- Short report
- Open Access
Ionizing radiation and inhibition of angiogenesis in a spontaneous mammary carcinoma and in a syngenic heterotopic allograft tumor model: a comparative study
- Oliver Riesterer†1,
- Christoph Oehler-Jänne†1,
- Wolfram Jochum2, 3,
- Angela Broggini-Tenzer1,
- Van Vuong1 and
- Martin Pruschy†1Email author
© Riesterer et al; licensee BioMed Central Ltd. 2011
Received: 26 January 2011
Accepted: 8 June 2011
Published: 8 June 2011
The combined treatment modality of ionizing radiation (IR) with inhibitors of angiogenesis (IoA) is a promising treatment modality based on preclinical in vivo studies using heterotopic xeno- and allograft tumor models. Nevertheless reservations still exist to translate this combined treatment modality into clinical trials, and more advanced, spontaneous orthotopic tumor models are required for validation to study the efficacy and safety of this treatment modality.
We therefore investigated the combined treatment modality of IR in combination with the clinically relevant VEGF receptor (VEGFR) tyrosine kinase inhibitor PTK787 in the MMTV/c-neu induced mammary carcinoma model and a syngenic allograft tumor model using athymic nude mice. Mice were treated with fractionated IR, the VEGFR-inhibitor PTK787/ZK222584 (PTK787), or in combination, and efficacy and mechanistic-related endpoints were probed in both tumor models. Overall the treatment response to the IoA was comparable in both tumor models, demonstrating minimal tumor growth delay in response to PTK787 and PTK787-induced tumor hypoxia. Interestingly spontaneously growing tumors were more radiosensitive than the allograft tumors. More important combined treatment of irradiation with PTK787 resulted in a supraadditive tumor response in both tumor models with a comparable enhancement factor, namely 1.5 and 1.4 in the allograft and in the spontaneous tumor model, respectively.
These results demonstrate that IR in combination with VEGF-receptor tyrosine kinase inhibitors is a valid, promising treatment modality, and that the treatment responses in spontaneous mammary carcinomas and syngenic allografts tumor models are comparable.
Preclinical studies have demonstrated that the combined treatment of IR with IoA is highly effective in xeno- and allograft tumor models of breast cancer [1–3]. It is generally agreed that IR and IoA interact on the level of the tumor microenvironment, although the exact mechanism of synergistic action of these two treatment modalities is still a matter of debate. For example, IoAs can either improve tumor oxygenation by a mechanism termed vascular normalization  and thereby sensitize for IR, or IoAs can increase tumor hypoxia [3, 5–7], which is counteracted by combined treatment with IR. The cause for these different treatment responses to IoAs is unknown but might be related to differences in the mode of action of the IoAs and the treatment regimens including doses and scheduling, and the tumor models used on the preclinical level . With respect to the tumor models used on the preclinical level, most studies were performed with either orthotopic or heterotopic xenograft [4–6] or heterotopic allograft tumor models . Though, little is known about the relevance of a differential microenvironment in xenograft versus allograft and heterotopic versus orthotopic tumors with regard to the treatment response to IoAs and in particular to a combined treatment modality of IoAs with IR [9, 10]
We previously demonstrated that the risk of enhanced tumor hypoxia in response to the inhibitor of vascular endothelial growth factor receptor 2 (VEGFR2) PTK787/ZK222584 (PTK787) exists, but is minimal when PTK787 is combined with IR . Our experiments were originally performed in a classic allograft tumor model derived from NF9006 tumor cells, which were originally established from spontaneous murine MMTV/c-neu driven mammary carcinomas. These fast-growing allografts and their fast-developing tumor vasculature might not represent the tumor microenvironment in a spontaneously growing tumor. We therefore revisited the potential drawback of this artificial fast-growing tumor model in the corresponding MMTV/(c-neu)-driven spontaneously growing mammary tumor model and compared the treatment-dependent responses with those achieved in the syngenic allografts.
The female, heterozygous offspring of female FVB-wild type mice, which were mated with male FVB-Tg(MMTV/c-neu) mice (Charles River), developed mammary carcinomas within 100 days after a first littering. To generate the corresponding allograft tumor model, mammary carcinoma cells (NF9006), which were established from the spontaneous tumor model, were subcutaneously injected (4 × 106 cells) on the back of athymic nude mice. Spontaneous tumors and allografts were allowed to grow to 200 mm3 ± 10% before start of treatment. Mice carrying allograft tumors on their backs were irradiated using a customized shielding device whereas mice with spontaneous tumors in the mouse breast were given upper-half-body radiotherapy. All mice were treated with a minimally fractionated locoregional radiotherapy regimen of 4 × 3 Gy during 4 consecutive days, using a Pantak Therapax 300-kV X-ray unit at 0.7 Gy/min. PTK787 (dissolved in 5% DMSO, 1% Tween-80 and 94% H2O) was applied orally either alone (100 mg/kg) or 1 hour prior to irradiation. Immunohistochemical stainings of tumor sections were performed after tumor excision at day 4 of treatment. Detailed descriptions of the experimental procedures are given elsewhere . The Student's t-test was used to statistically analyse the differences between the treatment groups.
Results of Growth Delay Assays
Time in days for tumors to grow from 200 to 600 mm3
8.8 ± 0.9
10.5 ± 0.5
17.2 ± 0.5
22.8 ± 1.4
11.5 ± 0.8
14.3 ± 1.5
31.6 ± 2.8
41.9 ± 1.8
Here, we have examined the effects of the combined treatment modality of ionizing radiation with the VEGF-receptor tyrosine kinase inhibitor PTK787 in both a spontaneous and a strongly related allograft mammary carcinoma model. Little is known about differences in the make-up of the tumor microenvironment between allografts and xenografts, orthotopic and heterotopic tumors. In the models used in this study, major differences with regard to the tumor biology, and eventually to the treatment response, would rather be expected on the level of the tumor microenvironment than on the level of the syngenic tumor cells. Interestingly we observed the strongest difference between the two tumor models on the level of radiation sensitivity. The NF9006 cell line, which is derived from a spontaneous murine MMTV/c-neu driven mammary carcinoma, may have acquainted additional mutations during the in vitro establishment, and these genetic mutations might contribute to the increased radiation resistant phenotype of allografts derived from this cell line. On the other hand, increased radiation sensitivity of spontaneous tumors in comparison to allograft tumors may be linked to differences in the tumor vasculature as well as immunomodulatory effects in the immunocompetent host . PTK787 exerts its antivascular effects by targeting the VEGF receptor, which is almost exclusively located on endothelial cells. The treatment responses to PTK787 alone were similar in both tumor models, which indicate a similar phenotype and treatment sensitivity of the respective endothelial cells. This is further supported by a similar treatment-dependent reduction of microvessel densities and a treatment-dependent increase of tumor hypoxia.
We previously demonstrated that IoA induce tumor hypoxia in allografts, which is counteracted by combined treatment with irradiation . Eventually combined treatment results in a supraadditive treatment response. Insofar our studies are of high clinical interest since PTK787 exerted a similar treatment response in the allograft and the spontaneously growing tumor model with potent radiation enhancement to a similar extent in both tumor models. Thereby the data strengthen the evidence to overcome a major obstacle translating such a treatment combination into the clinics, i.e. the supposition that a potential IoA-dependent increase of tumor hypoxia might impair the treatment response to ionizing radiation. Obtaining preclinical data with spontaneous tumor models is highly laborious and cost-effective. Our comparative study furthermore demonstrates that an allograft tumor model is adequate and represents a valid tumor model to investigate the combined treatment modality of IR with IoA.
We would like to acknowledge Marion Bawohl for excellent technical support and we acknowledge the following sources of funding: Oncosuisse, the Sassella, the Novartis and Swiss National Foundations (to M.P.).
- Dudek AZ, Zwolak P, Jasinski P, Terai K, Gallus NJ, Ericson ME, Farassati F: Protein kinase C-beta inhibitor enzastaurin (LY317615.HCI) enhances radiation control of murine breast cancer in an orthotopic model of bone metastasis. Invest New Drugs 2008, 26: 13-24. 10.1007/s10637-007-9079-yView ArticlePubMedGoogle Scholar
- Maggiorella L, Aubel C, Haton C, Milliat F, Connault E, Opolon P, Deutsch E, Bourhis J: Cooperative effect of roscovitine and irradiation targets angiogenesis and induces vascular destabilization in human breast carcinoma. Cell Prolif 2009, 42: 38-48. 10.1111/j.1365-2184.2008.00570.xView ArticlePubMedGoogle Scholar
- Riesterer O, Honer M, Jochum W, Oehler C, Ametamey S, Pruschy M: Ionizing radiation antagonizes tumor hypoxia induced by antiangiogenic treatment. Clin Cancer Res 2006, 12: 3518-3524. 10.1158/1078-0432.CCR-05-2816View ArticlePubMedGoogle Scholar
- Winkler F, Kozin SV, Tong RT, Chae SS, Booth MF, Garkavtsev I, Xu L, Hicklin DJ, Fukumura D, di Tomaso E, et al.: Kinetics of vascular normalization by VEGFR2 blockade governs brain tumor response to radiation: role of oxygenation, angiopoietin-1, and matrix metalloproteinases. Cancer Cell 2004, 6: 553-563.PubMedGoogle Scholar
- Fenton BM, Paoni SF: The addition of AG-013736 to fractionated radiation improves tumor response without functionally normalizing the tumor vasculature. Cancer Res 2007, 67: 9921-9928. 10.1158/0008-5472.CAN-07-1066View ArticlePubMedGoogle Scholar
- Franco M, Man S, Chen L, Emmenegger U, Shaked Y, Cheung AM, Brown AS, Hicklin DJ, Foster FS, Kerbel RS: Targeted anti-vascular endothelial growth factor receptor-2 therapy leads to short-term and long-term impairment of vascular function and increase in tumor hypoxia. Cancer Res 2006, 66: 3639-3648. 10.1158/0008-5472.CAN-05-3295View ArticlePubMedGoogle Scholar
- Williams KJ, Telfer BA, Brave S, Kendrew J, Whittaker L, Stratford IJ, Wedge SR: ZD6474, a potent inhibitor of vascular endothelial growth factor signaling, combined with radiotherapy: schedule-dependent enhancement of antitumor activity. Clin Cancer Res 2004, 10: 8587-8593. 10.1158/1078-0432.CCR-04-1147View ArticlePubMedGoogle Scholar
- Jain RK: Normalization of tumor vasculature: an emerging concept in antiangiogenic therapy. Science 2005, 307: 58-62. 10.1126/science.1104819View ArticlePubMedGoogle Scholar
- Gasparini G, Longo R, Fanelli M, Teicher BA: Combination of antiangiogenic therapy with other anticancer therapies: results, challenges, and open questions. J Clin Oncol 2005, 23: 1295-1311. 10.1200/JCO.2005.10.022View ArticlePubMedGoogle Scholar
- Izumi Y, di Tomaso E, Hooper A, Huang P, Huber J, Hicklin DJ, Fukumura D, Jain RK, Suit HD: Responses to antiangiogenesis treatment of spontaneous autochthonous tumors and their isografts. Cancer Res 2003, 63: 747-751.PubMedGoogle Scholar
- Oehler-Janne C, Jochum W, Riesterer O, Broggini-Tenzer A, Caravatti G, Vuong V, Pruschy M: Hypoxia modulation and radiosensitization by the novel dual EGFR and VEGFR inhibitor AEE788 in spontaneous and related allograft tumor models. Mol Cancer Ther 2007, 6: 2496-2504. 10.1158/1535-7163.MCT-07-0253View ArticlePubMedGoogle Scholar
- Taghian AG, Suit HD: Animal systems for translational research in radiation oncology. Acta Oncol 1999, 38: 829-838. 10.1080/028418699432518View 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.