., Hexal/Sandoz, A.X.Z., Sanofi-Aventis, Eisai, Exelixis; Advisory arrangements – T.R., MSD, Gilead; Speakers' Bureau – none; Grants/Contracts: Research – T.R., Roche, Phenex, MSD, Gilead, Janssen, R.K.J., Dyax, MedImmune, Roche, A.X.Z., Bayer, Onyx, Lilly; Grants/Contracts: Unrestricted – none; Travel Grants – T.R., Roche, MSD, Gilead, Janssen; Intellectual Property Rights – none; Other Interests – R.K.J., Board of Directors of XTuit and Boards of Trustees of H&Q Healthcare Investors and H&Q Life Sciences Investors. This study was supported by the National Institutes of Health grants no.: P01-CA080124, R01-CA159258, R21-CA139168, R01-CA126642 and National Cancer Institute/Proton Beam Federal Share Program awards (to D.G.D. and R.K.J.), by the American Cancer Society grant 120733-RSG-11-073-01-TBG (to D.G.D.) and by a Max Kade Fellowship (to T.R.). AboutSectionsPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Abstract Sorafenib—a broad kinase inhibitor—is a standard therapy for advanced hepatocellular carcinoma (HCC) and has been shown to exert antifibrotic effects in liver cirrhosis, a precursor of HCC. However, the effects of sorafenib on tumor desmoplasia—and its consequences on treatment resistance—remain unknown. We demonstrate that sorafenib has differential effects on tumor fibrosis versus liver fibrosis in orthotopic models of HCC in mice. Sorafenib intensifies tumor hypoxia, which increases stromal-derived factor 1 alpha (SDF-1α) expression in cancer and stromal cells and, subsequently, myeloid differentiation antigen–positive (Gr-1+) myeloid cell infiltration. The SDF-1α/C-X-C receptor type 4 (CXCR4) pathway directly promotes hepatic stellate cell (HSC) differentiation and activation through the mitogen-activated protein kinase pathway. This is consistent with the association between SDF-1α expression with fibrotic septa in cirrhotic liver tissues as well as with desmoplastic regions of human HCC samples. We demonstrate that after treatment with sorafenib, SDF-1α increased the survival of HSCs and their alpha-smooth muscle actin and collagen I expression, thus increasing tumor fibrosis. Finally, we show that Gr-1+ myeloid cells mediate HSC differentiation and activation in a paracrine manner. CXCR4 inhibition, using AMD3100 in combination with sorafenib treatment, prevents the increase in tumor fibrosis—despite persistently elevated hypoxia—in part by reducing Gr-1+ myeloid cell infiltration and inhibits HCC growth. Similarly, antibody blockade of Gr-1 reduces tumor fibrosis and inhibits HCC growth when combined with sorafenib treatment. Conclusion: Blocking SDF-1α/CXCR4 or Gr-1+ myeloid cell infiltration may reduce hypoxia-mediated HCC desmoplasia and increase the efficacy of sorafenib treatment. (Hepatology 2014;59:1435-1447) Abbreviations Ab antibody Ad-Cre Cre-expressing adenovirus AKT protein kinase B APC allophycocyanin CAIX carbonic anhydrase IX CXCL12 C-X-C ligand 12 CXCR4 C-X-C receptor type 4 DAPI 4′,6-diamidino-2-phenylindole ECM extracellular matrix ERK extracellular signal-related kinase FCM flow cytometry FGFR basic fibroblast growth factor receptor Gr-1 myeloid differentiation antigen HCC hepatocellular carcinoma HSC hepatic stellate cell IHC immunohistochemical IV intravenous 5-LO 5-lipoxygenase MAPK mitogen-activated protein kinase MCP-1 monocyte chemoattractant protein-1 MMP matrix metalloproteinase MTT 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide p-AKT phosphorylated AKT PBS phosphate-buffered solution PCR polymerase chain reaction PDGFR platelet-derived growth factor receptor p-ERK phosphorylated ERK r recombinant SDF-1α stromal-derived factor 1 alpha siRNA small interfering RNA α-SMA alpha smooth muscle actin TGF transforming growth factor TKI tyrosine kinase inhibitor VEGF vascular endothelial growth factor VEGFR vascular endothelial growth factor receptor Hepatocellular carcinoma (HCC) almost exclusively arises in cirrhotic livers as well as preexisting chronic inflammation and fibrosis fuel hepatocarcinogenesis and HCC growth.1-3 Fibrosis is the consequence of hepatic stellate cell (HSC) activation and proliferation as well as myofibroblast differentiation leading to increased collagen deposition.4 This dual pathology of the liver contributes to an aggressive, systemic treatment-refractory characteristic in HCCs.1 Recently, the tyrosine kinase inhibitor (TKI), sorafenib, has emerged as the first systemic therapy for HCC. Sorafenib is an antiangiogenic drug that has a broad tyrosine kinase inhibition spectrum.5 However, despite this progress, the mortality rate from HCC remains high, making this disease the third-leading cause of cancer-related death worldwide.6-10 Sorafenib is widely considered as an antiangiogenic/-vascular drug through inhibition of vascular endothelial growth factor (VEGF) receptors (VEGFRs) and platelet-derived growth factor receptors (PDGFRs). However, more-potent and -selective anti-VEGF agents or more-broad antiangiogenic agents (e.g., VEGFR/basic fibroblast growth factor receptor [FGFR] and anti-VEGFR/PDGFR inhibitors) have thus far failed to match the efficacy of sorafenib in phase III trials in HCC.10-13 Moreover, antiangiogenic therapy has not led to tumor regression in patients or in experimental models in mice: The benefit noted with sorafenib in HCC patients is likely the result of a transient delay in HCC growth, after which most tumors resume their growth.10 Whereas the mechanisms of acquired resistance to sorafenib and other anti-VEGF inhibitors in HCC remain unknown, it is likely that tumor stroma-mediated survival pathways might play a key role.1, 10 Of these, increased hypoxia has been proposed as a mechanism of resistance to multitargeted TKI therapy.14-17 The challenge is to identify the key molecular pathways regulating stroma-mediated resistance to sorafenib treatment in HCC. Hypoxia and other cellular stresses can promote the expression of the chemokine, stromal-derived factor 1 alpha (SDF-1α or C-X-C ligand 12 [CXCL12]), and of its receptor, C-X-C receptor type 4 (CXCR4).18-22 In clinical studies, we showed that SDF-1α level increased in plasma circulation in HCC patients after treatment with sunitinib or cediranib (both anti-VEGFR and anti-PDGFR TKIs).23, 24 Moreover, we showed that elevated circulating levels of SDF-1α correlated with poor treatment outcome in HCC patients after sunitinib treatment.23 Systemic activation of the SDF-1α/CXCR4 axis is known to mediate intratumoral infiltration of inflammatory cells, including myeloid differentiation antigen–positive (Gr-1+) myeloid (CD11b+) cells.25-28 Gr-1+ myeloid cells can drive tumor recurrence after anti-VEGF therapy in various tumor models.29 Finally, clinical correlative data also strongly suggest that the effects on multitargeted TKI treatment on tumor vasculature and myeloid cells may mediate response and resistance therapy in HCC patients.23, 30 However, a causal role of Gr-1+ myeloid cells in HCC resistance to antiangiogenic treatment has not been characterized. Furthermore, a mechanistic understanding of the interplay between treatment-induced hypoxia, SDF-1α/CXCR4 pathway activation, and Gr-1+ myeloid cell infiltration and tumor fibrosis in HCC is currently lacking. Here, we examined, in orthotopic HCC models, whether the SDF-1α/CXCR4 pathway is activated and causally related to Gr-1+ myeloid cell infiltration and tumor-associated fibrosis and, ultimately, to sorafenib resistance. Materials and Methods Cells and Materials We used the C3H mouse-derived HCC cell line, HCA-1.31 Human HSCs (catalog no.: 5300) were purchased from ScienCell Research Laboratories (San Diego, CA). These primary HSCs have been previously characterized.32 We purchased AMD3100 and FR180204 from Sigma-Aldrich (St. Louis, MO), AZD6244 and sorafenib from Selleck Chemicals (Houston, TX), recombinant PDGF-B and SDF-1α from R&D Systems (Minneapolis, MN), and anti-Gr-1 antibody (Ab; Leaf purified anti-mouse Gr-1 Ab) from BioLegend, Inc. (San Diego, CA). For coculture experiments, we isolated Gr-1+ myeloid cells from enzymatically digested HCA-1 tumors. Animals To induce liver fibrosis, we treated 5-week-old male C3H or Mst1–/–Mst2F/– mice with CCl4 (16% [v/v] in olive oil, 100-µL gavage, 3 times per week) before tumor implantation and induction. HCA-1 cells were orthotopically implanted in mice 2 weeks after the last CCl4 treatment, as previously described.33 Mst1–/–Mst2F/– mice develop spontaneous HCCs after intravenous (IV) injection of Cre-expressing adenovirus (Ad-Cre)34 and were a kind gift from Dr. Nabeel Bardeesy (Massachusetts General Hospital, Boston, MA). All animals received humane care according to the criteria outlined in the "Guide for the Care and Use of Laboratory Animals" prepared by the National Academy of Sciences and published by the National Institutes of Health (publication 86-23, revised 1985). Treatment Studies Mice were treated daily by gavage with sorafenib (40 mg/kg) or vehicle (phosphate-buffered saline; PBS) alone. AMD3100 (10 mg/kg/day) was delivered continuously using Alzet micro-osmotic pumps (DURECT Corporation, Cupertino, CA) over 2 weeks. Cell Viability Assays We assessed cell viability using the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) assay (Sigma-Aldrich). Western Blotting Analysis Western blotting was performed using Abs against collagen I and alpha-smooth muscle actin (α-SMA; Abcam, Cambridge, MA) as well as phosphorylated (p-) extracellular signal-related kinase (p-ERK), ERK, phosphorylated (p-) protein kinase B (p-AKT), and AKT (Cell Signaling Technology, Danvers, MA). Small Interfering RNA Knockdown ON-TARGETplus CXCR4 small interfering RNA (siRNA) and nontargeting control siRNA were purchased from Dharmacon (Lafayette, CO) and used for transfection of HCA-1 cells. Immunohistochemistry For murine samples, we used Abs against α-SMA, carbonic anydrase IX (CAIX; Abcam), collagen I (LF-67; kindly provided by Dr. L. Fisher, National Institute of Dental and Craniofacial Research, Bethesda, MD), and SDF-1α (BioVision, Inc., Milpitas, CA). For apoptosis detection, frozen tumor sections were stained using the TACS TdT Kit (R&D Systems). To assess expression level, we measured the fluorescently stained area for each marker and normalized it to the 4′,6-diamidino-2-phenylindole (DAPI) area (used as a measure of cellularity in viable tumor regions), as previously described.28 In addition, we performed immunostaining for SDF-1α in tumor samples from patients who underwent HCC resection. Hepatic fibrosis in the non-HCC liver area was evaluated according to the Laennec system, as previously described.35 Colocalization between SDF-1α expression and desmoplasia was assessed in 10 randomly selected high-power fields at 4× magnification. Flow Cytometry We used fluorescently labeled rat monoclonal Abs anti-mouse CD45/phycoerythrin/Cy7, Gr-1/allophycocyanin (APC), and CD11b/APC/Cy7 (BD Biosciences, San Jose, CA) to perform flow cytometric (FCM) analysis in digested tumor tissue, as previously described.36 Quantitative Reverse-Transcription Polymerase Chain Reaction We determined relative gene expression of SDF-1α, CXCR4, monocyte chemoattractant protein 1 (MCP-1), 5-lipoxygenase (5-LO), transforming growth factor (TGF)-α, TGF-β, PDGF-α, PDGF-β, matrix metalloproteinase (MMP)−9, MMP-13, and β-actin in tumor-infiltrating Gr-1+ cells using specific primers (Supporting Table 1), Real-Time SYBR Green PCR master mix (Applied Biosystems, Branchburg, NJ) and the Stratagene Mx3000P QPCR System, as previously described.36 Statistical Analysis Comparisons between treatment groups were performed using Mann-Whitney's U-test. A P value of less than 0.05 was considered to denote statistical significance. Further details on materials and methods used are described in the Supporting Experimental Methods section of the Supporting Materials. Results Sorafenib Treatment Increases Hypoxia and SDF-1α Expression in Orthotopic HCC Models To model the clinical features of HCC, we first induced liver fibrosis in mice by CCl4 treatment for 11 weeks (Supporting Fig. 1). Then, we generated orthotopic tumors by intrahepatic HCA-1 cell implantation in C3H mice or by inducing spontaneous HCC using Ad-Cre IV injection in Mst1–/–Mst2F/– mice (Supporting Fig. 2). When tumors became established, we treated mice with sorafenib for 14 days and then measured the changes in tissue oxygenation. Sorafenib treatment significantly increased the hypoxic tissue fraction—measured by CAIX immunostaining—when compared to control-treated tumors (Fig. 1A,B). In contrast, hypoxic tissue surface area did not increase in surrounding fibrotic liver tissue after treatment (Fig. 1E). Figure 1Open in figure viewerPowerPoint Tumor hypoxia and SDF-1α expression are increased after sorafenib treatment in orthotopic HCC. (A) Representative immunofluorescence staining of CAIX and SDF-1α in HCA-1 tumor-implanted in mice with liver fibrosis. Images are 636 µm across. (B and C) Hypoxic tumor tissue fraction (B) and SDF-1α expression (C) increased significantly after sorafenib treatment in orthotopic HCA-1 tumors in mice (n = 5-9). (D) Immunofluorescence imaging analysis showed that SDF-1α expression is increased after 14 days of sorafenib treatment in spontaneous HCCs in Mst1–/–Mst2F/– transgenic mice. Increased hypoxia and SDF-1α expression persisted when sorafenib treatment was combined with AMD3100 (n = 5-9). (E and F) Expression of CAIX (E) and SDF-1α (F) in the liver was not significantly changed after sorafenib, AMD3100, or combination treatment (n = 4-7). The number of random regions of interest used for quantification is shown in parentheses. Data are presented as mean ± standard error of the mean. *P < 0.05; **P < 0.01. To examine the consequence of treatment-induced increase in tumor hypoxia, we next evaluated whether the increase in SDF-1α expression—suggested by studies in HCC patients23, 24—is recapitulated in these HCC models. Indeed, we found a 2-fold elevation in SDF-1α expression after sorafenib treatment in HCA-1 tumor grafts and in spontaneous HCCs in Mst1–/–Mst2F/– mice (Fig. 1A-D and Supporting S3). In contrast, SDF-1α expression did not significantly increase in the fibrotic liver after sorafenib treatment (Fig. 1F). To confirm that hypoxia is the driver of SDF-1α expression in cancer and stromal cells, we cultured HCA-1 cells as well as HSCs for 48 hours in hypoxic (1% O2) or normoxic (21% O2) conditions and measured SDF-1α expression by quantitative polymerase chain reaction (PCR). Exposure to hypoxic conditions increased SDF-1α expression in both HCC cells and HSCs (Supporting Fig. 4). To evaluate the effect of SDF-1α/CXCR4 pathway inhibition on tumor tissue oxygenation and SDF-1α expression after treatment, we combined sorafenib treatment with the CXCR4 inhibitor, AMD3100 (10 mg/kg/day; Sigma-Aldrich). Interestingly, the combination therapy further increased hypoxic tissue fraction and SDF-1α expression in orthotopic HCA-1 tumor grafts and spontaneous HCCs in Mst1–/–Mst2F/– mice, when compared to sorafenib treatment alone (Fig. 1 and Supporting Fig. 3). Inhibition of CXCR4 Prevents the Increase in HCC Fibrosis After Sorafenib In Vivo To establish whether fibrosis is associated with elevated SDF-1α expression in HCC patients, we examined surgical specimens for HCC patients with cirrhosis. We observed that SDF-1α expression colocalized with extracellular matrix (ECM) components and fibrotic septa in both HCC and cirrhotic liver tissues (Fig. 2). Thus, we next evaluated the effect of sorafenib treatment with or without inhibition of SDF-1α/CXCR4 pathway on fibrosis, a hallmark of HCC progression,4 in the mouse models. To this end, we measured expression of collagen I as well as the number of α-SMA+ myofibroblasts after sorafenib treatment, both in the tumor region and in surrounding liver tissue. Of note, the α-SMA+ myofibroblasts were also positive for glial fibrillary acidic protein (Supporting Fig. 5). Immunohistochemical (IHC) analyses revealed that the HCCs growing in the face of sorafenib treatment showed significantly increased tumor desmoplasia. Treatment resulted in increased intra- and peritumoral collagen I and α-SMA+ myofibroblast infiltration in orthotopic HCA-1 HCCs in mice with CCl4-induced liver fibrosis and in spontaneous HCCs in Mst1–/–Mst2F/– mice (Fig. 3A-E). Of note, sorafenib treatment reduced collagen I expression levels in surrounding liver tissue in mice with CCl4-induced liver fibrosis (Fig. 3F). Figure 2Open in figure viewerPowerPoint Immunostaining for SDF-1α in liver tissue samples from HCC patients. Paraffin-embedded tissue sections were stained for SDF-1α by IHC. (A and B) SDF-1α expression in nonmalignant liver tissue (A) was consistently lower than in the malignant HCC tumor area (B). Both in cirrhotic liver tissue (C) and in HCC nodules (D), SDF-1α expression was colocalized with ECM components/fibrous septa supporting the potential profibrotic role of SDF-1α in human hepatic fibrogenesis and HCC desmoplasia. (E) Specimens of tumors that reoccurred after transcatheter arterial chemoembolization and radiofrequency ablation—both of which are known to cause severe hypoxia—consistently showed high expression of SDF-1α, which was also associated with more pronounced desmoplasia in the HCC area. Error bars represent score ± standard error of the mean (n = 10 regions of interest per sample). Figure 3Open in figure viewerPowerPoint Sorafenib treatment selectively increases tumor-associated fibrosis in HCC in an SDF-1α/CXCR4 pathway-dependent manner. (A and B) Sorafenib significantly increased collagen I content (A) and the number of α-SMA+ myofibroblasts (B) in orthotopic HCA-1 tumors implanted in mice with fibrotic livers. Analysis was performed using immunofluorescence in tumor tissue, and the maker-positive area was normalized to the area of DAPI (nuclear stain; N = 5-7). (C and D) Sorafenib treatment significantly increased collagen I content (C and D) and expression of α-SMA (D) (detected by western blotting) in spontaneously arising HCCs (N = 7-8). (E) Representative images of immunofluorescence of collagen I staining in spontaneous HCCs. Images are 636 µm across. Addition of AMD3100 to sorafenib treatment prevented the increase in collagen I content and the number of α-SMA+ myofibroblasts in HCCs (A-E). (F) In tumor-bearing mice, sorafenib significantly reduced collagen I content in surrounding liver tissue in mice with underlying liver fibrosis to levels comparable to those noted in livers from non-CCl4-treated mice (N = 4-8). The number of random regions of interest used for quantification is shown in parentheses. *P < 0.05; **P < 0.01. Data are shown as mean ± standard error of the mean. To assess the effect of SDF-1α/CXCR4 pathway inhibition on tumor-associated fibrosis after sorafenib treatment, we tested the combined administration of sorafenib with the CXCR4 inhibitor, AMD3100. This combination therapy prevented the increase in collagen I expression and α-SMA+ myofibroblasts after sorafenib treatment in spontaneous and implanted HCCs, without significantly changing collagen I expression in fibrotic liver in CCl4-treated mice (Fig. 3). Of note, this effect was observed despite a persistent increase in hypoxia and SDF-1α expression in the HCCs treated with sorafenib and AMD3100 (Fig. 1A-D). Thus, CXCR4 inhibition can prevent the profibrotic effects of sorafenib treatment in HCC in vivo, despite persistent tumor hypoxia. SDF-1α/CXCR4 Axis Directly Mediates HSC Differentiation to Myofibroblasts in HCC Despite PDGFR Blockade by Sorafenib Next, we examined the role of the SDF-1α/CXCR4 axis in selective promotion of tumor fibrosis after sorafenib treatment in vitro. To determine whether increased SDF-1α expression in tumor could mediate the increase in tumor fibrosis after sorafenib treatment, we first exposed primary HSCs to recombinant (r)SDF-1α in the presence or absence of sorafenib or rPDGF-B. Consistent with previous reports,37 we found that rPDGF-B stimulated proliferation and α-SMA expression in HSCs (Fig. 4A,B). Sorafenib treatment prevented the effects of rPDGF-B and led to significant decrease in HSC viability (as evidenced by an increase in cleaved caspase-3 expression and in the number of apoptotic HSCs) and inhibited α-SMA and collagen I expression (Fig. 4A-C). These results indicate that sorafenib treatment may directly reduce liver fibrosis by blocking the PDGFR pathway in HSCs. Exposure to rSDF-1α induced HSC differentiation into myofibroblasts, as evidenced by dose-dependent increases in α-SMA and collagen I expression as well as in ERK and Akt activation (Fig. 4D). Then, we evaluated whether SDF-1α can drive HSC differentiation and activation of HSCs in the face of PDGFR blockade by sorafenib treatment. We found that SDF-1α increased cell proliferation and viability as well as promoted the differentiation of HSCs, despite sorafenib treatment (Fig. 4A-C). Figure 4Open in figure viewerPowerPoint SDF-1α/CXCR4 axis promotes HSC to myofibroblast differentiation in the face of PDGFR blockade by sorafenib. (A) Exposure to rPDGF-B increased HSC proliferation, whereas treatment with sorafenib reduced the viability of HSCs. Exposure to recombinant SDF1-α increased the viability of HSCs, despite PDGFR inhibition using sorafenib treatment, in a dose-dependent manner. HSC viability was measured by MTT assay (N = 6 experimental repeats). (B) Exposure to rSDF-1α increased expression of α-SMA and collagen I and reduced cleaved caspase-3 expression (evaluated by western blotting), despite sorafenib treatment, in a dose-dependent manner. (C) Exposure to rSDF-1α increased viability of HSCs, despite sorafenib treatment (N = 3-5 experimental repeats). (D) Exposure to rSDF-1α up-regulated collagen I and α-SMA expression levels as well as ERK and AKT activation in HSCs, consistent with their myofibroblast differentiation. Inhibition of CXCR4 with AMD3100 (D) or using siRNA (E) prevented the effects of SDF-1α. (F) ERK inhibition with FR-180204 (2 µM) decreased α-SMA expression in HSCs treated with rSDF-1α. Data are presented as mean ± standard error of the mean. Finally, to determine whether the SDF-1α/CXCR4 axis mediates these effects in HSCs, we used pharmacologic and genetic approaches to inhibit CXCR4. To this end, we cultured HSCs in the presence of rSDF-1α with or without the CXCR4 inhibitor, AMD3100, or with or without CXCR4 expression knockdown using siRNA. In both settings, inhibition of CXCR4 prevented the effects of SDF-1α on HSC differentiation and activation (i.e., prevented the increase in α-SMA and collagen I expression induced by SDF-1α in HSCs; Fig. 4D,E and Supporting Fig. 6). Inhibitory effects were mediated, in part, by preventing activation of mitogen-activated protein kinase (MAPK) pathway activation by SDF-1α, as shown by ERK inhibition with FR180204 (2 µM) or MAPK kinase inhibition with AZD6244 in HSCs treated with rSDF-1α (Fig. 4D-F and Supporting Fig. 7). Gr1+ Myeloid Cell Infiltration Increases in HCC AFTER Sorafenib in an SDF-1α/CXCR4-Dependent Manner Next, we examined the effects of treatment with sorafenib—with or without inhibition of CXCR4—on inflammatory cell infiltration in HCC. To this end, we evaluated enzymatically digested HCC tissue by FCM analysis. We found that the number of Gr-1+ myeloid cells increased by over 2-fold in HCA-1-transplanted HCC models and in spontaneous HCCs in Mst1–/–Mst2F/– mice after sorafenib treatment (Fig. 5A,B). In contrast, we found that sorafenib treatment reduced the accumulation of Gr-1+ myeloid cells in surrounding fibrotic liver tissue (Fig. 5C). Finally, inhibition of CXCR4, using AMD3100 in combination with sorafenib, decreased the number of tumor-infiltrating Gr-1+ myeloid cells to levels comparable to control-treated spontaneous and transplanted HCCs (Fig. 5A,B). Figure 5Open in figure viewerPowerPoint Intratumoral infiltration of Gr1+ myeloid cells is increased after sorafenib (SOR) treatment and is prevented by CXCR4 inhibition in HCC. (A and B) After SOR treatment, the number of 7-Aminoactinomycin D (7-AAD)/CD11b+Gr1+ monocytes (measured by FCM) significantly increased in HCA-1 tumors growing in C3H mice with liver fibrosis (A) as well as in spontaneous HCCs i