Quantifying Transient Hypoxia in Human Tumor Xenografts by Flow Cytometry

Bennewith, Kevin L.; Durand, Ralph E.

doi:10.1158/0008-5472.can-04-0289

Cited by 121 publications

(78 citation statements)

References 40 publications

(62 reference statements)

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“…Instead, there was a large variation from one tumor cord to another in the distance between Hoechst-stained perivascular regions and regions that expressed the hypoxia markers, a more subtle indicator of possible transient changes in perfusion in this tumor. These results are consistent with the finding that SiHa xenografts may undergo changes in perfusion but these typically occur over several hours (26). To eliminate transient changes in perfusion as a confounding factor in understanding the reason for lack of HIF-1a in perinecrotic regions, subsequent experiments were restricted to the SiHa tumor.…”

Section: Discussionsupporting

confidence: 86%

Reduced Expression of Hypoxia-Inducible Factor-1α in Perinecrotic Regions of Solid Tumors

et al. 2005

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Hypoxia that develops in solid tumors stabilizes the hypoxiainducible factor-1A (HIF-1A) subunit of the HIF-1 transcription factor, leading to up-regulation of dozens of hypoxia-regulated genes that increase glycolysis and oxygen delivery. HIF-1A and its downstream target gene CA9 have both been used as surrogate hypoxia markers, and, in general, high expression predicts for a poor response to treatment. Combinations of hypoxia markers offer the opportunity to measure changes in tumor oxygenation that may be relevant to tumor response to treatment. We compared the degree of colocalization of two endogenous markers for hypoxia, HIF-1A and carbonic anhydrase IX (CAIX), with a chemical marker for hypoxia, pimonidazole. Unexpectedly, expression of HIF-1A was reduced in the most hypoxic regions that border necrosis in xenograft tumors composed of SiHa cervical carcinoma, WiDr colon carcinoma, or M006 astrocytoma cells. Similar results were obtained for samples from three cervical cancer biopsies. However, CAIX was present in these perinecrotic cells that were also capable of metabolizing and binding a chemical marker for hypoxia, pimonidazole. In vitro experiments using tumor cells and tumor cubes incubated under anoxic conditions indicated that nutrient deprivation seems to be largely responsible for the lack of HIF-1A expression in perinecrotic regions. The half-life of CAIX was sufficiently long that, once formed, it remained for days in the absence of continued HIF-1A expression. These results have implications for the use of HIF-1A as an indicator of tumor hypoxia and aggressiveness as well as development of hypoxia-directed antitumor therapies based on the expression of HIF-1A. (Cancer Res 2005; 65(16): 7259-66)

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Section: Discussionsupporting

confidence: 86%

Reduced Expression of Hypoxia-Inducible Factor-1α in Perinecrotic Regions of Solid Tumors

et al. 2005

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“…To exclude the possibility that irradiation had resulted in deficient intracellular reductases, a separate set of tumors were clamped at 28 h after irradiation. The intracellular reduction and binding are similar for all 2-nitroimidazoles (51-54), as are the spatial distributions of pimonidazole and CCI-103F (36,55). Therefore, it was assumed that if pimonidazole could be reduced after irradiation, this would be similar for CCI-103F.…”

Section: Kinetics Of Hypoxic Cellsmentioning

confidence: 99%

Dynamics of Hypoxia, Proliferation and Apoptosis after Irradiation in a Murine Tumor Model

et al. 2006

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“…26 Recent cell separation techniques have provided many advantages for studying cell populations prepared from solid tumors. [27][28][29][30] However, current methods used to detect hypoxic cells are often technically complex, invasive, or require administration of chemicals to mark hypoxic cells. We have adapted the earlier work of Olive et al 27 and Vordermark et al 31 to devise a simple separation method for hypoxic cells on the basis of carbonic anhydrase-9 (CA-9) expression in an orthotopic xenograft cervical model.…”

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confidence: 99%

Increased expression of metastasis-related genes in hypoxic cells sorted from cervical and lymph nodal xenograft tumors

Chaudary

Hill

2009

Laboratory Investigation

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Solid tumors contain regions of poor oxygenation that relate to the abnormal vascular network. Clinical investigations in cervical carcinoma have shown that positive lymph node status in patients with cervical carcinoma correlates with hypoxia. Earlier, in an orthotopic cervical cancer model, we had shown that exposure to acute hypoxia enhances lymph node metastasis. This study describes a technique for sorting hypoxic cells directly from the cervical xenograft model and reports the expression of 'metastasis-related' genes in hypoxic cells from xenografted cervix and lymph node tumors. Tumor cells were sorted on the basis of DsRed fluorescence and the sub-population of hypoxic cells was sorted on the basis of carbonic anhydrase-9 (CA-9) expression. Quantitative RT-PCR was conducted to measure changes in gene expression in the hypoxic cells sorted from primary cervix tumors and lymph node metastases. Immunohistochemistry was used to track changes in protein expression in sections of the same tumors. Metastasis-related genes, CXCR4, uPAR, VEGFC, Hdm2, and OPN, were observed to be upregulated at gene and protein levels in the primary tumors and nodal metastasis from the orthotopic transplants. In particular, the hypoxic cells sorted from orthotopically transplanted cervix tumors and their lymph node metastases from mice exposed to cyclic (intermittent) hypoxia showed higher levels of expression of these genes. These results are consistent with the hypothesis that these genes may be involved in regulating lymph node metastasis in cervical cancers under hypoxic conditions and provide support to the concept cyclic hypoxia that plays an important role in this process. Our methodological study emphasizes the technique of cell sorting to identify hypoxic cells using CA-9, which may aid in improving prognostic capabilities and in designing rational therapeutic strategies by focusing on hypoxia-specific gene expression profiles of patients. The technique can be applied to identify other potential 'hypoxia-related' genes of interest for tumor growth and metastasis.

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Quantifying Transient Hypoxia in Human Tumor Xenografts by Flow Cytometry

Cited by 121 publications

References 40 publications

Reduced Expression of Hypoxia-Inducible Factor-1α in Perinecrotic Regions of Solid Tumors

Reduced Expression of Hypoxia-Inducible Factor-1α in Perinecrotic Regions of Solid Tumors

Dynamics of Hypoxia, Proliferation and Apoptosis after Irradiation in a Murine Tumor Model

Increased expression of metastasis-related genes in hypoxic cells sorted from cervical and lymph nodal xenograft tumors

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