Radioresistant mouse pheochromocytoma cell lines

Abstract

Objective: Patients diagnosed with metastatic pheochromocytoma/paraganglioma (PCC/PGL) have limited treatment options. In some cases, peptide receptor radionuclide therapy (PRRT) is followed by an eruption of metastases, possibly originating from tumor cells with a radioresistant phenotype. However, the underlying mechanisms of radioresistance in PCC/PGL remain largely unknown and appropriate models are missing.

Methods: Two genetically modified mouse pheochromocytoma (MPC) cell lines, one positive and one negative for hypoxia-inducible factor 2α expression (MPC+HIF-2α and MPC+EV [empty vector], respectively), were X-ray-conditioned through fractionated irradiation at sublethal doses. Two procedures were tested: one allowed for recovery between each irradiation step (recIR), while the other demanded daily irradiation (dayIR). Changes in cell morphology, growth rates, and DNA repair (γH2AX immunostaining) were characterized in response to irradiation.

Results: We generated two MPC+HIF-2α- and two MPC+EV-derived cell lines that tolerate irradiations with X-rays at dose fractions of 2 Gy per day without significant growth inhibition. All recIR-and dayIR-conditioned cell lines showed increased DNA repair capacity. Morphological changes toward stronger clustering and slower growth were more pronounced in dayIR-conditioned than in recIR-conditioned cell lines. X-ray-conditioned MPC+HIF-2α cells showed the highest increase in resistance to X-ray-treatment with dose fractions up to 5 Gy per day.

Conclusion: The herein established X-ray-conditioned MPC cell lines represent PCC/PGL models with a radioresistant phenotype. Further investigations on the radiation-induced genetic responses of these cell lines, their corresponding tumor spheroids, and tumor allografts in mice will help to elucidate the underlying mechanisms of acquired radioresistance and radionuclide therapy-induced metastatic eruption in PCC/PGL. Lastly, the suitability of advanced PRRT and complementary treatments can be tested to improve theranostic strategies.

Keywords: HIF-2α; X-ray; irradiation; paraganglioma; pheochromocytoma; pseudohypoxia; radioresistance.

Figure 1

Changes in morphology and proliferation during X-ray-conditioning of MPC+EV and MPC+HIF-2α cells. (A) Morphology of X-ray-conditioned cells two weeks after the last irradiation; recIR- and dayIR-conditioned sublines cumulated ~60 and ~200 Gy, respectively; upper panel (BF): bright field images, lower panel (H+P): ICC-IF staining with Hoechst33258 (blue) and Alexa-488 phalloidin (green); dayIR-conditioned cells with increased clustering; (B) Enhanced intercellular neurite-like networks (yellow arrows) during dayIR-conditioning at early stages (accumulated dose ~10 Gy) compared to endpoints (accumulated dose ~200 Gy); (C) Growth curves based on monolayer confluence two weeks after last irradiation; obtained from 3−5 independent experiments, each including n = 10 replicates, growth rates (k) determined by curve fitting using the logistic growth equation; significance of growth rate differences compared to corresponding MPC+EV sublines: **p<0.01; ***p<0.001.

Figure 2

Gradual loss of HIF-2α during dayIR-conditioning of genetically modified MPC+HIF-2α cells. While both unconditioned (noIR) and recIR-conditioned MPC+HIF-2α cells retained their cell line-specific HIF-2α-positive phenotype, dayIR-conditioning led to a gradual reduction in HIF-2α transgene expression in a dose-dependent manner that ultimately resulted in loss of HIF-2α; (A) Expression of the HIF-2α and neoR transgenes over the course of X-ray-conditioning at increasing intermediate and final accumulated doses (orange); relative quantity of RT-PCR products (RQ_RT-PCR, normalized to murine Actb) amplified from RNA isolates, n = 3; (B) Residual expression of the HIF-2α transgene detected by RNA sequencing of samples collected at the endpoints of X-ray-conditioning; relative quantity of transcripts (RQ_seq, normalized to murine Actb), n = 3; (C) HIF-2α protein levels at the endpoints of X-ray-conditioning; lysates prepared from cells exposed to CoCl₂ (HIF stabilizer) for 24 h, band intensities compared to β-actin and glyceraldehyde phosphate dehydrogenase (GAPDH).

Figure 3

X-ray-conditioning enabled sustained proliferation of MPC+HIF-2α cells during X-ray-treatment. Growth responses in monolayers during X-ray-treatment with increasing fractionated doses over four days; MPC+EV-noIR, -recIR, and -dayIR cell lines all showed similar growth responses; unconditioned MPC+HIF-2α-noIR cells stopped proliferating during X-ray-treatment, while both X-ray-conditioned MPC+HIF-2α-recIR and -dayIR sublines maintained proliferation; (A) Microscopic images with confluence masks (yellow) at the end of X-ray-treatment (day 4); (B) Changes in confluence over time; means of one experiment including n = 10 replicates; (gray box) duration of X-ray-treatment with fractionated doses in 24-hour intervals; (C) Relative growth at the end of X-ray-treatment (day 4) normalized to the corresponding untreated controls and to the respective confluence at treatment start (day 0), means of three independent experiments each including n = 10 replicates, significance of differences compared to unconditioned noIR controls: ***p<0.001; all experiments were conducted two weeks after last X-ray-conditioning.

Figure 4

X-ray-conditioning increases DNA double strand break repair and reduces proportions of dead MPC+EV and MPC+HIF-2α cells after X-ray-treatment. (A) Immunostaining of γH2AX foci (red) and nuclei counterstained with Hoechst 33258 (blue) after X-ray-treatment of cells with 2 Gy; (B) Quantitative image analysis of γH2AX foci per cell showing that irradiation induced DNA repair in both unconditioned MPC+EV and MPC+HIF-2α cells, while all recIR- and dayIR-conditioned sublines already showed increased prevalence of DNA repair; statistical distribution and means (dashed lines) from >20 nuclei; significance of differences compared to the respective noIR cell lines: ^### p<0.001 and compared to the same X-ray-conditioned cell line without acute irradiation: *p<0.05; ***p<0.001; (C) Proportions of dead cells after X-ray-treatment with 5 Gy; cells stained with propidium iodide 24 h after irradiation; relative fluorescence intensity of dead cells normalized to the maximum amount of dead cells determined from exposure to methanol; two independent experiments each including n = 9 replicates; significance of differences: *p<0.05; **°p<0.01; ***p<0.001; all experiments were conducted two weeks after last X-ray-conditioning.