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Journal of Clinical Oncology, Vol 26, No 11 (April 10), 2008: pp. 1836-1842 © 2008 American Society of Clinical Oncology. DOI: 10.1200/JCO.2007.11.6566 Effect of Molecular Therapeutics on Liver Regeneration in a Murine Model
From the Departments of Surgical Oncology and Cancer Biology, The University of Texas M.D. Anderson Cancer Center, Houston, TX; and ImClone Systems Inc, New York, NY Corresponding author: Lee M. Ellis, MD, Department of Surgical Oncology, Unit 444, The University of Texas M.D. Anderson Cancer Center, PO Box 301402, Houston, TX 77230-1402; e-mail: lellis{at}mdanderson.org
Purpose Unresectable metastatic colorectal cancer (CRC) can be rendered resectable with systemic chemotherapy in approximately 20% of cases. Most patients with metastatic CRC receive chemotherapy with the addition of targeted therapy with anti–vascular endothelial growth factor (VEGF) or anti–epidermal growth factor receptor (EGFR) antibodies. We sought to determine whether anti-VEGF receptor (VEGFR) or anti-EGFR therapy would impair liver regeneration after partial hepatectomy (PH) in mice. Materials and Methods Mice underwent either 66% PH or sham laparotomy. In the first experiment, mice in the PH group were randomly assigned to receive daily intraperitoneal injections of monoclonal antibodies (MoABs) to murine VEGFR-2 or nonspecific MoABs (control). In the second experiment, mice in the PH group were randomly assigned to receive intraperitoneal injections of antimurine EGFR or nonspecific (control) MoABs. In both experiments, therapy was initiated the day before surgery and continued until the mice were killed on day 5. Livers were collected and processed. Results Anti–VEGFR-2 therapy slightly impaired liver regeneration and hepatic cell proliferation compared with control. Hematoxylin and eosin staining showed no differences in liver morphology. CD105 staining showed decreased levels of activated endothelium in livers in the VEGFR-2 MoAB group. VEGFR-2 MoAB therapy decreased the levels of the cell cycle regulators cyclin D1 and cyclin D3 and the regenerative cytokine interleukin-6. Anti-EGFR therapy had no effect on liver regeneration or cellular proliferation. Conclusion Anti–VEGFR-2 therapy slightly impaired liver regeneration in this murine model, whereas anti-EGFR therapy had no effect on liver regeneration.
Recent studies have shown the value of chemotherapy for downsizing tumors in patients with colorectal cancer (CRC) that has metastasized to the liver.1-5 As many as 20% of CRCs initially deemed to be unresectable can be converted to resectable with the use of systemic therapy, and 5-year survival rates can be as high as 35%.1,2 Currently, approximately 70% of patients with metastatic CRC receive the anti–vascular endothelial growth factor (VEGF) monoclonal antibody (MoAB) bevacizumab as a component of first-line therapy. The anti–epidermal growth factor receptor (EGFR) MoABs cetuximab and panitumumab are also approved for use in subsequent lines of therapy, although in practice, these agents are likely to also be used as a component of first-line therapy.3 Both therapeutic strategies improve response rates to chemotherapy, thus raising the possibility that even more patients' cancer can be converted to resectable.4,5 Liver regeneration is an intricate process that results primarily from the proliferation of existing mature cellular populations.6 VEGF receptor (VEGFR)-2 is known to be the critical growth factor receptor in angiogenesis and endothelial cell proliferation. Preclinical findings have shown that levels of numerous growth factors and cytokines such as hepatocyte growth factor, interleukin-6 (IL-6), EGF, amphiregulin, and VEGF increase after partial hepatectomy (PH) and that regenerating livers depend on these growth factors for regeneration.7-17 In addition, growth factor receptors such as VEGFR-2 are also activated during liver regeneration.7,8 VEGF regulates both angiogenesis and the induction of growth factors in the liver after hepatic injury. In previous studies, activation of VEGFR-2 led to an increase in proliferation of hepatic endothelial cells after hepatic injury that, in turn, led to an increase in hepatocyte proliferation. This effect on hepatocyte proliferation was indirect, because an in vitro ligand selective for VEGFR-2 activation did not induce hepatocyte proliferation.9 VEGF upregulation through the adenoviral transduction of VEGF165 cDNA before hepatic resection has been shown to hasten functional hepatic recovery, possibly through preventing liver cell apoptosis.7 Activation of EGFR also plays a role in early hepatic regeneration.10 The EGFR ligand amphiregulin is upregulated during liver regeneration, and amphiregulin knockout mice show delays in liver regeneration after PH.11 Other EGFR ligands have also been noted to affect liver regeneration, but their effect is less pronounced than that of amphiregulin.11,12 Because EGFR and its ligands play a role in liver regeneration, it is possible that anti-EGFR therapy could inhibit liver regeneration. The dependence of the regenerating liver on growth factors and the effects of molecular therapeutics on wound healing13-16 led us to hypothesize that agents that target these pathways could impair liver regeneration. This issue is clinically relevant for patients being considered for hepatic resection during or shortly after being treated with bevacizumab or cetuximab/panitumumab containing regimens. However, no experimental studies have been published on the effect of anti-VEGF or anti-EGFR therapy on liver regeneration. VEGF ligands and receptors are characterized by specific binding of defined ligands to their cognate tyrosine kinase receptors that mediate specific functions within cells. VEGFR-2 is thought to be the major angiogenic receptor. Bevacizumab can inhibit the activity of the VEGF receptors and therefore may affect liver regeneration. In the first study, we tested the hypothesis that inhibition of murine VEGFR-2 specific MoABs would impair liver regeneration in a murine PH model. In the second study, we tested the hypothesis that inhibition of murine EGFR with a specific MoAB would impair liver regeneration in a murine PH model.
Reagents MoABs to murine VEGFR-2 (DC101) and EGFR were provided by ImClone Systems (New York, NY). The antibodies currently approved for treating patients with metastatic CRC recognize only human antigens and therefore cannot be used in experiments targeting murine receptors. In addition, antibodies targeting the tyrosine kinase receptors themselves as opposed to their ligands were used to help define what role each receptor plays in liver regeneration. Antibodies used for immunohistochemical and Western blot analyses were as follows: antibromodeoxyuridine (BrdU; BD Biosciences, San Jose, CA), anti-CD105/endoglin (Neomarkers, Fremont, CA), antiproliferating cell nuclear antigen (PCNA) PC-10 (DAKO, Carpinteria, CA), anticyclin D1 and anticyclin D3 antiphospho-EGFR-1173, antiphospho-EGFR-992, and anti-EGFR (all from Cell Signaling Technologies, Boston, MA).
Liver Regeneration Model The 66% PH technique was adapted from previously described techniques.17-19 Briefly, after anesthesia was induced with ketamine (30 mg/kg; Sigma Chemical Co, St Louis, MO), an upper midline abdominal incision was created. The right upper lobe was isolated first and ligated at the base with a 3-0 silk ligature. Next, the left upper and left lower lobes were isolated and ligated at the base with 3-0 silk. The isolated lobes were divided above the tie, removed, and weighed. Removing the three lobes resulted in removal of approximately 66% of the mouse liver volume.18,20 The incision was closed with 3-0 absorbable sutures. A sham surgery control group underwent an upper midline incision and gentle liver manipulation without liver resection. The experiment was terminated on postoperative day 5. The day 5 time point was selected on the basis of the knowledge that mouse livers exhibit nearly 100% regeneration 7 days after PH,6 and thus we wanted to capture changes in liver regeneration in actively regenerating liver. Livers were excised, weighed, and preserved. Tissue was snap-frozen in cryogenic vials (Corning, Corning, NY) for protein isolation, snap-frozen in optimum cutting temperature solution (Miles, Elkhart, IN) in preparation for immunohistochemical analyses, and placed in formaldehyde for the creation of paraffin tissue blocks.
Treatment Conditions Anti-EGFR therapy. The treatment model was similar to the anti-VEGFR therapy except for the MoAbs used. The MoAbs used were antimurine EGFR antibody (1 mg/dose) or nonspecific monoclonal antibodies (control; 1 mg/dose).
Quantification of Liver Regeneration
Immunohistochemical Staining Because the sinusoidal vascular architecture of the liver makes obtaining accurate blood vessel counts difficult, we stained livers for CD105, a marker of activated endothelium,26 to grossly assess the relative levels of activated endothelium among different groups. An investigator who did not participate in the staining procedure and who had no knowledge of treatment group assignment blindly compared specimens from the various groups in the second study. To examine the expression of activated EGFR in the liver specimens, formalin-fixed, paraffin-embedded tissues were stained with an antibody to phosphorylated EGFR. Images were obtained in four different quadrants of each liver section (x100 magnification). An investigator without knowledge of treatment group assignment blindly evaluated the specimens. For EGFR activation studies, tumors derived from A431 human epidermoid squamous cell carcinoma served as a positive control. Negative controls were created by omission of the primary antibody. Paraffin-embedded sections were also stained with hematoxylin and eosin by standard methods. Digital images of immunostaining were captured by an Axioskop microscope, Axiocam digital camera, and Axiovision 4.0 software (Zeiss, Thornwood, NY).
Western Blot Analysis
IL-6 Enzyme-Linked Immunosorbent Assay
Statistical Analysis
Effect of Anti-VEGFR Therapy on Hepatic Regeneration Gross measures of liver regeneration. Mouse body mass and resected liver mass did not differ among groups (Table 1). Anti–VEGFR-2 treatment led to impairment of liver regeneration (22% v the nonspecific MoAB–treated control group; P < .01; Fig 1A). Hematoxylin and eosin staining revealed no significant differences in liver morphology among the groups (data not shown).
Hepatic cell proliferation: BrdU immunocytochemical analysis29 (Fig 2). Rare proliferating cells were observed in the normal liver specimens of the sham surgery control group (Fig 2). The control group that underwent 66% PH and was treated with nonspecific MoAB showed a more than three-fold increase in the number of proliferating cells per high-power field (P < .0006). Anti–VEGFR-2 treatment led to a 68% reduction in the number of proliferating cells as compared with the nonspecific MoAB control (P = .0002).
IL-6 Levels. IL-6 is an important cytokine mediating liver regeneration. We performed an ELISA for IL-6 on whole-cell protein isolated from snap-frozen liver specimens. The control group that underwent 66% PH and was treated with nonspecific MoAB showed an approximately 70% increase in the amount of IL-6 present as compared with the sham PH group (an increase from 140 ± 3 pg/mL to 240 ±14 pg/mL; P < .05). Anti–VEGFR-2 treatment blocked this induction of IL-6 (102 ± 6 pg/mL; P < .05 v PH/nonspecific MoAB group) in regenerating liver. Activated endothelium in the regenerating liver. CD105 staining, as a marker of activated endothelium,26 was increased in the PH group compared with the sham laparotomy group. CD105 staining was less intense in livers from the anti–VEGFR-2 therapy group as compared with the PH control group (Fig 3).
Cell cycle mediators in the regenerating liver. Western blotting of whole-cell protein lysates extracted from snap-frozen liver specimens was performed to assess the levels of the cell cycle regulators cyclin D1 and cyclin D3 in the groups treated with VEGFR-2 MoABs (Fig 4). Cyclin D1 was increased after PH compared with the sham surgery control group. Cyclin D3 was not significantly changed after PH compared with the sham surgery control group. Anti–VEGFR-2 therapy decreased both cyclin D1 and D3 relative to that in the nonspecific MoAB control.
Effect of Anti-EGFR Therapy on Hepatic Regeneration Gross measures of liver regeneration. Mouse body mass, resected liver mass, liver remnant mass, and percentage of liver regeneration did not differ between the treatment and control groups (Table 2). Anti-EGFR MoAB therapy did not affect liver regeneration (P > .1; Fig 1B). Hematoxylin and eosin staining revealed no significant differences in liver morphology among the groups (data not shown).
EGFR phosphorylation in the regenerating liver. On Western blotting, phosphorylated EGFR (Tyr992) levels were slightly higher in the nonspecific MoAB group than in the sham surgery control group, and phosphorylated EGFR levels were lower in the anti-EGFR group than in the nonspecific MoAB control (Fig 5A). In blinded comparisons of immunohistochemical staining for phosphorylated EGFR (Tyr1173) in liver specimens, phosphorylated EGFR levels were slightly higher in the nonspecific MoAB group than in the sham surgery control group, and phosphorylated EGFR levels were lower in the anti-EGFR MoAB group than in the nonspecific MoAB control (Fig 5B).
Hepatic cell proliferation: PCNA immunocytochemical analysis (data not shown). Rare proliferating cells were observed in the normal liver specimens of the sham surgery control group. The control group that underwent 66% PH and was treated with nonspecific MoAB showed a more than eight-fold increase in the number of proliferating cells per high-power field (P < .00001). The anti-EGFR treatment group showed no significant reduction in hepatocyte proliferation as compared with the nonspecific MoAB control.
We investigated the effects of molecular therapeutics on liver regeneration in mice after PH. Our studies showed that anti–VEGFR-2 therapy slightly impaired liver regeneration after PH, whereas anti-EGFR therapy had no effect on liver regeneration. We were also able to show that anti–VEGFR-2 therapy blocked the following responses in regenerating liver: cell proliferation, activation of endothelial cells, levels of IL-6, and activation of early-phase cell cycle regulators. Anti-VEGF therapy is primarily an antiangiogenic therapy and is known to impair endothelial cell proliferation. Because of the fact that endothelial cells comprise a large fraction of the liver architecture owing to its inherent function, vessel counts, as is typically done in tumor studies, was not feasible. Instead, we sought to assess the level of activated endothelium within regenerating livers. CD105, also known as endoglin, is a validated marker of activated endothelium in tumors26; therefore, we selected this marker for study in regenerating liver. Immunohistochemical staining of regenerating liver demonstrated an increase in vessel staining intensity for endoglin compared with sham-operated mice, and this increase was abrogated by the VEGFR-2 MoAB. By examining the early-phase cell cycle regulators cyclin D1 and cyclin D3 in mice treated with anti–VEGFR-2 therapy, we were able to gain insight into the cellular processes affected by VEGFR-2 activation in regenerating liver. Cyclin D1, and to a lesser extent, cyclin D3, was activated in the regenerating liver in mice treated with nonspecific MoAB as compared with the sham control. Treatment with anti–VEGFR-2 MoAB led to a relative downregulation of both cyclin D1 and D3 relative to nonspecific MoAB. Furthermore, we also investigated an upstream regulator of cyclin D1 and D3, glycogen synthase kinase–3 beta. We found that phosphorylated glycogen synthase kinase–3 beta was increased with PH and anti–VEGFR-2 therapy blunted this induction (data not shown). Similar results were observed with important mediators of hepatic regeneration, such as IL-6 levels and activation of c-Met (data not shown).28 These findings in a murine model raise several issues for patients receiving anti-VEGF therapy who undergo liver resection. Compared with traditional chemotherapeutic agents, the half-life of antibodies, bevacizumab in particular, is relatively long (approximately 20 days).30 The dose of bevacizumab approved by the US Food and Drug Administration for patients with metastatic CRC is 5 to 10 mg/kg every 2 weeks. However, even a relatively low dose of bevacizumab (0.3 mg/kg) can lead to undetectable levels of free VEGF in the systemic circulation.30 Theoretically, at three half-lives (approximately 6 weeks) after administering a 5-mg/kg dose, approximately 0.67 mg/kg of bevacizumab would remain in circulation—a level higher than that necessary to remove all detectable free VEGF from circulation,30 implying that liver regeneration may be affected in patients who undergo liver resection in the face of anti-VEGF therapy. However, whether circulating VEGF levels are in fact an accurate predictor of the biologic effects of bevacizumab is still unknown. Another issue to be addressed is the optimal interval between cessation of neoadjuvant chemotherapy with bevacizumab and hepatic resection. Our findings showed that anti–VEGFR-2 therapy impaired liver regeneration in a murine model, albeit to a lesser extent than we expected. These findings imply that we must carefully consider the timing of cessation of anti-VEGF therapy relative to the interval to liver resection. These recommendations are particularly important for patients undergoing resection of more than 30% of their baseline liver volume (the so-called threshold for the regenerative response) and for patients with chemotherapy-induced or preexisting hepatic dysfunction. We reiterate that the impairment of liver regeneration with anti–VEGFR-2 therapy was less than we had expected, but one must keep in mind that murine models may not reflect underlying comorbidities of patients, including obesity and chemotherapy, both of which may impair baseline hepatic function. Although it has been reported that liver resection can be performed safely after bevacizumab administration, the number of patients studied to date has been relatively small.31 The use of registries tracking patients receiving bevacizumab for metastatic CRC (Bevacizumab Regimen–Investigation of Treatment Effect in the United States and Bevacizumab Expanded Access Trial in other countries) and prospective trials will provide more insight into the timing and safety of liver resection for patients receiving bevacizumab-containing regimens. In contrast to anti–VEGFR-2 therapy, anti-EGFR therapy did not impair liver regeneration or hepatic cellular proliferation. Although EGFR ligands are upregulated in liver regeneration, their blockade may not significantly impede the processes critical to liver regeneration, perhaps because of the existence of numerous alternative pathways that can affect the same downstream signals as does EGFR. We showed that anti-EGFR therapy decreased EGFR phosphorylation in the liver by both Western blotting and immunohistochemical analysis. Our findings indicate that anti-EGFR therapy does not affect liver regeneration. In addition to the data presented here, we also evaluated the effects of inhibition of VEGFR-2 and EGFR at day 3 after partial hepatectomy. Similar to the findings reported, anti–VEGFR-2 antibodies modestly impaired liver regeneration and hepatic proliferation (data not shown). Also similar to the current study, anti-EGFR therapy did not affect liver regeneration (data not shown). The introduction of targeted therapies into first-line chemotherapy regimens for CRC metastatic to the liver has led to improved rates of response and overall survival.32 However, enthusiasm for these new agents must be tempered with caution, given their side effects. As stated in a previous commentary, "Although the era of targeted therapies has provided us with new opportunities, we are also confronted with new challenges. It is critical for surgeons and oncologists to understand biologic principles of targeted therapies if we are to maximize treatment efficacy while minimizing morbidity."33 Prospective clinical studies will better determine the safety profiles of neoadjuvant targeted therapies before hepatic resection.
Although all authors completed the disclosure declaration, the following authors or their immediate family members indicated a financial interest. No conflict exists for drugs or devices used in a study if they are not being evaluated as part of the investigation. For a detailed description of the disclosure categories, or for more information about ASCO's conflict of interest policy, please refer to the Author Disclosure Declaration and the Disclosures of Potential Conflicts of Interest section in Information for Contributors. Employment: Yan Wu, ImClone Systems Inc; Daniel J. Hicklin, ImClone Systems Inc Leadership: N/A Consultant: Lee M. Ellis, ImClone Systems Inc Stock: Yan Wu, ImClone Systems Inc; Daniel J. Hicklin, ImClone Systems Inc Honoraria: N/A Research Funds: Lee M. Ellis, Funds, ImClone Systems Inc Testimony: N/A Other: N/A
Conception and design: George Van Buren II, Anthony D. Yang, Yan Wu, Daniel J. Hicklin, Lee M. Ellis Provision of study materials or patients: Yan Wu, Daniel J. Hicklin Collection and assembly of data: George Van Buren II, Anthony D. Yang, Nikolaos A. Dallas, Michael J. Gray, Sherry J. Lim, Ling Xia, Fan Fan, Ray Somcio, Yan Wu, Daniel J. Hicklin, Lee M. Ellis Data analysis and interpretation: George Van Buren II, Anthony D. Yang, Nikolaos A. Dallas, Michael J. Gray, Sherry J. Lim, Ling Xia, Fan Fan, Ray Somcio, Daniel J. Hicklin, Lee M. Ellis Manuscript writing: George Van Buren II, Anthony D. Yang, Lee M. Ellis Final approval of manuscript: Daniel J. Hicklin, Lee M. Ellis
We thank Corazon D. Bucana, PhD, and Donna Reynolds for their contributions to the immunohistochemical imaging studies. We also thank Christine Wogan, Department of Scientific Publications, and Rita Hernandez, Department of Surgical Oncology, for editorial assistance.
Supported by National Institutes of Health Grants No. T-32 09599 (G.V.B., A.D.Y., N.A.D., and S.J.L.) and ImClone Systems (Y.W., D.J.H., and L.M.E.). Both G.V.B. II and A.D.Y. contributed equally to this work. Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.
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Copyright © 2008 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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ABSTRACT
INTRODUCTION
