Perspectives on label-free microscopy of heterogeneous and dynamic biological systems.

Significance: Advancements in label-free microscopy could provide real-time, non-invasive imaging with unique sources of contrast and automated standardized analysis to characterize heterogeneous and dynamic biological processes. These tools would overcome challenges with widely used methods that are destructive (e.g., histology, flow cytometry) or lack cellular resolution (e.g., plate-based assays, whole animal bioluminescence imaging). Aim: This perspective aims to (1) justify the need for label-free microscopy to track heterogeneous cellular functions over time and space within unperturbed systems and (2) recommend improvements regarding instrumentation, image analysis, and image interpretation to address these needs. Approach: Three key research areas (cancer research, autoimmune disease, and tissue and cell engineering) are considered to support the need for label-free microscopy to characterize heterogeneity and dynamics within biological systems. Based on the strengths (e.g., multiple sources of molecular contrast, non-invasive monitoring) and weaknesses (e.g., imaging depth, image interpretation) of several label-free microscopy modalities, improvements for future imaging systems are recommended. Conclusion: Improvements in instrumentation including strategies that increase resolution and imaging speed, standardization and centralization of image analysis tools, and robust data validation and interpretation will expand the applications of label-free microscopy to study heterogeneous and dynamic biological systems.

Naive primary neutrophils play a dual role in the tumor microenvironment.

The tumor microenvironment (TME) is characterized by a network of cancer cells, recruited immune cells and extracellular matrix (ECM) in a hypoxic microenvironment. However, the specific role of neutrophils during tumor development, and their interactions with other immune cells is still not well understood. Thus, there is a need to investigate the interaction between primary neutrophils and natural killer cells and the resulting effects on tumor development. Here we use both standard well plate culture and an under oil microfluidic (UOM) assay with an integrated extracellular cell matrix (ECM) bridge to elucidate how naive primary neutrophils respond to both patient derived tumor cells and tumor cell lines. Our data demonstrated that both patient derived head and neck squamous cell carcinoma (HNSCC) tumor cells and MDA-MB-231 breast cancer cells trigger cluster formation in neutrophils, and the swarm of neutrophils restricts tumor invasion through the generation of reactive oxygen species (ROS) and neutrophil extracellular trap (NETs) release within the neutrophil cluster. However, we also observed that the presence of neutrophils downregulates granzyme B in NK-92 cells and the resulting NETs can obstruct NK cells from penetrating the tumor mass in vitro suggesting a dual role for neutrophils in the TME. Further, using label-free optical metabolic imaging (OMI) we observed changes in the metabolic activities of primary neutrophils during the different swarming phases when challenged with tumor cells. Finally, our data demonstrates that neutrophils in direct contact, or in close proximity, with tumor cells exhibit greater metabolic activities (lower nicotinamide adenine dinucleotide phosphate (NAD(P)H) mean lifetime) compared to non-contact neutrophils.

Interactions with stromal cells promote a more oxidized cancer cell redox state in pancreatic tumors.

Access to electron acceptors supports oxidized biomass synthesis and can be limiting for cancer cell proliferation, but how cancer cells overcome this limitation in tumors is incompletely understood. Nontransformed cells in tumors can help cancer cells overcome metabolic limitations, particularly in pancreatic cancer, where pancreatic stellate cells (PSCs) promote cancer cell proliferation and tumor growth. However, whether PSCs affect the redox state of cancer cells is not known. By taking advantage of the endogenous fluorescence properties of reduced nicotinamide adenine dinucleotide and oxidized flavin adenine dinucleotide cofactors we use optical imaging to assess the redox state of pancreatic cancer cells and PSCs and find that direct interactions between PSCs and cancer cells promote a more oxidized state in cancer cells. This suggests that metabolic interaction between cancer cells and PSCs is a mechanism to overcome the redox limitations of cell proliferation in pancreatic cancer.

Metabolic mapping of glioblastoma stem cells reveals NADH fluxes associated with glioblastoma phenotype and survival.

SIGNIFICANCE: Glioblastoma multiforme (GBM) is the most frequently diagnosed adult primary brain malignancy with poor patient prognosis. GBM can recur despite aggressive treatment due to therapeutically resistant glioblastoma stem cells (GSCs) that may exhibit metabolic plasticity. AIM: Intrinsic nicotinamide adenine dinucleotide (NADH) fluorescence can be acquired with fluorescence lifetime imaging microscopy (FLIM) to examine its bound and free metabolic states in GSC and GBM tissues. APPROACH: We compared the mean NADH fluorescence lifetime in live human GSCs and normal neural stem cells and validated those results by measuring oxygen consumption rates (OCRs). We also examined the role that invasive versus less-invasive GSCs had on tumor metabolism by measuring the mean NADH lifetimes and the relative amount of the longer-lived component of NADH and correlated these results with survival in an orthotopic mouse xenograft model. RESULTS: Mean NADH lifetime, amount of bound NADH, and OCR were increased in GSCs. Compared with normal mouse brain, mean NADH lifetimes were longer for all GBM tissues. Invasive xenografts had higher relative amounts of the longer-lived NADH component, and this correlated with decreased survival. CONCLUSIONS: FLIM offers cellular resolution quantification of metabolic flux in GBM phenotypes, potentially informing biomedical researchers on improved therapeutic approaches.

3D microtumors in vitro supported by perfused vascular networks.

There is a growing interest in developing microphysiological systems that can be used to model both normal and pathological human organs in vitro. This "organs-on-chips" approach aims to capture key structural and physiological characteristics of the target tissue. Here we describe in vitro vascularized microtumors (VMTs). This "tumor-on-a-chip" platform incorporates human tumor and stromal cells that grow in a 3D extracellular matrix and that depend for survival on nutrient delivery through living, perfused microvessels. Both colorectal and breast cancer cells grow vigorously in the platform and respond to standard-of-care therapies, showing reduced growth and/or regression. Vascular-targeting agents with different mechanisms of action can also be distinguished, and we find that drugs targeting only VEGFRs (Apatinib and Vandetanib) are not effective, whereas drugs that target VEGFRs, PDGFR and Tie2 (Linifanib and Cabozantinib) do regress the vasculature. Tumors in the VMT show strong metabolic heterogeneity when imaged using NADH Fluorescent Lifetime Imaging Microscopy and, compared to their surrounding stroma, many show a higher free/bound NADH ratio consistent with their known preference for aerobic glycolysis. The VMT platform provides a unique model for studying vascularized solid tumors in vitro.

Metabolic changes associated with methionine stress sensitivity in MDA-MB-468 breast cancer cells.

BACKGROUND: The majority of cancer cells have a unique metabolic requirement for methionine that is not observed in normal, non-tumorigenic cells. This phenotype is described as "methionine dependence" or "methionine stress sensitivity" in which cancer cells are unable to proliferate when methionine has been replaced with its metabolic precursor, homocysteine, in cell culture growth media. We focus on the metabolic response to methionine stress in the triple negative breast cancer cell line MDA-MB-468 and its methionine insensitive derivative cell line MDA-MB-468res-R8. RESULTS: Using a variety of techniques including fluorescence lifetime imaging microscopy (FLIM) and extracellular flux assays, we identified a metabolic down-regulation of oxidative phosphorylation in both MDA-MB-468 and MDA-MB-468res-R8 cell types when cultured in homocysteine media. Untargeted metabolomics was performed by way of gas chromatography/time-of-flight mass spectrometry on both cell types cultured in homocysteine media over a period of 2 to 24 h. We determined unique metabolic responses between the two cell lines in specific pathways including methionine salvage, purine/pyrimidine synthesis, and the tricarboxylic acid cycle. Stable isotope tracer studies using deuterium-labeled homocysteine indicated a redirection of homocysteine metabolism toward the transsulfuration pathway and glutathione synthesis. This data corroborates with increased glutathione levels concomitant with increased levels of oxidized glutathione. Redirection of homocysteine flux resulted in reduced generation of methionine from homocysteine particularly in MDA-MB-468 cells. Consequently, synthesis of the important one-carbon donor S-adenosylmethionine (SAM) was decreased, perturbing the SAM to S-adenosylhomocysteine ratio in MDA-MB-468 cells, which is an indicator of the cellular methylation potential. CONCLUSION: This study indicates a differential metabolic response between the methionine sensitive MDA-MB-468 cells and the methionine insensitive derivative cell line MDA-MB-468res-R8. Both cell lines appear to experience oxidative stress when methionine was replaced with its metabolic precursor homocysteine, forcing cells to redirect homocysteine metabolism toward the transsulfuration pathway to increase glutathione synthesis. The methionine stress resistant MDA-MB-468res-R8 cells responded to this cellular stress earlier than the methionine stress sensitive MDA-MB468 cells and coped better with metabolic demands. Additionally, it is evident that S-adenosylmethionine metabolism is dependent on methionine availability in cancer cells, which cannot be sufficiently supplied by homocysteine metabolism under these conditions.

Label-free identification of macrophage phenotype by fluorescence lifetime imaging microscopy.

Macrophages adopt a variety of phenotypes that are a reflection of the many functions they perform as part of the immune system. In particular, metabolism is a phenotypic trait that differs between classically activated, proinflammatory macrophages, and alternatively activated, prohealing macrophages. Inflammatory macrophages have a metabolism based on glycolysis while alternatively activated macrophages generally rely on oxidative phosphorylation to generate chemical energy. We employ this shift in metabolism as an endogenous marker to identify the phenotype of individual macrophages via live-cell fluorescence lifetime imaging microscopy (FLIM). We demonstrate that polarized macrophages can be readily discriminated with the aid of a phasor approach to FLIM, which provides a fast and model-free method for analyzing fluorescence lifetime images.

Fluorescence lifetime imaging of endogenous biomarker of oxidative stress.

Presence of reactive oxygen species (ROS) in excess of normal physiological level results in oxidative stress. This can lead to a range of pathological conditions including inflammation, diabetes mellitus, cancer, cardiovascular and neurodegenerative disease. Biomarkers of oxidative stress play an important role in understanding the pathogenesis and treatment of these diseases. A number of fluorescent biomarkers exist. However, a non-invasive and label-free identification technique would be advantageous for in vivo measurements. In this work we establish a spectroscopic method to identify oxidative stress in cells and tissues by fluorescence lifetime imaging (FLIM). We identified an autofluorescent, endogenous species with a characteristic fluorescent lifetime distribution as a probe for oxidative stress. To corroborate our hypothesis that these species are products of lipid oxidation by ROS, we correlate the spectroscopic signals arising from lipid droplets by combining FLIM with THG and CARS microscopy which are established techniques for selective lipid body imaging. Further, we performed spontaneous Raman spectral analysis at single points of the sample which provided molecular vibration information characteristics of lipid droplets.