Macrophage mediated mesoscale brain mechanical homeostasis mechanically imaged via optical tweezers and Brillouin microscopy in vivo.

Tissues are active materials where epithelial turnover, immune surveillance, and remodeling of stromal cells such as macrophages all regulate form and function. Scattering modalities such as Brillouin microscopy (BM) can non-invasively access mechanical signatures at GHz. However, our traditional understanding of tissue material properties is derived mainly from modalities which probe mechanical properties at different frequencies. Thus, reconciling measurements amongst these modalities remains an active area. Here, we compare optical tweezer active microrheology (OT-AMR) and Brillouin microscopy (BM) to longitudinally map brain development in the larval zebrafish. We determine that each measurement is able to detect a mechanical signature linked to functional units of the brain. We demonstrate that the corrected BM-Longitudinal modulus using a density factor correlates well with OT-AMR storage modulus at lower frequencies. We also show that the brain tissue mechanical properties are dependent on both the neuronal architecture and the presence of macrophages. Moreover, the BM technique is able to delineate the contributions to mechanical properties of the macrophage from that due to colony stimulating factor 1 receptor (CSF1R) mediated stromal remodeling. Here, our data suggest that macrophage remodeling is instrumental in the maintenance of tissue mechanical homeostasis during development. Moreover, the strong agreement between the OT-AM and BM further demonstrates that scattering-based technique is sensitive to both large and minute structural modification in vivo.

YAP localization mediates mechanical adaptation of human cancer cells during extravasation in vivo.

Biophysical profiling of primary tumors has revealed that individual tumor cells fall along a highly heterogeneous continuum of mechanical phenotypes. One idea is that a subset of tumor cells is "softer" to facilitate detachment and escape from the primary site, a step required to initiate metastasis. However, it has also been postulated that cells must be able to deform and generate sufficient force to exit into distant sites. Here, we aimed to dissect the mechanical changes that occur during extravasation and organ colonization. Using multiplexed methods of intravital microscopy and optical tweezer based active microrheology, we obtained longitudinal images and mechanical profiles of cells during organ colonization in vivo. We determined that cells were softer, more liquid like upon exit of the vasculature but stiffened and became more solid like once in the new organ microenvironment. We also determined that a YAP mediated mechanogenotype influenced the global dissemination in our in vivo and in vitro models and that reducing mechanical heterogeneity could reduce extravasation. Moreover, our high throughput analysis of mechanical phenotypes of patient samples revealed that this mechanics was in part regulated by the external hydrodynamic forces that the cancer cells experienced within capillary mimetics. Our findings indicate that disseminated cancer cells can keep mutating with a continuum landscape of mechano-phenotypes, governed by the YAP-mediated mechanosensing of hydrodynamic flow.

Preclinical models for drug discovery for metastatic disease.

Despite many advances, metastatic disease remains essentially uncurable. Thus, there is an urgent need to better understand mechanisms that promote metastasis, drive tumor evolution, and underlie innate and acquired drug resistance. Sophisticated preclinical models that recapitulate the complex tumor ecosystem are key to this process. We begin with syngeneic and patient-derived mouse models that are the backbone of most preclinical studies. Second, we present some unique advantages of fish and fly models. Third, we consider the strengths of 3D culture models for resolving remaining knowledge gaps. Finally, we provide vignettes on multiplexed technologies to advance our understanding of metastatic disease.

Rapid biomechanical imaging at low irradiation level via dual line-scanning Brillouin microscopy.

Brillouin microscopy is a technique for mechanical characterization of biological material without contact at high three-dimensional resolution. Here, we introduce dual line-scanning Brillouin microscopy (dLSBM), which improves acquisition speed and reduces irradiation dose by more than one order of magnitude with selective illumination and single-shot analysis of hundreds of points along the incident beam axis. Using tumor spheroids, we demonstrate the ability to capture the sample response to rapid mechanical perturbations as well as the spatially resolved evolution of the mechanical properties in growing spheroids.

An in vivo phosphoregulation paradox for focal adhesions.

Focal adhesions (FAs) dynamics regulate single cell migration. In this issue, Xue et al. (2023. J. Cell Biol.https://doi.org/10.1083/jcb.202206078) show that Y118 phosphorylation on Paxilin, a key FA protein, limits migration of cells in vivo. Unphosphorylated Paxilin is necessary for FA disassembly and cell motility. Their findings directly contradict results from in vitro experiments, emphasizing the need for recreating the in vivo complexity to understand how cells behave in their native environments.

Cell-specific cargo delivery using synthetic bacterial spores.

Delivery of cancer therapeutics to non-specific sites decreases treatment efficacy while increasing toxicity. In ovarian cancer, overexpression of the cell surface marker HER2, which several therapeutics target, relates to poor prognosis. We recently reported the assembly of biocompatible bacterial spore-like particles, termed "SSHELs." Here, we modify SSHELs with an affibody directed against HER2 and load them with the chemotherapeutic agent doxorubicin. Drug-loaded SSHELs reduce tumor growth and increase survival with lower toxicity in a mouse tumor xenograft model compared with free drug and with liposomal doxorubicin by preferentially accumulating in the tumor mass. Target cells actively internalize and then traffic bound SSHELs to acidic compartments, whereupon the cargo is released to the cytosol in a pH-dependent manner. We propose that SSHELs represent a versatile strategy for targeted drug delivery, especially in cancer settings.

Biophysical determinants of cancer organotropism.

Metastasis remains the leading cause of cancer lethality. The 'seed/soil' hypothesis provides the framework to explain this cancer phenomenon where the concept of organotropism has been in part mechanistically explained by the properties of the tumor cells and their compatibility with the stromal environment of the distal site. The 'mechanical' hypothesis counters that non-random seeding is driven solely by the circulation patterns and vascular networks of organ systems. We incorporate concepts of mechanobiology and revisit the two hypotheses to provide additional insights into the mechanisms that regulate organ selection during metastatic outgrowth. We focus on the latter stages of the metastatic cascade and examine the role of the endothelium in regulating organ selectivity.

Vascular regulation of disseminated tumor cells during metastatic spread.

Cancer cells can travel to other organs via interconnected vascular systems to form new lesions in a process known as metastatic spread. Unfortunately, metastasis remains the leading cause of patient lethality. In recent years, it has been demonstrated that physical cues are just as important as chemical and genetic perturbations in driving changes in gene expression, cell motility, and survival. In this concise review, we focus on the physical cues that cancer cells experience as they migrate through the lymphatic and blood vascular networks. We also present an overview of steps that may facilitate organ specific metastasis.

Organ specific microenvironmental MR1 expression in cutaneous melanoma.

The microenvironment is an important regulator of intertumoral trafficking and activity of immune cells. Understanding how the immune system can be tailored to maintain anti-tumor killing responses in metastatic disease remains an important goal. Thus, immune mediated eradication of metastasis requires the consideration of organ specific microenvironmental cues. Using a xenograft model of melanoma metastasis in adult zebrafish, we perturbed the dynamic balance between the infiltrating immune cells in the metastatic setting using a suite of different transgenic zebrafish. We employed intravital imaging coupled with metabolism imaging (FLIM) to visualize and map the organ specific metabolism with near simultaneity in multiple metastatic lesions. Of all the MHC complexes examined for brain and skeletal metastases, we determined that there is an organ specific expression of mhc1uba (human ortholog, MR1) for both the melanoma cells and the resident and infiltrating immune cells. Specifically, immune clusters did not express mhc1uba in brain metastatic lesions in immune competent fish. Finally, the differential immune response drove organ specific metabolism where tumor glycolysis was increased in brain metastases compared to skeletal and parental lines as measured using fluorescence lifetime imaging microscopy (FLIM). As MR1 belongs to the MHC class I molecules and is a target of immunotherapeutic drugs, we believe that our data presents an opportunity to understand the relationship between organ specific tumor metabolism and drug efficacy in the metastatic setting.

Multimodal microscale mechanical mapping of cancer cells in complex microenvironments.

The mechanical phenotype of the cell is critical for survival following deformations due to confinement and fluid flow. One idea is that cancer cells are plastic and adopt different mechanical phenotypes under different geometries that aid in their survival. Thus, an attractive goal is to disrupt cancer cells' ability to adopt multiple mechanical states. To begin to address this question, we aimed to quantify the diversity of these mechanical states using in vitro biomimetics to mimic in vivo two-dimensional (2D) and 3D extracellular matrix environments. Here, we used two modalities Brillouin microscopy (∼GHz) and broadband frequency (7-15 kHz) optical tweezer microrheology to measure microscale cell mechanics. We measured the response of intracellular mechanics of cancer cells cultured in 2D and 3D environments where we modified substrate stiffness, dimensionality (2D versus 3D), and presence of fibrillar topography. We determined that there was good agreement between two modalities despite the difference in timescale of the two measurements. These findings on cell mechanical phenotype in different environments confirm a correlation between modalities that employ different mechanisms at different temporal scales (Hz-kHz versus GHz). We also determined that observed heterogeneity in cell shape is more closely linked to the cells' mechanical state. Moreover, individual cells in multicellular spheroids exhibit a lower degree of mechanical heterogeneity when compared with single cells cultured in monodisperse 3D cultures. The observed decreased heterogeneity among cells in spheroids suggested that there is mechanical cooperativity between cells that make up a single spheroid.

The material properties of chromatin in vivo.

Chromatin is fluidlike within the crowded nucleus when probed in a living cell.

Identifying drivers of breast cancer metastasis in progressively invasive subpopulations of zebrafish-xenografted MDA-MB-231.

Cancer metastasis is the primary cause of the high mortality rate among human cancers. Efforts to identify therapeutic agents targeting cancer metastasis frequently fail to demonstrate efficacy in clinical trials despite strong preclinical evidence. Until recently, most preclinical studies used mouse models to evaluate anti-metastatic agents. Mouse models are time-consuming and expensive. In addition, an important drawback is that mouse models inadequately model the early stages of metastasis which plausibly leads to the poor correlation with clinical outcomes.Here, we report an in vivo model based on xenografted zebrafish embryos where we select for progressively invasive subpopulations of MDA-MB-231 breast cancer cells. A subpopulation analogous to circulating tumor cells found in human cancers was selected by injection of MDA-MB-231 cells into the yolk sacs of 2 days post-fertilized zebrafish embryos and selecting cells that migrated to the tail. The selected subpopulation derived from MDA-MB-231 cells were increasingly invasive in zebrafish. Isolation of these subpopulations and propagation in vitro revealed morphological changes consistent with activation of an epithelial-mesenchymal transition program. Differential gene analysis and knockdown of genes identified gene-candidates (DDIT4, MT1X, CTSD, and SERPINE1) as potential targets for anti-metastasis therapeutics. Furthermore, RNA-splicing analysis reinforced the importance of BIRC5 splice variants in breast cancer metastasis. This is the first report using zebrafish to isolate and expand progressively invasive populations of human cancer cells. The model has potential applications in understanding the metastatic process, identification and/or development of therapeutics that specifically target metastatic cells and formulating personalized treatment strategies for individual cancer patients.

Characterization and tissue localization of zebrafish homologs of the human ABCB1 multidrug transporter.

Capillary endothelial cells of the human blood-brain barrier (BBB) express high levels of P-glycoprotein (P-gp, encoded by ABCB1) and ABCG2 (encoded by ABCG2). However, little information is available regarding ATP-binding cassette transporters expressed at the zebrafish BBB, which has emerged as a potential model system. We report the characterization and tissue localization of two genes that are similar to ABCB1, zebrafish abcb4 and abcb5. When stably expressed in HEK293 cells, both Abcb4 and Abcb5 conferred resistance to P-gp substrates; however, Abcb5 poorly transported doxorubicin and mitoxantrone compared to zebrafish Abcb4. Additionally, Abcb5 did not transport the fluorescent P-gp probes BODIPY-ethylenediamine or LDS 751, while they were transported by Abcb4. High-throughput screening of 90 human P-gp substrates confirmed that Abcb4 has an overlapping substrate specificity profile with P-gp. In the brain vasculature, RNAscope probes for abcb4 colocalized with staining by the P-gp antibody C219, while abcb5 was not detected. The abcb4 probe also colocalized with claudin-5 in brain endothelial cells. Abcb4 and Abcb5 had different tissue localizations in multiple zebrafish tissues, potentially indicating different functions. The data suggest that zebrafish Abcb4 functionally phenocopies P-gp and that the zebrafish may serve as a model to study the role of P-gp at the BBB.

Emerging principles of cancer biophysics.

Cancer is a multi-step process where normal cells become transformed, grow, and may disseminate to establish new lesions within the body. In recent years, the physical properties of individual cells and the tissue microenvironment have been shown to be potent determinants of cancer progression. Biophysical tools have long been used to examine cell and tissue mechanics, morphology, and migration. However, exciting developments have linked these physical traits to gene expression changes that drive metastatic seeding, organ selectivity, and tumor growth. Here, we present some vignettes to address recent studies to show progress in harnessing biophysical tools and concepts to gain insights into metastasis.

Tissue Architectural Cues Drive Organ Targeting of Tumor Cells in Zebrafish.

Tumor cells encounter a myriad of physical cues upon arrest and extravasation in capillary beds. Here, we examined the role of physical factors in non-random organ colonization using a zebrafish xenograft model. We observed a two-step process by which mammalian mammary tumor cells showed non-random organ colonization. Initial homing was driven by vessel architecture, where greater numbers of cells became arrested in the topographically disordered blood vessels of the caudal vascular plexus (CVP) than in the linear vessels in the brain. Following arrest, bone-marrow- and brain-tropic clones exhibited organ-specific patterns of extravasation. Extravasation was mediated by ß1 integrin, where knockdown of ß1 integrin reduced extravasation in the CVP but did not affect extravasation of a brain-tropic clone in the brain. In contrast, silencing myosin 1B redirected early colonization from the brain to the CVP. Our results suggest that organ selectivity is driven by both vessel topography and cell-type-dependent extravasation.

High-frequency microrheology in 3D reveals mismatch between cytoskeletal and extracellular matrix mechanics.

Mechanical homeostasis describes how cells sense physical cues from the microenvironment and concomitantly remodel both the cytoskeleton and the surrounding extracellular matrix (ECM). Such feedback is thought to be essential to healthy development and maintenance of tissue. However, the nature of the dynamic coupling between microscale cell and ECM mechanics remains poorly understood. Here we investigate how and whether cells remodel their cortex and basement membrane to adapt to their microenvironment. We measured both intracellular and extracellular viscoelasticity, generating a full factorial dataset on 5 cell lines in 2 ECMs subjected to 4 cytoskeletal drug treatments at 2 time points. Nonmalignant breast epithelial cells show a similar viscoelasticity to that measured for the local ECM when cultured in 3D laminin-rich ECM. In contrast, the malignant counterpart is stiffer than the local environment. We confirmed that other mammary cancer cells embedded in tissue-mimetic hydrogels are nearly 4-fold stiffer than the surrounding ECM. Perturbation of actomyosin did not yield uniform responses but instead depended on the cell type and chemistry of the hydrogel. The observed viscoelasticity of both ECM and cells were well described by power laws in a frequency range that governs single filament cytoskeletal dynamics. Remarkably, the intracellular and extracellular power law parameters for the entire dataset collectively fall onto 2 parallel master curves described by just 2 parameters. Our work shows that tumor cells are mechanically plastic to adapt to many environments and reveals dynamical scaling behavior in the microscale mechanical responses of both cells and ECM.

Model systems for studying the blood-brain barrier: Applications and challenges.

The blood-brain barrier (BBB) poses a serious impediment to the delivery of effective therapies to the central nervous system (CNS). Over time, various model systems have been crafted and used to evaluate the complexities of the BBB, which includes an impermeable physical barrier and a series of energy-dependent efflux pumps. Models of the BBB have mainly sought to assess changes in endothelial cell permeability, the role of ATP-dependent efflux transporters in drug disposition, and alterations in communication between BBB cells and the microenvironment. In the context of disease, various animal models have been utilized to examine real time BBB drug permeability, CNS dynamic changes, and overall treatment response. In this review, we outline the use of these in vitro and in vivo blood-brain barrier model systems to study normal physiology and diseased states. These current models each have their own advantages and disadvantages for studying the response of biologic processes to physiological and pathological conditions. Additional models are needed to mimic more closely the dynamic quality of the BBB, with the goal focused on potential clinical applications.

Human macrophages survive and adopt activated genotypes in living zebrafish.

The inflammatory response, modulated both by tissue resident macrophages and recruited monocytes from peripheral blood, plays a critical role in human diseases such as cancer and neurodegenerative disorders. Here, we sought a model to interrogate human immune behavior in vivo. We determined that primary human monocytes and macrophages survive in zebrafish for up to two weeks. Flow cytometry revealed that human monocytes cultured at the physiological temperature of the zebrafish survive and differentiate comparable to cohorts cultured at human physiological temperature. Moreover, key genes that encode for proteins that play a role in tissue remodeling were also expressed. Human cells migrated within multiple tissues at speeds comparable to zebrafish macrophages. Analysis of gene expression of in vivo educated human macrophages confirmed expression of activated macrophage phenotypes. Here, human cells adopted phenotypes relevant to cancer progression, suggesting that we can define the real time immune modulation of human tumor cells during the establishment of a metastatic lesion in zebrafish.