Role of Mitochondrial Optical Redox Imaging in Modern Cancer Research (2024 Peter Vaupel Honorary Lecture)

Abstract

The mitochondrial optical redox imaging (ORI), pioneered by the late Britton Chance and his coworkers starting from the 1950s, is mainly based on the intrinsic fluorescence of reduced nicotinamide adenine dinucleotide (NADH) and oxidized flavoproteins (Fp) containing flavin adenine dinucleotide (FAD). These two important metabolic coenzymes regulate many intracellular biochemical reactions including glycolysis, the Krebs cycle, and oxidative phosphorylation, and participate in the intricate interplay between metabolism and signaling. The optical redox ratio (ORR) of these two fluorescence signals is an indicator of mitochondrial metabolic and redox status. Chance and his coworkers not only studied redox status in cells and tissues based on the intrinsic fluorescence but also developed the Chance redox scanner in the 1970s–80s, which images the 3D distribution of NADH, Fp, and ORR ex vivo in snap-frozen tissues. The repertoire of ORI has been further strengthened and enriched with the modern technical advancements including state-of-the-art fluorescence, confocal, two-photon, and lifetime imaging microscopy. ORI has been extensively applied to (pre)cancer cells and tissues including clinical biopsies and patient tumor-derived organoids, to develop metabolic biomarkers for (pre)cancer diagnosis, prognosis, and treatment response. In this chapter, I illustrate the basic principles, main concepts and progresses, and future directions of ORI in cancer research.

1.1. Introduction

It is a great honor for me to receive this Peter Vaupel Lecture Award at the 2024 Annual Meeting of the International Society on Oxygen Transport to Tissue (ISOTT), organized by Dr. Sally Pias at the New Mexico Institute of Mining and Technology, Socorro, New Mexico, USA. My personal career development has benefited greatly from participating in the ISOTT society. My experience with ISOTT started in the 2006 ISOTT meeting organized by Dr. Kyung Kang at the University of Louisville, Louisville, Kentucky, USA. The late Dr. Britton Chance, a long-time senior member and persistent participant of the ISOTT society since the very first meeting in 1973 (Bruley and Thiessen 2016), encouraged me to attend the Louisville meeting. Since then, I have become an ISOTT member and a regular participant of ISOTT meetings. In the 2007 Meeting in Uppsala, I met personally with Dr. Duane Bruley, cofounder of the society, with impressive vision, generosity, wisdom, and kindness. For years, Duane and Kyung mentored me about the ISOTT history, administrative structures, and functions and guided me through the process of bidding for and organizing the 2015 ISOTT meeting in Wuhan, China, as a co-president. The 2015 ISOTT president was Dr. Qingming Luo, a former postdoc of Dr. Chance and the Director of Britton Chance Center for Biomedical Photonics in Huazhong University of Science and Technology, Wuhan, China. Dr. Luo generously allocated local resources and manpower to organize the first ISOTT conference in China very successfully and fulfilled the wish of Dr. Chance. This conference was also a good learning and growing experience for me, with much thanks to the work of local organizing staffs and the support of ISOTT senior members. Throughout the past two decades, I have benefited tremendously from the mentorship of ISOTT senior members including Drs. Peter Vaupel, Harold Swartz, Joseph LaManna, and the help from many other friends and colleagues. Dr. Vaupel is a long-time senior member of ISOTT, and a renowned cancer biologist pioneered in studying tumor oxygenation and hypoxia. I had numerous personal interactions with him at ISOTT meetings as well as the Britton Chance Commemorative Symposia at the University of Pennsylvania. My research benefited from his lectures on tumor hypoxia and the Warburg effect presented at these events.

My first ISOTT presentation was about mitochondrial optical redox imaging (ORI) of human melanoma xenografts under the mentorship of Drs. Jerry D. Glickson and Britton Chance. ORI or optical metabolic imaging (OMI) as referred by some publications, has been developed by the Chance lab since the 1950s. The first detailed study of cellular NADH by fluorescence spectrophotometry in vitro was conducted by Duysens and Amesz in 1957 (Duysens and Amesz 1957). In the next year, Chance and coworkers conducted the measurement of NADH fluorescence in isolated mitochondria and identified how NADH fluorescence changes under different mitochondrial redox states depending on the supply of substrates and ADP (Chance and Baltscheffsky 1958). Within the next several years, Chance et al. reported ex vivo and in vivo measurements of NADH in tissues including muscle, brain, and kidney (Chance and Jobsis 1959; Chance et al. 1962a, b). In the late 1960s, Chance and coworkers studied and identified the fluorescence of oxidized flavoproteins (Fp containing flavin adenine dinucleotide, i.e., FAD) in the mitochondrial respiratory chain (Hassinen and Chance 1968; Garland et al. 1967; Chance et al. 1967). During the 1970s–80s, Chance’s lab built the cryogenic redox scanner, imaging the 3D distribution of NADH, Fp, and the redox ratio in tissue (Quistorff et al. 1985; Barlow et al. 1979). Chance predicted that the fluorescence from mitochondrial NADH and Fp would open “a new field of metabolic research” (Li et al. 2014). Indeed, ORI has become an established, valuable metabolic imaging tool for biomedical research including cancer, neurodegenerative diseases, and other disorders. Due to the limited scope of this manuscript, I will only touch upon some main concepts as well as key applications of this technique in cancer so that beginners can quickly grasp the essence. For more in-depth and detailed information, readers should check for more comprehensive reviews (Li and Sun 2014; Li et al. 2009a; Georgakoudi and Quinn 2023; Gu et al. 2002) and original literatures.

1.2. Basic Principles of Optical Redox Imaging

ORI is mainly based on the optical signals of NADH and FAD (or Fp) which have fluorescence in the range of visible light, whereas NAD⁺ and FADH₂ do not have. Fluorescence of NADH (excitation ~370 nm, emission ~450 nm) and FAD (excitation ~425 nm, emission ~525 nm) has been found to be mainly coming from the mitochondria within cells other than cytosol and nuclei, with the mitochondrial compartment accounting for more than 90% and 60% of the Fp and NADH signals, respectively, in normal and cancer cells (Yu and Heikal 2009; Heikal 2010). Although the cellular NADPH concentration can be several times less than NADH, NADPH has the same fluorescence property as NADH, and currently NAD(P)H is a more commonly used terminology in the field of ORI. The oxidized flavin signal can be contributed by both free and protein-bound flavins including flavinadenine mononucleotide (FMN) and FAD. However, it was shown that FAD dominates the oxidized flavin signal in tissue (Bessey et al. 1949; Kunz and Gellerich 1993; Kunz and Kunz 1985). Thus, here FAD and Fp are used interchangeably.

The ratio of the two fluorescence signals, i.e., optical redox ratio (ORR), is an indicator of mitochondrial metabolic or redox status (Chance et al. 1979). Often, the normalized ORR, defined as Fp/(NAD(P)H+Fp), is adopted to have a value between 0 and 1 with an improved statistical, normal distribution. A higher ORR represents a more oxidative state and a lower ORR represents a more reductive state. Such definition is aligned with the common definition of redox potential in the mitochondrial electron transport train, by which a higher redox potential represents a more oxidized redox state and tends to grab electrons from the environment (Mobilian and Craft 2022; Alberts et al. 2002). It has been demonstrated that ORR correlates linearly with the redox potential of NAD(H), i.e., NAD⁺/(NADH+NAD⁺), as measured by liquid chromatography–mass spectrometry (LC-MS) in normal cells and precancerous tissues (Quinn et al. 2013; Varone et al. 2014). Note that a higher ORR is not necessarily an indicator of oxidative stress or a higher level of reactive oxygen species (ROS). Both ORR increase and decrease can be associated with a higher ROS level as shown by studies of breast cancer cells in vitro (Podsednik et al. 2020, 2024). Thus, the redox status probed by ORI is expected to provide independent information that cannot be simply represented by ROS or oxidative stress.

ORI can be acquired with various optical imaging tools including the cryogenic Chance redox scanning, regular fluorescence microscopy or macroscopy, confocal and multiphoton microscopy, etc. Lifetime imaging microscopy is also developed to measure the lifetimes of NAD(P)H and FAD, which are sensitive to the chemical environment and metabolic status and can separate the measurements of free and bound NAD (P)H or FAD (Datta et al. 2020). Single photon fluorescence imaging with visible light is constrained to the surface imaging due to the limited light penetration depth in tissue. Two-photon imaging utilizes the near-infrared light for NAD(P)H and FAD excitation with a larger light penetration depth. Recently, with 3-photon 1100 nm excitation, NAD(P)H imaging has been demonstrated with good signal contrast in murine skin in vivo (Lee et al. 2019). Nevertheless, despite longer wavelengths used, the multiphoton light penetration depth is still less than 1 mm. It remains difficult to image deep organ tissues.

The cryogenic Chance redox scanner is a unique instrument that can image the 3D distribution of NAD(P)H, Fp, and ORR in snap-frozen tissue blocks under low temperature of liquid nitrogen (Quistorff et al. 1985; Li et al. 2009a). Fresh harvested tissue specimens are snap-frozen ex vivo and kept in liquid nitrogen during the storage and imaging to maintain the metabolic status the same as or as close as possible to the in vivo state. During the imaging, the tissue blocks are shaved by a grinder to expose a flat plane at a certain depth, and then line scanned by a moving coaxial fibro-optic probe that excites tissue and collects emitted light to generate 2D fluorescence images. Such procedure is repeated multiple times at different tissue depths to generate 3D maps of NADH, Fp, and ORR. Chance redox scanner has been applied to image organ tissues including brain, heart, liver, and pancreas ex vivo (Barlow et al. 1979; Haselgrove et al. 1990; Xu et al. 2013b, 2014; Xu and Li 2014). Despite its invasiveness, Chance scanner remains one of the gold standards for studying the 3D distribution and heterogeneity of the mitochondrial metabolic/redox status in tissues.

1.3. Optical Redox Imaging for Cancer Diagnosis and Prognosis

To develop imaging biomarkers for cancer diagnosis and prognosis, a general approach is to compare the differences between normal tissue, precancer, and cancer or between cancers with different aggressiveness (invasive/metastatic potential) so that potential biomarkers can be identified. ORI has been applied to image (pre)cancer cell cultures and organoids, animal models, and clinical tumor specimens, as having been reviewed and summarized (Li and Sun 2014). Here, I would take breast cancer as a representative example to highlight some key concepts, applications, and results for cancer diagnosis and prognosis. Readers can also refer to other publications for detecting precancer (Xu et al. 2013b; Pouli et al. 2020).

It has been shown that ORR is sensitive to breast cancer cell metabolism (Hou et al. 2016) and can differentiate between breast cancer subtypes (Walsh et al. 2014). A review of literature data indicated that higher ORR and more oxidized redox status appear to correlate with higher breast cancer cell aggressiveness in vitro (Li and Sun 2014). Within the triple-generative breast cancer (TNBC) subtype, ORR (=Fp/(NADH+Fp)) was shown to positively correlate with the invasive potential of four human cell lines in vitro (Feng et al. 2021), whereas the intracellular ROS level does not correlate with the invasiveness (data not published). It was also found that the change of ORR due to reoxygenation after acute hypoxia correlates with the rank order of metastatic potential in four murine isogenic TNBC lines (Alhallak et al. 2016). In addition, ORI of the highly metastatic MDA-MB-231 cells responded to the knockdown of oncogene DEK or PGC1α with a concomitant decrease of both ORR and invasive potential (Wen et al. 2019; Lin et al. 2026). All these data support the potential of using ORR as a biomarker for TNBC invasive/metastatic potential.

In breast cancer mouse xenografts, ORI by the cryogenic Chance redox scanner showed significant intratumor redox heterogeneity. The more invasive/metastatic MDA-MB-231 tumors exhibit a distinct bimodal distribution with a more oxidized region and a more reduced region, whereas the more indolent MCF-7 tumors are less heterogeneous (Xu et al. 2010). The ORR in the more oxidized region can differentiate the MDA-MB-231 tumors from the MCF-7 tumors significantly better than the globally averaged ORR of whole tumors. We also obtained data showing that the ORR of the more oxidized region correlates better with the aggressiveness of human breast cancer xenografts than the globally averaged ORR (manuscripts in preparation). These data are consistent with the results from the redox scanning of mouse xenografts of human melanoma cell lines with different invasive/metastatic potential. It was found that the ORR in the oxidized region correlates significantly with the invasive potential of the five melanoma xenografts, whereas the ORR averaged over the whole tumor does not (Li et al. 2009b). Furthermore, the NAD(P)H in the oxidized region is also more effective than the globally averaged NAD (P)H to differentiate melanoma xenografts with different aggressiveness, although its differentiating power is less than the ORR in the oxidized region (Peng et al. 2024). In addition, the ORR in the oxidized tumor region is significantly higher in a p53-null xenograft model of human colon cancer than the corresponding wildtype model, whereas the whole-tumor averaged redox indices do not show significant differences between the two models (Xu et al. 2013a). These results demonstrate the importance of imaging intratumor redox heterogeneity and that ORR of the oxidized region can potentially be a good indicator of tumor aggressiveness. H&E staining, TUNEL assay, and the imaging of glucose analog uptake indicated that there were viable cells in the oxidized regions, and the ORR differences observed were more likely due to differential cell metabolism, rather than variations in cell density (Peng et al. 2024; Xu et al. 2012, 2013, 2013a, 2009).

ORI with the Chance redox scanning also identified intrasample redox heterogeneity in clinical specimens of breast cancer patients, which are more heterogeneous than adjacent normal breast tissues (Xu et al. 2016, 2013c). ORR, NADH, and Fp can differentiate the cancerous and normal breast tissue with sensitivity and specificity in the range of 69–81%. Work is in progress to test the value of ORI of clinical specimens for predicting the prognosis in breast cancer patients.

Regarding the metabolic state of mitochondria in cancer cells within the oxidized region, Drs. Jerry Glickson and Britton Chance had a debate for years. Mitochondrial metabolism can be classified into five states from 1 to 5 depending on the availability of ADP, substrate, and oxygen (Xu et al. 2010, Li et al. 2009b). A general working hypothesis proposed by Dr. Glickson for tumor ORI studies is that the oxidized tumor region with higher ORR indicates a State 2, which has limited blood perfusion and oxygen/nutrient supply and favors the survival of cancer cells with higher metastatic potential (Li et al. 2009b). An alternative hypothesis was proposed by Dr. Chance that the optically oxidized region represents high oxidative metabolism of more aggressive cancer cells with ample supply of ADP and substrate, which is characteristic of a State 3 (Xu et al. 2010). This Glickson-Chance debate remains to be resolved by future investigations.

1.4. Optical Redox Imaging Predicting Treatment Response

ORI indices were identified to predict the response to chemotherapy and targeted therapy in breast cancer cell cultures. For example, the ORR of ER⁺ breast cancer cell line MCF-7 was responsive to antiestrogen drugs in vitro, whereas the MCF-7-derived drug-resistant line LCC9 and TNBC line MDA-MB-231 were not responsive with insignificant alterations in ORR (Ostrander et al. 2010). Furthermore, the breast cancer Her2⁺ BT474 mouse xenograft, more responsive to anti-Her2 trastuzumab than a HR6 xenograft with acquired resistance to trastuzumab, was associated with a significant change of ORR as early as day 2 post treatment compared to the control, whereas no significant changes were found in the resistant HR6 xenograft (Walsh et al. 2013). In contrast, FDG-PET detected no changes in both responsive and non-responsive xenografts until day 12. Association between ORR change and trastuzumab sensitivity was also observed for the same cell models in vitro in both 2D cell cultures (Walsh et al. 2013, 2012) and organoids derived from mouse models (Walsh et al. 2014).

The sensitivity of ORI to treatment response was also demonstrated in patient-derived organoids of human breast cancer (Walsh et al. 2014; Sharick et al. 2020) and pancreatic cancer (Sharick et al. 2020). Taking the data from an ER⁺ organoid as reported in reference Walsh et al. (2014), the optical redox index is shown to correlate significantly with the cell death index when the organoids were under tamoxifen (a hormone therapy drug), paclitaxel (a standard chemotherapy drug), or in combination (R²=0.97, p = 0.01) (Fig. 1.1). In contrast, the more aggressive TNBC organoids exhibited no significant response in ORI indices under anti-estrogen or anti-Her2 therapy or combination (Walsh et al. 2014). Under the settings of standard chemotherapy (adriamycin/cyclophosphamide+paclitaxel), the organoids from two TNBC patients with less residual disease burden exhibited larger alterations of ORI index in response to the chemotherapy in vitro compared to the organoids from a TNBC patient with higher disease burden (Sharick et al. 2020). Furthermore, in seven pancreatic cancer patients, a larger effect size on the ORI index induced by in vitro chemotherapy in patient-derived organoids was associated with the responsiveness to adjuvant chemotherapy and a longer relapse-free survival (Sharick et al. 2020).

Fig. 1.1.

Optical redox imaging indices correlate with therapeutic responses in organoids derived from an ER⁺ human breast tumor. (a) Optical redox index; (b) cell death index (%); and (c) their correlation under different 72-hour treatments, i.e., P (paclitaxel), T (tamoxifen), and combination therapy (P+T plus trastuzumab which showed no effect as a single agent). Optical redox index is the inverse of OMI index and conceptually equivalent to ORR. Cell death index is based on the percent of caspase-3 cleavage. OMI index and caspase-3 data were taken from Fig. 5A-B in reference Walsh et al. (2014)

Xu et al. also compared the treatment response of two TNBC cell cultures, i.e., more invasive/metastatic MDA-MB-231 cells versus less invasive/metastatic HCC1806 cells, to paclitaxel. The more responsiveness with higher proliferation inhibition in HCC1806 is associated with a more significant alteration in ORR induced by paclitaxel compared to MDA-MB-231 (personal communication with Xu HN). ORI of HCC1806 cells in vitro was found to be sensitive to the treatment of paclitaxel, FK866 (an inhibitor of the salvage pathway of NAD biosynthesis), and their combinations (Podsednik et al. 2021). ORI indices such as ORR and Fp in HCC1806 cells were found to correlate positively with the cell growth inhibition induced by such treatment 2021, which is consistent with Fig. 1.1c conceptually. Similarly, the positive correlations of ORR with the growth inhibition were demonstrated in a murine breast cancer line under the FK866 treatment in vitro (Kollmar et al. 2024).

In summary, in response to drug treatment, a common observation is that a higher sensitivity to a treatment is associated with a larger responsiveness in ORI indices. This is a hypothesis that needs further validation by future studies. ORI indices are likely to reflect treatment responses regardless of cancer (sub)types, which is consistent with NAD(H) metabolome alteration as a general hallmark of cancer (Chiarugi et al. 2012) and mitochondrial redox status as a central player in cell death.

1.5. Conclusion and Future Directions

Mitochondrial optical redox imaging is sensitive to perturbation of metabolism and signaling pathways and can provide potential biomarkers for cancer diagnosis, prognosis, and treatment response. Future directions are numerous in both technical developments and biological/clinical applications. Before the in vivo ORI of deep tissue ever becomes feasible in the future, it is worthwhile to explore the feasibility and value of room-temperature ORI of clinical tissue slides, which are more readily available under clinical settings than frozen tissue blocks. It has been under investigation how fixation may affect the NAD(P)H and Fp fluorescence signals and whether the room-temperature ORI of fresh or fixed but unstained tissue slices can generate any biologically useful information (Xu et al. 2000, 2019; Sánchez-Hernández et al. 2023). Other future directions of interest, to name several, include: (i) in-depth study of the relationship between ORI indices and ROS levels; (ii) ORI of cancer cells and tissues under hypoxia, which is known to drive the Warburg effect and cancer progression; (iii) more application and validation of ORI biomarkers under clinical settings of cancer.