Fluorescent mouse pheochromocytoma spheroids expressing hypoxia-inducible factor 2 alpha: Morphologic and radiopharmacologic characterization

Abstract

BACKGROUND:

Pheochromocytomas and paragangliomas (PPGLs) are rare catecholamine-producing tumors arising from chromaffin tissue. In a PPGL subgroup, dysregulation of hypoxia signaling pathways, in particular mediated through stabilization of hypoxia-inducible factor 2 alpha (HIF2α), have been suggested to drive tumorigenesis through altering downstream transcriptional activity.

OBJECTIVE:

This study evaluated the use of mCherry-transgenic mouse pheochromocytoma (MPC^mCherry) spheroids as in vitro models for investigating consequences of HIF2α expression on aggregation behavior, morphology, growth, glucose consumption, amino acid uptake, and somatostatin type 2 receptors under stable hypoxic conditions.

METHODS:

MPC^mCherry spheroids were monitored using confocal laser scanning microscopy. Hypoxic regions were detected using pimonidazole. Radiotracer incubation was performed using 2-[¹⁸F]fluoro-2-deoxyglucose ([¹⁸F]FDG), O-3-(2[¹⁸F]fluoroethoxy)-4-hydroxyphenylalanine ([¹⁸F]OFED), and [⁶⁸Ga]Ga-(Tyr³)octreotate ([⁶⁸Ga]Ga-DOTA-TATE).

RESULTS:

Both HIF2α-expressing and empty vector (EV) control spheroids showed regions of stable cellular hypoxia. Expression of HIF2α in MPC^mCherry spheroids was associated with less symmetric morphology, faster growth, and decreased uptake of [⁶⁸Ga]Ga-DOTA-TATE (somatostatin type 2 receptors) compared to controls, whereas, uptake of [¹⁸F]FDG (glucose transporter 1 and hexokinases) and [¹⁸F]OFED (system L amino acid transporter 1) remained unaffected.

CONCLUSIONS:

The recent study proved MPC^mCherry spheroids to be complex three-dimensional tumor cell models for investigating morphologic and metabolic consequences of dysregulated hypoxia pathways under hypoxic conditions.

Keywords

Amino acid transporters confocal laser scanning microscopy neuroendocrine tumors paraganglioma pimonidazole positron emitters radiotracer uptake somatostatin receptors

Abbreviations

DOTA

1,4,7,10-tetraazacyclododecane-N,N′,N’,N”′-tetraacetic acid

[¹⁸F]FDG

2-[¹⁸F]fluoro-2-deoxyglucose

GLUT

Glucose transporter

HIF

Hypoxia inducible factor

LAT1

System L amino acid transporter 1

MPC

Mouse pheochromocytoma cell line

MTT

Mouse tumor tissue-derived cell line

[¹⁸F]OFED

O-3-(2-[¹⁸F]fluoroethyl)-3,4,-dihydroxyphenylalanine; 3-O-Methyl-6-[¹⁸F]fluoro-L-dopa ([¹⁸F]OMFD

TATE

(Tyr³)octreotate

PPGL

Pheochromocytoma and paraganglioma

SSTR2

Somatostatin type 2 receptor

1 Introduction

Pheochromocytomas and Paragangliomas (PPGLs) are rare catecholamine-producing tumors arising from adrenomedullary or extra-adrenal chromaffin tissue, respectively, with an incidence of only 2-8 cases per 1 million per year [1]. Most cases of PPGLs are benign [2, 3] with a moderate risk of tumor relapse after surgical removal [4, 5]. One subgroup of metastatic PPGLs is related to mutations in certain tumor susceptibility genes encoding van Hippel-Lindau tumor suppressor (VHL), various succinate dehydrogenase subunits (SDHx), or hypoxia-inducible factor 2 alpha (HIF2α, encoded by the endothelial PAS domain-containing protein 1 (EPAS1)-gene) [6].

Catecholamine-producing PPGLs are commonly diagnosed based on increased levels of catecholamines and their respective O-methylated metabolites in plasma and urine [1, 7]. Taking advantage of their neuroendocrine character, metastatic PPGLs can be diagnosed based on positron emission tomography (PET) imaging using radiotracers such as meta-[¹²³I]iodobenzylguanidine ([¹²³I]MIBG) visualizing norepinephrine transport [8, 9], and 3,4-dihydroxy-6-[¹⁸F]fluorophenylalanine ([¹⁸F]FDOPA) visualizing system L amino acid transporter 1 (LAT1)-dependent amino acid transport [10 –12]. Referring to the latter, an experimental LAT1-specific PET radiotracer, O-3-(2-[¹⁸F]fluoroethyl)-3,4,-dihydroxyphenylalanine ([¹⁸F]OFED), with similar characteristics to the clinically established tracer 3-O-Methyl-6-[¹⁸F]fluorodopa ([¹⁸F]OMFD), has recently been developed in our group and is currently under preclinical evaluation [13 –16]. A considerable number of PPGLs are known to express somatostatin type 2 receptors (SSTR2) and can be visualized using radiolabeled somatostatin analogs such as [⁶⁸Ga]Ga-DOTA(Tyr³)octreotate ([⁶⁸Ga]Ga-DOTA-TATE) [17]. Furthermore, metastatic, in particular SDHB-mutated, PPGLs show high detection rates using 2-[¹⁸F]fluoro-2-deoxyglucose ([¹⁸F]FDG) visualizing glucose turnover [17, 18]. Unfortunately, all currently recommended treatment options for metastatic PPGLs are considered as palliative [4, 19]. SSTR2-targeting endoradiotherapy offers potential for treating metastatic PPGLs using amongst others [¹⁷⁷Lu]Lu-DOTA-TATE [20].

Hypoxia is a common characteristic of solid tumors including PPGLs. Key mediators of the metabolic response are HIFα proteins involved in regulation of oxygen-dependent pathways affecting glucose and amino acid metabolism, cell proliferation and survival, but also metastasis and angiogenesis [21]. It has been described that functional defects in VHL, SDHx, and HIF2α are involved in dysregulation of hypoxic signaling pathways via stabilization of HIFα proteins, although, there is no actual lack of oxygen [6 , 23]. This metabolic state is referred to as pseudohypoxia. Under normoxic conditions, HIFα proteins are oxygen-dependently ubiquitin-tagged and degraded in proteasomes. Under hypoxic or pseudohypoxic conditions, stabilized HIFα subunits associate with HIFβ (also referred to as aryl hydrocarbon receptor nuclear translocator) forming active transcription factors that bind to HIF-responsive elements in promotors [21]. Additionally, HIF2α may also aggregate with avian myelocytomatosis viral oncogene homolog/myc-associated factor X (MYC/MAX)-complexes activating correlated gene expressions [24, 25]. In PPGLs, but also in other tumor entities, dysregulated hypoxia signaling pathways, in particular mediated through oxygen-independent stabilization of HIF2α, have been suggested to drive tumorigenesis through altering downstream transcriptional activity [26, 27].

In order to investigate effects of dysregulated hypoxia signaling pathways in PPGLs in vitro, it is mandatory to develop reliable cell culture models. Unfortunately, no fully differentiated human PPGL cell line is available to date. However, the mouse pheochromocytoma (MPC) cell line that has been developed from an adrenal pheochromocytoma of a neurofibromin 1 knockout mouse provides a suitable alternative [28]. Furthermore, a red-fluorescent MPC cell line is available continuously expressing mCherry (MPC^mCherry) in order to allow for fluorescence imaging in vitro and in vivo [29]. MPC cells and tumor allografts have been described to resemble at least in part biochemical features and molecular characteristics of human PPGLs, in particular in terms of catecholamine production and high SSTR2 density [20]. Because MPC cells only produce the HIF1α isoform [30], the genetically modified MPC+HIF2α cell line has recently been introduced in order to mimic a pseudohypoxic metabolic state through overexpression of HIF2α resulting in elevated growth [31].

In order to characterize morphologic, metabolic, and molecular effects of increased HIFα protein levels in MPC cells in vitro, it is mandatory to cultivate them under hypoxic conditions. Therefore, cells are most commonly cultivated in hypoxia chambers, however, experiments are often performed under normoxic conditions. Thus, MPC cell culture models with stable hypoxic conditions may be preferable. Three-dimensional tumor spheroids are models for avascular tumors, micrometastases, and tumor microregions between functional blood vessels. Since the supply of nutrients and oxygen occurs only via diffusion, tumor cell spheroids develop nutrient and oxygen gradients as well as outward catabolite gradients caused by increasing distance of the inner cells from the surrounding medium [32 –35].

Hence, we hypothesized that in particular HIF2α-expressing MPC^mCherry spheroids allow for characterizing morphologic and functional effects of increased HIF2α activity under tumor-like stable hypoxic conditions, in vitro.

To address the above hypothesis our objectives were to (1) genetically induce HIF2α expression in red-fluorescent MPC^mCherry cells; (2) characterize three-dimensional morphology, growth, and hypoxic regions of HIF2α-expressing MPC^mCherry spheroids and; (3) measure uptake of the radiotracers [¹⁸F]FDG, [¹⁸F]OFED, and [⁶⁸Ga]Ga-DOTA-TATE in HIF2α-expressing MPC^mCherry spheroids compared to monolayer cell cultures in order to functionally characterize glucose consumption, LAT-1-dependent amino acid uptake and SSTR2 status, respectively.

2 Materials and methods

2.1 Cell lines and monolayer cultivation

Red-fluorescent MPC^mCherry cells (passage 10) were routinely cultured on collagen-coated flasks as previously described [29]. In order to induce HIF2α expression (codon-optimized murine EPAS1), cells were genetically modified as described elsewhere [31]. In brief, MPC^mCherry cells were transfected with pcDNA3.1+ carrying a codon-optimized version of the murine Epas1 gene (MPC^mCherry+HIF2α, Genescript) using nucleofection (4D-Nucleofector™ System, Lonza). Genetically modified cells were selected with 500 μg/mL geneticin (Thermo Fisher Scientific, Waltham, MA, USA). An empty-vector control was generated at the same time (MPC^mCherry+EV). HIF2α-expressing MPC^mCherry+HIF2α cells and empty vector MPC^mCherry+EV control cells were maintained with 250 μg/mL geneticin. Cells were supplied with new medium every 2-3 days and passaged every 7 days. HIF2α expression in MPC^mCherry+HIF2α cells and lack of HIF2α expression in MPC^mCherry+EV cells was confirmed by quantitative reverse transcriptase polymerase chain reaction and immunoblotting.

2.2 Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR)

Gene expression was measured via qRT-PCR using the CFX Connect Real-Time PCR Detection System (BioRad, Hercules, CA, USA) as described previously [31]. The following primers were employed: β-actin forward 5’-AAGGCCAACCGTGAAAAGAT-3’ and reverse 5’-GTGGTACGACCAGAGGCATAC-3’; HIF2α forward 5’-TCGACTCCTCTGACGATGTG-3’ and reverse 5’-CAGAGGGCTCGTCAAAGTTC-3’; NeoR forward 5’-AGACAATCGGCTGCTCTGAT-3’ and reverse 5’-CTCGTCCTGCAGTTCATTCA-3’.

2.3 Immunoblotting

For HIF2α stabilization, cells were treated with CoCl₂ (100 and 200 μM). After 24 h cells were detached from the flasks by incubation with 2 mmol/L ethylenediaminetetraacetic acid at 4°C and washed with phosphate-buffered saline. Cells were lysed on ice in radioimmuno precipitation assay buffer supplemented with 1 mmol/L phenylmethylsulfonyl fluoride, 7 μg/mL leupeptin, 1 mmol/L dithiotreitol, 7 mmol/L NaF, 1 mmol/L Na₃VO₄ followed by ultrasonic treatment (20 %, 15 s). Lysates were heated to 99°C and proteins (20 μg per lane) were separated on a 8.5% SDS-polyacrylamide gel and transferred to a polyvinylidene membrane. Lysate of HIF2α-overexpressing HEK293 cells (NBL1-10286; 20 μg per lane, Novus Biologicals, Littleton, CO, USA) served as positive control. Non-specific binding was blocked for 1 h at room temperature in wash buffer (Tris-buffered saline containing 0.05 % Tween-20) supplemented with 2 % bovine serum albumin and 5 % nonfat dried milk. HIF2α was detected using the primary antibody NB100-122 (Novus Biologicals; 1:500) and the secondary antibody A5045 (Sigma-Aldrich, St. Louis, MS, USA; 1:5000) diluted in wash buffer supplemented with bovine serum albumin. For loading control, ß-actin was detected using the primary antibody A5316 (Sigma-Aldrich; 1:1000) and secondary antibody A9044 (Sigma-Aldrich; 1:10000) diluted in blocking buffer. Specific binding was detected using SuperSignal West chemiluminescent substrates (Thermo Fisher Scientific).

2.4 Spheroid cultivation

Cells were detached from the culture flask using 1 mg/mL trypsin in Dulbecco’s phosphate-buffered saline and resuspended in cell culture medium. Concave bottom ultra-low attachment plates (Perkin Elmer Life Sciences, Waltham, MA, USA) were filled with 1000 cells per cavity. In order to enhance formation of spheroids, plates were centrifuged at 300 rpm for 5 min and maintained at 37 °C in a humidified 5 % CO₂/95 % O₂ atmosphere. Cell culture medium was replaced every 4 days. Size and shape of spheroids were documented every 2–4 days using the microscope Axiovert 40 CFL (Carl Zeiss, Oberkochen, Germany).

2.5 Confocal laser scanning microscopy

In order to investigate the three-dimensional morphology of spheroids, distribution of red-fluorescent mCherry protein (intrinsic) and blue-fluorescent DNA stain (140 min incubation with 10 μmol/L Hoechst 33258 diluted in phosphate-buffered saline) were imaged simultaneously (n = 2). Three-dimensional surfaces of spheroids were reconstructed from a series of z-stack images (slice distance = 7.28 μm) using Imaris 7.6.1 (Bitplane AG, Belfast, UK) applying the following parameters: surface grain size = 3.18 μm, manual threshold value A = 1110, manual threshold value B = 3990, voxels = 10. For spheroid imaging Silica-Green® nanoparticles with diameters of 1 μm (Micromod Partikeltechnologie GmbH, Rostock, Germany) were added to visualize plate bottom.

2.6 Immunohistochemistry

Hypoxic regions of spheroids (n = 3) were stained using the Hypoxyprobe-Kit (Hypoxyprobe, Burlington, MA, USA). At 18 days of cultivation, spheroids were incubated with pimonidazole (20 μg/ml in phosphate-buffered saline, 140 min). Formalin-fixed and paraffin-embedded spheroids were dewaxed in RotiClear (Carl Roth GmbH & Co. KG, Karlsruhe, Germany) and rehydrated in a graded series of ethanol (100, 80, 70, 60, 50 %, H₂O). For de-masking, spheroid sections were incubated in citric acid buffer (10 mmol/L, pH 6, 100 °C, 20 min). Endogenous peroxidase was quenched using hydrogen peroxide (3 % in Tris-buffered saline, 10 min). Non-specific binding sites were blocked with fetal bovine serum (10 % (v/v) in Tris-buffered saline, 1 h). Spheroid sections were incubated with the primary anti-pimonidazole antibody PAb2627 (1:100 in blocking solution, 60 min) and the secondary biotinylated donkey anti-rabbit antibody RPN1004V1 (Amersham Biosciences, Little Chalfont, UK; 1:200 in blocking solution). Spheroid sections treated with rabbit IgG ab37415 (Abcam, Cambridge, UK) instead of primary antibody served as isotype control. Specific binding was detected using extra-avidin-peroxidase E2886 (Sigma-Aldrich, St. Louis, MS; 1:50 in Tris-buffered saline, 30 min) followed by incubation with 3-amino-9-ethylcarbazole (Thermo Fisher Scientific) and counterstaining with hematoxylin.

2.7 Radiotracer uptake of monolayer cell cultures and spheroids

Three different radiotracer incubation media (volume activity at incubation start (A _V ) = 0.4 MBq/mL) were prepared: (i) glucose-free Dulbecco’s Modified Eagle’s medium (Thermo Fisher Scientific) containing [¹⁸F]FDG (molar activity (A _M ) = 10 GBq/μmol), (ii) phosphate-buffered saline supplemented with 0.9 mol/L CaCl₂ and 0.5 mol/L MgCl₂ containing [¹⁸F]OFED (A _M = 40 GBq/μmol), and (iii) MPC cell routine culture medium [29] containing [⁶⁸Ga]Ga-DOTA-TATE (A _M = 25 GBq/μmol) at 37°C.

Radiotracer uptake of monolayer cell cultures was measured as described previously [20], normalized to total cellular protein as determined using the Qbit protein assay (Thermo Fisher Scientific), and calculated as % initial dose (ID)/mg of protein.

Radiotracer uptake of spheroids was measured at 18 d of cultivation (MPC^mCherry+HIF2α: d = 788±34 μm, n = 12; MPC^mCherry+EV: d = 560±22 μm, n = 12) following a protocol published elsewhere with some modifications [36]. In order to enhance signal-to-background ratios, two spheroids were pooled in one cavity of a 96-well ultra-low attachment microplate. Culture medium was replaced by 200 μL of radiotracer incubation medium. Spheroids were incubated for 60 min and soaked into 96-well filter plates (Perkin Elmer Life Sciences, Waltham, MA, USA). Filters were washed in ice-cold phosphate-buffered saline supplemented with 0.9 mol/L CaCl₂ and 0.5 mol/L MgCl₂, transferred into tubes containing 300 mL H₂O, and A _V was measured using the gamma detector Wizard² 2480 (Perkin Elmer Life Sciences). Tubes containing incubation media served as standards. Background activities were measured accordingly in spheroid-free cavities containing radiotracer incubation medium only. Relative uptake of radiotracers was normalized to spheroid volume and calculated as % ID/mm³.

2.8 Statistics

Statistical analyses were performed using Prism 6 (GraphPad, La Jolla, CA, USA). All data are presented as mean±standard error of the mean. Significance of differences was tested using the t-test. Differences were considered significant at p-values > 0.05. For documentation of spheroid growth, diameters were determined from microscopic images (n = 25) at different time points using the AxioVision software version 4.8.2.0 (Carl Zeiss). Circularity of spheroids was determined at diameters between 600 and 700 μm (n = 25) using the polygon selection and fit spline tool of ImageJ version 1.51 m (National Institute of Health, Bethesda, MD, USA). Progression of spheroid diameters (n = 25) was fitted with linear growth equation (between 6 and 18 d of cultivation) and growth rates were calculated as μm/d.

3 Results

3.1 Confirmation of HIF2α gene expression in MPC^mCherry cells

Quantitative reverse transcriptase polymerase chain reaction showed that HIF2α (codon-optimized endothelial PAS domain-containing protein 1 (EPAS1) gene) was expressed in MPC^mCherry+HIF2α cells but not in the corresponding empty vector control cell line MPC^mCherry+EV (Table 1). Furthermore, immunoblotting showed a specific 118 kDa protein band for HIF2α only in MPC^mCherry+HIF2α cells treated with CoCl₂ as a hypoxia mimetic. HIF2α protein was not detected in CoCl₂-treated MPC^mCherry+EV cells and under normoxic conditions in both cell lines (Fig. 1).

Table 1
Gene expression of MPC^mCherry+HIF2α cells compared to MPC^mCherry+EV cells; gene expression data from quantitative reverse transcriptase polymerase chain reaction; (Ct) cycle threshold, (EV) empty vector (HIF2α) codon-optimized EPAS1-expression cassette ligated into the expression vector, (NeoR) neomycin resistance expression cassette, (RQ) mean expression level for the gene in question, (SD) standard deviation

Gene MPC^mCherry cell line Ct SD β-actin Δ Ct (β-actin) ΔΔ Ct (HIF2α) RQ RQ-SD RQ+SD

HIF2α +HIF2α 21.82 0.18 16.85 4.97 0.00 1.00 0.88 1.14

+EV 0.00 0.00 16.58 –16.58 –21.55 0.00

NeoR +HIF2α 20.65 0.21 16.85 3.80 0.00 1.00 0.87 1.16

+EV 20.47 0.04 16.58 3.88 0.08 0.95 0.92 0.97

Gene	MPC^mCherry cell line	Ct	SD	β-actin	Δ Ct (β-actin)	ΔΔ Ct (HIF2α)	RQ	RQ-SD	RQ+SD
HIF2α	+HIF2α	21.82	0.18	16.85	4.97	0.00	1.00	0.88	1.14
	+EV	0.00	0.00	16.58	–16.58	–21.55	0.00
NeoR	+HIF2α	20.65	0.21	16.85	3.80	0.00	1.00	0.87	1.16
	+EV	20.47	0.04	16.58	3.88	0.08	0.95	0.92	0.97

Fig.1

Immunoblot of stabilized HIF2α in mCherry-transgenic MPC cells; HIF2α and β-actin immunoblots of cell lysates from cultures treated with different concentrations of CoCl₂ as a hypoxia mimetic; lysate of HIF2α-overexpressing HEK293 cells served as positive control.

3.2 Morphology of monolayer and spheroid cultures

HIF2α-expressing cells cultured as collagen-supported monolayers exhibited a lower tendency for aggregation compared to controls that preferentially formed cell clusters (Fig. 2A). Confocal laser scanning microscopy confirmed that expression of red-fluorescent mCherry protein was maintained in HIF2α-expressing and control cell lines (Fig. 2B).

Fig.2

Effects of HIF2α expression on aggregation behavior of MPC^mCherry cells in monolayer cultures under normoxic conditions; (A) phase contrast micrographs showing decreased aggregation in HIF2α-expressing cells; (B) fluorescence micrographs showing mCherry protein in red channel (λ_ex/em = 543/618 nm) and the DNA stain Hoechst 33258 in blue channel (λ_ex/em = 405/455 nm); scale bars: 200 μm.

Both cell lines reproducibly formed spheroids using liquid overlay cultivation in concave bottom ultra-low attachment microplates. Phase contrast microscopy (Fig. 3A, left columns) showed that both HIF2α-expressing spheroids and controls appeared fully translucent at diameters below 450 μm and developed an opaque core region surrounded by a translucent layer (d = 120±30 μm) at diameters higher than 450 μm. HIF2α-expressing spheroids exhibited a less symmetric morphology compared to controls. Three-dimensional surface reconstructions (right columns) from z-stacks of mCherry confocal laser scanning micrographs showed that HIF2α-expressing spheroids exhibited a more rippled surface with characteristic bulges compared to the smooth surface of controls. Of note, non-fluorescent surface regions resulted from clonal expansion of residing mCherry-negative cells.

Fig.3

Effects of HIF2α expression on morphology and growth of MPC^mCherry spheroids; (A) phase contrast micrographs (left columns) and three-dimensional surface reconstructions (right columns) from z-stacks of confocal laser scanning micrographs (mCherry,λ_ex/em = 543/618 nm); bottom-up views; scale bars: 200 μm; (B) circularity of spheroids; (C) monitoring of spheroid growth presented as diameter versus time plots; dotted line indicates appearance of opaque core regions at diameters higher than 450 μm; significance of differences: (†) p < 0.01; (‡) p < 0.001.

In line with microscopic observations, circularity values of HIF2α-expressing spheroids (0.94±0.002) were significantly lower compared to controls (0.99±0.001) as determined at diameters between 600 and 750 μm, respectively (Fig. 3B).

3.3 Characterization of spheroid growth

Monitoring spheroid diameters between 4 and 20 d of cultivation showed that HIF2α expression accelerated the growth of MPC^mCherry spheroids (Fig. 3C). At day 4, comparable diameters of HIF2α-expressing spheroids (242±14 μm) and controls (224±13 μm) were detected. Between 6 and 18 d of cultivation diameters for both HIF2α-expressing spheroids (R² = 0.96) and controls (R² = 0.96) increased linearly with significantly higher growth rates of HIF2α-expressing spheroids (44±1.5 μm/d) compared to controls (28±1.2 μm/d). At day 18, HIF2α-expressing spheroids reached significantly larger diameters (753±35 μm) compared to controls (561±24 μm). After 18 d of cultivation, diameters of all spheroids remained static or decreased due to incipient disintegration.

3.4 Surface reconstruction, protein biosynthesis and DNA integrity of spheroids

Three-dimensional reconstructions of MPC^mCherry spheroid surfaces from z-stacks of mCherry confocal laser scanning micrographs resulted in bowl-like shapes (Fig. 4A). After adding green nanoparticles for visualizing the orientation of spheroids with reference to plate bottom, coronal images showed that mCherry was only excitable up to a maximum depth of 119±3 μm from the spheroid surface at a maximum focal plane distance of 185 μm above the plate bottom (Fig. 4B-D). Of note, maximum excitation depths in spheroids were significantly higher compared to the widths of mCherry positive regions between spheroid surface and necrotic core (55±4 μm). Therefore, fluorophore distribution in MPC^mCherry spheroids was subsequently analyzed in confocal laser scanning micrographs captured at a transversal focal plane distance of 130 μm.

Fig.4

Visualization of MPC^mCherry spheroids using confocal laser scanning microscopy; (A) three-dimensional mCherry distribution in an MPC^mCherry+EV spheroid appearing as bowl-like shape (red, λ_ex/em = 543/603 nm) and sedimented nanoparticles visualizing plate bottom (green, λ_ex/em = 488/510 nm) as reconstructed from z-stacks of inverse confocal laser scanning micrographs; scale bar 200 μm; (B) scheme of spheroid orientation and visualization with reference to plate bottom; (C) coronal view of mCherry excitation/detection in an MPC^mCherry+EV spheroid; (a) maximum excitation depth; (b) width of mCherry-positive regions between spheroid surface and necrotic core; (fp_max) focal plane at a maximum distance of 185 μm from plate bottom; (fp_opt) optimal focal plane at a distance of 130 μm from plate bottom for characterizing fluorophore distribution in transversal micrographs; scale bar 200 μm; (D) comparison between maximum excitation depths (n = 24) and width of mCherry-positive regions (n = 12); significance of differences: (‡) p < 0.001.

Confocal laser scanning microscopy of mCherry distribution in spheroids showed protein biosynthesis to a depth of 55±4 μm (10–12 cellular layers) in both HIF2α-expressing MPC^mCherry spheroids and controls (Fig. 5A). All spheroids showed decreasing fluorescence intensities of mCherry with increasing distance to the nutritional medium. Fluorescence signals of mCherry were absent in necrotic core regions.

Microscopic images showed that Hoechst 33258 accumulated selectively in nuclei of the most peripheral layer up to 55±4 μm (10–12 cellular layers) in HIF2α-expressing MPC^mCherry spheroids and controls (Fig. 5B). Hoechst 33258 also remained detectable in core regions of HIF2α-expressing spheroids showing a diffuse pattern, but was absent in core regions of control spheroids.

Fig.5

Distribution of mCherry, Hoechst 33258, and pimonidazole in HIF2α-expressing and control MPC^mCherry spheroids; (A) confocal laser scanning micrographs of intrinsic mCherry distribution (red, λ_ex/em = 543/618 nm) showing regions of active protein biosynthesis; scale bars: 400 μm; (B) confocal laser scanning micrographs of Hoechst 33258 distribution (blue, λ_ex/em = 405/455 nm) showing nuclei-selective accumulation in peripheral regions and diffuse accumulation in core regions, scale bars: 400 μm; (C) immunohistochemistry of pimonidazole accumulation (red) on spheroid sections showing narrow and asymmetric regions of stable intrinsic hypoxia including 4–6 cellular layers surrounding core regions; upper-right schemes illustrate the position of sectioning planes; scale bars: 100 μm.

3.5 Hypoxic regions in spheroids

Pimonidazole immunohistochemistry showed a comparable distribution of hypoxic regions in both HIF2α-expressing MPC^mCherry spheroids and controls, as investigated at diameters between 700 and 1000 μm (Fig. 5C). In all spheroids, hypoxia staining was graded and intensified with increasing distance to the nutritional medium showing highest intensities at 4–6 layers of vital cells surrounding the necrotic core region. Cells within this region changed rapidly from a hypoxic into a necrotic state. Of note, widths of hypoxic regions differed on opposite poles of spheroids.

3.6 Radiotracer uptake in monolayer and spheroid cultures

In the applied experimental settings, signal-to-background ratios of [¹⁸F]FDG, [¹⁸F]OFED, and [⁶⁸Ga]Ga-DOTA-TATE incubation showed that in MPC^mCherry cell- or spheroid-containing cavities measured activities were always significantly higher compared to cavities containing medium only (Table 2). Signal-to-noise-ratios were between 5 and 100-fold higher in monolayer compared to spheroid cultures depending on utilized radiotracer. The results demonstrate that the applied methodologies allowed for specific measurement of radiotracer uptake in both monolayer and spheroid cultures.

Table 2
Signal-to-noise-ratios of radiotracer incubation in monolayer and spheroid cultures of MPC^mCherry cells with differential HIF2α expression; ratios were calculated against cell-free cavities; significance of differences: () p < 0.05; (†) p < 0.01; (‡) p < 0.001

Radiotracer Monolayer Spheroids

MPC^mCherry MPC^mCherry MPC^mCherry MPC^mCherry

+HIF2α +EV +HIF2α +EV

[¹⁸F]FDG ‡72±3 ‡177±9 ‡12±1,9 ‡6.0±1.1

[¹⁸F]OFED ‡245±21 ‡204±17 †3.4±0.3 2.3±0.4

[⁶⁸Ga]Ga-DOTA-TATE †9.4±1.6 ‡33±2 ‡1.9±0.2 †2.8±0.1

Radiotracer	Monolayer	Spheroids
[¹⁸F]FDG	‡72±3	‡177±9	‡12±1,9	‡6.0±1.1
[¹⁸F]OFED	‡245±21	‡204±17	†3.4±0.3	* 2.3±0.4
[⁶⁸Ga]Ga-DOTA-TATE	†9.4±1.6	‡33±2	‡1.9±0.2	†2.8±0.1

HIF2α-expressing cells showed significantly decreased [¹⁸F]FDG uptake and [⁶⁸Ga]Ga-DOTA-TATE uptake (22±1 % ID/mg of protein and 12±2 % ID/mg of protein, respectivey) compared to controls (56±9 % ID/mg of protein and 45±3 % ID/mg of protein, respectively) in monolayer culture (Fig. 6A). Uptake of [¹⁸F]OFED, however, was slightly but not significantly increased in HIF2α-expressing cells (59±5 % ID/mg of protein) compared to controls (49±4 % ID/mg of protein).

Fig.6

Uptake of [¹⁸F]FDG, [¹⁸F]OFED, and [⁶⁸Ga]Ga-DOTA-TATE in HIF2α-expressing and control MPC^mCherry cells grown as monolayers and spheroids; (A) radiotracer uptake in monolayer cultures normalized to protein content; (B) radiotracer uptake in spheroids normalized to spheroid volume; (ID) initial dose at incubation start; significance of differences: (‡) p < 0.001.

In contrast, HIF2α-expressing spheroids showed unchanged uptake of [¹⁸F]FDG and [¹⁸F]OFED (0.14±0.03 % ID/mm³ and 0.07±0.02 % ID/mm³, respectively) compared to controls (0.13±0.03 % ID/mm³ and 0.07±0.01 % ID/mm³, respectively) (Fig. 6B). Uptake of [⁶⁸Ga]Ga-DOTA-TATE was significantly decreased in HIF2α-expressing spheroids (0.03±0.01 % ID/mm³) compared to controls (0.16±0.01 % ID/mm).

4 Discussion

This study characterized hypoxia-inducible factor 2 alpha (HIF2α)-related morphologic and metabolic alterations in mCherry-transgenic mouse pheochromocytoma (MPC^mCherry) cells grown as monolayers and spheroids. Cultivation of MPC^mCherry cells as monolayers showed that under normoxic conditions HIF2α expression (codon-optimized endothelial PAS domain-containing protein 1 (EPAS1) gene) was associated with reduced aggregation as well as with altered glucose consumption, system L amino acid transport (LAT1), and somatostatin type 2 receptor (SSTR2) presence. In spheroids, expression of HIF2α was associated with less symmetric morphology, faster growth, and altered SSTR2 status, but glucose consumption and LAT1-dependent amino acid transport were unchanged compared to control. Herein, we demonstrate that MPC^mCherry cell monolayers and spheroids are complementary in vitro models for PPGLs with HIF2α gain-of-function with potential use for drug screening or testing.

Cultivation of MPC^mCherry spheroids provided red-fluorescent chromaffin tissue models with regions of stable intrinsic cellular hypoxia allowing for time-resolved monitoring. Three-dimensional confocal laser scanning microscopy and radiotracer incubation were successfully adjusted in order to provide time-resolved morphologic and functional metabolic readouts from MPC^mCherry spheroid cultures, respectively.

Previous investigations have shown that in the original non-fluorescent MPC cell line that lacks Hif2α, continuous expression of the gene led to immature phenotypic features and increased proliferation [31]. Following up on these initial investigations, the recent study demonstrates that HIF2α expression affected the morphology of both monolayer and spheroid cultures. In monolayer cultures HIF2α-expressing MPC^mCherry cells showed a lower tendency to aggregate and spheroids were less compact and symmetric compared to controls. Since HIF2α has been suggested to drive metastatic signaling pathways in pheochromocytes [21], this phenotype may indicate weakened cell-cell or cell-matrix contacts associated with increased cell motility.

Interestingly, morphologic alterations in monolayer cultures of HIF2α-expressing MPC^mCherry cells already occurred under normoxic conditions suggesting that a fraction of functional HIF2α proteins may constantly evade proteasomal degradation resulting in a HIF2α-related pseudohypoxic phenotype. Supporting this, other studies have shown that proteasomal degradation of HIF2α is less efficient compared to HIF1α under both normoxic and hypoxic conditions [37, 38]. However, it is currently unknown what minimum cytoplasmic concentration of stabilized HIF2α is required to initiate a cellular response in chromaffin cells.

In keeping with accelerated growth in monolayer [31], HIF2α expression also increased growth rates of MPC^mCherry spheroids suggesting that HIF2α drives proliferative pathways in pheochromocytes. Alongside with silenced SDHB, found to increase tumor cell migration and invasion in murine pheochromocytoma cell spheroids, overexpressed HIF2α may play a role in enhancing the tumor metastasizing potential [39].

In terms of monitoring spheroid growth it has to be considered that many but not all spheroid types reach a maximum size of 1-1.3 mm³ under optimal nutrient supply [34, 40]. The size of HIF2α-expressing and control MPC^mCherry spheroids remained static or decreased already at diameters below 1 mm and later than 18 d of cultivation due to incipient disintegration. Taking into account the different growth rates of HIF2α-expressing and control MPC^mCherry spheroids and the appearance of opaque cores at diameters around 450 μm, the optimal time frame for experiments comparing both spheroid lines is between 14 and 18 d of cultivation.

Within the opaque core regions of both HIF2α-expressing and control MPC^mCherry spheroids, loss of intrinsic mCherry and increasingly diffuse accumulation of Hoechst 33258 revealed lack of protein biosynthesis and off-nucleus DNA, respectively, indicating cells in a necrotic state. These findings are consistent with other studies, showing that spheroids deriving from other cancer cell lines also develop a central necrotic region at diameters between 400 and 500 μm [33, 41].

Within the translucent outer shells of both HIF2α-expressing and control MPC^mCherry spheroids, continuous biosynthesis of mCherry and nucleus-specific accumulation of Hoechst 33258 indicate that cells remained viable up to 55 μm from the spheroid surface. With further increasing distance to the nutritional medium, cells lost their biosynthetic capacity and changed rapidly from a normoxic into a hypoxic state. These observations suggest that MPC^mCherry spheroids may impede diffusion of oxygen more efficiently compared to spheroids deriving from other cancer cell lines where diffusion of oxygen is nearly unlimited up to diameters of 200 μm [42, 43]. Furthermore, hypoxic regions in both HIF2α-expressing and control MPC^mCherry spheroids were found to be very narrow involving only 4–6 cellular layers followed by immediate necrosis. These results indicate that MPC^mCherry cells are very sensitive to hypoxia.

Widths of hypoxic regions differed on opposite poles of MPC^mCherry spheroids indicating that an asymmetric oxygen gradient developed most likely due to close contact between spheroid surface and plate bottom. For fine-tuning of spheroid cultivation, hanging-drop techniques or adjustments of liquid overlay culture conditions such as soft agar coating of microplates or the use of methylcellulose as viscosity-increasing cell culture medium additive have been described [44, 45] and may prevent asymmetric oxygen and nutrient gradients in spheroids. However, in terms of MPC spheroid cultivation, advantages and disadvantages of the aforementioned techniques remain to be elucidated.

Confocal laser scanning microscopy and phase contrast microscopy are common imaging techniques for monitoring spheroid growth [46 –48]. In our recent study, mCherry distribution in MPC^mCherry spheroids was detected in order to reconstruct three-dimensional surface models from z-stacks of inverse confocal laser scanning micrographs. Applying this methodology, spheroids appeared as bowl-like shapes reflecting the limitations of confocal laser scanning microscopy in terms of optical tissue penetration. However, image analyses showed that excitation depth in MPC^mCherry spheroids was sufficient in order to visualize the distribution of fluorophores up to a transversal focal plane that includes all cellular regions between spheroid surface and necrotic core. Prospectively, two-photon confocal laser scanning microscopy could be applied in order to achieve deeper optical penetration [49].

The recent study also evaluated a methodology for functional metabolic characterization of MPC^mCherry cell monolayers and spheroids using radiotracers. Signal-to-background ratios of radiotracer incubation indicate that precision of uptake values was considerably lower in spheroids compared to monolayers. This limitation is mostly due to the small fraction of viable cells in MPC^mCherry spheroids that contribute to radiotracer uptake in only the most peripheral regions. Thus, the current methodology is most appropriate for spheroids at diameters above 550 μm. Further optimization is required to enable dynamic measurement of radiotracer uptake in smaller spheroids at earlier time points and measurements. Pooling a higher number of spheroids prior to radiotracer incubation may be a possibility to further enhance signal-to-background ratios.

Radiotracer incubation showed that HIF2α-expression in MPC^mCherry cells was associated with decreased 2-[¹⁸F]fluoro-2-deoxyglucose ([¹⁸F]FDG) uptake in normoxic monolayer cultures, but remained unaffected in spheroid cultures. Taking into account that HIF2α-related morphologic alterations occurred in monolayer cultures already under normoxic conditions, these observations suggest that pseudohypoxia due to single-acting HIF2α leads to downregulation of glucose turnover in monolayers, whereas, additional stabilization of HIF1α under hypoxic conditions in spheroids may counteract this regulation. These findings are in line with other reports showing that stabilization of HIF1α or HIF2α can have opposing effects on target genes [21, 27].

It is well known that HIF1α activates glucose metabolism [27 , 51]. On the other hand, HIF2α has been reported not to directly interfere with glycolysis in most cancer types [21]. Thus, in the present study, downregulation of glucose turnover in HIF2α-expressing MPC^mCherry cells may rather have occurred as a consequence of HIF2α-independent metabolic alterations. On the other hand, actions of HIF2α have also been described to be highly cell type-specific [52].

Uptake of O-3-(2-fluoroethyl)-3,4,-dihydroxyphenylalanine ([¹⁸F]OFED) tended to be increased in HIF2α-expressing MPC^mCherry cells grown as monolayers under normoxic conditions, but remained unaffected in spheroids. These observations point towards upregulation of LAT1-dependent amino acid transport in response to HIF2α-related pseudohypoxia, whereas additional stabilization of the HIF1α isoform under truly hypoxic conditions may counteract this regulation. Supporting this, other reports have shown that LAT1 expression is impaired by hypoxia [53]. Increased expression of LAT1 is known to be associated with elevated cell proliferation during cancer progression [15 , 55]. Therefore, LAT1 may be essential for amino acid supply in HIF2α-expressing MPC^mCherry cells in order to maintain proliferation under pseudohypoxic metabolic conditions with reduced glucose turnover, whereas, this regulatory response apparently does not occur in spheroids in complete hypoxic state.

HIF2α-expressing MPC^mCherry cells showed considerably decreased uptake of [⁶⁸Ga]Ga-DOTA-(Tyr³)octreotate ([⁶⁸Ga]Ga-DOTA-TATE) in both monolayer and spheroid cultures indicating that SSTR2 is downregulated by HIF2α under both pseudohypoxic and hypoxic conditions, respectively. These findings are in accordance with clinical reports demonstrating a poor detection rate of [⁶⁸Ga]Ga-DOTA-TATE in EPAS1-mutated PPGLs due to impaired degradation of HIF2α [56].

5 Conclusion

The present study demonstrates that HIF2α expression in MPC^mCherry cells and spheroids leads to various changes in morphology and metabolism depending on whether the cells are exposed to normoxic or hypoxic conditions. In spheroids, mCherry expression facilitated precise three-dimensional and time-resolved morphologic characterization using confocal laser scanning microscopy. Furthermore, radiotracer uptake techniques have been successfully applied providing functional metabolic readouts from MPC^mCherry monolayer and spheroid cultures on glucose consumption, LAT1-dependent amino acid uptake and SSTR2 status. Thus, cultivation of HIF2α-expressing and control MPC^mCherry cells both as monolayers and spheroids provide complementary in vitro models to further investigate the complex involvement of HIF proteins in regulating tumor progression and metabolism in PPGLs. Furthermore, the introduced cell lines will allow for fluorescence imaging of tumor allografts in PPGL mouse models and, therefore, are considered prerequisites for preclinical investigations on HIF2α-related tumor effects in vivo. In addition, spheroids represent an excellent model for evaluating new radiotracers and radiotherapeutics, as well as small molecule inhibitors for potential effects as chemotherapeutics or radiosensitzers.

Declaration of interest

All authors have contributed to the work and agree with the presented findings. The recent work has not been published before nor is being considered for publication in another journal.

Disclosure statement

The authors have nothing to disclose.

Conflict of interest

The authors have declared that no conflict of interest exists. The funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Competing interests

The authors have declared that no competing interest exists. The funding sponsors had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to publish the results.

Funding

This work was supported by the Collaborative Research Center Transregio 205 “The Adrenal: Central Relay in Health and Disease” (CRC/TRR 205/1; V.S., S.R., N.B., G.E., J.P. & M.U.), and the Paradifference Foundation (Consortium for Personalized Targeted Therapy for SDHB-mutated Metastatic PPGLs; S.R., N.B., G.E., J.P. & M.U.).

Footnotes

Acknowledgments

The authors thank the staff of the GMP radiopharmaceuticals production for providing PET tracers [¹⁸F]FDG, [¹⁸F]OFED and [⁶⁸Ga]Ga-DOTA-TATE. The excellent technical assistance of Andrea Suhr, Regina Herrlich, Sonja Lehnert, Uta Lenkeit and Sebastian Meister is greatly acknowledged. We further thank Prof. Arthur Tischler, Prof. Karel Pacak and Dr. James Powers for providing MPC 4/30PRR cells.

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Fluorescent mouse pheochromocytoma spheroids expressing hypoxia-inducible factor 2 alpha: Morphologic and radiopharmacologic characterization

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSIONS:

Keywords

Abbreviations

1 Introduction

2 Materials and methods

2.1 Cell lines and monolayer cultivation

2.2 Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR)

2.3 Immunoblotting

2.4 Spheroid cultivation

2.5 Confocal laser scanning microscopy

2.6 Immunohistochemistry

2.7 Radiotracer uptake of monolayer cell cultures and spheroids

2.8 Statistics

3 Results

3.1 Confirmation of HIF2α gene expression in MPCmCherry cells

3.4 Surface reconstruction, protein biosynthesis and DNA integrity of spheroids

3.6 Radiotracer uptake in monolayer and spheroid cultures

5 Conclusion

Declaration of interest

Disclosure statement

Conflict of interest

Competing interests

Funding

Footnotes

Acknowledgments

References

3.1 Confirmation of HIF2α gene expression in MPC^mCherry cells