In Vivo Autofluorescence Imaging of Tumor Heterogeneity in Response to Treatment

Shah, Amy T.; Diggins, Kirsten E.; Walsh, Alex J.; Irish, Jonathan M.; Skala, Melissa C.

doi:10.1016/j.neo.2015.11.006

Cited by 88 publications

(109 citation statements)

References 35 publications

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“…Images were analyzed using SPCImage (Becker and Hickl), as described previously [28]. Spatial binning included each pixel and the surrounding 8 pixels.…”

Section: Methodsmentioning

confidence: 99%

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Metabolic Imaging of Head and Neck Cancer Organoids

2017

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Head and neck cancer patients suffer from toxicities, morbidities, and mortalities, and these ailments could be minimized through improved therapies. Drug discovery is a long, expensive, and complex process, so optimized assays can improve the success rate of drug candidates. This study applies optical imaging of cell metabolism to three-dimensional in vitro cultures of head and neck cancer grown from primary tumor tissue (organoids). This technique is advantageous because it measures cell metabolism using intrinsic fluorescence from NAD(P)H and FAD on a single cell level for a three-dimensional in vitro model. Head and neck cancer organoids are characterized alone and after treatment with standard therapies, including an antibody therapy, a chemotherapy, and combination therapy. Additionally, organoid cellular heterogeneity is analyzed quantitatively and qualitatively. Gold standard measures of treatment response, including cell proliferation, cell death, and in vivo tumor volume, validate therapeutic efficacy for each treatment group in a parallel study. Results indicate that optical metabolic imaging is sensitive to therapeutic response in organoids after 1 day of treatment (p<0.05) and resolves cell subpopulations with distinct metabolic phenotypes. Ultimately, this platform could provide a sensitive high-throughput assay to streamline the drug discovery process for head and neck cancer.

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“…Images were analyzed using SPCImage (Becker and Hickl), as described previously [28]. Spatial binning included each pixel and the surrounding 8 pixels.…”

Section: Methodsmentioning

confidence: 99%

“…Heterogeneity analysis was performed as described previously [28]. Briefly, per-cell data was plotted as frequency distributions and fit to 1, 2, or 3 Gaussian curves based on the Akaike Information Criterion, where each Gaussian curve represented a cell subpopulation.…”

Section: Methodsmentioning

confidence: 99%

Metabolic Imaging of Head and Neck Cancer Organoids

2017

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“…These are intrinsically fluorescent [2], a phenomenon that has been exploited as a label-free method for monitoring the intracellular redox state of living cells and tissues for more than 60 years [3]. The approach represents a potentially powerful tool for interrogating the state of biochemical pathways in cells and tissues, but interpreting changes in autofluorescence in relation to the activity of metabolic pathways remains a challenge [4], [5], [6].…”

Section: Introductionmentioning

confidence: 99%

Investigating mitochondrial redox state using NADH and NADPH autofluorescence

Blacker

Duchen

2016

Free Radical Biology and Medicine

315

276

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The redox states of the NAD and NADP pyridine nucleotide pools play critical roles in defining the activity of energy producing pathways, in driving oxidative stress and in maintaining antioxidant defences. Broadly speaking, NAD is primarily engaged in regulating energy-producing catabolic processes, whilst NADP may be involved in both antioxidant defence and free radical generation. Defects in the balance of these pathways are associated with numerous diseases, from diabetes and neurodegenerative disease to heart disease and cancer. As such, a method to assess the abundance and redox state of these separate pools in living tissues would provide invaluable insight into the underlying pathophysiology. Experimentally, the intrinsic fluorescence of the reduced forms of both redox cofactors, NADH and NADPH, has been used for this purpose since the mid-twentieth century. In this review, we outline the modern implementation of these techniques for studying mitochondrial redox state in complex tissue preparations. As the fluorescence spectra of NADH and NADPH are indistinguishable, interpreting the signals resulting from their combined fluorescence, often labelled NAD(P)H, can be complex. We therefore discuss recent studies using fluorescence lifetime imaging microscopy (FLIM) which offer the potential to discriminate between the two separate pools. This technique provides increased metabolic information from cellular autofluorescence in biomedical investigations, offering biochemical insights into the changes in time-resolved NAD(P)H fluorescence signals observed in diseased tissues.

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“…NAD(P)H and FAD are autofluorescent metabolic co-enzymes. The ratio of the fluorescence intensities of NAD(P)H to FAD is the "optical redox ratio," which is sensitive to malignancy, tumor aggressiveness, and drug response [12][13][14][15][16][17][18]. In fact, changes in the redox ratio induced by anti-cancer drugs predict in vivo tumor response long before changes in tumor volume can be measured [15].…”

Section: Introductionmentioning

confidence: 99%

Autofluorescence imaging captures heterogeneous drug response differences between 2D and 3D breast cancer cultures

Cannon

Shah

Skala

2017

Biomed. Opt. Express

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Two-photon microscopy of cellular autofluorescence intensity and lifetime (optical metabolic imaging, or OMI) is a promising tool for preclinical drug development. OMI, which exploits the endogenous fluorescence from the metabolic coenzymes NAD(P)H and FAD, is sensitive to changes in cell metabolism produced by drug treatment. Previous studies have shown that drug response, genetic expression, cell-cell communication, and cell signaling in 3D culture match those of the original in vivo tumor, but not those of 2D culture. The goal of this study is to use OMI to quantify dynamic cell-level metabolic differences in drug response in 2D cell lines vs. 3D organoids generated from xenograft tumors of the same cell origin. BT474 cells and Herceptin-resistant BT474 (HR6) cells were tested. Cells were treated with vehicle control, Herceptin, XL147 (PI3K inhibitor), and the combination. The OMI index was used to quantify response, and is a linear combination of the redox ratio (intensity of NAD(P)H divided by FAD), mean NADH lifetime, and mean FAD lifetime. The results confirm that the OMI index resolves significant differences (p<0.05) in drug response for 2D vs. 3D cultures, specifically for BT474 cells 24 hours after Herceptin treatment, for HR6 cells 24 and 72 hours after combination treatment, and for HR6 cells 72 hours after XL147 treatment. Cell-level analysis of the OMI index also reveals differences in the number of cell sub-populations in 2D vs. 3D culture at 24, 48, and 72 hours post-treatment in control and treated groups. Finally, significant increases (p<0.05) in the mean lifetime of NADH and FAD were measured in 2D vs. 3D for both cell lines at 72 hours post-treatment in control and all treatment groups. These whole-population differences in the mean NADH and FAD lifetimes are supported by differences in the number of cell sub-populations in 2D vs. 3D. Overall, these studies confirm that OMI is sensitive to differences in drug response in 2D vs. 3D, and provides further information on dynamic changes in the relative abundance of metabolic cell sub-populations that contribute to this difference.

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In Vivo Autofluorescence Imaging of Tumor Heterogeneity in Response to Treatment

Cited by 88 publications

References 35 publications

Metabolic Imaging of Head and Neck Cancer Organoids

Metabolic Imaging of Head and Neck Cancer Organoids

Investigating mitochondrial redox state using NADH and NADPH autofluorescence

Autofluorescence imaging captures heterogeneous drug response differences between 2D and 3D breast cancer cultures

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