Unique fractal evaluation and therapeutic implications of mitochondrial morphology in malignant mesothelioma

Abstract

Malignant mesothelioma (MM), is an intractable disease with limited therapeutic options and grim survival rates. Altered metabolic and mitochondrial functions are hallmarks of MM and most other cancers. Mitochondria exist as a dynamic network, playing a central role in cellular metabolism. MM cell lines display a spectrum of altered mitochondrial morphologies and function compared to control mesothelial cells. Fractal dimension and lacunarity measurements are a sensitive and objective method to quantify mitochondrial morphology and most importantly are a promising predictor of response to mitochondrial inhibition. Control cells have high fractal dimension and low lacunarity and are relatively insensitive to mitochondrial inhibition. MM cells exhibit a spectrum of sensitivities to mitochondrial inhibitors. Low mitochondrial fractal dimension and high lacunarity correlates with increased sensitivity to the mitochondrial inhibitor metformin. Lacunarity also correlates with sensitivity to Mdivi-1, a mitochondrial fission inhibitor. MM and control cells have similar sensitivities to cisplatin, a chemotherapeutic agent used in the treatment of MM. Neither oxidative phosphorylation nor glycolytic activity, correlated with sensitivity to either metformin or mdivi-1. Our results suggest that mitochondrial inhibition may be an effective and selective therapeutic strategy in mesothelioma, and identifies mitochondrial morphology as a possible predictor of response to targeted mitochondrial inhibition.

Figure 1. Fractal dimension and lacunarity of mesothelioma subtypes.

(a) Hematoxylin and eosin-stained sections of hyperplastic mesothelium and various malignant pleural mesothelioma subtypes, the (b) fractal dimension and (c) lacunarity of hyperplastic/benign, biphasic and epithelioid tumor samples was calculated using the FracLac ImageJ plug-in. Significant differences were detected between hyperplasia/benign control tissues and the biphasic and epithelioid subtypes, p ≤ 0.001. No significant different was detected between the biphasic and epithelioid subtypes. Results were analyzed via ANOVA with Tukey post-test. and are shown as the mean and SEM of each subtype.

Figure 2. Mitochondrial morphology in malignant mesothelioma cell lines.

Mesothelioma cell lines were fixed in 4%paraformaldehyde and stained with TOM20 and Alexa647. Confocal Z-stack images were captured using a Leica SP5 II and a maximum projection image was generated using ImageJ. Scale bar shown is 10 μm. Detailed image from highlighted area is shown for each cell type.

Figure 3. Fractal dimension and lacunarity of mitochondria.

(a)Examples of typical mitochondrial morphologies obsereved in mesothelial cell lines classified via fractal dimension and lacunarity. Maximum projection of Z-stack image series of TOM20 mitochondrial marker stain was used to quantify (b) mitochondrial fractal dimensions and (c) lacunarity. Images were converted to grayscale and analysed using the FracLac plugin from ImageJ. The entire mitochondria of each cell was analyzed. Data are presented as mean ± SEM, significance was calculated using an ANOVA with Tukey post test (*p < 0.05, **p < 0.005, ***p < 0.0001) n ≥ 40 for each cell type (c). (d) Profile of mesothelioma mitochondrial morphologies classified via fractal dimension and lacunarity.

Figure 4. Altered expression of mitochondrial proteins in malignant mesothelioma cell lines.

(a) Representative immunoblots showing expression of mitochondrial and focal adhesion marker proteins in a panel of mesothelioma cell lines and a control transformed but non-tumorgenic cell line, MeT-5A. (b) Ratio of DRP1 to MFN2 expression in total cell lysates as measured via densitometry (c) Representative immunoblots showing expression of DRP1, MFN2 and VDAC in the mitochondrial fraction of mesothelioma cell lysates (d) Ratio of DRP1 to MFN2 expression in the mitochondrial fraction of mesothelioma cell lysates as measured via densitometry. 20 μg of protein was loaded per sample. Results are expressed as mean ± the standard deviation of 3 independent measurements.

Figure 5. Mitochondrial oxidative and glycolytic activity in malignant mesothelioma cell lines.

Oxygen consumption rate (OCR), an indicator of mitochondrial oxidative phosphorylation, and extracellular acidification rate (ECAR) an indicator of glycolytic activity measured over time via Seahorse extracellular flux analyzer. The mitochondrial stress test was used to obtain the bioenergetic parameters. (a) shows an example of one mitochondrial stress assay, arrows indicate the addition of 0.5 μM oligomycin A (oligo), 0.5 μM FCCP, and 1 μM Rotenone. (b) Basal mitochondrial OCR, (c) mitochondrial respiratory reserve capacity, obtained by subtracting basal OCR from maximum OCR, (d) basal ECAR (e) ratio of OCR to ECAR. Results are shown as the mean ± SEM of 5 independent experiments and analyzed via ANOVA with Tukey’s post-test (*p < 0.05, **p < 0.005, ***p < 0.0001).

Figure 6. Bioenergetic profile of malignant mesothelioma cell lines.

Plot of basal ECAR and OCR levels in mesothelioma and control cell lines, mean values ± SEM are shown. From the plot we can easily see that H28 and H2452 are the least energetic cell lines, while MeT-5A and H513 have relatively high levels of both oxidative phosphorylation and glycolysis and are therefore more energetic. H2461 and H2373 are more aerobic, while H2461 and H2596 are more glycolytic.

Figure 7. Cytotoxicity of mesothelioma cell lines to cisplatin, metformin and mdivi-1.

Mesothelioma and control cells in 1% serum media were treated with increasing concentrations of (a) Cisplatin, (b) Metformin, or (c) mdivi-1 as indicated for 72 hrs and cell viability was then assessed via Calcein-AM uptake as described. The results were normalized as a percentage of the untreated controls and analyzed via a sigmoidal dose response curve using Prism software (version 5.0) to calculate the EC50 value for each drug, n = 6 for each concentration shown ± the standard error of the mean.

Figure 8. Correlation between EC50 and mitochondrial morphology.

Scatter plot sowing correlation between (a) Fractal dimension and (b) Lacunarity and inhibitor sensitivities (EC50 values) for cisplatin, metformin and mdivi-1. Significant correlation is observed between fractal dimension and metformin EC50 and between lacunarity and metformin and mdivi-1 EC50. Pearson correlation coefficient was calculated for each data pair as indicated.