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1. Autofluorescence imaging of endogenous metabolic cofactors in response to cytokine stimulation of classically activated macrophages

   https://doi.org/10.1186/s40170-023-00325-z  

   Bess, Shelby N ; Igoe, Matthew J ; Denison, Abby C ; Muldoon, Timothy J ( December 2023 , Cancer & Metabolism)

   Macrophages are one of the most prevalent subsets of immune cells within the tumor microenvironment and perform a range of functions depending on the cytokines and chemokines released by surrounding cells and tissues. Recent research has revealed that macrophages can exhibit a spectrum of phenotypes, making them highly plastic due to their ability to alter their physiology in response to environmental cues. Recent advances in examining heterogeneous macrophage populations include optical metabolic imaging, such as fluorescence lifetime imaging (FLIM), and multiphoton microscopy. However, the method of detection for these systems is reliant upon the coenzymes NAD(P)H and FAD, which can be affected by factors other than cytoplasmic metabolic changes. In this study, we seek to validate these optical measures of metabolism by comparing optical results to more standard methods of evaluating cellular metabolism, such as extracellular flux assays and the presence of metabolic intermediates.

   Here, we used autofluorescence imaging of endogenous metabolic co-factors via multiphoton microscopy and FLIM in conjunction with oxygen consumption rate and extracellular acidification rate through Seahorse extracellular flux assays to detect changes in cellular metabolism in quiescent and classically activated macrophages in response to cytokine stimulation.

   Based on our Seahorse XFP flux analysis, M0 and M1 macrophages exhibit comparable trends in oxygen consumption rate (OCR) and extracellular acidification rate (ECAR). Autofluorescence imaging of M0 and M1 macrophages was not only able to show acute changes in the optical redox ratio from pre-differentiation (0 hours) to 72 hours post-cytokine differentiation (M0: 0.320 to 0.258 and M1: 0.316 to 0.386), mean NADH lifetime (M0: 1.272 ns to 1.379 ns and M1: 1.265 ns to 1.206 ns), and A1/A2 ratio (M0: 3.452 to ~ 4 and M1: 3.537 to 4.529) but could also detect heterogeneity within each macrophage population.

   Overall, the findings of this study suggest that autofluorescence metabolic imaging could be a reliable technique for longitudinal tracking of immune cell metabolism during activation post-cytokine stimulation.

    

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2. Fluorescence Lifetime Imaging Microscopy (FLIM) reveals spatial-metabolic changes in 3D breast cancer spheroids

   https://doi.org/10.1038/s41598-023-30403-7  

   Karrobi, Kavon ; Tank, Anup ; Fuzail, Mohammad Ahsan ; Kalidoss, Madhumathi ; Tilbury, Karissa ; Zaman, Muhammad ; Ferruzzi, Jacopo ; Roblyer, Darren ( March 2023 , Scientific Reports)

   Cancer cells are mechanically sensitive to physical properties of the microenvironment, which can affect downstream signaling to promote malignancy, in part through the modulation of metabolic pathways. Fluorescence Lifetime Imaging Microscopy (FLIM) can be used to measure the fluorescence lifetime of endogenous fluorophores, such as the metabolic co-factors NAD(P)H and FAD, in live samples. We used multiphoton FLIM to investigate the changes in cellular metabolism of 3D breast spheroids derived from MCF-10A and MD-MB-231 cell lines embedded in collagen with varying densities (1 vs. 4 mg/ml) over time (Day 0 vs. Day 3). MCF-10A spheroids demonstrated spatial gradients, with the cells closest to the spheroid edge exhibiting FLIM changes consistent with a shift towards oxidative phosphorylation (OXPHOS) while the spheroid core had changes consistent with a shift towards glycolysis. The MDA-MB-231 spheroids had a large shift consistent with increased OXPHOS with a more pronounced change at the higher collagen concentration. The MDA-MB-231 spheroids invaded into the collagen gel over time and cells that traveled the farthest had the largest changes consistent with a shift towards OXPHOS. Overall, these results suggest that the cells in contact with the extracellular matrix (ECM) and those that migrated the farthest had changes consistent with a metabolic shift towards OXPHOS. More generally, these results demonstrate the ability of multiphoton FLIM to characterize how spheroids metabolism and spatial metabolic gradients are modified by physical properties of the 3D ECM.

    

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3. Fibroblasts Promote Proliferation and Matrix Invasion of Breast Cancer Cells in Co‐Culture Models

   https://doi.org/10.1002/adtp.201900121  

   Plaster, Madison ; Singh, Sunil ; Tavana, Hossein ( September 2019 , Advanced Therapeutics)

   Fibroblasts are an abundant cell type in tumor microenvironments. Activated fibroblasts, known as carcinoma‐associated fibroblasts (CAFs), interact with cancer cells through biochemical signaling and render cancer cells proliferative, invasive, and resistant to therapeutics. Targeting CAFs–cancer cells interactions offers a strategy to block cancer progression. 2D and 3D co‐cultures of human mammary fibroblasts and triple negative breast cancer (TNBC) cells are used to investigate the impact of heterotypic cellular interactions on the proliferation of matrix invasion of TNBC cells. The results show that fibroblasts secreting a chemokine, CXCL12, significantly enhance proliferation of TNBC cells expressing the chemokine receptor, CXCR4. Disrupting this interaction with a receptor antagonist normalizes cancer cell proliferation to that of a co‐culture model lacking this signaling. When co‐culture spheroids are embedded in collagen, fibroblasts producing CXCL12 promote collagen invasion of TNBC cells. Although co‐cultures containing normal fibroblasts also lead to TNBC cell spreading into the matrix, a morphological analysis of cells and inhibition of chemokine‐receptor signaling shows that this spreading is due to the incompatibility of fibroblasts and cancer cells leading to the segregation of the two cell types from the spheroid.

    

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4. Activation of Wnt/β‐catenin signalling and HIF1α stabilisation alters pluripotency and differentiation/proliferation properties of human‐induced pluripotent stem cells

   https://doi.org/10.1111/boc.202000055  

   Wei, Hua ; Beeson, Gyda C. ; Ye, Zhiwei ; Zhang, Jie ; Yao, Hai ; Damon, Brooke ; Morad, Martin ( December 2020 , Biology of the Cell)

   Wnt/β‐catenin signalling, in the microenvironment of pluripotent stem cells (PSCs), plays a critical role in their differentiation and proliferation. Contradictory reports on the role of Wnt/β‐catenin signalling in PSCs self‐renewal and differentiation, however, render these mechanisms largely unclear.

   Wnt/β‐catenin signalling pathway in human‐induced pluripotent stem cells (hiPSCs) was activated by inhibiting glycogen synthase kinase 3 (GSK3), driving the cells into a mesodermal/mesenchymal state, exhibiting proliferative, invasive and anchorage‐independent growth properties, where over 70% of cell population became CD 44 (+)/CD133 (+). Wnt/β‐catenin signalling activation also altered the metabolic state of hiPSCs from aerobic glycolysis to oxidative metabolism and changed their drug and oxidative stress sensitivities. These effects of GSK3 inhibition were suppressed in HIF1α‐stabilised cells.

   Persistent activation of Wnt/β‐catenin signalling endows hiPSCs with proliferative/invasive ‘teratoma‐like’ states, shifting their metabolic dependence and allowing HIF1α‐stabilisation to inhibit their proliferative/invasive properties.

   The hiPSC potential to differentiate into ‘teratoma‐like’ cells suggest that stem cells may exist in two states with differential metabolic and drug dependency.

    

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5. Fluorescence lifetime shifts of NAD(P)H during apoptosis measured by time‐resolved flow cytometry

   https://doi.org/10.1002/cyto.a.23606  

   Alturkistany, Faisal ; Nichani, Kapil ; Houston, Kevin D. ; Houston, Jessica P. ( October 2018 , Cytometry Part A)

   Autofluorescence from the intracellular metabolite, NAD(P)H, is a biomarker that is widely used and known to reliably screen and report metabolic activity as well as metabolic fluctuations within cells. As a ubiquitous endogenous fluorophore, NAD(P)H has a unique rate of fluorescence decay that is altered when bound to coenzymes. In this work we measure the shift in the fluorescence decay, or average fluorescence lifetime (1–3 ns), of NAD(P)H and correlate this shift to changes in metabolism that cells undergo during apoptosis. Our measurements are made with a flow cytometer designed specifically for fluorescence lifetime acquisition within the ultraviolet to violet spectrum. Our methods involved culture, treatment, and preparation of cells for cytometry and microscopy measurements. The evaluation we performed included observations and quantification of the changes in endogenous emission owing to the induction of apoptosis as well as changes in the decay kinetics of the emission measured by flow cytometry. Shifts in NAD(P)H fluorescence lifetime were observed as early as 15 min post‐treatment with an apoptosis inducing agent. Results also include a phasor analysis to evaluate free to bound ratios of NAD(P)H at different time points. We defined the free to bound ratios as the ratio of ‘short‐to‐long’ (S/L) fluorescence lifetime, where S/L was found to consistently decrease with an increase in apoptosis. With a quantitative framework such as phasor analysis, the short and long lifetime components of NAD(P)H can be used to map the cycling of free and bound NAD(P)H during the early‐to‐late stages of apoptosis. The combination of lifetime screening and phasor analyses provides the first step in high throughput metabolic profiling of single cells and can be leveraged for screening and sorting for a range of applications in biomedicine. © 2018 The Authors. Cytometry Part A published by Wiley Periodicals, Inc. on behalf of International Society for Advancement of Cytometry.

    

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