A Computational Method for the Visualization of Nitrated Hsp90 Distribution in 3D Culture Models
Authors: Sathler Alexandre R. , Nguyen Kyle T. , Marean‐Reardon Carrie L. , Estevez Alvaro G. , Franco Maria C. 
Published: May 2022

Protein tyrosine (Y) nitration is an oxidative post‐translational modification that occurs in pathological conditions but is absent in normal tissue. We previously showed that nitrated proteins support tumor cell survival and migration, and recently identified the chaperone Heat Shock Protein 90 (Hsp90) as the first protein that, when nitrated, acquires a novel tumorigenic activity in glioblastoma multiforme (GBM). While Hsp90 is expressed in all cells, we showed that nitration of Hsp90 on Y33 and/or Y56 induces a pathological gain‐of‐function. Nitration on Y33 (Hsp90NY33) decreases mitochondrial metabolism by inhibiting complex IV activity, while nitration on Y56 (Hsp90NY56) activates the P2X7 receptor (P2X7R), which is linked to increased glycolysis in tumors. Together, these metabolic changes are characteristics of the Warburg effect, a metabolic phenotype common amongst tumors, suggesting that nitrated Hsp90 may play a role as a metabolic switch driving tumor progression. Here, we developed a computational approach to visualize the subcellular and spatial distribution of differentially nitrated Hsp90 in 3D cell culture models of GBM (tumoroids) using confocal Z‐Stack images. Our automated, high‐throughput method takes Z‐stack outputs and generates a 3D heatmap of a differentially localized protein of interest, normalized to a housekeeping fluorescence signal. These normalized heatmaps can be generated from any fluorescence image and serve as an unbiased visualization tool to use in conjunction with fluorescence microscopy images. GBM tumoroids were fluorescently labelled for Hsp90NY33or Hsp90NY56using specific and selective in‐house monoclonal antibodies, and for mitochondrial complex IV and P2X7R. DAPI was used as nuclear stain and tumoroids were imaged using confocal laser scanning microscopy. We observed a ~50% loss in fluorescence signal across the depth of tumoroid Z‐stacks, a common problem related to tissue penetration of laser light that leads to an underrepresented fluorescent signal at the core and bottom of imaged tumoroids. To overcome this problem, Python was used to analyze each focal plane within a confocal Z‐stack, normalizing the signal corresponding to the protein of interest by the mean DNA content (DAPI signal) at each focal plane. This normalized signal was used to generate a 3D heatmap of the protein of interest within the tumoroids. Method‐generated heatmaps confirmed the intracellular localization of Hsp90NY33in mitochondria and cytosol, and a distinct localization in the tumoroid periphery. In contrast, Hsp90NY56 was also localized in the nucleus, and was homogeneously distributed throughout the tumoroid. P2X7R showed a clear cell membrane localization, with increased expression in the tumoroid periphery. This suggests that Hsp90NY33may reduce aerobic respiration in the periphery of the tumoroid, allowing oxygen diffusion into the core, while Hsp90NY56 supports glycolysis throughout the tumoroid. Our results provide a novel fluorescence image analysis tool with potential to be expanded for several other 2D and 3D imaging modalities.