Abstract PR06: VEGF-targeted therapy induces extracellular matrix remodeling and increases mechanical barriers to therapy in colorectal cancer liver metastases
Authors: Rahbari Nuh N. , Kedrin Dmitriy , Incio Joao , Liu Hao , Ho William T. , Nia Hadi T. , Edrich Christina M. , Jung Keehoon , Daubriac Julien , Chen Ivy , Heishi Takahiro , Martin John , Huang Yuhui , Maimon Nir , Reissfelder Christoph , Weitz Juergen , Boucher Yves , Clark Jeffrey W. , Grodzinsky Alan J. , Duda Dan G. , Jain Rakesh K. , Fukumura Dai 
Published: March 2017

Abstract
Introduction: The anti-VEGF antibody bevacizumab in combination with chemotherapy is a standard treatment for metastatic colorectal cancer (mCRC) today, based on an overall survival improvement. However, this survival benefit is modest and mCRC ultimately progresses. The underlying mechanism of resistance to anti-angiogenic therapy remains unclear. Recent preclinical studies have shown that anti-angiogenic therapy increases collagen expression in tumors as a consequence of hypoxia. The extracellular matrix (ECM) play an important role in solid stress-induced blood vessel collapse as they transmit the mechanical stress created by proliferating cells within the confined space of a tumor. In this study, we investigated the effects of anti-angiogenic therapy on extracellular matrix expression, both collagenous and non-collagenous and perfusion as a novel mechanism of acquired resistance to anti-angiogenic therapy in liver metastases from CRC.
Experimental Design: We used C57BL/6 and BALB/c, as well as CCR2-/- and ATR1-/- mice for this study. To generate liver metastasis, we exteriorized and transected spleen, inject syngenic CRC SL4 or CT26 cells (0.1 million cells) in one hemispleen and then, removed it. The other hemispleen remained intact. We monitored tumor burden by measuring blood Gaussia luciferase (Gluc) activity from Gluc transduced tumors and/or by high-frequency ultrasound imaging. Treatments include anti-VEGF monoclonal antibody B20.4-1.1 (5 mg/kg or 1 mg/kg i.p. 2x/week, Genentech), an anti-Ly6G antibody (5 mg/kg i.p. every 2days), pegylated hyaluronidase (PEGHAse, 4.5 mg/kg i.v. 2x/week 24 hour prior to administration of chemotherapy), 5-Fluorouracil (50 mg/kg i.v. 2x/week). We determined ECM–hyaluronic acid (HA), sulfated glycosaminoglycan (sGAG), and collagen contents—by ELISA, the Blyscan Proteoglycan and Glycosaminoglycan assay, and hydroxyproline assay, respectively. We further determined ECM, activated fibroblasts, vasculature, perfusion (Hechst33342), and hypoxia (pimonidazole) by immunohistochemistry, and immune cell profile using flow cytometry. We then, determined stiffness (Young's modulus) of tumors by unconfined compression tests (Cancer Res 60: 2497-503, 2000) and solid stress using a recently established method (PNAS 109: 15101-8, 2012).
We obtained patient samples from the Heidelberg University Hospital and analyzed surgical specimens of patients who underwent liver resection for colorectal liver metastases without preoperative chemotherapy, after preoperative chemotherapy without bevacizumab or after preoperative chemotherapy with bevacizumab.
Results: Here, we show that anti-VEGF therapy induces remodeling of the extracellular matrix with subsequent alteration of physical properties of liver metastases. We find that preoperative treatment with bevacizumab in patients with colorectal liver metastases resulted in a significant increase in HA deposition within the tumor. Moreover, in two syngeneic mouse models of mCRC, we show that anti-VEGF therapy markedly increased expression of HA and sGAG, without significant changes in collagen deposition. The density of these matrix components correlates with tumor stiffness; the latter was also significantly increased after anti-VEGF therapy. In time-course and immunofluorescence studies, treatment-induced hypoxia appeared as the driving force for enhanced extracellular matrix expression. Enzymatic depletion of HA partially reversed compromised perfusion in liver metastases after anti-VEGF therapy and prolonged survival in combination with anti-VEGF therapy and systemic chemotherapy.
Conclusion: These findings suggest that extracellular matrix components such as HA could be a potential therapeutic target for reducing physical barriers to systemic treatments in patients with mCRC who receive anti-VEGF therapy.
This abstract is also being presented as Poster B27.
Citation Format: Nuh N. Rahbari, Dmitriy Kedrin, Joao Incio, Hao Liu, William T. Ho, Hadi T. Nia, Christina M. Edrich, Keehoon Jung, Julien Daubriac, Ivy Chen, Takahiro Heishi, John Martin, Yuhui Huang, Nir Maimon, Christoph Reissfelder, Juergen Weitz, Yves Boucher, Jeffrey W. Clark, Alan J. Grodzinsky, Dan G. Duda, Rakesh K. Jain, Dai Fukumura. VEGF-targeted therapy induces extracellular matrix remodeling and increases mechanical barriers to therapy in colorectal cancer liver metastases. [abstract]. In: Proceedings of the AACR Special Conference on Engineering and Physical Sciences in Oncology; 2016 Jun 25-28; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2017;77(2 Suppl):Abstract nr PR06.