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. 2021 May 26;13(11):2602.
doi: 10.3390/cancers13112602.

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

Pernille B Elming  1 , Thomas R Wittenborn  1 , Morten Busk  2 , Brita S Sørensen  1   2 , Mathilde Borg Houlberg Thomsen  3   4 , Trine Strandgaard  3   4 , Lars Dyrskjøt  3   4 , Steffen Nielsen  1 , Michael R Horsman  1
Affiliations

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

Pernille B Elming et al. Cancers (Basel). .

Abstract

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.

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Conflict of interest statement

B.S. Sørensen is a co-inventor on a patent on a method (gene expression profile) for determining clinically relevant hypoxia in cancer (WO2012146259A1). All other authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
Illustration of the set-up for the in vivo experiment. A nude mouse with a xenograft prostate tumor growing on the flank was intraperitoneally injected with both FAZA and pimonidazole. Approximately four hours later, the mouse was sacrificed, and the tumor was excised and fresh frozen. The tumor was immediately cryo-sectioned in three layers, each layer consisting of 10 consecutive 10 μm sections. Then, these sections were stained for either hematoxylin and eosin (HE) or pimonidazole binding, or used for FAZA autoradiography. The autoradiogram was used as a template for laser micro dissection, where the hypoxic (H) and the non-hypoxic (NH) areas were identified and cut. HE staining was used to evaluate and avoid necrotic areas. This dissected tissue was immediately lysed and real-time PCR (qPCR) performed. The images of the laser micro dissection technique are from Arcturus Bioscience (Mountain View, CA, USA).
Figure 2
Figure 2
[18F]-FAZA autoradiogram (A) and corresponding raw (B) and segmented (C) pimonidazole staining of the same section (each image is approximately three times larger than the original tumor section), showing that the two hypoxia markers distribute similarly. This was further verified by assessing the spatial relationship between FAZA signal intensity (PSL) and hypoxic fraction (HF), which is derived by placing a grid consisting of 1 mm2 squares covering the autoradiogram and the segmented pimonidazole image. Only squares within the tumor periphery and largely free of necrosis were used in the final analysis. (D) The resulting correlation between FAZA signal and HF.
Figure 3
Figure 3
The increased degree of gene expression in vitro under different oxygen concentrations compared to that found under 21% oxygen. Results show means (± 1 S.E.) for n = 3 measurements in PC3 (A) or DU-145 (B) cells. Cells were exposed to the different oxygen concentrations as indicated for 24 h. A greater degree of up-regulation with decreasing oxygen concentration is seen for almost every gene in both cell lines.
Figure 4
Figure 4
Representative histograms showing the oxygen partial pressure (pO2) profiles obtained with the Eppendorf oxygen electrode in PC3 (A) or DU-145 (B) xenografts. Results show the means (±1 S.E.) of the relative frequency of the various pO2 values measured in individual flank tumors from eight (PC3) or 10 (DU-145) tumor-bearing mice. Both tumor types were very hypoxic.
Figure 5
Figure 5
Relative in vivo expression of the 15 hypoxia genes in hypoxic areas when compared to measurements made in non-hypoxic areas. Results are for PC3 (A) or DU-145 (B) flank xenografts. For the PC3 tumors, the results are based on 43 samples from non-hypoxic areas and 33 samples from hypoxic areas distributed over 17 tumors in nine animals (one animal only had one flank tumor). The results for the DU-145 xenografts consisted of 34 samples from non-hypoxic areas and 43 samples from hypoxic areas (from a total of 12 flank tumors in eight mice). All values are listed as means ± 1 S.E.
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