Refinement of an Established Procedure and Its Application for Identification of Hypoxia in Prostate Cancer Xenografts

Authors Elming PB, Wittenborn TR, Busk M, Sørensen BS, Thomsen MBH, Strandgaard T, Dyrskjøt L, Nielsen S, Horsman MR
Source Cancers (Basel) . 2021 May 26;13(11):2602 Publicationdate 01 Jan 0001


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.

Keywords: DU-145; PC3; hypoxia; hypoxia gene signature; pre-clinical models; prostate cancer.