Patient-specific vascularized tumor model: Blocking monocyte recruitment with multispecific antibodies targeting CCR2 and CSF-1R.

Tumor-associated inflammation drives cancer progression and therapy resistance, often linked to the infiltration of monocyte-derived tumor-associated macrophages (TAMs), which are associated with poor prognosis in various cancers. To advance immunotherapies, testing on immunocompetent pre-clinical models of human tissue is crucial. We have developed an in vitro model of microvascular networks with tumor spheroids or patient tissues to assess monocyte trafficking into tumors and evaluate immunotherapies targeting the human tumor microenvironment. Our findings demonstrate that macrophages in vascularized breast and lung tumor models can enhance monocyte recruitment via CCL7 and CCL2, mediated by CSF-1R. Additionally, a multispecific antibody targeting CSF-1R, CCR2, and neutralizing TGF-ß (CSF1R/CCR2/TGF-ß Ab) repolarizes TAMs towards an anti-tumoral M1-like phenotype, reduces monocyte chemoattractant protein secretion, and blocks monocyte migration. This antibody also inhibits monocyte recruitment in patient-specific vascularized tumor models. In summary, this vascularized tumor model recapitulates the monocyte recruitment cascade, enabling functional testing of innovative therapeutic antibodies targeting TAMs in the tumor microenvironment.

Utilizing convolutional neural networks for discriminating cancer and stromal cells in three-dimensional cell culture images with nuclei counterstain.

Significance: Accurate cell segmentation and classification in three-dimensional (3D) images are vital for studying live cell behavior and drug responses in 3D tissue culture. Evaluating diverse cell populations in 3D cell culture over time necessitates non-toxic staining methods, as specific fluorescent tags may not be suitable, and immunofluorescence staining can be cytotoxic for prolonged live cell cultures. Aim: We aim to perform machine learning-based cell classification within a live heterogeneous cell culture population grown in a 3D tissue culture relying only on reflectance, transmittance, and nuclei counterstained images obtained by confocal microscopy. Approach: In this study, we employed a supervised convolutional neural network (CNN) to classify tumor cells and fibroblasts within 3D-grown spheroids. These cells are first segmented using the marker-controlled watershed image processing method. Training data included nuclei counterstaining, reflectance, and transmitted light images, with stained fibroblast and tumor cells as ground-truth labels. Results: Our results demonstrate the successful marker-controlled watershed segmentation of 84% of spheroid cells into single cells. We achieved a median accuracy of 67% (95% confidence interval of the median is 65-71%) in identifying cell types. We also recapitulate the original 3D images using the CNN-classified cells to visualize the original 3D-stained image's cell distribution. Conclusion: This study introduces a non-invasive toxicity-free approach to 3D cell culture evaluation, combining machine learning with confocal microscopy, opening avenues for advanced cell studies.

Polyploidy of MDA-MB-231 cells drives increased extravasation with enhanced cell-matrix adhesion.

Metastasis, the leading cause of cancer-related deaths, involves a complex cascade of events, including extravasation. Despite extensive research into metastasis, the mechanisms underlying extravasation remain unclear. Molecular targeted therapies have advanced cancer treatment, yet their efficacy is limited, prompting exploration into novel therapeutic targets. Here, we showed the association of polyploidy in MDA-MB-231 breast cancer cells and their extravasation, using microfluidic systems to reproduce the in vivo microvascular environment. We observed enhanced extravasation in polyploid cells alongside upregulated expression of genes involved in cell-substrate adhesion and cell mechanical dynamics. These findings offer insights into the relationship between polyploidy and extravasation, highlighting potential targets for cancer therapy.

Personalized Vascularized Models of Breast Cancer Desmoplasia Reveal Biomechanical Determinants of Drug Delivery to the Tumor.

Desmoplasia in breast cancer leads to heterogeneity in physical properties of the tissue, resulting in disparities in drug delivery and treatment efficacy among patients, thus contributing to high disease mortality. Personalized in vitro breast cancer models hold great promise for high-throughput testing of therapeutic strategies to normalize the aberrant microenvironment in a patient-specific manner. Here, tumoroids assembled from breast cancer cell lines (MCF7, SKBR3, and MDA-MB-468) and patient-derived breast tumor cells (TCs) cultured in microphysiological systems including perfusable microvasculature reproduce key aspects of stromal and vascular dysfunction causing impaired drug delivery. Models containing SKBR3 and MDA-MB-468 tumoroids show higher stromal hyaluronic acid (HA) deposition, vascular permeability, interstitial fluid pressure (IFP), and degradation of vascular HA relative to models containing MCF7 tumoroids or models without tumoroids. Interleukin 8 (IL8) secretion is found responsible for vascular dysfunction and loss of vascular HA. Interventions targeting IL8 or stromal HA normalize vascular permeability, perfusion, and IFP, and ultimately enhance drug delivery and TC death in response to perfusion with trastuzumab and cetuximab. Similar responses are observed in patient-derived models. These microphysiological systems can thus be personalized by using patient-derived cells and can be applied to discover new molecular therapies for the normalization of the tumor microenvironment.

Engineering microvascular networks using a KLF2 reporter to probe flow-dependent endothelial cell function.

Shear stress generated by the flow of blood in the vasculature is a potent regulator of endothelial cell function and vascular structure. While vascular responses to flow are complex and context-dependent, endothelial cell signaling in response to shear stress induced by laminar flows is coordinated by the transcription factor KLF2. The flow-dependent expression of KLF2 in endothelial cells is associated with a quiescent, anti-inflammatory phenotype and has been well characterized in two-dimensional systems but has not been studied in three-dimensional in vitro systems. Here we develop engineered microvascular networks (MVNs) that incorporate a KLF2-based endothelial cell flow sensor within a microfluidic chip, apply continuous flow using an attached microfluidic pump, and study the effects of this flow on vascular structure and function. We found that application of flow to MVNs for 48 h resulted in increased expression of the KLF2 reporter, larger vessel diameters, and decreased vascular branching and resistance. Notably, vessel diameters after the application of flow were independent of initial MVN morphologies. Finally, we found that MVNs exposed to flow have improved vascular barrier function and decreased platelet adhesion. MVNs with KLF2-based flow sensors represent a novel, powerful tool for evaluating the structural and functional effects of flow on engineered three-dimensional vascular systems.

Challenges and Future Perspectives in Modeling Neurodegenerative Diseases Using Organ-on-a-Chip Technology.

Neurodegenerative diseases (NDDs) affect more than 50 million people worldwide, posing a significant global health challenge as well as a high socioeconomic burden. With aging constituting one of the main risk factors for some NDDs such as Alzheimer's disease (AD) and Parkinson's disease (PD), this societal toll is expected to rise considering the predicted increase in the aging population as well as the limited progress in the development of effective therapeutics. To address the high failure rates in clinical trials, legislative changes permitting the use of alternatives to traditional pre-clinical in vivo models are implemented. In this regard, microphysiological systems (MPS) such as organ-on-a-chip (OoC) platforms constitute a promising tool, due to their ability to mimic complex and human-specific tissue niches in vitro. This review summarizes the current progress in modeling NDDs using OoC technology and discusses five critical aspects still insufficiently addressed in OoC models to date. Taking these aspects into consideration in the future MPS will advance the modeling of NDDs in vitro and increase their translational value in the clinical setting.

Transmural Flow Upregulates PD-L1 Expression in Microvascular Networks.

Endothelial programmed death-ligand 1 (PD-L1) expression is higher in tumors than in normal tissues. Also, tumoral vasculatures tend to be leakier than normal vessels leading to a higher trans-endothelial or transmural fluid flow. However, it is not clear whether such elevated transmural flow can control endothelial PD-L1 expression. Here, a new microfluidic device is developed to investigate the relationship between transmural flow and PD-L1 expression in microvascular networks (MVNs). After treating the MVNs with transmural flow for 24 h, the expression of PD-L1 in endothelial cells is upregulated. Additionally, CD8 T cell activation by phytohemagglutinin (PHA) is suppressed when cultured in the MVNs pre-conditioned with transmural flow. Moreover, transmural flow is able to further increase PD-L1 expression in the vessels formed in the tumor microenvironment. Finally, by utilizing blocking antibodies and knock-out assays, it is found that transmural flow-driven PD-L1 upregulation is controlled by integrin αVß3. Overall, this study provides a new biophysical explanation for high PD-L1 expression in tumoral vasculatures.

Ether lipids influence cancer cell fate by modulating iron uptake.

Cancer cell fate has been widely ascribed to mutational changes within protein-coding genes associated with tumor suppressors and oncogenes. In contrast, the mechanisms through which the biophysical properties of membrane lipids influence cancer cell survival, dedifferentiation and metastasis have received little scrutiny. Here, we report that cancer cells endowed with a high metastatic ability and cancer stem cell-like traits employ ether lipids to maintain low membrane tension and high membrane fluidity. Using genetic approaches and lipid reconstitution assays, we show that these ether lipid-regulated biophysical properties permit non-clathrin-mediated iron endocytosis via CD44, leading directly to significant increases in intracellular redox-active iron and enhanced ferroptosis susceptibility. Using a combination of in vitro three-dimensional microvascular network systems and in vivo animal models, we show that loss of ether lipids also strongly attenuates extravasation, metastatic burden and cancer stemness. These findings illuminate a mechanism whereby ether lipids in carcinoma cells serve as key regulators of malignant progression while conferring a unique vulnerability that can be exploited for therapeutic intervention.

Modeling early pathophysiological phenotypes of diabetic retinopathy in a human inner blood-retinal barrier-on-a-chip.

Diabetic retinopathy (DR) is a microvascular disorder characterized by inner blood-retinal barrier (iBRB) breakdown and irreversible vision loss. While the symptoms of DR are known, disease mechanisms including basement membrane thickening, pericyte dropout and capillary damage remain poorly understood and interventions to repair diseased iBRB microvascular networks have not been developed. In addition, current approaches using animal models and in vitro systems lack translatability and predictivity to finding new target pathways. Here, we develop a diabetic iBRB-on-a-chip that produces pathophysiological phenotypes and disease pathways in vitro that are representative of clinical diagnoses. We show that diabetic stimulation of the iBRB-on-a-chip mirrors DR features, including pericyte loss, vascular regression, ghost vessels, and production of pro-inflammatory factors. We also report transcriptomic data from diabetic iBRB microvascular networks that may reveal drug targets, and examine pericyte-endothelial cell stabilizing strategies. In summary, our model recapitulates key features of disease, and may inform future therapies for DR.

Linking cell mechanical memory and cancer metastasis.

Metastasis causes most cancer-related deaths; however, the efficacy of anti-metastatic drugs is limited by incomplete understanding of the biological mechanisms that drive metastasis. Focusing on the mechanics of metastasis, we propose that the ability of tumour cells to survive the metastatic process is enhanced by mechanical stresses in the primary tumour microenvironment that select for well-adapted cells. In this Perspective, we suggest that biophysical adaptations favourable for metastasis are retained via mechanical memory, such that the extent of memory is influenced by both the magnitude and duration of the mechanical stress. Among the mechanical cues present in the primary tumour microenvironment, we focus on high matrix stiffness to illustrate how it alters tumour cell proliferation, survival, secretion of molecular factors, force generation, deformability, migration and invasion. We particularly centre our discussion on potential mechanisms of mechanical memory formation and retention via mechanotransduction and persistent epigenetic changes. Indeed, we propose that the biophysical adaptations that are induced by this process are retained throughout the metastatic process to improve tumour cell extravasation, survival and colonization in the distant organ. Deciphering mechanical memory mechanisms will be key to discovering a new class of anti-metastatic drugs.

Microfluidic-Based Reconstitution of Functional Lymphatic Microvasculature: Elucidating the Role of Lymphatics in Health and Disease.

The knowledge of the blood microvasculature and its functional role in health and disease has grown significantly attributable to decades of research and numerous advances in cell biology and tissue engineering; however, the lymphatics (the secondary vascular system) has not garnered similar attention, in part due to a lack of relevant in vitro models that mimic its pathophysiological functions. Here, a microfluidic-based approach is adopted to achieve precise control over the biological transport of growth factors and interstitial flow that drive the in vivo growth of lymphatic capillaries (lymphangiogenesis). The engineered on-chip lymphatics with in vivo-like morphology exhibit tissue-scale functionality with drainage rates of interstitial proteins and molecules comparable to in vivo standards. Computational and scaling analyses of the underlying transport phenomena elucidate the critical role of the three-dimensional geometry and lymphatic endothelium in recapitulating physiological drainage. Finally, the engineered on-chip lymphatics enabled studies of lymphatic-immune interactions that revealed inflammation-driven responses by the lymphatics to recruit immune cells via chemotactic signals similar to in vivo, pathological events. This on-chip lymphatics platform permits the interrogation of various lymphatic biological functions, as well as screening of lymphatic-based therapies such as interstitial absorption of protein therapeutics and lymphatic immunomodulation for cancer therapy.

Patient-Specific Vascularized Tumor Model: Blocking TAM Recruitment with Multispecific Antibodies Targeting CCR2 and CSF-1R.

Tumor-associated inflammation drives cancer progression and therapy resistance, with the infiltration of monocyte-derived tumor-associated macrophages (TAMs) associated with poor prognosis in diverse cancers. Targeting TAMs holds potential against solid tumors, but effective immunotherapies require testing on immunocompetent human models prior to clinical trials. Here, we develop an in vitro model of microvascular networks that incorporates tumor spheroids or patient tissues. By perfusing the vasculature with human monocytes, we investigate monocyte trafficking into the tumor and evaluate immunotherapies targeting the human tumor microenvironment. Our findings demonstrate that macrophages in vascularized breast and lung tumor models can enhance monocyte recruitment via TAM-produced CCL7 and CCL2, mediated by CSF-1R. Additionally, we assess a novel multispecific antibody targeting CCR2, CSF-1R, and neutralizing TGF-ß, referred to as CSF1R/CCR2/TGF-ß Ab, on monocytes and macrophages using our 3D models. This antibody repolarizes TAMs towards an anti-tumoral M1-like phenotype, reduces monocyte chemoattractant protein secretion, and effectively blocks monocyte migration. Finally, we show that the CSF1R/CCR2/TGF-ß Ab inhibits monocyte recruitment in patient-specific vascularized tumor models. Overall, this vascularized tumor model offers valuable insights into monocyte recruitment and enables functional testing of innovative therapeutic antibodies targeting TAMs in the tumor microenvironment (TME).

Engineering microvascular networks using a KLF2 reporter to probe flow-dependent endothelial cell function.

Shear stress generated by the flow of blood in the vasculature is a potent regulator of endothelial cell phenotype and vascular structure. While vascular responses to flow are complex and context-dependent, endothelial cell signaling in response to shear stress induced by laminar flows is coordinated by the transcription factor KLF2. The expression of KLF2 in endothelial cells is associated with a quiescent, anti-inflammatory phenotype and has been well characterized in two-dimensional systems, but has not been studied in three-dimensional in vitro systems. Here we develop engineered microvascular networks (MVNs) with a KLF2-based endothelial cell sensor within a microfluidic chip, apply continuous flow using an attached microfluidic pump, and study the effects of this flow on vascular structure and function. We found that culture of MVNs exposed to flow for 48 hours that resulted in increased expression of the KLF2-GFP-reporter display larger vessel diameters and decreased vascular branching and resistance. Additionally, vessel diameters after the application of flow were independent of initial MVN morphologies. Finally, we found that MVNs exposed to flow have improved vascular barrier function and decreased platelet adhesion. The MVNs with KLF2-based flow sensors represent a powerful tool for evaluating the structural and functional effects of flow on engineered three-dimensional vascular systems.

Accelerating the in vitro emulation of Alzheimer's disease-associated phenotypes using a novel 3D blood-brain barrier neurosphere co-culture model.

High failure rates in clinical trials for neurodegenerative disorders such as Alzheimer's disease have been linked to an insufficient predictive validity of current animal-based disease models. This has created an increasing demand for alternative, human-based models capable of emulating key pathological phenotypes in vitro. Here, a three-dimensional Alzheimer's disease model was developed using a compartmentalized microfluidic device that combines a self-assembled microvascular network of the human blood-brain barrier with neurospheres derived from Alzheimer's disease-specific neural progenitor cells. To shorten microfluidic co-culture times, neurospheres were pre-differentiated for 21 days to express Alzheimer's disease-specific pathological phenotypes prior to the introduction into the microfluidic device. In agreement with post-mortem studies and Alzheimer's disease in vivo models, after 7 days of co-culture with pre-differentiated Alzheimer's disease-specific neurospheres, the three-dimensional blood-brain barrier network exhibited significant changes in barrier permeability and morphology. Furthermore, vascular networks in co-culture with Alzheimer's disease-specific microtissues displayed localized ß-amyloid deposition. Thus, by interconnecting a microvascular network of the blood-brain barrier with pre-differentiated neurospheres the presented model holds immense potential for replicating key neurovascular phenotypes of neurodegenerative disorders in vitro.

A three-dimensional method for morphological analysis and flow velocity estimation in microvasculature on-a-chip.

Three-dimensional (3D) imaging techniques (e.g., confocal microscopy) are commonly used to visualize in vitro models, especially microvasculature on-a-chip. Conversely, 3D analysis is not the standard method to extract quantitative information from those models. We developed the µVES algorithm to analyze vascularized in vitro models leveraging 3D data. It computes morphological parameters (geometry, diameter, length, tortuosity, eccentricity) and intravascular flow velocity. µVES application to microfluidic vascularized in vitro models shows that they successfully replicate functional features of the microvasculature in vivo in terms of intravascular fluid flow velocity. However, wall shear stress is lower compared to in vivo references. The morphological analysis also highlights the model's physiological similarities (vessel length and tortuosity) and shortcomings (vessel radius and surface-over-volume ratio). The addition of the third dimension in our analysis produced significant differences in the metrics assessed compared to 2D estimations. It enabled the computation of new indices, such as vessel eccentricity. These µVES capabilities can find application in analyses of different in vitro vascular models, as well as in vivo and ex vivo microvasculature.

Development of a perfusable, hierarchical microvasculature-on-a-chip model.

Several methods have been developed for generating 3D, in vitro, organ-on-chip models of human vasculature to study vascular function, transport, and tissue engineering. However, many of these existing models lack the hierarchical nature of the arterial-to-capillary-to-venous architecture that is key to capturing a more comprehensive view of the human microvasculature. Here, we present a perfusable, multi-compartmental model that recapitulates the three microvascular compartments to assess various physiological properties such as vessel permeability, vasoconstriction dynamics, and circulating cell arrest and extravasation. Viscous finger patterning and passive pumping create the larger arterial and venular lumens, while the smaller diameter capillary bed vessels are generated through self-assembly. These compartments anastomose and form a perfusable, hierarchical system that portrays the directionality of blood flow through the microvasculature. The addition of collagen channels reduces the apparent permeability of the central capillary region, likely by reducing leakage from the side channels, enabling more accurate measurements of vascular permeability-an important motivation for this study. Furthermore, the model permits modulation of fluid flow and shear stress conditions throughout the system by using hydrostatic pressure heads to apply pressure differentials across either the arteriole or the capillary. This is a pertinent system for modeling circulating tumor or T cell dissemination and extravasation. Circulating cells were found to arrest in areas conducive to physical trapping or areas with the least amount of shear stress, consistent with hemodynamic or mechanical theories of metastasis. Overall, this model captures more features of human microvascular beds and is capable of testing a broad variety of hypotheses.

Targeting CCR7-PI3Kγ overcomes resistance to tyrosine kinase inhibitors in ALK-rearranged lymphoma.

Anaplastic lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs) show potent efficacy in several ALK-driven tumors, but the development of resistance limits their long-term clinical impact. Although resistance mechanisms have been studied extensively in ALK-driven non-small cell lung cancer, they are poorly understood in ALK-driven anaplastic large cell lymphoma (ALCL). Here, we identify a survival pathway supported by the tumor microenvironment that activates phosphatidylinositol 3-kinase γ (PI3K-γ) signaling through the C-C motif chemokine receptor 7 (CCR7). We found increased PI3K signaling in patients and ALCL cell lines resistant to ALK TKIs. PI3Kγ expression was predictive of a lack of response to ALK TKI in patients with ALCL. Expression of CCR7, PI3Kγ, and PI3Kδ were up-regulated during ALK or STAT3 inhibition or degradation and a constitutively active PI3Kγ isoform cooperated with oncogenic ALK to accelerate lymphomagenesis in mice. In a three-dimensional microfluidic chip, endothelial cells that produce the CCR7 ligands CCL19/CCL21 protected ALCL cells from apoptosis induced by crizotinib. The PI3Kγ/δ inhibitor duvelisib potentiated crizotinib activity against ALCL lines and patient-derived xenografts. Furthermore, genetic deletion of CCR7 blocked the central nervous system dissemination and perivascular growth of ALCL in mice treated with crizotinib. Thus, blockade of PI3Kγ or CCR7 signaling together with ALK TKI treatment reduces primary resistance and the survival of persister lymphoma cells in ALCL.

Age-related alterations in meningeal immunity drive impaired CNS lymphatic drainage.

The meningeal lymphatic network enables the drainage of cerebrospinal fluid (CSF) and facilitates the removal of central nervous system (CNS) waste. During aging and in Alzheimer's disease, impaired meningeal lymphatic drainage promotes the buildup of toxic misfolded proteins in the CNS. Reversing this age-related dysfunction represents a promising strategy to augment CNS waste clearance; however, the mechanisms underlying this decline remain elusive. Here, we demonstrate that age-related alterations in meningeal immunity underlie this lymphatic impairment. Single-cell RNA sequencing of meningeal lymphatic endothelial cells from aged mice revealed their response to IFNγ, which was increased in the aged meninges due to T cell accumulation. Chronic elevation of meningeal IFNγ in young mice via AAV-mediated overexpression attenuated CSF drainage-comparable to the deficits observed in aged mice. Therapeutically, IFNγ neutralization alleviated age-related impairments in meningeal lymphatic function. These data suggest manipulation of meningeal immunity as a viable approach to normalize CSF drainage and alleviate the neurological deficits associated with impaired waste removal.

Impact of Fibrinogen, Fibrin Thrombi, and Thrombin on Cancer Cell Extravasation Using In Vitro Microvascular Networks.

A bidirectional association exists between metastatic dissemination and the hypercoagulable state associated with many types of cancer. As such, clinical studies have provided evidence that markers associated with elevated levels of coagulation and fibrinolysis correlate with decreased patient survival. However, elucidating the mechanisms underpinning the effects of different components of the coagulation system on metastasis formation is challenging both in animal models and 2D models lacking the complex cellular interactions necessary to model both thrombosis and metastasis. Here, an in vitro, 3D, microvascular model for observing the formation of fibrin thrombi is described, which is in turn used to study how different aspects of the hypercoagulable state associated with cancer affect the endothelium. Using this platform, cancer cells expressing ICAM-1 are shown to form a fibrinogen-dependent bridge and transmigrate through the endothelium more effectively. Cancer cells are also demonstrated to interact with fibrin thrombi, using them to adhere, spread, and enhance their extravasation efficiency. Finally, thrombin is also shown to enhance cancer cell extravasation. This system presents a physiologically relevant model of fibrin clot formation in the human microvasculature, enabling in-depth investigation of the cellular interactions between cancer cells and the coagulation system affecting cancer cell extravasation.

Priming a vascular-selective cytokine response permits CD8+ T-cell entry into tumors.

Targeting DNA methyltransferase 1 (DNMT1) has immunomodulatory and anti-neoplastic activity, especially when paired with cancer immunotherapies. Here we explore the immunoregulatory functions of DNMT1 in the tumor vasculature of female mice. Dnmt1 deletion in endothelial cells (ECs) impairs tumor growth while priming expression of cytokine-driven cell adhesion molecules and chemokines important for CD8+ T-cell trafficking across the vasculature; consequently, the efficacy of immune checkpoint blockade (ICB) is enhanced. We find that the proangiogenic factor FGF2 promotes ERK-mediated DNMT1 phosphorylation and nuclear translocation to repress transcription of the chemokines Cxcl9/Cxcl10 in ECs. Targeting Dnmt1 in ECs reduces proliferation but augments Th1 chemokine production and extravasation of CD8+ T-cells, suggesting DNMT1 programs immunologically anergic tumor vasculature. Our study is in good accord with preclinical observations that pharmacologically disrupting DNMT1 enhances the activity of ICB but suggests an epigenetic pathway presumed to be targeted in cancer cells is also operative in the tumor vasculature.