In cancer, the extensive methylation found in the bulk of chromatin is reduced, while the normally unmethylated CpG islands become hypermethylated. Regions of solid tumors are transiently and/or chronically exposed to ischemia (hypoxia) and reperfusion, conditions known to contribute to cancer progression. We hypothesized that hypoxic microenvironment may influence local epigenetic alterations, leading to inappropriate silencing and re-awakening of genes involved in cancer. We cultured human colorectal and melanoma cancer cell lines under severe hypoxic conditions, and examined their levels of global methylation using HPLC to quantify 5-methylcytosine (5-mC), and found that hypoxia induced losses of global methylation. This was more extensive in normal human fibroblasts than cancer cell lines. Cell lines from metastatic colorectal carcinoma or malignant melanoma were found to be markedly more hypomethylated than cell lines from their respective primary lesions, but they did not show further reduction of 5-mC levels under hypoxic conditions. To explore these epigenetic changes in vivo, we established xenografts of the same cancer cells in immune deficient mice. We used Hypoxyprobe to assess the magnitude of tissue hypoxia, and immunostaining for 5-mC to evaluate DNA methylation status in cells from different regions of tumors. We found an inverse relationship between the presence of extensive tumor hypoxia and the incidence of methylation, and a reduction of 5-mC in xenografts compared to the levels seen in the same cancer cell lines in vitro, verifying that methylation patterns are also modulated by hypoxia in vivo. This suggests that epigenetic events in solid tumors may be modulated by microenvironmental conditions such as hypoxia.

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BackgroundTargeting tumor vasculature is a strategy with great promise in the treatment of many cancers. However, anti-angiogenic reagents that target VEGF/VEGFR2 signaling have met with variable results clinically. Among the possible reasons for this may be heterogeneous expression of the target protein.MethodsDouble immunofluorescent staining was performed on formalin-fixed paraffin embedded sections of treated and control SW480 (colorectal) and WM239 (melanoma) xenografts, and tissue microarrays of human colorectal carcinoma and melanoma. Xenografts were developed using RAG1-/- mice by injection with WM239 or SW480 cells and mice were treated with 20 mg/kg/day of cyclophosphamide in their drinking water for up to 18 days. Treated and control tissues were characterized by double immunofluorescence using the mural cell marker α-SMA and CD31, while the ratio of desmin/CD31 was also determined by western blot. Hypoxia in treated and control tissues were quantified using both western blotting for HIF-1α and immunohistochemistry of CA-IX.ResultsVEGFR2 is heterogeneously expressed in tumor vasculature in both malignant melanoma and colorectal carcinoma. We observed a significant decrease in microvascular density (MVD) in response to low dose metronomic cyclophosphamide chemotherapy in both malignant melanoma (with higher proportion VEGFR2 positive blood vessels; 93%) and colorectal carcinoma (with lower proportion VEGFR2 positive blood vessels; 60%) xenografts. This reduction in MVD occurred in the absence of a significant anti-tumor effect. We also observed less hypoxia in treated melanoma xenografts, despite successful anti-angiogenic blockade, but no change in hypoxia of colorectal xenografts, suggesting that decreases in tumor hypoxia reflect a complex relationship with vascular density. Based on α-SMA staining and the ratio of desmin to CD31 expression as markers of tumor blood vessel functionality, we found evidence for increased stabilization of colorectal microvessels, but no such change in melanoma vessels.ConclusionsOverall, our study suggests that while heterogeneous expression of VEGFR2 is a feature of human tumors, it may not affect response to low dose metronomic cyclophosphamide treatment and possibly other anti-angiogenic approaches. It remains to be seen whether this heterogeneity is partly responsible for the variable clinical success seen to date with targeted anti-VEGFR2 therapy.

These studies aimed to advance understanding of the functions of pregnancy-associated uterinelymphocytes of the natural killer (NK) cell lineage. The approach was morphometric analysis of implantation sites from timed pregnancies in genetically modified mice deficient in NK cells or in signaling associated with their major product, the cytokine interferon-γ. In four different strains of pregnant, NK cell-deficient mice, the major decidual arterioles failed to undergo modifications to their smooth-muscle coats and displayed endothelial cell damage. Decidua lacked normal cell density. This pathology was observed by the end of the first trimester, before placental differentiation. By midgestation in these strains, placentas were smaller than in control strains. In normal mice, many uterine NK cells are perivascular in location and appear to be activated because they are the major sources of interferon-γ and of the interferon-γ-regulated enzyme inducible nitric oxide synthase. During pregnancy in mice genetically ablated for interferon-γ, the interferon-γ receptor chain-α or the transcription factor interferon regulatory factor-1, uterine NK cells differentiate but appear to be abnormal both morphologically and functionally. In these three strains, failure of pregnancy-induced vascular modifications and overt necrosis of decidua occur. Thus, in mice, lymphocytes of the NK cell lineage make specialized contributions to pregnancy-associated modification of the uterine vasculature and to maintenance of decidua. These contributions are achieved through interferon-γ-mediated gene regulation and appear to enhance subsequent placental growth. Human CD56 bright decidual lymphocytes may have analogous functions. If so, changes in numbers or levels of activity of human uterine NK cells or mutations in genes regulated by uterine interferon-γ could contribute to initiation of preeclampsia.