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.

Microphysiological head and neck cancer model identifies novel role of lymphatically secreted monocyte migration inhibitory factor in cancer cell migration and metabolism.

Regional metastasis of head and neck cancer (HNC) is prevalent (approximately 50% of patients at diagnosis), yet the underlying drivers and mechanisms of lymphatic spread remain unclear. The complex tumor microenvironment (TME) of HNC plays a crucial role in disease maintenance and progression; however, the contribution of the lymphatics remains underexplored. We created a primary patient cell derived microphysiological system that incorporates cancer-associated-fibroblasts from patients with HNC alongside a HNC tumor spheroid and a lymphatic microvessel to create an in vitro TME platform to investigate metastasis. Screening of soluble factor signaling identified novel secretion of macrophage migration inhibitory factor (MIF) by lymphatic endothelial cells conditioned in the TME. Importantly, we also observed patient-to-patient heterogeneity in cancer cell migration similar to the heterogeneity observed in clinical disease. Optical metabolic imaging at the single cell level identified a distinct metabolic profile of migratory versus non-migratory HNC cells in a microenvironment dependent manner. Additionally, we report a unique role of MIF in increasing HNC reliance on glycolysis over oxidative phosphorylation. This multicellular, microfluidic platform expands the tools available to explore HNC biology in vitro through multiple orthogonal outputs and establishes a system with enough resolution to visualize and quantify patient-to-patient heterogeneity.

Touch-free optical technologies to streamline the production of T cell therapies.

Currently approved adoptive T cell therapy relies on autologous (obtained from the same patient) T cells, which often suffer from poor quality that diminishes treatment efficacy. Due to the heterogeneous nature of T cell quality between and within patients, significant efforts are aimed at optimizing cell manipulation and growth conditions for potent T cell products. We believe that touch-free imaging and sensing technologies are critical to monitor single-cell features during T cell manufacturing to ensure consistent and optimally timed methods for cell manipulation and growth. Here, we discuss emerging label-free optical imaging and sensing methods, along with machine learning techniques that could enable in-line feedback to optimize T cell quality at multiple stages during manufacturing. These methods have the potential to streamline current workflow, accelerate the manufacture of safe high-quality T cell therapies, and improve our understanding of the dynamic, heterogeneous processes of T cell manufacturing.

Biodegradable magnesium materials regulate ROS-RNS balance in pro-inflammatory macrophage environment.

The relationship between reactive oxygen and nitrogen species (ROS-RNS) secretion and the concomitant biocorrosion of degradable magnesium (Mg) materials is poorly understood. We found that Mg foils implanted short term in vivo (24 h) displayed large amounts of proinflammatory F4/80+/iNOS + macrophages at the interface. We sought to investigate the interplay between biodegrading Mg materials (98.6% Mg, AZ31 & AZ61) and macrophages (RAW 264.7) stimulated with lipopolysaccharide (RAW 264.7LPS) to induce ROS-RNS secretion. To test how these proinflammatory ROS-RNS secreting cells interact with Mg corrosion in vitro, Mg and AZ61 discs were suspended approximately 2 mm above a monolayer of RAW 264.7 cells, either with or without LPS. The surfaces of both materials showed acute (24 h) changes when incubated in the proinflammatory RAW 264.7LPS environment. Mg discs incubated with RAW 264.7LPS macrophages showed greater corrosion pitting, while AZ61 showed morphological and elemental bulk product changes via scanning electron microscopy-energy dispersive X-ray spectroscopy (SEM-EDX). X-ray photoelectron spectroscopy (XPS) analysis showed a reduction in the Ca/P ratio of the surface products for AZ61 disc incubated with RAW 264.7LPS, but not the Mg discs. Moreover, RAW 264.7LPS macrophages were found to be more viable in the acute biodegradative environment generated by Mg materials, as demonstrated by calcein-AM and cleaved (active) caspase-3 staining (CC3). LPS stimulation caused an increase in ROS-RNS, and a decrease in antioxidant peroxidase activity. Mg and AZ61 were found to change this ROS-RNS balance, independently of physiological antioxidant mechanisms. The findings highlight the complexity of the cellular driven acute inflammatory responses to different biodegradable Mg, and how it can potentially affect performance of these materials.

Prostate cancer cells demonstrate unique metabolism and substrate adaptability acutely after androgen deprivation therapy.

BACKGROUND: Androgen deprivation therapy (ADT) has been the standard of care for advanced hormone-sensitive prostate cancer (PC), yet tumors invariably develop resistance resulting in castrate-resistant PC. The acute response of cancer cells to ADT includes apoptosis and cell death, but a large fraction remains arrested but viable. In this study, we focused on intensively characterizing the early metabolic changes that result after ADT to define potential metabolic targets for treatment. METHODS: A combination of mass spectrometry, optical metabolic imaging which noninvasively measures drug responses in cells, oxygen consumption rate, and protein expression analysis was used to characterize and block metabolic pathways over several days in multiple PC cell lines with variable hormone response status including ADT sensitive lines LNCaP and VCaP, and resistant C4-2 and DU145. RESULTS: Mass spectrometry analysis of LNCaP pre- and postexposure to ADT revealed an abundance of glycolytic intermediates after ADT. In LNCaP and VCaP, a reduction in the optical redox ratio [NAD(P)H/FAD], extracellular acidification rate, and a downregulation of key regulatory enzymes for fatty acid and glutamine utilization was acutely observed after ADT. Screening several metabolic inhibitors revealed that blocking fatty acid oxidation and synthesis reversed this stress response in the optical redox ratio seen with ADT alone in LNCaP and VCaP. In contrast, both cell lines demonstrated increased sensitivity to the glycolytic inhibitor 2-Deoxy- d-glucose(2-DG) and maintained sensitivity to electron transport chain inhibitor Malonate after ADT exposure. ADT followed by 2-DG results in synergistic cell death, a result not seen with simultaneous administration. CONCLUSIONS: Hormone-sensitive PC cells displayed altered metabolic profiles early after ADT including an overall depression in energy metabolism, induction of a quiescent/senescent phenotype, and sensitivity to selected metabolic inhibitors. Glycolytic blocking agents (e.g., 2-DG) as a sequential treatment after ADT may be promising.

Inhibition of B-cell lymphoma 2 family proteins alters optical redox ratio, mitochondrial polarization, and cell energetics independent of cell state.

SIGNIFICANCE: The optical redox ratio (ORR) [autofluorescence intensity of the reduced form of nicotinamide adenine dinucleotide (phosphate) (NAD(P)H)/flavin adenine dinucleotide (FAD)] provides a label-free method to quantify cellular metabolism. However, it is unclear whether changes in the ORR with B-cell lymphoma 2 (Bcl-2) family protein inhibition are due to metabolic stress alone or compromised cell viability. AIM: Determine whether ABT-263 (navitoclax, Bcl-2 family inhibitor) changes the ORR due to changes in mitochondrial function that are independent of changes in cell viability. APPROACH: SW48 colon cancer cells were used to investigate changes in ORR, mitochondrial membrane potential, oxygen consumption rates, and cell state (cell growth, viability, proliferation, apoptosis, autophagy, and senescence) with ABT-263, TAK-228 [sapanisertib, mammalian target of rapamycin complex 1/2 (mTORC 1/2) inhibitor], and their combination at 24 h. RESULTS: Changes in the ORR with Bcl-2 inhibition are driven by increases in both NAD(P)H and FAD autofluorescence, corresponding with increased basal metabolic rate and increased mitochondrial polarization. ABT-263 treatment does not change cell viability or induce autophagy but does induce a senescent phenotype. The metabolic changes seen with ABT-263 treatment are mitigated by combination with mTORC1/2 inhibition. CONCLUSIONS: The ORR is sensitive to increases in mitochondrial polarization, energetic state, and cell senescence, which can change independently from cell viability.

Impact of baseline culture conditions of cancer organoids when determining therapeutic response and tumor heterogeneity.

Representative models are needed to screen new therapies for patients with cancer. Cancer organoids are a leap forward as a culture model that faithfully represents the disease. Mouse-derived cancer organoids (MDCOs) are becoming increasingly popular, however there has yet to be a standardized method to assess therapeutic response and identify subpopulation heterogeneity. There are multiple factors unique to organoid culture that could affect how therapeutic response and MDCO heterogeneity are assessed. Here we describe an analysis of nearly 3500 individual MDCOs where individual organoid morphologic tracking was performed. Change in MDCO diameter was assessed in the presence of control media or targeted therapies. Individual organoid tracking was identified to be more sensitive to treatment response than well-level assessment. The impact of different generations of mice of the same genotype, different regions of the colon, and organoid specific characteristics including baseline size, passage number, plating density, and location within the matrix were examined. Only the starting size of the MDCO altered the subsequent growth. These results were corroborated using ~ 1700 patient-derived cancer organoids (PDCOs) isolated from 19 patients. Here we establish organoid culture parameters for individual organoid morphologic tracking to determine therapeutic response and growth/response heterogeneity for translational studies.

Multimodal assessment of autophagy in mammalian cells with a novel, LC3-based tandem reporter.

Autophagy is an important intracellular pathway for the degradation of superfluous or harmful subcellular materials, thereby playing a critical role in the maintenance of cell health under normal and stress-related conditions. Researchers interrogating autophagic activity in mammalian cell lines often leverage complementary assay technologies to confirm observations. The Autophagy LC3 HiBiT Reporter assay system utilizes a tandem reporter module, HiBiT-HaloTag, fused to a key marker of autophagic activity, LC3B protein, to enable multiple, cell-based assay modalities. This novel autophagy reporter expressed in a single cell line supports (a) a bioluminescent, homogeneous, plate-reader assay for rapid and quantitative assessment of changes in the level of the LC3-based reporter, (b) a fluorescence-based imaging approach to monitor reporter subcellular distribution in live cells, and (c) an antibody-free, protein blotting method to detect the relative amounts of the LC3-I and LC-II forms of the reporter associated with modulation of autophagic flux. Here we detail protocols for all three assay modalities applied to a U2OS human osteosarcoma cell line stably expressing the novel autophagy reporter, enabling the identification of modulators of autophagic activity and subsequent confirmation of mechanism of action.

Autofluorescence Imaging of Treatment Response in Neuroendocrine Tumor Organoids.

Gastroenteropancreatic neuroendocrine tumors (GEP-NET) account for roughly 60% of all neuroendocrine tumors. Low/intermediate grade human GEP-NETs have relatively low proliferation rates that animal models and cell lines fail to recapitulate. Short-term patient-derived cancer organoids (PDCOs) are a 3D model system that holds great promise for recapitulating well-differentiated human GEP-NETs. However, traditional measurements of drug response (i.e., growth, proliferation) are not effective in GEP-NET PDCOs due to the small volume of tissue and low proliferation rates that are characteristic of the disease. Here, we test a label-free, non-destructive optical metabolic imaging (OMI) method to measure drug response in live GEP-NET PDCOs. OMI captures the fluorescence lifetime and intensity of endogenous metabolic cofactors NAD(P)H and FAD. OMI has previously provided accurate predictions of drug response on a single cell level in other cancer types, but this is the first study to apply OMI to GEP-NETs. OMI tested the response to novel drug combination on GEP-NET PDCOs, specifically ABT263 (navitoclax), a Bcl-2 family inhibitor, and everolimus, a standard GEP-NET treatment that inhibits mTOR. Treatment response to ABT263, everolimus, and the combination were tested in GEP-NET PDCO lines derived from seven patients, using two-photon OMI. OMI measured a response to the combination treatment in 5 PDCO lines, at 72 h post-treatment. In one of the non-responsive PDCO lines, heterogeneous response was identified with two distinct subpopulations of cell metabolism. Overall, this work shows that OMI provides single-cell metabolic measurements of drug response in PDCOs to guide drug development for GEP-NET patients.

A bioengineered organotypic prostate model for the study of tumor microenvironment-induced immune cell activation.

The prostate tumor microenvironment (TME) is strongly immunosuppressive; it is largely driven by alteration in cell phenotypes (i.e. tumor-associated macrophages and exhausted cytotoxic T cells) that result in pro-tumorigenic conditions and tumor growth. A greater understanding into how these altered immune cell phenotypes are developed and could potentially be reversed would provide important insights into improved treatment efficacy for prostate cancer. Here, we report a microfluidic model of the prostate TME that mimics prostate ducts across various stages of prostate cancer progression, with associated stroma and immune cells. Using this platform, we exposed immune cells to a benign prostate TME or a metastatic prostate TME and investigated their metabolism, gene and cytokine expression. Immune cells exposed to the metastatic TME showed metabolic differences with a higher redox ratio indicating a switch to a more glycolytic metabolic profile. These cells also increased expression of pro-tumor response cytokines that have been shown to increase cell migration and angiogenesis such as Interleukin-1 (IL-1) a and Granulocyte-macrophage colony-stimulating factor (GM-CSF). Lastly, we observed decreased TLR, STAT signaling and TRAIL expression, suggesting that phenotypes derived from exposure to the metastatic TME could have an impaired anti-tumor response. This platform could provide a valuable tool for studying immune cell phenotypes in in vitro tumor microenvironments.

Fluorescence lifetime imaging microscopy: fundamentals and advances in instrumentation, analysis, and applications.

SIGNIFICANCE: Fluorescence lifetime imaging microscopy (FLIM) is a powerful technique to distinguish the unique molecular environment of fluorophores. FLIM measures the time a fluorophore remains in an excited state before emitting a photon, and detects molecular variations of fluorophores that are not apparent with spectral techniques alone. FLIM is sensitive to multiple biomedical processes including disease progression and drug efficacy. AIM: We provide an overview of FLIM principles, instrumentation, and analysis while highlighting the latest developments and biological applications. APPROACH: This review covers FLIM principles and theory, including advantages over intensity-based fluorescence measurements. Fundamentals of FLIM instrumentation in time- and frequency-domains are summarized, along with recent developments. Image segmentation and analysis strategies that quantify spatial and molecular features of cellular heterogeneity are reviewed. Finally, representative applications are provided including high-resolution FLIM of cell- and organelle-level molecular changes, use of exogenous and endogenous fluorophores, and imaging protein-protein interactions with Förster resonance energy transfer (FRET). Advantages and limitations of FLIM are also discussed. CONCLUSIONS: FLIM is advantageous for probing molecular environments of fluorophores to inform on fluorophore behavior that cannot be elucidated with intensity measurements alone. Development of FLIM technologies, analysis, and applications will further advance biological research and clinical assessments.

Patient-Derived Cancer Organoid Cultures to Predict Sensitivity to Chemotherapy and Radiation.

PURPOSE: Cancer treatment is limited by inaccurate predictors of patient-specific therapeutic response. Therefore, some patients are exposed to unnecessary side effects and delays in starting effective therapy. A clinical tool that predicts treatment sensitivity for individual patients is needed. EXPERIMENTAL DESIGN: Patient-derived cancer organoids were derived across multiple histologies. The histologic characteristics, mutation profile, clonal structure, and response to chemotherapy and radiation were assessed using bright-field and optical metabolic imaging on spheroid and single-cell levels, respectively. RESULTS: We demonstrate that patient-derived cancer organoids represent the cancers from which they were derived, including key histologic and molecular features. These cultures were generated from numerous cancers, various biopsy sample types, and in different clinical settings. Next-generation sequencing reveals the presence of subclonal populations within the organoid cultures. These cultures allow for the detection of clonal heterogeneity with a greater sensitivity than bulk tumor sequencing. Optical metabolic imaging of these organoids provides cell-level quantification of treatment response and tumor heterogeneity allowing for resolution of therapeutic differences between patient samples. Using this technology, we prospectively predict treatment response for a patient with metastatic colorectal cancer. CONCLUSIONS: These studies add to the literature demonstrating feasibility to grow clinical patient-derived organotypic cultures for treatment effectiveness testing. Together, these culture methods and response assessment techniques hold great promise to predict treatment sensitivity for patients with cancer undergoing chemotherapy and/or radiation.

Protein-bound NAD(P)H Lifetime is Sensitive to Multiple Fates of Glucose Carbon.

While NAD(P)H fluorescence lifetime imaging (FLIM) can detect changes in flux through the TCA cycle and electron transport chain (ETC), it remains unclear whether NAD(P)H FLIM is sensitive to other potential fates of glucose. Glucose carbon can be diverted from mitochondria by the pentose phosphate pathway (via glucose 6-phosphate dehydrogenase, G6PDH), lactate production (via lactate dehydrogenase, LDH), and rejection of carbon from the TCA cycle (via pyruvate dehydrogenase kinase, PDK), all of which can be upregulated in cancer cells. Here, we demonstrate that multiphoton NAD(P)H FLIM can be used to quantify the relative concentrations of recombinant LDH and malate dehydrogenase (MDH) in solution. In multiple epithelial cell lines, NAD(P)H FLIM was also sensitive to inhibition of LDH and PDK, as well as the directionality of LDH in cells forced to use pyruvate versus lactate as fuel sources. Among the parameters measurable by FLIM, only the lifetime of protein-bound NAD(P)H (τ2) was sensitive to these changes, in contrast to the optical redox ratio, mean NAD(P)H lifetime, free NAD(P)H lifetime, or the relative amount of free and protein-bound NAD(P)H. NAD(P)H τ2 offers the ability to non-invasively quantify diversions of carbon away from the TCA cycle/ETC, which may support mechanisms of drug resistance.

Corrosion Characteristics Dictate the Long-Term Inflammatory Profile of Degradable Zinc Arterial Implants.

There has been considerable recent interest to develop a feasible bioresorbable stent (BRS) metal. Although zinc and its alloys have many potential advantages, the inflammatory response has not been carefully examined. Using a modified wire implantation model, we characterize the inflammatory response elicited by zinc at high purity (4N) [99.99%], special high grade (SHG)[∼99.7%], and alloyed with 1 wt % (Zn-1Al), 3% (Zn-3Al), and 5.5% (Zn-5Al) aluminum. We found that inflammatory cells were able to penetrate the thick and porous corrosion layer that quickly formed around SHG, Zn-1Al, Zn-3Al, and Zn-5Al implants. In contrast, a delayed entrance of inflammatory cells into the corrosion layer around 4N zinc due to a significantly lower corrosion rate was associated with greater fibrous encapsulation, appearance of necrotic regions, and increased macrophage labeling. Interestingly, cell viability at the interface decreased from SHG, to Zn-1Al, and then Zn-3Al, a trend associated with an increased CD68 and CD11b labeling and capsule thickness. Potentially, the shift to intergranular corrosion due to the aluminum addition increased the activity of macrophages. We conclude that the ability of macrophages to penetrate and remain viable within the corrosion layer may be of fundamental importance for eliciting biocompatible inflammatory responses around corrodible metals.

Fibrosis worsens chronic lymphedema in rodent tissues.

Secondary lymphedema in humans is a common consequence of lymph node dissection (LND) to treat breast cancer. A peculiar characteristic of the disease is that lifelong swelling often precipitously appears several years after the surgical treatment, often due to an inflammatory stimulus. Although the incidence of secondary lymphedema dramatically increases after radiation therapy, the relationship between fibrotic scarring and the eventual appearance of lymphedema remains unclear. To clarify the role of fibrosis in secondary lymphedema initiation, we chemically increased fibrosis in rodent tissues with bleomycin and assessed the ability of the local lymphatic system to prevent lymphedema, either acutely or in a chronic state induced by inflammation. We found that bleomycin injections exacerbated fibrotic matrix deposition in an acute mouse tail lymphedema model (P < 0.005), reduced wound closure (P < 0.005), and impaired the ability of tail lymphatics to regenerate (P < 0.005) and reduce the swelling (P < 0.05). When fibrosis was worsened with bleomycin after axillary LND in the rat foreleg, the ability of the foreleg lymphatic system to reduce the chronic state swelling induced by stimulated inflammation was severely impaired (P < 0.005). Indocyanine green lymphography in axillary LND-recovered rat forelegs revealed a worsened lymphatic drainage due to inflammation and bleomycin pretreatment. Although inflammation reduced the drainage of dextran fluid tracer from control forelegs (P < 0.05), the reduction in fluid drainage was more severe after axillary LND when fibrosis was first increased (P < 0.005). These findings demonstrate that fibrosis reduces the lymphatic capacity to functionally regenerate and prevent the chronic appearance of lymphedema.

Shaping the microenvironment: evidence for the influence of a host galaxin on symbiont acquisition and maintenance in the squid-Vibrio symbiosis.

Most bacterial species make transitions between habitats, such as switching from free living to symbiotic niches. We provide evidence that a galaxin protein, EsGal1, of the squid Euprymna scolopes participates in both: (i) selection of the specific partner Vibrio fischeri from the bacterioplankton during symbiosis onset and, (ii) modulation of V. fischeri growth in symbiotic maintenance. We identified two galaxins in transcriptomic databases and showed by quantitative reverse-transcriptase polymerase chain reaction that one (esgal1) was dominant in the light organ. Further, esgal1 expression was upregulated by symbiosis, a response that was partially achieved with exposure to symbiont cell-envelope molecules. Confocal immunocytochemistry of juvenile animals localized EsGal1 to the apical surfaces of light-organ epithelia and surrounding mucus, the environment in which V. fischeri cells aggregate before migration into the organ. Growth assays revealed that one repeat of EsGal1 arrested growth of Gram-positive bacterial cells, which represent the cell type first 'winnowed' during initial selection of the symbiont. The EsGal1-derived peptide also significantly decreased the growth rate of V. fischeri in culture. Further, when animals were exposed to an anti-EsGal1 antibody, symbiont population growth was significantly increased. These data provide a window into how hosts select symbionts from a rich environment and govern their growth in symbiosis.

The secret languages of coevolved symbioses: insights from the Euprymna scolopes-Vibrio fischeri symbiosis.

Recent research on a wide variety of systems has demonstrated that animals generally coevolve with their microbial symbionts. Although such relationships are most often established anew each generation, the partners associate with fidelity, i.e., they form exclusive alliances within the context of rich communities of non-symbiotic environmental microbes. The mechanisms by which this exclusivity is achieved and maintained remain largely unknown. Studies of the model symbiosis between the Hawaiian squid Euprymna scolopes and the marine luminous bacterium Vibrio fischeri provide evidence that the interplay between evolutionarily conserved features of the innate immune system, most notably MAMP/PRR interactions, and a specific feature of this association, i.e., luminescence, are critical for development and maintenance of this association. As such, in this partnership and perhaps others, symbiotic exclusivity is mediated by the synergism between a general animal-microbe 'language' and a 'secret language' that is decipherable only by the specific partners involved.