Impact of Extrinsic and Intrinsic Hypoxia on Catecholamine Biosynthesis in Absence or Presence of Hif2α in Pheochromocytoma Cells

Pheochromocytomas and paragangliomas (PPGLs) with activated pseudohypoxic pathways are associated with an immature catecholamine phenotype and carry a higher risk for metastasis. For improved understanding of the underlying mechanisms we investigated the impact of hypoxia and pseudohypoxia on catecholamine biosynthesis in pheochromocytoma cells naturally lacking Hif2α (MPC and MTT) or expressing both Hif1α and Hif2α (PC12). Cultivation under extrinsic hypoxia or in spheroid culture (intrinsic hypoxia) increased cellular dopamine and norepinephrine contents in all cell lines. To distinguish further between Hif1α- and Hif2α-driven effects we expressed Hif2α in MTT and MPC-mCherry cells (naturally lacking Hif2α). Presence of Hif2α resulted in similarly increased cellular dopamine and norepinephrine under hypoxia as in the control cells. Furthermore, hypoxia resulted in enhanced phosphorylation of tyrosine hydroxylase (TH). A specific knockdown of Hif1α in PC12 diminished these effects. Pseudohypoxic conditions, simulated by expression of Hif2α under normoxia resulted in increased TH phosphorylation, further stimulated by extrinsic hypoxia. Correlations with PPGL tissue data led us to conclude that catecholamine biosynthesis under hypoxia is mainly mediated through increased phosphorylation of TH, regulated as a short-term response (24–48 h) by HIF1α. Continuous activation of hypoxia-related genes under pseudohypoxia leads to a HIF2α-mediated phosphorylation of TH (permanent status).

. Gene expression of MTT H2A cells compared to the MTT control cells was analyzed by reverse transcriptase polymerase chain reaction. Three independent experiments were performed (n = 3). Mean ± SEM. Cycle threshold (Ct).

Cell Line
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Spheroid Characterization and Method Comparison
For spheroid cultivation, three different methods were compared within the study to find optimal conditions and to investigate the impact of different cultivation methods on chromaffin cell features. In general, cells were trypsinized from monolayer culture and an optimized cell number of 500 cells per spheroid was used for spheroid generation with one of the three following methods. The methyl cellulose (MC) method (A) uses the addition of methyl cellulose stock solution to prevent subsidence of cells as previously described by us [28]. For the medium method (B), cell suspensions without addition of methyl cellulose were seeded in cell culture plates for suspension culture (No.650185,Greiner Bio One,Kremsmünster,Austria). For the third method, termed the NunClon method (C), cell suspensions were added to Nunclon Sphera 96U-well plates (No. 174925, ThermoFisher Scientific, Waltham, MA, USA). Spheroids were grown under standard culture conditions (5% CO2, at 37 °C). Spheroid formation was considered complete four days after seeding ( Figure S1); thereafter medium was replaced by fresh complete medium with or without addition of 0.24% methyl cellulose after 3-4 days of cultivation. Spheroids were harvested 11 and 18 days after generation. No spheroid formation was achieved for PC12 cells using any of the three methods as well as the liquid overlay technique. Size of each spheroid was measured using the inverse microscope Axiovert 200M (Software: AxioVision 4.8, Zeiss, Göttingen, Germany). Area (A) of each spheroid was determined using the software package Fiji (ImageJ, Bethesda, MD, USA, www.imagej.net). Diameters (d) were calculated assuming an approximately spherical shape of the spheroids (d = 2 ×√(A/π)).

Spheroid Formation and Optimal Cell Number
Spheroid formation was monitored over a time period of four days via microscopy (Figures S1 and S2). Optimal cell numbers were evaluated for all three conditions by seeding 500-10,000 cells in each well of a 96-well plate with growth monitoring over 18 days ( Figure S3). The optimal cell numbers for MPC and MTT cells were determined at 500 cells per spheroid. These numbers of cells were required to generate uniform single spheroids without detectable outgrowth over 18 days. Figure S1. MPC cell spheroid formation. MPC cell spheroid formation has been monitored for four days. Only cultivation in presence of methyl cellulose provides spheroids with a smooth shape already after 24 h incubation. Scale bar: 100 µ m. Figure S2. MTT cell spheroid formation. MTT cell spheroid formation has been monitored for four days. Cultivation in presence of methyl cellulose and using NunClon plate provides spheroids with a smooth shape already after 24 h incubation. Scale bar: 100 µ m. Figure S3. Determining the optimal cell number to generate MTT cell spheroids. In each well of an ultra-low binding 96-well plate (NunClon) 500-10,000 MTT cells were seeded and growth was monitored. For the other methods, similar results were obtained. Scale bar: 200 µ m.

Spheroid Growth Pattern and Morphology
All three methods enabled formation of uniform, circular spheroids with uniform surface characteristics ( Figure S4A,B) that were resistant to mechanical stress. The diameters of generated spheroids were comparable between all three methods. The total amount of protein in spheroids generated using NunClon plates was significantly lower compared to cultivation in monolayer culture ( Figure S4C). These results indicate that the NunClon plates generate less compact spheroids, a result of their larger size but associated with reduced total cell material. The trend was that generation of spheroids was associated with reduced amounts of protein, which can be explained by the structure of spheroids. As a consequence of the diminished oxygen and nutrient supply a necrotic core surrounded by a hypoxic ring was formed within spheroids (Figures 1 and S5). Only outer cell layers are able to proliferate. Subsequently proliferation is reduced in comparison to monolayer culture. The necrotic area developed within 11 days ( Figure S4D) in both cell lines. Hematoxylin and Eosin staining showed considerable differences between different methods that could be related to various section planes. Spheroids cultivated with the medium methods showed a more diffuse structure especially in MPC cell spheroids. Spheroids generated using the NunClon plates demonstrated a solid structure but the outer proliferating cell layer seems to be inconsistent with regard numbers of cell layers (marked with an arrow, Figure S4D). In comparison, spheroids generated using methyl cellulose showed a solid structure and uniform distribution of the proliferating outer cell layer in both cell lines. Figure S5. Pheochromocytoma cell spheroids stained with pimonidazole to visualize hypoxic regions. To visualize hypoxic regions within the spheroid pimonidazole was used as hypoxia marker. In comparison to 18 days old spheroids, 11 days old spheroids showed a pimonidazole localization in the spheroid core. Once spheroids get bigger (18 days of cultivation), the hypoxic area surrounded the necrotic core clearly. Representative picture of three independent experiments.
A characteristic of chromaffin cells involves the biosynthesis of catecholamines. Both cell lines produced dopamine (DA) and norepinephrine under monolayer conditions. Cellular levels of DA were noticeably increased under extrinsic hypoxia. All three conditions for spheroid cultivation resulted in a significant increase in cellular DA contents ( Figure S6A,B). Cultivation under MC and medium conditions lead to significantly increased DA contents after 18 days cultivation in comparison to day 11. This effect was not detectable using the NunClon method. Cultivation under extrinsic and intrinsic conditions resulted in a strengthened phosphorylation of tyrosine hydroxylase (TH), whereas TH protein levels remained unaffected ( Figure S6C).

MALDI-MSI Measurements
MALDI-MSI measurements were performed on a solariX 7 T FTICR mass spectrometer (Bruker, Billerica, MA, USA) in the mass range of m/z 50−1000 in negative ion mode with an estimated resolution of 35,000 at m/z 400 and a raster width of 50 μm at a frequency of 1000 Hz, using 100 laser shots, and "small" laser focus. Spectra were recorded on Compass solariXcontrol software with 1 M data points, a transient length of 0.26 s, and an ion cooling time of 0.01 s. Source conditions were as follows: deflector plate at −220 V, funnel 1 at −150 V, skimmer 1 at −15 V, and funnel rf amplitude of 150 Vpp. Calibration of the mass spectrometer was conducted using L-arginine in the electrospray ionization (ESI) mode. Two non-tissue measurement regions were included as background controls, one in the beginning and another in the end of the measurement. After MALDI-MSI, matrix was removed with 70% ethanol, stained with Hematoxylin and Eosin, mounted with a coverslip, scanned with a Mirax Desk scanner (Zeiss, Göttingen, Germany) with 20-fold magnification objective, and co-registered to respective MSI data using FlexImaging (version 4.0) (Bruker, Billerica, MA, USA). FlexImaging (Bruker, Billerica, MA, USA, www.bruker.com) was also used to analyze the ion maps and for the export of images.

Distribution of Selected Metabolites within MTT Cell Spheroids
Two different sections of an MTT cell spheroid were analyzed with regard to distribution of hexose-monophosphate (HMP) and phosphatidylinositol (PIP). Hematoxylin and Eosin staining were used to confirm the cut in the proliferating cell zone (first section) or in the middle of the spheroid (second section), displaying the necrotic and hypoxic core surrounded by proliferating cells ( Figure S7). Originally, this analysis was designed to measure the distribution of catecholamines within spheroids, but dopamine, norepinephrine and epinephrine contents within the spheroid were below the MALDI/MS imaging technique's limit of detection. Figure S7. Distribution of hexose-monophosphate (HMP) and phosphatidylinositol (PIP) in MTT cell spheroids. Two different sections of the MTT cell spheroid were generated via cryosectioning. Hematoxylin and Eosin staining confirmed the cross section in the proliferating cell layer (upper pictures) while the second section (lower pictures) showed a cross section of the necrotic/hypoxic core surrounded by a proliferating cell zone. MALDI-MSI records of HMP and PIP, demonstrated an accumulation of these metabolites only in the proliferating cell zone.
The activity of the hexose monophosphate shunt, also known as pentose phosphate pathway, was analyzed. This pathway occurs in parallel to glycolysis and generates NADPH, pentoses and ribose 5-phosphate. A higher activity in outer cell layers confirmed that more activity is shunted in this pathway. In comparison, the inner core of the spheroids showed no HMP activity. Phosphatidylinositol and its phosphorylated derivatives form a minor component of the cell