Enhancement of radiation response with bevacizumab
© Hoang et al.; licensee BioMed Central Ltd. 2012
Received: 24 March 2012
Accepted: 16 April 2012
Published: 26 April 2012
Vascular endothelial growth factor (VEGF) plays a critical role in tumor angiogenesis. Bevacizumab is a humanized monoclonal antibody that neutralizes VEGF. We examined the impact on radiation response by blocking VEGF signaling with bevacizumab.
Human umbilical vein endothelial cell (HUVEC) growth inhibition and apoptosis were examined by crystal violet assay and flow cytometry, respectively. In vitro HUVEC tube formation and in vivo Matrigel assays were performed to assess the anti-angiogenic effect. Finally, a series of experiments of growth inhibition on head and neck (H&N) SCC1 and lung H226 tumor xenograft models were conducted to evaluate the impact of bevacizumab on radiation response in concurrent as well as sequential therapy.
The anti-angiogenic effect of bevacizumab appeared to derive not only from inhibition of endothelial cell growth (40%) but also by interfering with endothelial cell function including mobility, cell-to-cell interaction and the ability to form capillaries as reflected by tube formation. In cell culture, bevacizumab induced a 2 ~ 3 fold increase in endothelial cell apoptosis following radiation. In both SCC1 and H226 xenograft models, the concurrent administration of bevacizumab and radiation reduced tumor blood vessel formation and inhibited tumor growth compared to either modality alone. We observed a siginificant tumor reduction in mice receiving the combination of bevacizumab and radiation in comparison to mice treated with bevacizumab or radiation alone. We investigated the impact of bevacizumab and radiation treatment sequence on tumor response. In the SCC1 model, tumor response was strongest with radiation followed by bevacizumab with less sequence impact observed in the H226 model.
Overall, these data demonstrate enhanced tumor response when bevacizumab is combined with radiation, supporting the emerging clinical investigations that are combining anti-angiogenic therapies with radiation.
KeywordsAnti-angiogenesis VEGF Bevacizumab Radiation
Tumor angiogenesis is critical for tumors to grow and spread. Four decades ago, Folkman proposed targeting the tumor vasculature as a strategy to treat cancer. Since then advances in biology have provided new tools and knowledge in the area of angiogenesis. A key discovery was the identification of vascular endothelial growth factor (VEGF), a key angiogenic protein critical for the growth of endothelial cells and development of tumor blood vessels[2–4]. VEGF herein emerged as an attractive target for anticancer therapy. It has been demonstrated in animal models that neutralization VEGF could inhibit the growth of primary tumor and metastases. In small 1–2 mm foci of tumor cells, blocking the VEGF pathway inhibited the “angiogenic switch”, i.e. preventing tumor transformation from an avascular to vascular phase, thus maintaining a quiescent state. Bevacizumab is a humanized monoclonal antibody which neutralizes the VEGF ligand. Since its development in the late 1990s, the anti-tumor effects of this anti-VEGF antibody have been studied in various preclinical cancer models as well as in clinical trials. The combination of bevacizumab and cytotoxic chemotherapy prolongs survival in patients with advanced colorectal, lung or breast cancer. Bevacizumab is currently approved for use in combination with chemotherapy in those diseases, as well as monotherapy in recurrent glioblastoma.
Another potential treatment strategy is to combine bevacizumab with radiation to enhance the therapeutic index. Radiation dose escalation is limited in most anatomic sites by normal tissue toxicities. Therefore, combining radiation with targeted agents such as anti-angiogenic in an effort to augment radiation impact and improve tumor control is desirable. It has been shown that blocking VEGF with recombinant human anti-VEGF antibody can enhance radiation response in preclinical studies. Augmentation of tumor response was also observed when radiation was combined with other anti-angiogenic or vascular disrupting drugs[8–16].
The primary objective of this study was to investigate the anti-angiogenic and anti-tumor activity of bevacizumab in combination with radiation in human endothelial cells as well as in H&N and lung tumor models. We also explored the sequencing treatment of bevacizumab and radiation.
Chemicals, cell lines and animals
Bevacizumab was provided by Genentech (South San Francisco, CA). SCC1, a human head and neck squamous carcinoma cell line was kindly provided by Dr. Tom Carey (University of Michigan). The lung cancer cell line H226 was from the laboratory of Dr. Minna and Dr. Gazdar (University of Texas Southwestern Medical School). Supplement of all materials used in our experiments can be found in our previous publication[].
HUVEC growth inhibition assay
In this crystal violet assay, growing HUVEC seeded in 6-well plates (50,000 cells/well) were treated with bevacizumab in EGM-2 at various concentrations (0–10 μM). After 3 days, cells were stained with crystal violet. The method of this assay was described in detail in previous publication. The relative percentage of cell growth was calculated by comparison between the bevacizumab-treated and control wells.
Flow cytometry analysis of HUVEC apoptosis
Growing HUVEC were treated with EGM-2 (control), bevacizumab 0.1 μM, radiation 6 Gy, or combined bevacizumab and radiation. After 24 and 48 hours of incubation, cells were harvested, prepared, and stained with propidium iodide (PI) prior to flow cytometry analysis. The procedure was described in detail in previous publication. DNA distributions were analyzed by Modfit for the proportion of apoptotic cells.
In vitro angiogenesis (HUVEC tube formation) assay
In this assay, HUVEC (40,000 cells) were seeded atop of matrigel membrane in the absence (control) or presence of bevacizumab (0.5 μM and 5 μM). The method of this assay was described in detail in previous publication. The plate was examined and photographed for the formation of capillary-like endotubes under a phase-contrast microscopy at 3 h, 6 h and 22 h.
In vivo angiogenesis (matrigel plug) assay
The method of this assay was described in detail in previous publication. In brief, 4 groups of mice with H226-containing matrigel plugs were treated with IgG (control), bevacizumab alone (1 mg/kg intraperitoneally), radiation alone (2 Gy/fraction), or combination treatment in which bevacizumab was administered immediately following radiation, twice a week for 2 weeks. At the end of week 2, mice were injected with FITC-Dextran solution. The plugs were removed and examined for the perfused blood vessels. The intensity of fluorescence in captured images was quantified by Adobe Photoshop software.
Growth inhibition assay in tumor xenograft models
A series of in vivo experiments in athymic mice bearing SCC1 and H226 xenografts were conducted to examine the anti-tumor activity of bevacizumab, radiation and combined therapy in concurrent and sequential fashion. Design and treatment schedule of those experiments are described in the Results Section. Details on xenografts, animal care, tumor measurement and radiation delivery were described in previous publication.
Analysis of variance (ANOVA) was performed to compare tumor volume in groups of mice treated with bevacizumab and/or radiation with the control group. Treatment interaction and linear contrasts were used to evaluate the synergistic effect of the bevacizumab and radiation therapy combination. Tumor volume was log-transformed to meet the assumption of normality. Effects of bevacizumab and radiation on tumor growth in mice bearing SCC1 and H226 xenografts were analyzed using ANOVA and linear mixed-effects models. An autoregressive correlation structure was assumed to account for correlations between repeated measurements within an experimental unit. Tukey’s HSD method was used to control the type 1 error for the pairwise comparisons between treatment groups. All p values were two sided and considered significant when ≤0.05. Statistical analyses were performed with SAS statistical software (version 8.2; SAS Institute, Cary, NC).
Bevacizumab inhibits HUVEC proliferation in vitro and tumor growth in vivo
Bevacizumab inhibits the formation of HUVEC capillary-like network
Bevacizumab enhanced radiation-induced apoptosis in HUVEC
Concurrent administration of bevacizumab and radiation inhibits in vivo tumor vascularization
Bevacizumab augments tumor response to radiation
Impact of treatment sequence of bevacizumab and radiation
In this current study, we confirm the ability of the anti-VEGF monoclonal antibody bevacizumab to inhibit endothelial cell proliferation and disrupt the formation of capillary-like networks in culture. In the H&N and lung cancer xenograft models, treatment with bevacizumab inhibited tumor vascularization and inhibited volume growth of both SCC1 and H226 tumors. However, the growth inhibitory effect of bevacizumab is not complete, suggesting the potential value of combining bevacizumab with other cytotoxic modalities, such as radiation to achieve more potent therapeutic effects.
In this work, we demonstrate that radiation combined with bevacizumab reduced the formation of tumor vasculature and inhibited tumor growth in SCC1 and H226 cancer xenograft models more strongly than either modality alone (Figure6). This is consistent with prior work using the recombinant human monoclonal anti-VEGF 165 antibody in mouse models bearing other human cancers. Our findings confirm that neutralizing the VEGF ligand with bevacizumab can augment tumor response to radiation. Works from our laboratory and others have previously demonstrated that radiation response is enhanced by blocking the VEGF signaling pathway using small molecule VEGF receptor tyrosine kinase inhibitors such as ZD6474, SU6668 and PTK787/ZK222584, or by directly targeting tumor blood vessels with vascular targeting agents such as ZD6126[14, 15] and combretastatin.
The anti-tumor effect of this combination approach is consistent with the two-compartment model described by Folkman. According to this model, tumors are comprised of distinct compartments including tumor cells and endothelial cells. By targeting the endothelial cell compartment, bevacizumab not only inhibits the supply of oxygen and nutrients to the tumor, but also interrupts the “paracrine effect” by inhibiting endothelial secretion of growth factors such as IGF1, bFGF, and HB-EGF, which can stimulate tumor proliferation. In parallel, by targeting the tumor compartment, radiation kills cancer cells and thereby shuts down their production of “pro-angiogenic” factors, thus indirectly affecting the endothelial compartment. We have also observed that treatment with radiation can inhibit endothelial cell proliferation and stimulate apoptosis and G2/M arrest (nonpublished data), suggesting direct inhibitory effects of radiation on this compartment.
A current question of interest in clinical trial design regards the optimal sequencing of radiation and anti-angiogenic drugs to achieve maximal benefit. A valid concern is whether targeting the tumor vasculature will decrease tumor blood perfusion, resulting in tumor hypoxia, and thereby diminishing the effects of radiation. To investigate the impact of treatment sequencing on tumor response, we designed sequence experiments as described in Figure7. In the SCC-1 model, it appeared that tumor control was best achieved with the regimen of radiation followed by bevacizumab. This result supports the hypothesis that hypoxia induced by bevacizumab may hinder radiation effect. However, we found no clear difference between sequence regimens in the H226 tumors.
Consistent with our observation in the SCC-1 tumors, preclinical studies have shown that delivering ZD6126 prior to radiation to U87 glioblastoma xenografts resulted in acute drop in tumor oxygen tension and attenuation of the killing effects of radiation. Further, in KHT sarcoma models, the strongest anti-tumor activity was achieved when ZD6126 was administered one hour following radiation. These observations suggest a negative impact of ZD6126-induced hypoxia on radiation effect. However, the concept of normalization of tumor vasculature proposed by Jain et al. supports a strategy of using anti-angiogenic drugs to improve efficacy of radiation. This theory suggests that short term treatment of anti-angiogenic agents may “normalize” the network of abundant but chaotic, leaky and dysfunctional tumor vasculature, thus restore the integrity and function of the blood vessels, leading to a decrease in interstitial fluid pressure and the improvement in tumor oxygenation. Therefore, this process of vascular normalization could enhance the tumor killing activity of radiation as well as improve drug delivery into the tumor. Although the induction of vascular normalization by anti-angiogenic agents has been supported by preclinical studies, it remains a challenge to capture the transient “tumor oxygenation window” for the delivery of radiation. We are commencing real-time imaging of tumor hypoxia profiles in animals during treatment to help explore optimal strategies for this combined therapy.
In the clinic, several clinical phase I/II studies have been conducted to investigate the safety and efficacy of radiation and bevacizumab in cancer patients. The first report came from a series of 6 patients with locally advanced rectal carcinoma who were treated in a phase I trial with induction therapy of bevacizumab (5 mg/kg x 1 dose) followed by radiation in combination with bevacizumab and 5-fluorouracil, then surgical resection. This pilot study demonstrated that a single dose of bevacizumab induction lead to a significant decrease in interstitial fluid pressure, tumor blood perfusion, and microvascular density on day 12. The subsequent phase II trial in the same patient population demonstrated that bevacizumab induction therapy followed by concurrent bevacizumab and chemoradiation appeared safe and active with a 5-year local control and overall survival of 100%. The combination of bevacizumab with radiation was also investigated in early clinical studies in other diseases including pancreatic cancer and head and neck cancer, in which bevacizumab was started either prior or concurrently with chemoradiation.
In conclusion, the current study demonstrates enhanced tumor response when bevacizumab is combined with radiation. These data support the strategy of blocking the VEGF signaling pathway and targeting tumor blood vessels to improve the therapeutic index of radiation. Important questions remain including optimization of modality sequencing to achieve best outcome. Further molecular and genetic knowledge regarding angiogenesis, interaction between radiation and tumor, blood vessels as well as microenvironment are needed. New imaging tools that capture real time changes in tumor oxygenation may provide further guidance regarding optimal sequencing of combined antiangiogenic therapies and radiation. Further studies of anti-angiogenic drugs and irradiation in non-squamous carcinoma lung and squamous carcinoma H&N models are warranted.
Vascular endothelial growth factor
Human umbilical vein endothelial cell
Head and neck
- Folkman J: Tumor angiogenesis: therapeutic implications. N Engl J Med. 1971, 285: 1182-6. 10.1056/NEJM197111182852108.View ArticlePubMedGoogle Scholar
- Ferrara N, Henzel WJ: Pituitary follicular cells secrete a novel heparin-binding growth factor specific for vascular endothelial cells. Biochem Biophys Res Commun. 1989, 161: 851-8. 10.1016/0006-291X(89)92678-8.View ArticlePubMedGoogle Scholar
- Connolly DT, Olander JV, Heuvelman D, et al: Human vascular permeability factor. Isolation from U937 cells. J Biol Chem. 1989, 264: 20017-24.PubMedGoogle Scholar
- Ferrara N: VEGF and the quest for tumour angiogenesis factors. Nat Rev Cancer. 2002, 2: 795-803. 10.1038/nrc909.View ArticlePubMedGoogle Scholar
- Hanahan D, Folkman J: Patterns and emerging mechanisms of the angiogenic switch during tumorigenesis. Cell. 1996, 86: 353-64. 10.1016/S0092-8674(00)80108-7.View ArticlePubMedGoogle Scholar
- Ferrara N, Hillan KJ, Gerber HP, Novotny W: Discovery and development of bevacizumab, an anti-VEGF antibody for treating cancer. Nat Rev Drug Discov. 2004, 3: 391-400. 10.1038/nrd1381.View ArticlePubMedGoogle Scholar
- Gorski DH, Beckett MA, Jaskowiak NT, et al: Blockage of the vascular endothelial growth factor stress response increases the antitumor effects of ionizing radiation. Cancer Res. 1999, 59: 3374-8.PubMedGoogle Scholar
- Mauceri HJ, Hanna NN, Beckett MA, et al: Combined effects of angiostatin and ionizing radiation in antitumour therapy. Nature. 1998, 394: 287-91. 10.1038/28412.View ArticlePubMedGoogle Scholar
- Hanna NN, Seetharam S, Mauceri HJ, et al: Antitumor interaction of short-course endostatin and ionizing radiation. Cancer J. 2000, 6: 287-93.PubMedGoogle Scholar
- Li J, Huang S, Armstrong EA, Fowler JF, Harari PM: Angiogenesis and radiation response modulation after vascular endothelial growth factor receptor-2 (VEGFR2) blockade. Int J Radiat Oncol Biol Phys. 2005, 62: 1477-85. 10.1016/j.ijrobp.2005.04.028.View ArticlePubMedGoogle Scholar
- Hoang T, Huang S, Armstrong E, Harari PM: Augmentation of radiation response with the VEGFR-tyrosine kinase inhibitor ZD6474. Pro Am Assoc Cancer Res. 2004, 45: 955-Google Scholar
- Griffin RJ, Williams BW, Wild R, Cherrington JM, Park H, Song CW: Simultaneous inhibition of the receptor kinase activity of vascular endothelial, fibroblast, and platelet-derived growth factors suppresses tumor growth and enhances tumor radiation response. Cancer Res. 2002, 62: 1702-6.PubMedGoogle Scholar
- Zips D, Hessel F, Krause M, et al: Impact of adjuvant inhibition of vascular endothelial growth factor receptor tyrosine kinases on tumor growth delay and local tumor control after fractionated irradiation in human squamous cell carcinomas in nude mice. Int J Radiat Oncol Biol Phys. 2005, 61: 908-14. 10.1016/j.ijrobp.2004.11.007.View ArticlePubMedGoogle Scholar
- Siemann DW, Rojiani AM: Enhancement of radiation therapy by the novel vascular targeting agent ZD6126. Int J Radiat Oncol Biol Phys. 2002, 53: 164-71. 10.1016/S0360-3016(02)02742-6.View ArticlePubMedGoogle Scholar
- Hoang T, Huang S, Armstrong E, Eickhoff JC, Harari PM: Augmentation of radiation response with the vascular targeting agent ZD6126. Int J Radiat Oncol Biol Phys. 2006, 64: 1458-65. 10.1016/j.ijrobp.2005.11.017.View ArticlePubMedGoogle Scholar
- Li L, Rojiani A, Siemann DW: Targeting the tumor vasculature with combretastatin A-4 disodium phosphate: effects on radiation therapy. Int J Radiat Oncol Biol Phys. 1998, 42: 899-903. 10.1016/S0360-3016(98)00320-4.View ArticlePubMedGoogle Scholar
- Folkman J: Tumor angiogenesis and tissue factor. Nat Med. 1996, 2: 167-8. 10.1038/nm0296-167.View ArticlePubMedGoogle Scholar
- Wachsberger PR, Burd R, Marero N, et al: Effect of the tumor vascular-damaging agent, ZD6126, on the radioresponse of U87 glioblastoma. Clin Cancer Res. 2005, 11: 835-42.PubMedGoogle Scholar
- Jain RK: Normalizing tumor vasculature with anti-angiogenic therapy: a new paradigm for combination therapy. Nat Med. 2001, 7: 987-9. 10.1038/nm0901-987.View ArticlePubMedGoogle Scholar
- Tong RT, Boucher Y, Kozin SV, Winkler F, Hicklin DJ, Jain RK: Vascular normalization by vascular endothelial growth factor receptor 2 blockade induces a pressure gradient across the vasculature and improves drug penetration in tumors. Cancer Res. 2004, 64: 3731-6. 10.1158/0008-5472.CAN-04-0074.View ArticlePubMedGoogle Scholar
- Willett CG, Boucher Y, di Tomaso E, et al: Direct evidence that the VEGF-specific antibody bevacizumab has antivascular effects in human rectal cancer. Nat Med. 2004, 10: 145-7. 10.1038/nm988.PubMed CentralView ArticlePubMedGoogle Scholar
- Willett CG, Duda DG, di Tomaso E, et al: Efficacy, safety, and biomarkers of neoadjuvant bevacizumab, radiation therapy, and fluorouracil in rectal cancer: a multidisciplinary phase II study. J Clin Oncol. 2009, 27: 3020-6. 10.1200/JCO.2008.21.1771.PubMed CentralView ArticlePubMedGoogle Scholar
- Crane CH, Ellis LM, Abbruzzese JL, et al: Phase I trial evaluating the safety of bevacizumab with concurrent radiotherapy and capecitabine in locally advanced pancreatic cancer. J Clin Oncol. 2006, 24: 1145-51. 10.1200/JCO.2005.03.6780.View ArticlePubMedGoogle Scholar
- Seiwert TY, Haraf DJ, Cohen EE, et al: Phase I study of bevacizumab added to fluorouracil- and hydroxyurea-based concomitant chemoradiotherapy for poor-prognosis head and neck cancer. J Clin Oncol. 2008, 26: 1732-41. 10.1200/JCO.2007.13.1706.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.