Primary fibroblast co-culture stimulates growth and metabolism in Sdhb-impaired mouse pheochromocytoma MTT cells

Abstract

Pheochromocytomas and paragangliomas (PGLs) due to mutations of succinate dehydrogenase (SDH) B, a subunit of the SDH complex with a role in the Krebs cycle and the respiratory chain, tend to be larger at diagnosis and more prone to metastatic disease than other tumors. This presentation contrasts with the behavior of some cell line models of SDHB impairment, which show reduced growth compared to wild type. We hypothesize that reduced growth of SDHB-impaired monolayer culture models might reflect lack of support from sources within the tumor microenvironment. The present study therefore investigates how the microenvironment, modeled here by fibroblast co-culture, modulates cell metabolism, growth and invasion in an Sdhb-impaired mouse pheochromocytoma cell line. We employed two different constructs of short hairpin RNA to knockdown Sdhb and compared growth in a monolayer with and without fibroblast co-culture. Sdhb-silenced cells showed functional impairment of SDH with elevated succinate to fumarate ratio and decreased oxidative capacity. Cell growth was delayed with an increase in doubling time of 2 h or 20 h. Clonogenic cell survival and viability, on the other hand, were either unchanged or increased compared to control. In standard monolayer culture, no differences in pro-metastatic features were present. Co-culture with primary mouse fibroblast reversed the difference of proliferation between control and Sdhb knockdown but was unable to significantly influence invasiveness under these culture conditions. Metabolic studies identified that lactate secreted by fibroblasts was taken up preferentially by Sdhb-silenced cells. In summary, the present study identified a potential role for the tumor microenvironment in influencing phenotypic features of SDHB-mutated PGLs, providing a basis for the use of therapies targeted towards the tumor microenvironment.

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

“…MPC-mCherry H2A, expressing Hif2α , and their counterpart cell line MPC-mCherry control, transfected with an empty vector, were cultivated in collagen coated flask with antibiotic selection as previously described [22,44,45]. For the generation of Hif2α expressing MTT cells we used cells with stable expression of a non-targeting shRNA construct (SHC002V) [46]. These cells were transfected with pcDNA3.1+ carrying a codon-optimized version of the murine Epas1 gene (MTT H2A; Genescript, Piscataway, NJ, USA) via nucleofection (4D-Nucleofector™ System, Lonza).…”

Prodromal Intestinal Events in Alzheimer’s Disease (AD): Colonic Dysmotility and Inflammation Are Associated with Enteric AD-Related Protein Deposition

“…The colonic tissues were weighed and homogenised in lysis buffer, using a polytron homogeniser, as described by Richter et al [60]. Proteins were quantified with the Bradford assay.…”

Advances in paraganglioma–pheochromocytoma cell lines and xenografts

“…While the development of mouse and cell models was originally 'plan A', as in any disease-related field of investigation, over the last two decades this task has proved more challenging than expected and has thus effectively been relegated to 'plan B' status by many groups. Nevertheless, seasoned figures in the field continue their efforts (Powers et al 2017 and others are adapting existing models to new circumstances (Richter et al 2018, Ullrich et al 2018, so research using these models continues and in light of hopeful recent developments from the Tischler/Powers lab, the coming years will hopefully see the introduction of new models from both rodent, and more importantly, human tumor sources. Do we even need a model in a field in which the primary clinical challenge is metastatic SDHB-mutated paraganglioma?…”