Surgically excised PPGLs vary considerably in their gross appearance.

Pliant, highly vascular tumours (A) digest readily, whereas firm, dark tumours (B) generally require more rigorous treatment to release tumour cells. Digested tissue features single cells and large and small groups of tumour cells (C, Tu57_10x obj. phase contrast). Tumour-derived cells show an irregular light halo around a darker central area, in contrast to the ‘doughnut’ appearance of erythrocytes (D, Tu57_40x obj. phase contrast). Long-term cultures occasionally develop areas that show roughly circular lattice structures (E, Tu11_4 months_5% FBS_10x obj.) that resemble sustentacular cell networks found in tumours but more likely represent connective tissue cells specific to PPGLs, perhaps suggesting that these cells have self-organizing capabilities specific to this tissue. Paraganglioma cell cultures may also develop extensive networks of processes (F, Tu21_4 months_5% FBS_5x obj.), usually but not always after an extended period in culture. These networks can become macroscopically visible in cell culture flasks and usually interconnect discrete cell masses. Cells of tumour culture 23, a paraganglioma without a variant in any commonly-mutated gene, exhibited short, eccentrically branching cell processes (G, Tu23_20 days_5% FBS_20x obj.), occasionally accumulating around a single, flattened cell. Tumour cells originating from a PPGL bone metastasis showed a semi-differentiated morphology in culture (H, Tu26_6 days_5% FBS_10x obj.), a morphology occasionally seen in other non-metastatic tumours.</p

Lactate supplementation enhances chromaffin cell survival.

A representative experiment (tumour 44) illustrating the beneficial effect of lactate supplementation on the short-term survival of synaptophysin-positive cells of chromaffin morphology. A. Anti-synaptophysin immunohistochemical staining of cultures with 0.0, 5.0 and 20 mM lactate monitored at weeks 1, 2 and 4. B. Regression curves of average scorings of a series of lactate concentrations over the 3 time points (regression analysis, ** p < 0.01, ****p < 0.000001).</p

Summary of immunocytochemical staining of PPGL tumour cultures.

Both chromogranin A (Tu8_Chromogranin A_10x obj.) and neuron-specific enolase (Tu-28_Neuron-specific enolase (NSE)_10x obj.) are generally negative, even in very early cultures. Tyrosine hydroxylase staining (Tu36_Tyrosine hydroxylase (TH)_40x obj.) is often positive, even in long-term cultures, but as many HNPGL cultures are negative for TH at the outset this marker is not generally applicable. In our hands the most broadly reliable marker in PPGL tumour cultures proved to be synaptophysin (Tu18_Syn_40x obj.), as this marker can be found in both early and late cultures. To assess the survival of neuronal cells in PPGL tumour cell cultures we also stained for neurofilament protein (Tu-7_Neurofilament protein_20x obj.), which is expressed in nerve fibres and the sparse individual neurons seen in PPGL tumours. We found that even at 29 months of culture (Tu7), a few cells of neuronal morphology are still present in culture. CD56 (NCAM) is a cell surface marker for chromaffin cells and is found in most PPGLs, showing the distinct staining pattern of a cell surface protein. Of the few tumour cultures examined (Tu-42_CD56 (ab 123C3) _10x obj.), only sparse cells were positive for CD56. The classic markers for sustentacular cells in PPGLs are S100 and GFAP. S100 tends to show more widespread staining of these cells and is therefore favoured in IHC applications, while GFAP is expressed more sporadically in the same cells. Both marker proteins are expressed in tumour cultures but S100 expression (Tu-40_S100_20x obj.) appears to be short-lived, while GFAP expression (Tu44_thoracic PGL_GFAP_20x obj.) persists somewhat longer, both generally disappearing over the course of 4–8 weeks, along with cells showing sustentacular morphology. CD31 is a marker for endothelial cells and staining of PPGL FFPE tumour sections underscores the extensive vascularity of these tumours. Although few cultures were stained with CD31, those that were appeared uniformly negative (Tu-8_CD31_20x obj.), suggesting that endothelial cells do not persist in culture. Ki-67, a protein expressed in proliferating cells, was occasionally positive in cells of fibroblast morphology (Tu-52_Ki-67_20x obj.). Cytokeratin, a marker for epithelial cells, was occasionally positive (Tu-8_Cytokeratin_10x obj.) in a few cells. Cytoskeleton proteins such as smooth muscle actin (SMA), vimentin and fibronectin are present in most cells and as such are aspecific markers. However, they can usefully illustrate the general morphology of cell cultures. We found SMA (Tu-47_Smooth muscle actin (SMA)_40x obj._cytospin) and vimentin (Tu-28_Vimentin_10x obj.;Tu-8_Vimentin_10x obj.) to be the most useful, with fibronectin only sporadically positive in a few cells (Tu-17 _Fibronectin_20x obj.).</p

Antibody stainings of formalin-fixed paraffin-embedded (FFPE) PPGL tumors.

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Summary of the gene variants found in this study.

Abbreviations: SDHB, C & D, succinate dehydrogenase subunits B, C & D, respectively; HNPGL, head & neck paraganglioma; PPGL, pheochromocytoma-sympathetic paraganglioma; meta, metastatic.</p

Tumour explant culture to determine the ongoing cellularity of explants.

Tumour explants (1x1 mm tumour fragments) were cultured for up to 39 days to assess cellularity and remaining chromaffin tumour cells. At day 1, H&E staining of tumour 9 reveals the classic cell nest morphology in a highly stromal background (A, Tu9_CBT_SDHD_H&E_10x obj.). By around day 11 tumour fragments began to show clear signs of declining cellularity and an increase in eosin-avid (pink) acellular proteinaceous material (S2 Fig). By day 39, few cells of chromaffin appearance remained in the tumour fragments (B, Tu9_CBT_SDHD_H&E_10x obj.). Chromaffin cellularity was determined using immunohistochemistry (C, Tu44_ thoracic PGL_SDHD_Syn_10x obj.) to assess the proportion of synaptophysin-positive cells (‘chromaffin’ tumour cells) remaining at 1 week (C, Tu44, CBT_SDHD_Syn_10x obj.). At around 4 weeks, (D, Tu44_ thoracic PGL_SDHD_Syn_10x obj.) declining expression of synaptophysin (brown staining) was accompanied by visible shrinkage of cellular tumour areas. Residual chromaffin cell areas can be seen but the lack of nucleic acid (DNA) staining by haematoxylin suggests a loss of cellular integrity. Areas lightly staining for synaptophysin probably consist of cellular debris of chromaffin cells. Quantification of synaptophysin immunohistochemistry in three tumours (E, TU40-CBT, Tu42-PPGL, Tu43-CBT) showed a consistent decline in the expression of this marker protein in explants over a 4-week culture period.</p

Immunocytochemical scoring of control cell lines and a range of PGL tumour cultures based on morphological criteria.

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Comparison of five cell culture media.

Tested on Tu44 (thoracic PPGL, SDHD p.(D92Y)) and Tu46 (carotid body PGL, SDHD p.(L95P)), stained with 3 marker antibodies and scored double blinded in duplo. Percentages of positive cells, with standard error of the mean (SEM), are presented in 10log scale. P values calculated with one-way ANOVA (p S1 Table for details) when sufficient tissue was available. We assessed cultures for the presence of synaptophysin as a marker for chromaffin cells and GFAP as a marker for sustentacular cells. In addition, we estimated the proportion of proliferating cells based on Ki-67 expression. In general, serum-free medium supported the highest proportion of synaptophysin-positive cells, but no long-term serum-free cultures including these cells were noted, probably because serum-free culture medium leads to the slow depletion of cells. Chromaffin cells visibly fail to prosper in this medium, often typified by poor filopodia development and poor adhesion. Media including some bovine serum, at either 1% or 5%, seemed to achieve a better balance between proliferation of fibroblasts, which may have a supportive function in these cultures, and survival of chromaffin tumour cells. Richer media such as Izal and PC12 led to a predominance of fibroblasts that appeared detrimental to the survival of chromaffin cells, possibly due to the rapid consumption of available metabolites in the media.</p

Media, supplements, antibodies and immunocytochemical scoring method used in the study.

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Large and small cell masses are a common feature of paraganglioma cultures.

Cell masses appear to be anchored by fibroblast-like cells to the plastic substrate, and often ‘shed’ cells of chromaffin cell morphology during the first few weeks of culture (A, Tu42_35 days_5% FBS_20x obj. phase contrast). ‘Shed’ cells are often weakly adherent to the plastic or fibroblast underlayer and can be harvested by vigorous pipetting and re-cultured (B, Tu51_58 days _1% FBS_40x obj. phase contrast). These large cell masses consist of numerous synaptophysin-positive cells in the first weeks of culture (C, Tu44_ thoracic PGL_14 days_1% FBS_Syn_20x obj.). Vimentin antibody staining illustrates the general structure of these cell masses (D; Tu44_14 days_1% FBS_Vim_20x obj.). Cell masses often initially express synaptophysin intensely but gradually lose expression and by 4–6 weeks show synaptophysin-expressing cells only in the outermost layer (E, Tu44_4 weeks_5% FBS_Syn_40x obj.) Cells of sustentacular morphology often associate with cell masses when these cells are present in a culture (F, Tu44_4 weeks_5% FBS_GFAP_ 20x obj.). Cell proliferation is ongoing in cell masses (G, Tu44_4 wks_serum-free med_Ki-67_5x obj.) but does not appear to overlap with surviving synaptophysin-positive cells (H, Tu44_4 wks_serum-free med_Synaptophysin_5x obj.; inset Synaptophysin_40x obj.).</p