Collagen density regulates tip-stalk cell rearrangement during angiogenesis via cellular bioenergetics.

Tumor vasculature plays a crucial role in tumor progression, affecting nutrition and oxygen transportation as well as the efficiency of drug delivery. While targeting pro-angiogenic growth factors has been a significant focus for treating tumor angiogenesis, recent studies indicate that metabolism also plays a role in regulating endothelial cell behavior. Like cancer cells, tumor endothelial cells undergo metabolic changes that regulate rearrangement for tip cell position during angiogenesis. Our previous studies have shown that altered mechanical properties of the collagen matrix regulate angiogenesis and can promote a tumor vasculature phenotype. Here, we examine the effect of collagen density on endothelial cell tip-stalk cell rearrangement and cellular energetics during angiogenic sprouting. We find that increased collagen density leads to an elevated energy state and an increased rate of tip-stalk cell switching, which is correlated with the energy state of the cells. Tip cells exhibit higher glucose uptake than stalk cells, and inhibition of glucose uptake revealed that invading sprouts rely on glucose to meet elevated energy requirements for invasion in dense matrices. This work helps to elucidate the complex interplay between the mechanical microenvironment and the endothelial cell metabolic status during angiogenesis, which could have important implications for developing new anti-cancer therapies.

Obesity induces PD-1 on macrophages to suppress anti-tumour immunity.

Obesity is a leading risk factor for progression and metastasis of many cancers1,2, yet can in some cases enhance survival3-5 and responses to immune checkpoint blockade therapies, including anti-PD-1, which targets PD-1 (encoded by PDCD1), an inhibitory receptor expressed on immune cells6-8. Although obesity promotes chronic inflammation, the role of the immune system in the obesity-cancer connection and immunotherapy remains unclear. It has been shown that in addition to T cells, macrophages can express PD-19-12. Here we found that obesity selectively induced PD-1 expression on tumour-associated macrophages (TAMs). Type I inflammatory cytokines and molecules linked to obesity, including interferon-γ, tumour necrosis factor, leptin, insulin and palmitate, induced macrophage PD-1 expression in an mTORC1- and glycolysis-dependent manner. PD-1 then provided negative feedback to TAMs that suppressed glycolysis, phagocytosis and T cell stimulatory potential. Conversely, PD-1 blockade increased the level of macrophage glycolysis, which was essential for PD-1 inhibition to augment TAM expression of CD86 and major histocompatibility complex I and II molecules and ability to activate T cells. Myeloid-specific PD-1 deficiency slowed tumour growth, enhanced TAM glycolysis and antigen-presentation capability, and led to increased CD8+ T cell activity with a reduced level of markers of exhaustion. These findings show that obesity-associated metabolic signalling and inflammatory cues cause TAMs to induce PD-1 expression, which then drives a TAM-specific feedback mechanism that impairs tumour immune surveillance. This may contribute to increased cancer risk yet improved response to PD-1 immunotherapy in obesity.

Spatial distribution of tumor-associated macrophages in an orthotopic prostate cancer mouse model.

Mounting evidence suggests that the immune landscape within prostate tumors influences progression, metastasis, treatment response, and patient outcomes. In this study, we investigated the spatial density of innate immune cell populations within NOD.SCID orthotopic prostate cancer xenografts following microinjection of human DU145 prostate cancer cells. Our laboratory has previously developed nanoscale liposomes that attach to leukocytes via conjugated E-selectin (ES) and kill cancer cells via TNF-related apoptosis inducing ligand (TRAIL). Immunohistochemistry (IHC) staining was performed on tumor samples to identify and quantify leukocyte infiltration for different periods of tumor growth and E-selectin/TRAIL (EST) liposome treatments. We examined the spatial-temporal dynamics of three different immune cell types infiltrating tumors using QuPath image analysis software. IHC staining revealed that F4/80+ tumor-associated macrophages (TAMs) were the most abundant immune cells in all groups, irrespective of time or treatment. The density of TAMs decreased over the course of tumor growth and decreased in response to EST liposome treatments. Intratumoral versus marginal analysis showed a greater presence of TAMs in the marginal regions at 3 weeks of tumor growth which became more evenly distributed over time and in tumors treated with EST liposomes. TUNEL staining indicated that EST liposomes significantly increased cell apoptosis in treated tumors. Additionally, confocal microscopy identified liposome-coated TAMs in both the core and periphery of tumors, highlighting the ability of liposomes to infiltrate tumors by "piggybacking" on macrophages. The results of this study indicate that TAMs represent the majority of innate immune cells within NOD.SCID orthotopic prostate tumors, and spatial density varies widely as a function of tumor size, duration of tumor growth, and treatment of EST liposomes.

Mechano-metabolism of adherent cells in 2D and 3D microenvironments.

Cells regulate their shape and metabolic activity in response to the mechano-chemical properties of their microenvironment. To elucidate the impact of matrix stiffness and ligand density on a cell's bioenergetics, we developed a non-equilibrium, active chemo-mechanical model that accounts for mechanical energy of the cell and matrix, chemical energy from ATP hydrolysis, interfacial energy, and mechano-sensitive regulation of stress fiber assembly through signaling. By integrating the kinetics and energetics of these processes we introduce the concept of the metabolic potential of the cell that, when minimized, gives experimentally testable predictions of the cell contractility, shape, and the ATP consumption. Specifically, we show that MDA-MB-231 breast cancer cells in 3D collagen gels follow a spherical to spindle to spherical change in morphology with increasing matrix stiffness consistent with experimental observations. This biphasic transition in cell shape emerges from a competition between increased contractility accompanied by ATP hydrolysis enabled by mechano-sensitive signaling, which lowers the volumetric contribution to the metabolic potential of elongated cells and the interfacial energy which is lower for spherical shapes. On 2D hydrogels, our model predicts a hemispherical to spindle to disc shape transition with increasing gel stiffness. In both cases, we show that increasing matrix stiffness monotonically increases the cell's contractility as well as ATP consumption. Our model also predicts how the increased energy demand in stiffer microenvironments is met by AMPK activation, which is confirmed through experimental measurement of activated AMPK levels as a function of matrix stiffness carried out here in both 2D and 3D micro-environments. Further, model predictions of increased AMPK activation on stiffer micro-environments are found to correlate strongly with experimentally measured upregulation of mitochondrial potential, glucose uptake and ATP levels. The insights from our model can be used to understand mechanosensitive regulation of metabolism in physiological events such as metastasis and tumor progression during which cells experience dynamic changes in their microenvironment and metabolic state.

A call to uncover the availability, structure, and effects of parental leave policies at academic pharmacy institutions.

Parental leave is often an initial barrier to achieving family-career integration, and thus discussing this issue within the broader academic pharmacy community may have important implications for policy development and change. This commentary aims to reveal the implications of inadequate parental leave policies on faculty while highlighting the benefits well-developed policies can have for both parents and their children. Additionally, we put forth a call to action for additional research into the availability and structure of parental leave policies at pharmacy institutions and the effects such policies have on faculty wellbeing, retention, and job satisfaction.

The movement of mitochondria in breast cancer: internal motility and intercellular transfer of mitochondria.

As a major energy source for cells, mitochondria are involved in cell growth and proliferation, as well as migration, cell fate decisions, and many other aspects of cellular function. Once thought to be irreparably defective, mitochondrial function in cancer cells has found renewed interest, from suggested potential clinical biomarkers to mitochondria-targeting therapies. Here, we will focus on the effect of mitochondria movement on breast cancer progression. Mitochondria move both within the cell, such as to localize to areas of high energetic need, and between cells, where cells within the stroma have been shown to donate their mitochondria to breast cancer cells via multiple methods including tunneling nanotubes. The donation of mitochondria has been seen to increase the aggressiveness and chemoresistance of breast cancer cells, which has increased recent efforts to uncover the mechanisms of mitochondrial transfer. As metabolism and energetics are gaining attention as clinical targets, a better understanding of mitochondrial function and implications in cancer are required for developing effective, targeted therapeutics for cancer patients.

Confinement primes cells for faster migration by polarizing active mitochondria.

Mechanical cues in the tumor microenvironment interplay with internal cellular processes to control cancer cell migration. Microscale pores present in tumor tissue confer varying degrees of confinement on migrating cells, increasing matrix contact and inducing cytoskeletal rearrangement. Previously, we observed that increased collagen matrix contact significantly increased cell migration speed and cell-induced strains within the matrix. However, the effects of this confinement on future cell migration are not fully understood. Here, we use a collagen microtrack platform to determine the effect of confinement on priming MDA-MB-231 cancer cells for fast migration. We show that migration through a confined track results in increased speed and accumulation of migratory machinery, including actin and active mitochondria, in the front of migrating breast cancer cells. By designing microtracks that allow cells to first navigate a region of high confinement, then a region of low confinement, we assessed whether migration in high confinement changes future migratory behavior. Indeed, cells maintain their speed attained in high confinement even after exiting to a region of low confinement, indicating that cells maintain memory of previous matrix cues to fuel fast migration. Active mitochondria maintain their location at the front of the cell even after cells leave high confinement. Furthermore, knocking out vinculin to disrupt focal adhesions disrupts active mitochondrial localization and disrupts the fast migration seen upon release from confinement. Together, these data suggest that active mitochondrial localization in confinement may facilitate fast migration post-confinement. By better understanding how confinement contributes to future cancer cell migration, we can identify potential therapeutic targets to inhibit breast cancer metastasis.

Parametric information about eye movements is sent to the ears.

Eye movements alter the relationship between the visual and auditory spatial scenes. Signals related to eye movements affect neural pathways from the ear through auditory cortex and beyond, but how these signals contribute to computing the locations of sounds with respect to the visual scene is poorly understood. Here, we evaluated the information contained in eye movement-related eardrum oscillations (EMREOs), pressure changes recorded in the ear canal that occur in conjunction with simultaneous eye movements. We show that EMREOs contain parametric information about horizontal and vertical eye displacement as well as initial/final eye position with respect to the head. The parametric information in the horizontal and vertical directions can be modeled as combining linearly, allowing accurate prediction of the EMREOs associated with oblique (diagonal) eye movements. Target location can also be inferred from the EMREO signals recorded during eye movements to those targets. We hypothesize that the (currently unknown) mechanism underlying EMREOs could impose a two-dimensional eye-movement-related transfer function on any incoming sound, permitting subsequent processing stages to compute the positions of sounds in relation to the visual scene.

Individual similarities and differences in eye-movement-related eardrum oscillations (EMREOs).

We recently discovered a unique type of otoacoustic emission (OAE) time-locked to the onset (and offset) of saccadic eye movements and occurring in the absence of external sound (Gruters et al., 2018). How and why these eye-movement-related eardrum oscillations (EMREOs) are generated is unknown, with a role in visual-auditory integration being the likeliest candidate. Clues to both the drivers of EMREOs and their purpose can be gleaned by examining responses in normal hearing human subjects. Do EMREOs occur in all individuals with normal hearing? If so, what components of the response occur most consistently? Understanding which attributes of EMREOs are similar across participants and which show more variability will provide the groundwork for future comparisons with individuals with hearing abnormalities affecting the ear's various motor components. Here we report that in subjects with normal hearing thresholds and normal middle ear function, all ears show (a) measurable EMREOs (mean: 58.7 dB SPL; range 45-67 dB SPL for large contralateral saccades), (b) a phase reversal for contra- versus ipsilaterally-directed saccades, (c) a large peak in the signal occurring soon after saccade onset, (d) an additional large peak time-locked to saccade offset and (e) evidence that saccade duration is encoded in the signal. We interpret the attributes of EMREOs that are most consistent across subjects as the ones that are most likely to play an essential role in their function. The individual differences likely reflect normal variation in individuals' auditory system anatomy and physiology, much like traditional measures of auditory function such as auditory-evoked OAEs, tympanometry and auditory-evoked potentials. Future work will compare subjects with different types of auditory dysfunction to population data from normal hearing subjects. Overall, these findings provide important context for the widespread observations of visual- and eye-movement related signals found in cortical and subcortical auditory areas of the brain.

Cancer-associated fibroblasts in early-stage lung adenocarcinoma correlate with tumor aggressiveness.

Lung adenocarcinoma (LUAD) is the predominant type of lung cancer in the U.S. and exhibits a broad variety of behaviors ranging from indolent to aggressive. Identification of the biological determinants of LUAD behavior at early stages can improve existing diagnostic and treatment strategies. Extracellular matrix (ECM) remodeling and cancer-associated fibroblasts play a crucial role in the regulation of cancer aggressiveness and there is a growing need to investigate their role in the determination of LUAD behavior at early stages. We analyzed tissue samples isolated from patients with LUAD at early stages and used imaging-based biomarkers to predict LUAD behavior. Single-cell RNA sequencing and histological assessment showed that aggressive LUADs are characterized by a decreased number of ADH1B+ CAFs in comparison to indolent tumors. ADH1B+ CAF enrichment is associated with distinct ECM and immune cell signatures in early-stage LUADs. Also, we found a positive correlation between the gene expression of ADH1B+ CAF markers in early-stage LUADs and better survival. We performed TCGA dataset analysis to validate our findings. Identified associations can be used for the development of the predictive model of LUAD aggressiveness and novel therapeutic approaches.

Conserved features of eye movement related eardrum oscillations (EMREOs) across humans and monkeys.

Auditory and visual information involve different coordinate systems, with auditory spatial cues anchored to the head and visual spatial cues anchored to the eyes. Information about eye movements is therefore critical for reconciling visual and auditory spatial signals. The recent discovery of eye movement-related eardrum oscillations (EMREOs) suggests that this process could begin as early as the auditory periphery. How this reconciliation might happen remains poorly understood. Because humans and monkeys both have mobile eyes and therefore both must perform this shift of reference frames, comparison of the EMREO across species can provide insights to shared and therefore important parameters of the signal. Here we show that rhesus monkeys, like humans, have a consistent, significant EMREO signal that carries parametric information about eye displacement as well as onset times of eye movements. The dependence of the EMREO on the horizontal displacement of the eye is its most consistent feature, and is shared across behavioural tasks, subjects and species. Differences chiefly involve the waveform frequency (higher in monkeys than in humans) and patterns of individual variation (more prominent in monkeys than in humans), and the waveform of the EMREO when factors due to horizontal and vertical eye displacements were controlled for. This article is part of the theme issue 'Decision and control processes in multisensory perception'.

Phenotypically sorted highly and weakly migratory triple negative breast cancer cells exhibit migratory and metastatic commensalism.

BACKGROUND: Intratumor heterogeneity is a well-established hallmark of cancer that impedes cancer research, diagnosis, and treatment. Previously, we phenotypically sorted human breast cancer cells based on migratory potential. When injected into mice, highly migratory cells were weakly metastatic and weakly migratory cells were highly metastatic. The purpose of this study was to determine whether these weakly and highly migratory cells interact with each other in vitro or in vivo. METHODS: To assess the relationship between heterogeneity in cancer cell migration and metastatic fitness, MDA-MB-231 and SUM159PT triple negative breast cancer cells were phenotypically sorted into highly migratory and weakly migratory subpopulations and assayed separately and in a 1:1 mixture in vitro and in vivo for metastatic behaviors. Unpaired, two-tailed Student's t-tests, Mann-Whitney tests, ordinary, one-way ANOVAs, and Kruskal-Wallis H tests were performed as appropriate with p < 0.05 as the cutoff for statistical significance. RESULTS: When highly and weakly migratory cells are co-seeded in mixed spheroids, the weakly migratory cells migrated farther than weakly migratory only spheroids. In mixed spheroids, leader-follower behavior occurred with highly migratory cells leading the weakly migratory cells in migration strands. When cell suspensions of highly migratory, weakly migratory, or a 1:1 mixture of both subpopulations were injected orthotopically into mice, both the mixed cell suspensions and weakly migratory cells showed significant distal metastasis, but the highly migratory cells did not metastasize significantly to any location. Notably, significantly more distal metastasis was observed in mice injected with the 1:1 mixture compared to either subpopulation alone. CONCLUSIONS: This study suggests that weakly migratory cells interact with highly migratory cells in a commensal fashion resulting in increased migration and metastasis. Together, these findings indicate that cancer cell subpopulation migration ability does not correlate with metastatic potential and that cooperation between highly migratory and weakly migratory subpopulations can enhance overall metastatic fitness.

Cellular mechanosignaling for sensing and transducing matrix rigidity.

The mechanisms by which cells sense their mechanical environment and transduce the signal through focal adhesions and signaling pathways to the nucleus is an area of key focus for the field of mechanobiology. In the past two years, there has been expansion of our knowledge of commonly studied pathways, such as YAP/TAZ, FAK/Src, RhoA/ROCK, and Piezo1 signaling, as well as the discovery of new interactions, such as the effect of matrix rigidity of cell mitochondrial function and metabolism, which represent a new and exciting direction for the field as a whole. This review covers the most recent advances in the field of substrate stiffness sensing as well as perspective on future directions.

Mitochondrial transfer from cancer-associated fibroblasts increases migration in aggressive breast cancer.

Cancer-associated fibroblasts (CAFs) have distinct roles within the tumor microenvironment, which can impact the mode and efficacy of tumor cell migration. CAFs are known to increase invasion of less-aggressive breast cancer cells through matrix remodeling and leader-follower dynamics. Here, we demonstrate that CAFs communicate with breast cancer cells through the formation of contact-dependent tunneling nanotubes (TNTs), which allow for the exchange of cargo between cell types. CAF mitochondria are an integral cargo component and are sufficient to increase the 3D migration of cancer cells. This cargo transfer results in an increase in mitochondrial ATP production in cancer cells, whereas it has a negligible impact on glycolytic ATP production. Manually increasing mitochondrial oxidative phosphorylation (OXPHOS) by providing extra substrates for OXPHOS fails to enhance cancer cell migration unless glycolysis is maintained at a constant level. Together, these data indicate that tumor-stromal cell crosstalk via TNTs and the associated metabolic symbiosis is a finely controlled mechanism by which tumor cells co-opt their microenvironment to promote cancer progression and may become a potential therapeutic target.

Forces exerted and transduced by cancer-associated fibroblasts during cancer progression.

Although it is well-known that cancer-associated fibroblasts (CAFs) play a key role in regulating tumor progression, the effects of mechanical tissue changes on CAFs are understudied. Myofibroblastic CAFs (myCAFs), in particular, are known to alter tumor matrix architecture and composition, heavily influencing the mechanical forces in the tumor microenvironment (TME), but much less is known about how these mechanical changes initiate and maintain the myCAF phenotype. Additionally, recent studies have pointed to the existence of CAFs in circulating tumor cell clusters, indicating that CAFs may be subject to mechanical forces beyond the primary TME. Due to their pivotal role in cancer progression, targeting CAF mechanical regulation may provide therapeutic benefit. Here, we will discuss current knowledge and summarize existing gaps in how CAFs regulate and are regulated by matrix mechanics, including through stiffness, solid and fluid stresses, and fluid shear stress.

Matrix stiffness regulates tumor cell intravasation through expression and ESRP1-mediated alternative splicing of MENA.

During intravasation, cancer cells cross the endothelial barrier and enter the circulation. Extracellular matrix stiffening has been correlated with tumor metastatic potential; however, little is known about the effects of matrix stiffness on intravasation. Here, we utilize in vitro systems, a mouse model, specimens from patients with breast cancer, and RNA expression profiles from The Cancer Genome Atlas Program (TCGA) to investigate the molecular mechanism by which matrix stiffening promotes tumor cell intravasation. Our data show that heightened matrix stiffness increases MENA expression, which promotes contractility and intravasation through focal adhesion kinase activity. Further, matrix stiffening decreases epithelial splicing regulatory protein 1 (ESRP1) expression, which triggers alternative splicing of MENA, decreases the expression of MENA11a, and enhances contractility and intravasation. Altogether, our data indicate that matrix stiffness regulates tumor cell intravasation through enhanced expression and ESRP1-mediated alternative splicing of MENA, providing a mechanism by which matrix stiffness regulates tumor cell intravasation.

Individual similarities and differences in eye-movement-related eardrum oscillations (EMREOs).

We recently discovered a unique type of low-frequency otoacoustic emission (OAE) time-locked to the onset (and offset) of saccadic eye movements and occurring in the absence of external sound (Gruters et al., 2018). How and why these eye-movement-related eardrum oscillations (EMREOs) are generated is unknown, with a role in visual-auditory integration being the likeliest candidate. Clues to both the drivers of EMREOs and their purpose can be gleaned by examining responses in normal hearing human subjects. Do EMREOs occur in all individuals with normal hearing? If so, what components of the response occur most consistently? Understanding which attributes of EMREOs are similar across participants and which show more variability will provide the groundwork for future comparisons with individuals with hearing abnormalities affecting the ear's various motor components. Here we report that in subjects with normal hearing thresholds and normal middle ear function, all ears show (a) measurable EMREOs (mean: 58.7 dB SPL; range 45-67 dB SPL for large contralateral saccades), (b) a phase reversal for contra- versus ipsilaterally-directed saccades, (c) a large peak in the signal occurring soon after saccade onset, (d) an additional large peak time-locked to saccade offset and (e) evidence that saccade duration is encoded in the signal. We interpret the attributes of EMREOs that are most consistent across subjects as the ones that are most likely to play an essential role in their function. The individual differences likely reflect normal variation in individuals' auditory system anatomy and physiology, much like traditional measures of auditory function such as auditory-evoked OAEs, tympanometry and auditory-evoked potentials. Future work will compare subjects with different types of auditory dysfunction to population data from normal hearing subjects. Overall, these findings provide important context for the widespread observations of visual- and eye-movement related signals found in cortical and subcortical auditory areas of the brain.

Conserved features of eye movement related eardrum oscillations (EMREOs) across humans and monkeys.

Auditory and visual information involve different coordinate systems, with auditory spatial cues anchored to the head and visual spatial cues anchored to the eyes. Information about eye movements is therefore critical for reconciling visual and auditory spatial signals. The recent discovery of eye movement-related eardrum oscillations (EMREOs) suggests that this process could begin as early as the auditory periphery. How this reconciliation might happen remains poorly understood. Because humans and monkeys both have mobile eyes and therefore both must perform this shift of reference frames, comparison of the EMREO across species can provide insights to shared and therefore important parameters of the signal. Here we show that rhesus monkeys, like humans, have a consistent, significant EMREO signal that carries parametric information about eye displacement as well as onset times of eye movements. The dependence of the EMREO on the horizontal displacement of the eye is its most consistent feature, and is shared across behavioral tasks, subjects, and species. Differences chiefly involve the waveform frequency (higher in monkeys than in humans) and patterns of individual variation (more prominent in monkeys than humans), and the waveform of the EMREO when factors due to horizontal and vertical eye displacements were controlled for.

Matrix stiffness induces epithelial-to-mesenchymal transition via Piezo1-regulated calcium flux in prostate cancer cells.

Cells utilize calcium channels as one of the main signaling mechanisms to sense changes in the extracellular space and convert these changes to intracellular signals. Calcium regulates several key signaling networks, such as the induction of EMT. The current study expands on the understanding of how EMT is controlled via the mechanosensitive calcium channel Piezo1 in cancerous cells, which senses changes in the extracellular matrix stiffness. We model the biophysical environment of healthy and cancerous prostate tissue using polyacrylamide gels of different stiffnesses. Significant increases in calcium steady-state concentration, vimentin expression, and aspect ratio, and decreases in E-cadherin expression were observed by increasing matrix stiffness and also after treatment with Yoda1, a chemical agonist of Piezo1. Overall, this study concludes that Piezo1-regulated calcium flux plays a role in prostate cancer cell metastatic potential by sensing changes in ECM stiffness and modulating EMT markers.

Analysis of Energy-Driven Leader-Follower Hierarchy During Collective Cancer Cell Invasion.

Many solid tumors can invade the surrounding three-dimensional (3D) tissue in a collective manner, and increasing evidence suggests that collective migration makes cancer cell clusters more invasive and metastatic than individual cells. A cohesive cohort of cancer cells can have many advantages over individual cells, including more efficient bioenergetics that have been recently identified. Minimization of bioenergetic costs during collective cell migration drives leader-follower dynamics and contributes to enhanced cancer invasion. Hence, it is critical to understand the migratory and bioenergetic dynamics of cancer collective invasion. While analysis of structures and dynamics in a 3D space has been a challenging task, here we describe a widely applicable method to analyze the energy-driven leader-follower hierarchy during cancer collective invasion. An in vitro tumor spheroid model is employed to reproduce the in vivo collective behaviors of cancer cells while allowing high spatiotemporal resolution imaging, where the leader-follower dynamics can be analyzed by tracking nuclear positions. As glucose is one of the main energy sources that fuel cancer cell migration, the quantification of glucose uptake along the invading strands provides an estimate of the energy demand associated with collective invasion. Finally, we describe a method to quantify the dynamics of intracellular energy level using the PercevalHR ATP:ADP ratio biosensor.