HIF2alpha-Associated Pseudohypoxia Promotes Radioresistance in Pheochromocytoma: Insights from 3D Models

Simple Summary Low oxygen levels (hypoxia) as well as genetic defects activating hypoxia signaling pathways (pseudohypoxia) are known to contribute to tumorigenesis and therapy resistance in various cancers. The genetic background of pheochromocytomas and paragangliomas is well characterized and indicates that pseudohypoxia plays a role in tumor formation and metastatic spread in a subgroup of these tumors. It is, however, unknown how pseudohypoxia affects susceptibility to radiation treatments, which is of particular interest, since targeted radionuclide therapy is one of the few options used against metastatic pheochromocytomas and paragangliomas. To date, no curative treatment is available for metastatic disease. Here, we report on the radioprotective effects of pseudohypoxia against both external irradiation and beta particle-emitting lutetium-177 in a pheochromocytoma tumor spheroid model expressing hypoxia-inducible factor 2 alpha. Our findings highlight hypoxia signaling pathways as potential targets for neo-adjuvant—in particular, radiosensitizing—therapies in pseudohypoxic pheochromocytomas and paragangliomas. Abstract Pheochromocytomas and paragangliomas (PCCs/PGLs) are rare neuroendocrine tumors arising from chromaffin tissue located in the adrenal or ganglia of the sympathetic or parasympathetic nervous system. The treatment of non-resectable or metastatic PCCs/PGLs is still limited to palliative measures, including somatostatin type 2 receptor radionuclide therapy with [177Lu]Lu-DOTA-TATE as one of the most effective approaches to date. Nevertheless, the metabolic and molecular determinants of radiation response in PCCs/PGLs have not yet been characterized. This study investigates the effects of hypoxia-inducible factor 2 alpha (HIF2α) on the susceptibility of PCCs/PGLs to radiation treatments using spheroids grown from genetically engineered mouse pheochromocytoma (MPC) cells. The expression of Hif2α was associated with the significantly increased resistance of MPC spheroids to external X-ray irradiation and exposure to beta particle-emitting [177Lu]LuCl3 compared to Hif2α-deficient controls. Exposure to [177Lu]LuCl3 provided an increased long-term control of MPC spheroids compared to single-dose external X-ray irradiation. This study provides the first experimental evidence that HIF2α-associated pseudohypoxia contributes to a radioresistant phenotype of PCCs/PGLs. Furthermore, the external irradiation and [177Lu]LuCl3 exposure of MPC spheroids provide surrogate models for radiation treatments to further investigate the metabolic and molecular determinants of radiation responses in PCCs/PGLs and evaluate the effects of neo-adjuvant—in particular, radiosensitizing—treatments in combination with targeted radionuclide therapies.


References for approximation of absorbed β − dose
Since there are no simulations for absorbed dose in spheroids seeded in concave-bottomed microtiter plates available yet, we approximated dose based on administered initial activity using equation 1 and 2. To confirm our calculations, we applied them to examples in literature of irradiated cells in monolayer culture.

Impact of G418 and DMSO on external X-ray irradiation effects
Geneticin (G418) and dimethylsulfoxide (DMSO) are commonly used additives in cell culture studies. Here, we applied these agents during external X-ray irradiation of genetically modified MPC spheroids (Table S2). Supplementation with G418 or DMSO in concentrations of 250 µg/mL and 0.5%, respectively, influenced treatment outcome after external X-ray irradiation in MPC +HIF2α and MPC +EV spheroids. Especially dose-response to 20 and 25 Gy in Hif2α expressing MPC and to 16 and 20 Gy in empty vector controls was affected ( Figure S2A). G418 significantly decreased spheroid control dose, whereas DMSO had radioprotective properties and the necessary doses for growth arrest and sustained spheroid control were increased (Table S3, Figure S2B).  Figure S2. Impact of G418 and DMSO on single-dose X-ray irradiation treatment of MPC +HIF2α and MPC +EV spheroids; (A) Monitoring of spheroid growth as diameter versus time plots, (B) 6-day dose response plotted as spheroid growth (%SG) versus absorbed X-ray irradiation dose; (C) 35-day dose response plotted as spheroid control probability (%SCP) versus absorbed X-ray irradiation dose.

Additional investigations on experimental set-up and plate layout for radionuclide treatment with [ 177 Lu]LuCl3 in vitro
Lutetium-177 is a radionuclide emitting β − particles and γ photons. In order to assure a reliable experimental set-up, it was mandatory to exclude interferences of radiation and absorbed dose between wells. Therefore, we scanned diameters of MPC wt spheroids 6 days after treatment start in different plate layouts. These experiments showed no dependency of short-term response on spheroid positions in the microtiter plate ( Figure S3).

Impact of initial spheroid size on treatment effects of [ 177 Lu]LuCl3
Since generating similarly sized spheroids in independent experiments can be challenging, we evaluated the impact of initial spheroid diameter on long-term response to incubation with [ 177 Lu]LuCl3 in Hif2α expressing as well as empty vector control spheroids. Experimental parameters are summarized in Table S4. The dose necessary for sustained control of spheroids was significantly increased (p < 0.001) in larger spheroids and was overall significantly elevated (p < 0.001) in MPC +HIF2α compared to MPC +EV (Table S5, Figure S5A). SCD50 increased exponentially with initial spheroid diameter ( Figure S5B).
In respect to these findings, we chose a medium diameter range of 400 to 555 µm for all herein reported irradiation experiments, as size-dependent differences of irradiation response are negligible for this size range, but necrotic cores and hence intrinsic hypoxia have already developed. Sustained control was not achieved for very large MPC +HIF2α spheroids, even with the highest activity concentration of 2 MBq/mL (≈16 Gy). These spheroids have large hypoxic areas enhancing with stabilization of HIF2α and thereby driving radioresistance in Hif2α expressing cells.
Supplemental Figure S4. Impact of initial spheroid size on long-term response of MPC +HIF2α and MPC +EV spheroids to [ 177 Lu]LuCl3 treatment; (A) Spheroid control probability (%SCP) versus initial activity concentration of [ 177 Lu]LuCl3; (B) Size-dependency of long-term response plotted as halfmaximal spheroid control dose (SCD50) versus initial spheroid diameters ( † SCD50 of MPC +HIF2α spheroids > 590 µm could not be calculated as SCP = 0 for all tested initial activity concentrations predicting an SCD50 > 2 MBq/mL; ‡ SCD50 for MPC +EV spheroids < 370 µm could not be calculated with available data); (C) Categorization of spheroids depending on initial diameter.