A unique model for SDH-deficient GIST: an endocrine-related cancer

in Endocrine-Related Cancer
Correspondence should be addressed to J F Powers: jpowers1@tuftsmedicalcenter.org
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We describe a unique patient-derived xenograft (PDX) and cell culture model of succinate dehydrogenase-deficient gastrointestinal stromal tumor (SDH-deficient GIST), a rare mesenchymal tumor that can occur in association with paragangliomas in hereditary and non-hereditary syndromes. This model is potentially important for what it might reveal specifically pertinent to this rare tumor type and, more broadly, to other types of SDH-deficient tumors. The primary tumor and xenografts show a very high proliferative fraction, and distinctive morphology characterized by tiny cells with marked autophagic activity. It is likely that these characteristics resulted from the combination of the germline SDHB mutation and a somatic KRAS G12D mutation. The most broadly relevant findings to date concern oxygen and oxidative stress. In paragangliomas harboring SDHx mutations, both hypoxic signaling and oxidative stress are putative drivers of tumor growth. However, there are no models for SDH-deficient paragangliomas. This related model is the first from a SDHB-mutated human tumor that can be experimentally manipulated to study mechanisms of oxygen effects and novel treatment strategies. Our data suggest that tumor growth and survival require a balance between protective effects of hypoxic signaling vs deleterious effects of oxidative stress. While reduced oxygen concentration promotes tumor cell survival, a further survival benefit is achieved with antioxidants. This suggests potential use of drugs that increase oxidative stress as novel therapies. In addition, autophagy, which has not been reported as a major finding in any type of SDH-deficient tumor, is a potential target of agents that might trigger autophagic cell death.

Downloadable materials

  • Supplementary Figure.1 Histologic sections of the primary gastric tumor .A. Relatively well differentiated area of tumor showing loosely cohesive epithelioid cells and a minor component of spindle cells (at center). B. Poorly differentiated area of tumor showing numerous rounded tumor cells in a myxoid matrix. There is a gradient of cell size and appearance, ranging from epithelioid cells with conspicuous cytoplasm as in panel A to tiny round cells, some smaller than 10 um, with almost undetectable cytoplasm. Bar = 50 um
  • Supplementary Figure. 2 High magnification of epithelioid tumor cells showing marked pleomorphism and numerous mitoses (arrows). Bar = 50 um
  • Supplementary Figure. 3 Abdominal tumor deposit, consisting almost entirely of small rounded cells (A) and prominent pools of myxoid material (B). Bar = 50 um.
  • Supplementary Figure.4 DOG1 expression in the primary tumor. Staining is less overall than for KIT (cf. Fig. 1), and is absent in most of the small cells. Bar = 50 um
  • Supplementary Figure 5 representative 13C NMR spectra of Ian GIST (Panel A) compared to control SDH-intact GIST-T1 (Panel B) xenografts.. Select resonance multiplets are labeled with the corresponding molecule and carbon using IUPAC numbering.
  • Supplementary Table 1. Antibodies for Immunohistochemistry and Immunoblots
  • Supplementary Table 2. Medium Supplements
  • Supplementary Table 3 Complete list of RNA-Sequencing analysis of gene expression changed by ≥ tenfold between xenograft and cells cultured in 10%O2

 

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    Major characteristics of the primary gastric tumor (A, B, C and D) compared to passage 3 PDX (E, F, G and H). Bars = 50 µm. Tumor morphology (H&E stain, A and E). Primary tumor (A) showing relatively well-differentiated spindle and epithelioid tumor cells with conspicuous eosinophilic cytoplasm (top left) transitioning to loosely cohesive, poorly differentiated tiny round cells, some smaller than 10 µm, with almost undetectable cytoplasm (bottom right). PDX (E) consisting entirely of small to intermediate size cells, with prominent pools of extracellular myxoid material. KIT (CD117) (B and F). Primary tumor (B) showing strong KIT expression in the largest cells with the most cytoplasm and reduced or absent expression in smaller cells. In the PDX (F) KIT expression shows a comparable association with the amount of cytoplasm. SDHB (C and G), In both the primary tumor (C) and PDX (G) SDHB expression is lost in all tumor cells and retained in endothelial cells (arrows), which serve as positive controls. The PDX also contains scattered SDHB-positive mouse-derived inflammatory cells between tumor cells. Ki67 (D and H). Proliferative fraction assessed by Ki67 expression is approximately twice as high in the xenograft as in the primary tumor.

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    Ultrastructural features of the PDX cells. (A) Electron micrograph of xenograft cell showing markedly abnormal mitochondria with swelling and loss of cristae. (B) Xenograft cell with an autophagic vacuole containing a mitochondrion and amorphous material suggesting advanced degeneration of a mitochondrion or other cytoplasmic content. (C) Xenograft cell containing numerous cytoplasmic vacuoles.

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    Redox blot sequentially incubated with antibodies against human peroxiredoxin-2 (PRDX2) and peroxiredoxin-3 (PRDX3). The bands at approximately 22 kDa represent the reduced monomer PRDX2 or PRDX3 proteins, while bands at about 44 kDa represent the respective oxidized dimers. The amount of oxidized dimer relative to the total reduced monomer plus oxidized dimer in each lane is an indication of the relative amount of H2O2-induced stress (Cox et al. 2010). PRDX2 acts as a sensor for H2O2 stress in the cytosol of the cells, while PRDX3 acts in the mitochondria. Beta-tubulin is a loading control. Lanes 1–3 are from separate xenografts of SDH-deficient GIST. Lane 4, which shows the highest amounts of both oxidized dimers and reduced monomers, is from a primary conventional GIST with intact SDHB.

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    Effects of O2 concentration on GIST cell survival in culture. Cells were maintained at the indicated O2 concentrations for 3 days in complete medium in the presence or absence of N-acetylcysteine (NAC, 5 µM) or a proprietary antioxidant (SAO, Sigma antioxidant; 1 µL/mL). (A) Enhanced tumor cell survival in reduced O2 (representative experiment), each value mean ± s.e.m. of quadruplicate wells, P < .03, for 5 or 10% vs 20%, difference between 5 and 10% is not significant (Mann–Whitney U test). (B) Representative immunoblot showing effects of reduced O2 in cultures maintained concurrently with those in Panel A. A single immunoblot from a 4 to 15% gradient polyacrylamide gel was consecutively probed, stripped and re-probed for each of the indicated proteins. Increased survival and proliferative activity of GIST cells in both 10% and 5% vs 20% O2 are reflected by greater amounts of KIT and Ki67, and by decreased amounts of cleaved poly ADP-ribose polymerase (PARP), a marker of apoptosis (Kaufmann et al. 1993). (C and D) Phase contrast photomicrographs of GIST cells in 10% and 20% O2, respectively. (E) Immunocytochemical staining for Ki67 of GIST cells in a 10% O2 well represented in Panel A (arrows indicate stained nuclei). Note only a few scattered cells labeled, compared to nearly all cells in xenograft tissue.

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    Fold change in expression of selected genes in cell culture compared to the xenograft. RNA sequencing analysis was preformed from the xenograft and from cells cultures in 10% oxygen for 3 days and analysis was performed as described in ‘Methods’ section.

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    Representative immunoblot showing effects of MAPK inhibitor U0126 on the GIST cells in culture. Cells were maintained in 10% O2 for 3 days in complete medium with the indicated concentrations of U0126. A single immunoblot from a 4 to 15% gradient polyacrylamide gel was consecutively probed, stripped and re-probed for each of the indicated proteins. (Bands are arranged to highlight effects on KIT and Ki67, not by molecular weight. Anomalous migration accounts for artifactually low Ki67 in lane 3.)

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