Abstract
Simple SummaryRegions of low oxygen status (hypoxia) are a characteristic feature of solid tumors. This hypoxia is a major cause of resistance to treatment, especially radiation therapy, and it can increase the likelihood of metastatic spread. Being able to accurately identify tumor hypoxia will allow us to predict a patient’s response to therapy and find alternative approaches to improve outcome. We have refined a dissection method that involves autoradiography and laser-guided microdissection of hypoxic areas in tumors. Using this approach, we were able to test the feasibility of applying a 15-gene signature that was previously developed for head and neck cancer patients to identify hypoxia in pre-clinical models of prostate cancer. Our results demonstrated the potential of this method to identify hypoxia in this tumor type and suggest its applicability for use in patients with prostate cancer.Background: This pre-clinical study was designed to refine a dissection method for validating the use of a 15-gene hypoxia classifier, which was previously established for head and neck squamous cell carcinoma (HNSCC) patients, to identify hypoxia in prostate cancer. Methods: PC3 and DU-145 adenocarcinoma cells, in vitro, were gassed with various oxygen concentrations (0–21%) for 24 h, followed by real-time PCR. Xenografts were established in vivo, and the mice were injected with the hypoxic markers [18F]-FAZA and pimonidazole. Subsequently, tumors were excised, frozen, cryo-sectioned, and analyzed using autoradiography ([18F]-FAZA) and immunohistochemistry (pimonidazole); the autoradiograms used as templates for laser capture microdissection of hypoxic and non-hypoxic areas, which were lysed, and real-time PCR was performed. Results: In vitro, all 15 genes were increasingly up-regulated as oxygen concentrations decreased. With the xenografts, all 15 genes were up-regulated in the hypoxic compared to non-hypoxic areas for both cell lines, although this effect was greater in the DU-145. Conclusions: We have developed a combined autoradiographic/laser-guided microdissection method with broad applicability. Using this approach on fresh frozen tumor material, thereby minimizing the degree of RNA degradation, we showed that the 15-gene hypoxia gene classifier developed in HNSCC may be applicable for adenocarcinomas such as prostate cancer.
Highlights
Hypoxia is a characteristic feature of solid animal [1] and human [2] that occurs because of the structural and functional abnormalities in tumor vessels [2,3] resulting in an insufficient blood supply of oxygen to the growing tumor mass [4]
We investigated the usability of the head and neck squamous cell carcinoma (HNSCC) 15-gene signature to monitor hypoxia in PC3 and DU145 prostate cancer cell lines in vitro, and using our refined in vivo method, we investigated the potential of using this hypoxia-induced gene expression in both tumor types grown as xenografts in nude mice
In the DU-145 cell line, the increase is steeper for the genes EGLN3, KCTD11, P4HA1, and PDK1 when the oxygen tension is decreased from 0.5% to 0% compared to the same genes in the PC3 cell line
Summary
Hypoxia is a characteristic feature of solid animal [1] and human [2] that occurs because of the structural and functional abnormalities in tumor vessels [2,3] resulting in an insufficient blood supply of oxygen to the growing tumor mass [4]. Using human primary tumor biopsies from head and neck squamous cell carcinomas (HNSCC) patients with known oxygen status, that signature was refined to include a 15-gene “hypoxia gene expression classifier” [14]. This has retrospectively been shown to have prognostic and predictive impact for the radiosensitizer nimorazole in combination with radiotherapy [15]. We investigated the usability of the HNSCC 15-gene signature to monitor hypoxia in PC3 and DU145 prostate cancer cell lines in vitro, and using our refined in vivo method, we investigated the potential of using this hypoxia-induced gene expression in both tumor types grown as xenografts in nude mice. Reference genes were ACTR, NDFIP1, and RPL37A; these were previously established as robust reference genes for the 15 gene hypoxia profile [16]
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