Noninvasive assessment of mitochondrial organization in three-dimensional tissues reveals changes associated with cancer development

Abstract

Mitochondrial organization is often altered to accommodate cellular bioenergetic and biosynthetic demands. Changes in metabolism are a hallmark of a number of diseases, including cancer; however, the interdependence between mitochondrial metabolic function and organization is not well understood. Here, we present a noninvasive, automated and quantitative method to assess mitochondrial organization in three-dimensional (3D) tissues using exclusively endogenous two-photon excited fluorescence (TPEF) and show that mitochondrial organization reflects alterations in metabolic activities. Specifically, we examine the organization of mitochondria within live, engineered epithelial tissue equivalents that mimic normal and precancerous human squamous epithelial tissues. We identify unique patterns of mitochondrial organization in the different tissue models we examine, and we attribute these to differences in the metabolic profiles of these tissues. We find that mitochondria are clustered in tissues with high levels of glycolysis and are more highly networked in tissues where oxidative phosphorylation is more dominant. The most highly networked organization is observed within cells with high levels of glutamine consumption. Furthermore, we demonstrate that mitochondrial organization provides complementary information to traditional morphological hallmarks of cancer development, including variations in nuclear size. Finally, we present evidence that this automated quantitative analysis of endogenous TPEF images can identify differences in the mitochondrial organization of freshly excised normal and pre-cancerous human cervical tissue specimens. Thus, this method could be a promising new modality to assess the role of mitochondrial organization in the metabolic activity of 3D tissues and could be further developed to serve as an early cancer clinical diagnostic biomarker.

Imaging mitochondrial dynamics in human skin reveals depth-dependent hypoxia and malignant potential for diagnosis

“…Mitochondrial organization can be evaluated from NADH intensity images through a Fourier-based approach (28, 29) that capitalizes on the fact that the NADH fluorescence intensity yield is increased when NADH is enzymatically bound. The method provides a metric of mitochondrial organization, because most of the cellular bound NADH is contained within the mitochondria.…”

“…To extract this metric, we computed the power spectral density (PSD) from the 2D Fourier transform of each NADH intensity image. A simple inverse power law decay expression of the form R ( k ) = Ak − β was then fit to the PSD, with the magnitude of the exponent β being indicative of the levels of mitochondrial clustering (28, 29). Increased clustering values represented more fragmented/fissioned mitochondrial formations.…”

“…The third was the depth-dependent variation of the nuclear-to-cytoplasmic ratio, which is related to a standard histopathological morphological marker of healthy, differentiated squamous epithelia. Large variations in this ratio correlate with higher values of the PSD depth-dependent variance over scales that correspond to the size of nuclei and cell borders (29). Accordingly, the value of this third metric decreased with the loss of epithelial cell differentiation in cancerous lesions (fig.…”

“…These can be related to biochemical metabolic alterations but not necessarily to mitochondrial organization. Recently, we demonstrated that Fourier-based analysis is well suited to evaluate high-resolution NADH MPM images to assess intracellular texture in cell monolayers and three-dimensional (3D) engineered tissues (28, 29). In these studies, mitochondrial clustering was reliably quantified in cell layers with distinct nuclear-to-cytoplasmic ratios and used as a sensitive marker of metabolic changes in the relative levels of oxidative phosphorylation, glycolysis, and glutaminolysis, which often occur at the onset of cancer and may serve as diagnostic biomarkers or to guide therapy (28, 29).…”

Label-free optical imaging of live cells

“…Several reports focusing on NAD(P)H and flavin adenine dinucleotide (FAD) have shown differences between cancer cells and normal cells (Palmer et al, 2003;Yu and Heikal, 2009). Similarly to confocal microscopy, mitochondria can also be observed with TPEF microscopy, and it has shown that their distribution in cells and tissues seems to reflect the metabolic activity (Xylas et al, 2014). Similarly to confocal microscopy, mitochondria can also be observed with TPEF microscopy, and it has shown that their distribution in cells and tissues seems to reflect the metabolic activity (Xylas et al, 2014).…”