Abstract 528: A tumor-stroma interaction study in red mammary tumors in green mice with and without enhanced oxygenation
Authors: Moen Ingrid , Jevne Charlotte , Wang Jian , Kalland Karl-Henning , Chekenya Martha , Akslen Lars A. , Sleire Linda , Enger Per Øyvind , Reed Rolf K. , Øyan Anne M. , Stuhr Linda E.B. 
Published: June 2012

Abstract
Background: The tumor microenvironment is increasingly recognized as pivotal in tumor progression. Thus, by completely separating tumor cells from stromal cells it enables a thorough elucidation of gene-changes in the two compartments, and thereby offers the possibility to better understand the biological effects in tumors with and without treatment.
Since tumor hypoxia is considered to be relevant for several aspects of tumor pathophysiology, including activation of signalling pathways that regulate proliferation, angiogenesis and death, our hypothesis was that a reduction in the hypoxic state of the tumor, might have an inhibitory effect on tumor growth per se. Induction of hyperoxia by hyperbaric oxygen (HBO) exposure, enhance dissolved oxygen in the plasma and thereby the pO2 in the tumor tissue.
Methods: The murine mammary dsRed cell line 4T1 was implanted into the mouse mammary fat pad in eGFP expressing NOD/Scid mice. One group was exposed to repeated HBO treatment (2.5 bar, 100% O2, 3 exposures à 90 min), one to daily HBO treatment (2.5bar, 100% O2, 7 exposures à 90 min), whereas the control group was housed under normal atmosphere. Treatment effects were determined by assessment of tumor growth, tumor vascularisation, tumor cell proliferation and cell death. FACS was used to completely separate red tumor cells from green host cells, RNA was extracted and gene expression profiling performed.
Results: The model enabled us to completely separate the two compartments (tumor vs stroma), as verified by confocal microscopy and gene expression profiling. Highly upregulated genes in the untreated tumor stroma, included constituents of the ECM and MMP's, as well as genes related to cell-migration and motility. Significant changes in response to treatment between the tumor and stroma was found, especially concerning genes related to cell adhesion and angiogenesis. Furthermore, changes occurring within each of these compartments were found. Tumor growth was significantly inhibited (∼ 50%, after 8 days) after both repeated and daily HBO treatment compared to controls. Repeated HBO treatment showed a significant anti-angiogenic effect, while daily HBO treatment did not. The gene expression confirmed this. Neither morphology, proliferation nor the amount of cell death was significantly changed after the two HBO treatment regimes, despite the significant effect on tumor growth.
Conclusion: The present model provides a significant insight into how tumor cells communicate with the stroma, both before any treatment and after enhanced oxygenation. Furthermore, hyperoxia induced a significant inhibitory effect on the 4T1 mammary tumor growth, with a significant anti-angiogenic effect after repeated hyperoxic treatment.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 528. doi:10.1158/1538-7445.AM2011-528