Epidermal Growth Factor Receptor Inhibition Modulates the Microenvironment by Vascular Normalization to Improve Chemotherapy and Radiotherapy Efficacy

Abstract

BackgroundEpidermal growth factor receptor (EGFR) inhibitors have shown only modest clinical activity when used as single agents to treat cancers. They decrease tumor cell expression of hypoxia-inducible factor 1-α (HIF-1α) and vascular endothelial growth factor (VEGF). Hypothesizing that this might normalize tumor vasculature, we examined the effects of the EGFR inhibitor erlotinib on tumor vascular function, tumor microenvironment (TME) and chemotherapy and radiotherapy sensitivity.Methodology/Principal FindingsErlotinib treatment of human tumor cells in vitro and mice bearing xenografts in vivo led to decreased HIF-1α and VEGF expression. Treatment altered xenograft vessel morphology assessed by confocal microscopy (following tomato lectin injection) and decreased vessel permeability (measured by Evan's blue extravasation), suggesting vascular normalization. Erlotinib increased tumor blood flow measured by Power Doppler ultrasound and decreased hypoxia measured by EF5 immunohistochemistry and tumor O2 saturation measured by optical spectroscopy. Predicting that these changes would improve drug delivery and increase response to chemotherapy and radiation, we performed tumor regrowth studies in nude mice with xenografts treated with erlotinib and either radiotherapy or the chemotherapeutic agent cisplatin. Erlotinib therapy followed by cisplatin led to synergistic inhibition of tumor growth compared with either treatment by itself (p<0.001). Treatment with erlotinib before cisplatin led to greater tumor growth inhibition than did treatment with cisplatin before erlotinib (p = 0.006). Erlotinib followed by radiation inhibited tumor regrowth to a greater degree than did radiation alone, although the interaction between erlotinib and radiation was not synergistic.Conclusions/SignificanceEGFR inhibitors have shown clinical benefit when used in combination with conventional cytotoxic therapy. Our studies show that targeting tumor cells with EGFR inhibitors may modulate the TME via vascular normalization to increase response to chemotherapy and radiotherapy. These studies suggest ways to assess the response of tumors to EGFR inhibition using non-invasive imaging of the TME.

Vascular Normalization as a Therapeutic Strategy for Malignant and Nonmalignant Disease

“…As discussed below, many studies testing the effects of antiangiogenic drugs have shown a presence of a "normalization window"-a time period beginning with the appearance of a normalized vascular phenotype (typically within 1 -2 days of starting treatment), and ending when features of normalization are lost Winkler et al 2004;Jain 2005b;Batchelor et al 2007;Kamoun et al 2009 Xian et al 2006;McCarty et al 2007;Mazzone et al 2009. c This is controversial given recent reports of increased metastasis after antiangiogenic therapy in certain animal models (Ebos et al 2009;Paez-Ribes et al 2009 Gorski et al 1999;Hansen-Algenstaedt et al 2000;Lee et al 2000;Kozin et al 2001;Ader et al 2003;Winkler et al 2004;Ansiaux et al 2005Ansiaux et al , 2006Pore et al 2006a;Dings et al 2007;Kashiwagi et al 2008;Batra et al 2009;Cerniglia et al 2009;Tsukada et al 2009;McGee et al 2010. g Teicher et al 1995a,b;Wildiers et al 2003;Segers et al 2006;Dickson et al 2007a,b,c;Bhattacharya et al 2008Bhattacharya et al , 2009Zhou et al 2008;Juan et al 2009;Zhou and Gallo 2009;J Liu, S Liao, B Diop-Frimpong, et al, unpubl. data. section, we discuss the key molecules implicated in tumor vessel biology with a focus on their role in regulating vascular normalization.…”

“…Lee et al 2000;Winkler et al 2004;Dings et al 2007;Fischer et al 2007;Eichhorn et al 2008;Batra et al 2009;Skuli et al 2009;McGee et al 2010. j Teicher et al 1995bJain et al 1998;Bernsen et al 1999;Cohen-Jonathan et al 2001;Delmas et al 2003;Ansiaux et al 2005Ansiaux et al , 2006Pore et al 2006b;Segers et al 2006;Cerniglia et al 2009;Qayum et al 2009;Cham et al 2010. k Ader et al 2003;Hamzah et al 2008;Kashiwagi et al 2008;Stockmann et al 2008;Maione et al 2009;Mazzone et al 2009;Skuli et al 2009;Tsukada et al 2009;Rolny et al 2011. l Yuan et al 1996Lee et al 2000;Wildiers et al 2003;Tong et al 2004;Nakahara et al 2006;Dickson et al 2007b;Kurozumi et al 2007;Taguchi et al 2008;Zhou et al 2008;Kamoun et al 2009;Ohta et al 2009;Gallo 2009. m Jain et al 1998;Izumi et al 2002;…”

“…j Teicher et al 1995bJain et al 1998;Bernsen et al 1999;Cohen-Jonathan et al 2001;Delmas et al 2003;Ansiaux et al 2005Ansiaux et al , 2006Pore et al 2006b;Segers et al 2006;Cerniglia et al 2009;Qayum et al 2009;Cham et al 2010. k Ader et al 2003;Hamzah et al 2008;Kashiwagi et al 2008;Stockmann et al 2008;Maione et al 2009;Mazzone et al 2009;Skuli et al 2009;Tsukada et al 2009;Rolny et al 2011. l Yuan et al 1996Lee et al 2000;Wildiers et al 2003;Tong et al 2004;Nakahara et al 2006;Dickson et al 2007b;Kurozumi et al 2007;Taguchi et al 2008;Zhou et al 2008;Kamoun et al 2009;Ohta et al 2009;Gallo 2009. m Jain et al 1998;Izumi et al 2002;Ansiaux et al 2005;Salnikov et al 2005;Bhattacharya et al 2008Bhattacharya et al , 2009Schnell et al 2008;Cerniglia et al 2009. n Dickson et al 2007c;Kashiwagi et al 2008;di Tomaso et al 2009;…”

“…As a consequence, inhibitors of oncogene activity can indirectly normalize tumor vessels, potentially enhancing not only the action of chemotherapy and radiotherapy, but also their own antitumor cell activity. Investigators have reported various aspects of normalization in response to inhibition of several oncogenes including HER-2 (Izumi et al 2002), the PI3K-AKT-mTOR axis (Pore et al 2006a;Schnell et al 2008;Xue et al 2008;Qayum et al 2009), Ras (Cohen-Jonathan et al 2001Delmas et al 2003;Qayum et al 2009), and the epidermal growth factor receptor (EGFR) (Cerniglia et al 2009;Qayum et al 2009;Johns et al 2010). In the case of HER-2, we have shown that treatment of leptomeningeal HER-2 positive breast cancer with trastuzumab (a monoclonal antibody directed against HER-2) not only inhibits tumor cell proliferation, but also normalizes vessels.…”

“…EGFR inhibitors such as gefitinib and erlotinib normalize tumor vessel structure and function in several mouse models of solid tumors (Cerniglia et al 2009;Qayum et al 2009) and synergize with chemotherapy and/or radiotherapy to maximize tumor control. Importantly, these studies (and others pertaining to oncogene inhibitors) report normalization of longer duration than that observed with anti-VEGF therapy (Qayum et al 2009), potentially relating to specific suppression of oncogene-driven, excessive VEGF within a tumor rather than total VEGF suppression from anti-VEGF therapy, which ultimately causes vascular regression.…”