Label-free metabolic optical biomarkers track stem cell fate transition in real time.

Tracking stem cell fate transition is crucial for understanding their development and optimizing biomanufacturing. Destructive single-cell methods provide a pseudotemporal landscape of stem cell differentiation but cannot monitor stem cell fate in real time. We established a metabolic optical metric using label-free fluorescence lifetime imaging microscopy (FLIM), feature extraction and machine learning-assisted analysis, for real-time cell fate tracking. From a library of 205 metabolic optical biomarker (MOB) features, we identified 56 associated with hematopoietic stem cell (HSC) differentiation. These features collectively describe HSC fate transition and detect its bifurcate lineage choice. We further derived a MOB score measuring the "metabolic stemness" of single cells and distinguishing their division patterns. This score reveals a distinct role of asymmetric division in rescuing stem cells with compromised metabolic stemness and a unique mechanism of PI3K inhibition in promoting ex vivo HSC maintenance. MOB profiling is a powerful tool for tracking stem cell fate transition and improving their biomanufacturing from a single-cell perspective.

Cleanroom-Free Microfluidic Device for Natural Induction of Hypoxia in 2D and 3D Tumor Models.

Hypoxia is a common and critical feature of solid tumors that contributes to the plasticity and heterogeneity of the cancer cells. Cancer cell populations take on a region-specific adaptation induced by hypoxia, and each cancer cell population will show different levels of sensitivity and resistance to cancer therapeutics. Therefore, a faithful recapitulation of tumor hypoxia that allows for accurate assessments of hypoxia-induced adaptations, heterogeneity, and response to therapy is needed to develop new therapeutic approaches. The existing hypoxic tumor models rely on complex fabrication methods and external gas sources that make them unfavorable for the early-stage screening of new therapeutics. Here, we demonstrate how to establish a cleanroom-free microfluidic device that supports both 2D and 3D hypoxic tumor modeling through natural cancer cell metabolism and confirm the induction of the hypoxic gradient.

Ectopic Expression of a Truncated Isoform of Hair Keratin 81 in Breast Cancer Alters Biophysical Characteristics to Promote Metastatic Propensity.

Keratins are an integral part of cell structure and function. Here, it is shown that ectopic expression of a truncated isoform of keratin 81 (tKRT81) in breast cancer is upregulated in metastatic lesions compared to primary tumors and patient-derived circulating tumor cells, and is associated with more aggressive subtypes. tKRT81 physically interacts with keratin 18 (KRT18) and leads to changes in the cytosolic keratin intermediate filament network and desmosomal plaque formation. These structural changes are associated with a softer, more elastically deformable cancer cell with enhanced adhesion and clustering ability leading to greater in vivo lung metastatic burden. This work describes a novel biomechanical mechanism by which tKRT81 promotes metastasis, highlighting the importance of the biophysical characteristics of tumor cells.

Hypoxic 3D Tumor Model for Evaluating of CAR-T Cell Therapy In Vitro.

Solid tumors contain abnormal physical and biochemical barriers that hinder chimeric antigen receptor (CAR) T cell therapies. However, there is a lack of understanding on how the solid tumor microenvironment (e.g. hypoxia) modulates CAR-T cell function. Hypoxia is a common feature of many advanced solid tumors that contributes to reprogramming of cancer and T cell metabolism as well as their phenotypes and interactions. To gain insights into the activities of CAR-T cells in solid tumors and to assess the effectiveness of new combination treatments involving CAR-T cells, in vitro models that faithfully reflect CAR-T cell-solid tumor interactions under physiologically relevant tumor microenvironment is needed. Here we demonstrate how to establish a hypoxic 3-dimensional (3-D) tumor model using a cleanroom-free, micromilling-based microdevice and assess the efficacy of the combination treatment with CAR-T cells and PD-1/PD-L1 inhibition.

Intracellular and microenvironmental regulation of mitochondrial membrane potential in cancer cells.

Cancer cells have an abnormally high mitochondrial membrane potential (ΔΨm ), which is associated with enhanced invasive properties in vitro and increased metastases in vivo. The mechanisms underlying the abnormal ΔΨm in cancer cells remain unclear. Research on different cell types has shown that ΔΨm is regulated by various intracellular mechanisms such as by mitochondrial inner and outer membrane ion transporters, cytoskeletal elements, and biochemical signaling pathways. On the other hand, the role of extrinsic, tumor microenvironment (TME) derived cues in regulating ΔΨm is not well defined. In this review, we first summarize the existing literature on intercellular mechanisms of ΔΨm regulation, with a focus on cancer cells. We then offer our perspective on the different ways through which the microenvironmental cues such as hypoxia and mechanical stresses may regulate cancer cell ΔΨm . This article is categorized under: Cancer > Environmental Factors Cancer > Biomedical Engineering Cancer > Molecular and Cellular Physiology.

Faultline configurations affecting the entrepreneurial team performance of new generation of returning migrant workers in China: An empirical study based on fuzzy-set qualitative comparative analysis.

As the impact of faultlines is still without a consensus, to figure out how faultlines will hurt or promote the entrepreneurial performance can help the new generation of Chinese migrant workers to start their businesses successfully under the Rural Revitalization Strategy. This study addressed a fuzzy-set qualitative comparative analysis (fsQCA) based on 32 returning entrepreneurial teams from a complexity perspective. We firstly introduced three faultline categories for migrant workers and selected five of the faultlines with high factor loads in each category for further analysis. Then a scale was developed to measure the team performance. By conducting fsQCA, four types of faultline configurations were found: (1) background-experience actuation; (2) guidance-balance lacking; (3) role-cognition conflict; and (4) information-decision polarization. The "background-experience actuation" type will promote the entrepreneurial performance while the other types will hurt the performance. Theoretically, breaking through the limitations of traditional regressions in previous studies, fsQCA is used to explore the complex interactions and integrated effects among different categories of faultlines, demonstrates that the unstable impact is just a one-sided representation of the overall effect, and fills the general faultline theory with Chinese specific scenario and small-sized entrepreneurship. Practically, several implications are proposed to optimize the heterogeneity of the returning migrant workers' entrepreneurial teams and increase their performances, such as constructing the "balance" and "guidance" mechanism, enriching the background diversity of the members and solving the information-decision faultlines into individual diversity, etc., which can also be utilized by migrant worker entrepreneurs in other developing areas in the world.

Recapitulating Tumor Hypoxia in a Cleanroom-Free, Liquid-Pinning-Based Microfluidic Tumor Model.

In tumors, the metabolic demand of cancer cells often outpaces oxygen supply, resulting in a gradient of tumor hypoxia accompanied with heterogeneous resistance to cancer therapeutics. Models recapitulating tumor hypoxia are therefore essential for developing more effective cancer therapeutics. Existing in vitro models often fail to capture the spatial heterogeneity of tumor hypoxia or involve high-cost, complex fabrication/handling techniques. Here, we designed a highly tunable microfluidic device that induces hypoxia through natural cell metabolism and oxygen diffusion barriers. We adopted a cleanroom-free, micromilling-replica-molding strategy and a microfluidic liquid-pinning approach to streamline the fabrication and tumor model establishment. We also implemented a thin-film oxygen diffusion barrier design, which was optimized through COMSOL simulation, to support both two-dimensional (2-D) and three-dimensional (3-D) hypoxic models. We demonstrated that liquid-pinning enables an easy, injection-based micropatterning of cancer cells of a wide range of parameters, showing the high tunability of our design. Human breast cancer and prostate cancer cells were seeded and stained after 24 h of 2-D and 3-D culture to validate the natural induction of hypoxia. We further demonstrated the feasibility of the parallel microfluidic channel design to evaluate dual therapeutic conditions in the same device. Overall, our new microfluidic tumor model serves as a user-friendly, cost-effective, and highly scalable platform that provides spatiotemporal analysis of the hypoxic tumor microenvironments suitable for high-content biological studies and therapeutic discoveries.

Manipulation and Mechanical Deformation of Leukemia Cells by High-Frequency Ultrasound Single Beam.

Ultrasound single-beam acoustic tweezer system has attracted increasing attention in the field of biomechanics. Cell biomechanics play a pivotal role in leukemia cell functions. To better understand and compare the cell mechanics of the leukemia cells, herein, we fabricated an acoustic tweezer system in-house connected with a 50-MHz high-frequency cylinder ultrasound transducer. Selected leukemia cells (Jurkat, K562, and MV-411 cells) were cultured, trapped, and manipulated by high-frequency ultrasound single beam, which was transmitted from the ultrasound transducer without contacting any cells. The relative deformability of each leukemia cell was measured, characterized, and compared, and the leukemia cell (Jurkat cell) gaining the highest deformability was highlighted. Our results demonstrate that the high-frequency ultrasound single beam can be utilized to manipulate and characterize leukemia cells, which can be applied to study potential mechanisms in the immune system and cell biomechanics in other cell types.

A Pillar-Free Diffusion Device for Studying Chemotaxis on Supported Lipid Bilayers.

Chemotactic cell migration plays a crucial role in physiological and pathophysiological processes. In tissues, cells can migrate not only through extracellular matrix (ECM), but also along stromal cell surfaces via membrane-bound receptor-ligand interactions to fulfill critical functions. However, there remains a lack of models recapitulating chemotactic migration mediated through membrane-bound interactions. Here, using micro-milling, we engineered a multichannel diffusion device that incorporates a chemoattractant gradient and a supported lipid bilayer (SLB) tethered with membrane-bound factors that mimics stromal cell membranes. The chemoattractant channels are separated by hydrogel barriers from SLB in the cell loading channel, which enable precise control of timing and profile of the chemokine gradients applied on cells interacting with SLB. The hydrogel barriers are formed in pillar-free channels through a liquid pinning process, which eliminates complex cleanroom-based fabrications and distortion of chemoattractant gradient by pillars in typical microfluidic hydrogel barrier designs. As a proof-of-concept, we formed an SLB tethered with ICAM-1, and demonstrated its lateral mobility and different migratory behavior of Jurkat T cells on it from those on immobilized ICAM-1, under a gradient of chemokine CXCL12. Our platform can thus be widely used to investigate membrane-bound chemotaxis such as in cancer, immune, and stem cells.

E-Cadherin Regulates Mitochondrial Membrane Potential in Cancer Cells.

Epithelial cancer cells often have unusually higher mitochondrial membrane potential (ΔΨm) than their normal counterparts, which has been associated with increased invasiveness in vitro and higher metastatic potential in vivo. However, the mechanisms by which ΔΨm in cancer cells is regulated in tumor microenvironment (TME) remain unclear. In this study, we used an in vitro micropatterning platform to recapitulate biophysical confinement cues in the TME and investigated the mechanisms by which these regulate cancer cell ΔΨm. We found that micropatterning resulted in a spatial distribution of ΔΨm, which correlated with the level of E-cadherin mediated intercellular adhesion. There was a stark contrast in the spatial distribution of ΔΨm in the micropattern of E-cadherin-negative breast cancer cells (MDA-MB-231) compared to that of the high E-cadherin expressing (MCF-7) cancer cells. Disruption and knockout of E-cadherin adhesions rescued the low ΔΨm found at the center of MCF-7 micropatterns with high E-cadherin expression, while E-cadherin overexpression in MDA-MB-231 and MCF-7 cells lowered their ΔΨm at the micropattern center. These results show that E-cadherin plays an important role in regulating the ΔΨm of cancer cells in the context of biophysical cues in TME.

Engineering a Vascularized Hypoxic Tumor Model for Therapeutic Assessment.

Solid tumors in advanced cancer often feature a structurally and functionally abnormal vasculature through tumor angiogenesis, which contributes to cancer progression, metastasis, and therapeutic resistances. Hypoxia is considered a major driver of angiogenesis in tumor microenvironments. However, there remains a lack of in vitro models that recapitulate both the vasculature and hypoxia in the same model with physiological resemblance to the tumor microenvironment, while allowing for high-content spatiotemporal analyses for mechanistic studies and therapeutic evaluations. We have previously constructed a hypoxia microdevice that utilizes the metabolism of cancer cells to generate an oxygen gradient in the cancer cell layer as seen in solid tumor sections. Here, we have engineered a new composite microdevice-microfluidics platform that recapitulates a vascularized hypoxic tumor. Endothelial cells were seeded in a collagen channel formed by viscous fingering, to generate a rounded vascular lumen surrounding a hypoxic tumor section composed of cancer cells embedded in a 3-D hydrogel extracellular matrix. We demonstrated that the new device can be used with microscopy-based high-content analyses to track the vascular phenotypes, morphology, and sprouting into the hypoxic tumor section over a 7-day culture, as well as the response to different cancer/stromal cells. We further evaluated the integrity/leakiness of the vascular lumen in molecular delivery, and the potential of the platform to study the movement/trafficking of therapeutic immune cells. Therefore, our new platform can be used as a model for understanding tumor angiogenesis and therapeutic delivery/efficacy in vascularized hypoxic tumors.

Membrane-bound SCF and VCAM-1 synergistically regulate the morphology of hematopoietic stem cells.

Membrane-bound factors expressed by niche stromal cells constitute a unique class of localized cues and regulate the long-term functions of adult stem cells, yet little is known about the underlying mechanisms. Here, we used a supported lipid bilayer (SLB) to recapitulate the membrane-bound interactions between hematopoietic stem cells (HSCs) and niche stromal cells. HSCs cluster membrane-bound stem cell factor (mSCF) at the HSC-SLB interface. They further form a polarized morphology with aggregated mSCF under a large protrusion through a synergy with VCAM-1 on the bilayer, which drastically enhances HSC adhesion. These features are unique to mSCF and HSCs among the factors and hematopoietic populations we examined. The mSCF-VCAM-1 synergy and the polarized HSC morphology require PI3K signaling and cytoskeletal reorganization. The synergy also enhances nuclear retention of FOXO3a, a crucial factor for HSC maintenance, and minimizes its loss induced by soluble SCF. Our work thus reveals a unique role and signaling mechanism of membrane-bound factors in regulating stem cell morphology and function.

The complete mitochondrial genome of Hemitripterus villosus (Pallas, 1814) from Zhoushan archipelago.

In this study, we used whole genome sequencing to obtain the complete mitochondrial genome of Hemitripterus villosus. This mitochondrial genome, consisting of 17,449 base pairs (bp), contains 13 protein-coding genes, 2 ribosomal RNAs, 22 transfer RNAs and 2 noncoding control regions (control region and origin of light-strand replication) as those found in other vertebrates. Control region, of 1799 bp in length, is located between tRNAPro and tRNAPhe. We identified short tandem repeat sequences in the control region, which contributed largely to the relatively long mitogenome. The complete mitogenome data provides useful genetic markers for the studies on the molecular identification, phylogenetic analysis and conservation genetics.

The complete mitochondrial genome of Platycephalus sp.1 (Teleostei, Platycephalidae) obtained by whole genome sequencing.

In this study, we used next-generation sequencing to obtain the complete mitochondrial genome of Platycephalus sp.1. This mitochondrial genome, consisting of 16,552 base pairs (bp), contains 13 protein-coding genes, two ribosomal RNAs, 22 transfer RNAs, and two non-coding control regions (control region and origin of light-strand replication) as those found in other vertebrates. Control region, of 877 bp in length, is located between tRNAPro and tRNAPhe. Within the control region, typical conserved domains, such as the termination-associated sequence, central and conserved sequence blocks domains were identified. The overall base composition shows 25.83% of T, 29.98% of C, 27.01% of A, and 17.18% of G, with a slight A + T rich feature (52.84%). The complete mitogenome data provides useful genetic markers for the studies on the molecular identification, population genetics, phylogenetic analysis and conservation genetics.

Medulloblastoma uses GABA transaminase to survive in the cerebrospinal fluid microenvironment and promote leptomeningeal dissemination.

Medulloblastoma (MB) is a malignant pediatric brain tumor arising in the cerebellum. Although abnormal GABAergic receptor activation has been described in MB, studies have not yet elucidated the contribution of receptor-independent GABA metabolism to MB pathogenesis. We find primary MB tumors globally display decreased expression of GABA transaminase (ABAT), the protein responsible for GABA metabolism, compared with normal cerebellum. However, less aggressive WNT and SHH subtypes express higher ABAT levels compared with metastatic G3 and G4 tumors. We show that elevated ABAT expression results in increased GABA catabolism, decreased tumor cell proliferation, and induction of metabolic and histone characteristics mirroring GABAergic neurons. Our studies suggest ABAT expression fluctuates depending on metabolite changes in the tumor microenvironment, with nutrient-poor conditions upregulating ABAT expression. We find metastatic MB cells require ABAT to maintain viability in the metabolite-scarce cerebrospinal fluid by using GABA as an energy source substitute, thereby facilitating leptomeningeal metastasis formation.

Engineered in vitro tumor models for cell-based immunotherapy.

Tumor immunotherapy is rapidly evolving as one of the major pillars of cancer treatment. Cell-based immunotherapies, which utilize patient's own immune cells to eliminate cancer cells, have shown great promise in treating a range of malignancies, especially those of hematopoietic origins. However, their performance on a broader spectrum of solid tumor types still fall short of expectations in the clinical stage despite promising preclinical assessments. In this review, we briefly introduce cell-based immunotherapies and the inhibitory mechanisms in tumor microenvironments that may have contributed to this discrepancy. Specifically, a major obstacle to the clinical translation of cell-based immunotherapies is in the lack of preclinical models that can accurately assess the efficacies and mechanisms of these therapies in a (patho-)physiologically relevant manner. Lately, tissue engineering and organ-on-a-chip tools and microphysiological models have allowed for more faithful recapitulation of the tumor microenvironments, by incorporating crucial tumor tissue features such as cellular phenotypes, tissue architecture, extracellular matrix, physical parameters, and their dynamic interactions. This review summarizes the existing engineered tumor models with a focus on tumor immunology and cell-based immunotherapy. We also discuss some key considerations for the future development of engineered tumor models for immunotherapeutics. STATEMENT OF SIGNIFICANCE: Cell-based immunotherapies have shown great promise in treating hematological malignancies and some epithelial tumors. However, their performance on a broader spectrum of solid tumor types still fall short of expectations. Major obstacles include the inhibitory mechanisms in tumor microenvironments (TME) and the lack of preclinical models that can accurately assess the efficacies and mechanisms of cellular therapies in a (patho-)physiologically relevant manner. In this review, we introduce recent progress in tissue engineering and microphysiological models for more faithful recapitulation of TME for cell-based immunotherapies, and some key considerations for the future development of engineered tumor models. This overview will provide a better understanding on the role of engineered models in accelerating immunotherapeutic discoveries and clinical translations.

CCR2-targeted micelles for anti-cancer peptide delivery and immune stimulation.

Signaling between the CC chemokine receptor 2 (CCR2) with its ligand, monocyte chemoattractant protein-1 (MCP-1) promotes cancer progression by directly stimulating tumor cell proliferation and downregulating the expression of apoptotic proteins. Additionally, the MCP-1/CCR2 signaling axis drives the migration of circulating monocytes into the tumor microenvironment, where they mature into tumor-associated macrophages (TAMs) that promote disease progression through induction of angiogenesis, tissue remodeling, and suppression of the cytotoxic T lymphocyte (CTL) response. In order to simultaneously disrupt MCP-1/CCR2 signaling and target CCR2-expressing cancer cells for drug delivery, KLAK-MCP-1 micelles consisting of a CCR2-targeting peptide sequence (MCP-1 peptide) and the apoptotic KLAKLAK peptide were synthesized. In vitro, KLAK-MCP-1 micelles were observed to bind and induce cytotoxicity to cancer cells through interaction with CCR2. In vivo, KLAK-MCP-1 micelles inhibited tumor growth (34 ± 11%) in a subcutaneous B16F10 murine melanoma model despite minimal tumor accumulation upon intravenous injection. Tumors treated with KLAK-MCP1 demonstrated reduced intratumor CCR2 expression and altered infiltration of TAMs and CTLs as evidenced by immunohistochemical and flow cytometric analysis. These studies highlight the potential application of CCR2-targeted nanotherapeutic micelles in cancer treatment.

Bioengineering tools for probing intracellular events in T lymphocytes.

T lymphocytes are the central coordinator and executor of many immune functions. The activation and function of T lymphocytes are mediated through the engagement of cell surface receptors and regulated by a myriad of intracellular signaling network. Bioengineering tools, including imaging modalities and fluorescent probes, have been developed and employed to elucidate the cellular events throughout the functional lifespan of T cells. A better understanding of these events can broaden our knowledge in the immune systems biology, as well as accelerate the development of effective diagnostics and immunotherapies. Here we review the commonly used and recently developed techniques and probes for monitoring T lymphocyte intracellular events, following the order of intracellular events in T cells from activation, signaling, metabolism to apoptosis. The techniques introduced here can be broadly applied to other immune cells and cell systems. This article is categorized under: Immune System Diseases > Molecular and Cellular Physiology Immune System Diseases > Biomedical Engineering Infectious Diseases > Biomedical Engineering.

Non-invasive Optical Biomarkers Distinguish and Track the Metabolic Status of Single Hematopoietic Stem Cells.

Metabolism is a key regulator of hematopoietic stem cell (HSC) functions. There is a lack of real-time, non-invasive approaches to evaluate metabolism in single HSCs. Using fluorescence lifetime imaging microscopy, we developed a set of metabolic optical biomarkers (MOBs) from the auto-fluorescent properties of metabolic coenzymes NAD(P)H and FAD. The MOBs revealed the enhanced glycolysis, low oxidative metabolism, and distinct mitochondrial localization of HSCs. Importantly, the fluorescence lifetime of enzyme-bound NAD(P)H (τbound) can non-invasively monitor the glycolytic/lactate dehydrogenase activity in single HSCs. As a proof of concept for metabolism-based cell sorting, we further identified HSCs within the Lineage-cKit+Sca1+ (KLS) hematopoietic stem/progenitor population using MOBs and a machine-learning algorithm. Moreover, we revealed the dynamic changes of MOBs, and the association of longer τbound with enhanced glycolysis under HSC stemness-maintaining conditions during HSC culture. Our work thus provides a new paradigm to identify and track the metabolism of single HSCs non-invasively and in real time.