Quantitative mitochondrial redox imaging of breast cancer metastatic potential

Abstract

Predicting tumor metastatic potential remains a challenge in cancer research and clinical practice. Our goal was to identify novel biomarkers for differentiating human breast tumors with different metastatic potentials by imaging the in vivo mitochondrial redox states of tumor tissues. The more metastatic (aggressive) MDA-MB-231 and less metastatic (indolent) MCF-7 human breast cancer mouse xenografts were imaged with the low-temperature redox scanner to obtain multi-slice fluorescence images of reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavoproteins (Fp). The nominal concentrations of NADH and Fp in tissue were measured using reference standards and used to calculate the Fp redox ratio, Fp(NADH+Fp). We observed significant core-rim differences, with the core being more oxidized than the rim in all aggressive tumors but not in the indolent tumors. These results are consistent with our previous observations on human melanoma mouse xenografts, indicating that mitochondrial redox imaging potentially provides sensitive markers for distinguishing aggressive from indolent breast tumor xenografts. Mitochondrial redox imaging can be clinically implemented utilizing cryogenic biopsy specimens and is useful for drug development and for clinical diagnosis of breast cancer.

Figure 1

Typical pseudo-color redox images and their corresponding histograms of highly metastatic human breast tumor MDA-MB-231 mouse xenografts (tissue section depth 1740 μm). The color bars of the Fp and NADH images indicate the nominal concentrations in μM relative to the corresponding snap-frozen solution standards, and the color bar of the Fp and NADH ratio image indicates the ratio range from 0 to 1. Note that by definition, NADH ratio is merely 1−(Fp ratio). The x axes of the histograms represent the Fp or NADH redox ratio or concentration. The y axes represent the number of pixels in the tumor section having a specific value of Fp or NADH redox ratio or nominal concentration. The small round spots outside the tumor section are the images of Fp or NADH reference standards. The peaks corresponding to tumor core or rim are labeled in the histograms. The image matrix was 128×128, and the step size was 100 μm.

Figure 2

Typical redox images and corresponding histograms of indolent human breast tumor MCF-7 mouse xenografts (tissue section depth 980 μm). The x axes of the histograms represent the Fp or NADH redox ratio or nominal concentration. The y axes represent the number of pixels in the tumor section having a specific value of Fp or NADH redox ratio or nominal concentration. The small round spots outside the tumor section are Fp or NADH reference standards. The image matrix was 128×128, and the step size was 200 μm (twice that of the pixel size of MDA-MB-231 xenograft images in Fig. 1).

Figure 3

Redox imaging biomarkers distinguish between the MDA-MB-231 (aggressive, solid bars) tumor line and the MCF-7 (indolent, open bars) tumor line. Figure 3a shows that only the average of the Fp ratio (core) of the aggressive tumors can distinguish the aggressive from the indolent lines. Figures 3b, 3c show the average concentrations of Fp and NADH relative to the frozen-solution standards for the two tumor groups, respectively. Since there is no distinct core-rim difference identified for the MCF-7 tumors, only the average values of Fp (mean), NADH (mean), and Fp ratio (mean) of the tumor lines were plotted for the preceding figures.

Figure 4

TUNEL assays for tumor cell death in a representative MDA-MB-231 tumor. From left to right: tumor rim DAPI staining, tumor rim FITC staining, tumor core DAPI staining, and tumor core FITC staining. DAPI stains the cell nucleus, and FITC stains the TUNEL positive cells (Ref. 37).

Figure 5

TUNEL assays for tumor cell death in a representative MCF-7 tumor. From left to right: tumor rim DAPI staining, tumor rim FITC staining, tumor core DAPI staining, and tumor core FITC staining. DAPI stains the cell nucleus, and FITC stains the TUNEL positive cells (Ref. 37).