Abstract
Experimental models for pheochromocytoma and paraganglioma are needed for basic pathobiology research and for preclinical testing of drugs to improve treatment of patients with these tumors, especially patients with metastatic disease. The paucity of models reflects the rarity of the tumors, their slow growth, and their genetic complexity. While there are no human cell line or xenograft models that faithfully recapitulate the genotype or phenotype of these tumors, the past decade has shown progress in development and utilization of animal models, including a mouse and a rat model for SDH-deficient pheochromocytoma associated with germline Sdhb mutations. There are also innovative approaches to preclinical testing of potential treatments in primary cultures of human tumors. Challenges with these primary cultures include how to account for heterogeneous cell populations that will vary depending on the initial tumor dissociation and how to distinguish drug effects on neoplastic vs normal cells. The feasible duration for maintaining cultures must also be balanced against time required to reliably assess drug efficacy. Considerations potentially important for all in vitro studies include species differences, phenotype drift, changes that occur in the transition from tissue to cell culture, and the O2 concentration in which cultures are maintained.
Experimental models including xenografts and cell lines derived from multiple human tumors provide a critical foundation for preclinical cancer research. These are complemented by mouse models engineered to develop tumors that faithfully recapitulate their human counterparts' genotype and phenotype, thereby presenting targets for testing drugs and other interventions. While these types of models are readily available for common types of human cancer (Sajjad et al. 2021), they are generally lacking for pheochromocytoma and paraganglioma (PGL). There are no human cell lines that faithfully represent these tumors although many attempts have been made to establish them. The failure may be explained in large part simply by the extremely low proliferation rate of PGL, which typically have a doubling time measured in years or sometimes decades. That explanation is consistent with extensive evidence that human PGL cells in primary cultures can survive and maintain their differentiated features for at least a year with negligible evidence of proliferation (Bayley & Devilee 2020). Similarly, primary xenografts can survive but not grow for long periods in immunocompromised NSG (NOD scid gamma) mice (Powers et al. 2017). Both the development of valid animal models and their appropriate subsequent use must also take proliferation rate into account. Tumors must form fast enough and often enough for the model to be practical, but rapid proliferation makes a model innately different from what it is intended to represent. This proliferation paradox is likely to be particularly important in preclinical drug testing because proliferating cells show heightened sensitivity to many chemotherapeutic agents. It may ultimately be resolved by novel inducible regulators of proliferation (Gu et al. 2018, Misawa et al. 2021), but such solutions remain to be implemented.
The most frequent cause of death for patients with PGL is development and progression of metastasis (Dahia et al. 2020). Consequently, both human-derived and animal models are especially needed as tools to aid the development of new treatments for patients with metastatic disease. The task of establishing suitable models is particularly challenging because multiple levels of complexity exist under the PGL rubric. These embryologically and functionally (Fishbein et al. 2017) related tumors arise from neuroendocrine tissues called paraganglia associated with sympathetic nerves throughout the body and parasympathetic nerves in the head and neck (a pheochromocytoma is an intra-adrenal paraganglioma (Mete et al. 2022)). Despite their overall similarities, PGL are genetically, clinically, biochemically, and morphologically diverse. At least 40% of these tumors have a hereditary basis and at least 20 predisposing genes have been identified (Fishbein et al. 2017). Sporadic cases can be caused by somatic mutations of some of the same genes or other genes not associated with hereditary susceptibility (Flores et al. 2021). The phenotype of any individual tumor including its capacity for metastasis reflects an interplay between genotype, location and other factors that are poorly understood.
The World Health Organization currently considers all PGL to pose some risk of metastasis (Mete et al. 2022). However, risk is strongly correlated with genotype, ranging between approximately 30–40% with SDHB mutations and 2% with mutations of RET (Kumar et al. 2021). Consequently, efforts at model development have recently focused to a large extent on SDHB. However, there is currently no cure for metastatic disease associated with any genotype. Potential models for other pheochromocytomas and occasional paragangliomas were reported in mice rats, dogs, cats, and various domestic and wild animals as early as the 1950s and 1960s, but these were often considered to be curiosities and were generally not utilized as clinical disease models. In some instances, potentially important models were not maintained and were lost (Tischler et al. 2004).
A major catalyst for model development was the First International Symposium on Pheochromocytoma (ISP1) held at the US National Institutes of Health in Bethesda, Maryland, in 2007. That meeting brought clinicians together with basic and translational researchers in order to formulate clinical management guidelines, foster collaborative research, and chart future directions. The concluding sentence in the report from that meeting states ‘…new animal models of familial and metastatic pheochromocytoma or paraganglioma will provide essential tools to test new approaches for diagnosis and treatment of tumors and the eradication of tumor precursors.’ Subsequent publications and meetings including ISP6 held in Prague in 2022 show that considerable progress in development and utilization of models has indeed taken place although challenges remain. At the same time, the rapid development and implementation of single-cell sequencing and lineage-tracing techniques has dramatically increased our understanding of the cell populations in developing and adult human paraganglia (Kameneva et al. 2021) and in PGL (Zethoven et al. 2022). These studies show substantial variation between the neoplastic cells in tumors with different genotypes, and tumors within a given genotype may contain cell populations corresponding to different developmental stages (Zethoven et al. 2022). Multiple models are therefore needed to capture the nuance associated with any genotype (Fig. 1).
Schematical illustration of recent developments in PGL-related in vitro and in vivo experimental models.
Citation: Endocrine-Related Cancer 30, 5; 10.1530/ERC-22-0405
To date, most model-based studies pertaining to Sdhb have relied on knockdown models in which cultures or xenografts of cell lines with irrelevant drivers or incomplete neuroendocrine phenotype are used as surrogates for ‘true’ SDH-deficient tumors. The former include PC12 (Greene & Tischler 1976) or MPC (Powers et al. 2000) cells, respectively, harboring Max and Nf1 mutations and the MPC derivative MTT (Martiniova et al. 2009). The latter include immortalized chromaffin cells (Goncalves et al. 2021) or the putative human PC progenitor line hPheo1 (Ghayee et al. 2013, Matlac et al. 2021). These studies have yielded important information such as the critical importance of REDOX balance in promoting either death or survival of SDH-depleted cells, supporting suggestions for a possible role of ascorbate as a therapeutic agent (Liu et al. 2020, Goncalves et al. 2021). However, the overall relevance of these models to human disease may diminish in proportion to relevance of their cell of origin (Kluckova et al. 2020).
In addition, there are now two new SDH-deficient PGL models derived from rodents harboring germline Sdhb mutations similar to their human counterparts. The first called RS0 (for Rat Sdh null) is a rat xenograft and cell line model derived from a single primary pheochromocytoma that took almost 2 years to develop in a rat harboring a heterozygous germline Sdhb mutation (Powers et al. 2020). Loss of the wild-type allele and development of the tumor might have been facilitated by perinatal irradiation of the rat pup and the higher proclivity of rats to develop pheochromocytomas compared to mice and other species (Tischler et al. 2004). It should be noted that radiation also played a role in development of both PC12 and MPC cells, although this effect might have been more important in enabling the establishment of cell lines from the parent tumors than on initial tumor development.
In the second model derived from mice, multiple pheochromocytomas form in mice in which complete loss of SDHB was combined with loss of NF1 (Armstrong et al. 2022). In the process of model development, earlier findings that loss of SDHB alone is not sufficient for tumorigenesis in mice (Maher et al. 2011) were confirmed. There are no reported human PGL with double SDHB/NF1 mutations, and the precise mechanism by which NF1 contributes to tumorigenesis in this mouse model is still unclear (Armstrong et al. 2022). However, several intriguing observations associate NF1 with the pathobiology of human pheochromocytomas. These include frequent deletions in chromosome 1p and frequent somatic NF1 mutations in sporadic PGL (Flores et al. 2021) and the development of PGL in patients with neurofibromatosis type 1. In addition, neurofibromatosis type 1 is the most frequently identified genetic cause of composite tumors containing chromaffin cell and neuronal cell populations (Dhanasekar et al. 2022), suggesting that loss of function affects an early developmental window. Consistent with that suggestion, pheochromocytomas in Nf1 knockout mice express multiple genes involved in early development of the peripheral and central nervous systems (Powers et al. 2007). The RS0 and Sdhb/Nf1 tumors resemble SDHB-mutated human PGL to varying degrees and are likely to provide complementary information.
The RS0 cell line has already been used for preclinical drug testing while the Sdhb/Nf1 mouse has provided insights into early mechanisms of tumorigenesis, but derivative cell lines have not yet been established. Other new investigative tools include a mouse model in which multiple pheochromocytomas form in response to activation of a novel CDK5-signaling cascade initiated by succinate (Gupta et al. 2022) and the MENX rat that carries a Cdkn1b (p27) frameshift mutation and spontaneously develops pheochromocytomas. Intriguingly these tumors recapitulate most characteristics of SDH-related PGL including norepinephrine and dopamine secretion, a HIF2a-driven pseudohypoxic signature, metabolomic reprogramming associated with an accumulation of the oncometabolite 2-hydroxyglutarate, DNA hypermethylation and massive angiogenesis (Mohr et al. 2021). A zebrafish model in which loss of sdhb in homozygous larvae recapitulates metabolic characteristics of human PGL has also been generated, but tumors have not so far been observed in these animals (Dona et al. 2021). Finally, there is also renewed interest in canine pheochromocytomas as a fairly common naturally occurring model with up to 13% frequency of metastasis (Barthez et al. 1997) and genetic similarities to human PGL including a potentially pathogenic SDHD variant (Calsina et al. 2019).
All of the animal models for which cell lines exist have to some extent been studied in vitro both to investigate basic pathobiology and for preclinical drug testing. This research has not been exclusively focused on SDHB or on new drugs. Some studies may be relevant to a wide range of tumors or may aim to improve treatments with old drugs including conventional chemotherapy agents (Wang et al. 2022). However, important considerations sometimes overlooked include species differences, phenotype drift in cell lines maintained for years in different laboratories and changes that occur in transition from xenograft to cell line (Powers et al. 2020).
Given the absence of human cell lines, there is also increasing interest in primary cultures of human PGL which, tested in parallel with animal-derived cell lines, may add validity to the results (Wang et al. 2022). Primary cultures raise particular questions about how studies should optimally be performed and how cytotoxicity should be assessed. Approaches range from monolayer culture and counting of cells stained for neuroendocrine markers, a method first used effectively a decade ago (Giubellino et al. 2013), to co-cultures with stromal cells (Richter et al. 2018) or currently favored spheroid (Martinelli et al. 2022) or organoid cultures and high-throughput microcultures (Nguyen & Soragni 2020). Precise definitions distinguishing between ‘spheroid’, ‘organoid’, and loose floating aggregates have not been established. Challenges with these primary cultures include how to account for variable proportions of normal and neoplastic cells, which will depend on the nature and duration of the initial tumor dissociation, and how to distinguish drug effects on neoplastic vs normal cell populations. The feasible duration for maintaining the cultures must also be balanced against time required to reliably assess drug efficacy, which may be weeks rather than days (Eastman 2017).
A consideration potentially important for all in vitro studies of PGL is the O2 concentration in which cell cultures are maintained. It is well established that the concentration within solid tumors is well below the ~19% in traditional cell cultures (McKeown 2014) and that a low O2 environment preserves ‘stemness’ of various cell types (Hammarlund et al. 2018). However, except for RS0 cells, which would only grow in low O2 at their inception (Powers et al. 2020), almost all the cell lines and primary cultures to date were established, maintained, and studied under traditional conditions. It is unknown to what extent this may skew experimental data and whether the O2 concentration is equally important for cells representing different genotypes. The answer to this and other pressing questions will become clear as progress in the models field continues to advance.
Declaration of interest
We have no conflicts of interest to declare.
Funding
Arthur Tischler and Judith Favier are both grateful to The Paradifference Foundation for their support.
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