Pluripotency state of mouse ES cells determines their contribution to self-organized layer formation by mesh closure on microstructured adhesion-limiting substrates.

Assembly of pluripotent stem cells to initiate self-organized tissue formation on engineered scaffolds is an important process in stem cell engineering. Pluripotent stem cells are known to exist in diverse pluripotency states, with heterogeneous subpopulations exhibiting differential gene expression levels, but how such diverse pluripotency states orchestrate tissue formation is still an unrevealed question. In this study, using microstructured adhesion-limiting substrates, we aimed to clarify the contribution to self-organized layer formation by mouse embryonic stem cells in different pluripotency states: ground and naïve state. We found that while ground state cells as well as sorted REX1-high expression cells formed discontinuous cell layers with limited lateral spread, naïve state cells could successfully self-organize to form a continuous layer by progressive mesh closure within 3 days. Using sequential immunofluorescence microscopy to examine the mesh closure process, we found that KRT8+ cells were particularly localized around unfilled holes, occasionally bridging the holes in a manner suggestive of their role in the closure process. These results highlight that compared with ground state cells, naïve state cells possess a higher capability to contribute to self-organized layer formation by mesh closure. Thus, this study provides insights with implications for the application of stem cells in scaffold-based tissue engineering.

Analysis of Microstructure of Granules Prepared by Continuous Twin Screw Granulator Using X-Ray Micro-computed Tomography.

Granules prepared by a continuous twin screw granulator (TSG) were analyzed by X-ray micro-computed tomography (X-ray µCT) and the relationships between porosity of granules and granule properties were investigated. A model formulation containing ibuprofen, lactose monohydrate, microcrystalline cellulose, and hydroxypropyl cellulose was used. The porosity of granules was measured by X-ray µCT and mercury porosimetry. The data sets obtained by both methods showed linear correlation despite different values, which were attributed to the resolution of X-ray µCT and a low-signal-to-noise ratio of the original cross-sectional images. The porosity of granules measured by X-ray µCT decreased from 11-14 to 6-7% as liquid-to-solid ratio (L/S) increased, while the standard deviation (S.D.) of the porosity of individual granules decreased from 4-5 to 2%. L/S affected the porosity of granules. By contrast, the effect of screw speed was not significant. Pressure transmission, G, which indicates the liquid dispersion in wet kneaded masses, increased as the porosity of granules and the S.D. decreased. The cross-sectional images showed that granules were densified as L/S increased. Based on these results, the effect of L/S on the porosity of granules can be explained by liquid dispersion and densification of the wet granules. The porosity of granules measured by X-ray µCT showed good linear correlation with friability and drug dissolution rate (R2 = 0.9107 and 0.8834, respectively). This study revealed that the drug dissolution rate was regulated by a disintegration step in which the porosity of granules plays an important role.

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.

NOT gated T cells that selectively target EGFR and other widely expressed tumor antigens.

Here, we show that a NOT gated cell therapy (Tmod) can exploit antigens such as epidermal growth factor receptor (EGFR) and human leukocyte antigen-E (HLA-E) which are widely expressed on cancer cells. Noncancerous cells-despite high expression of these antigens-are protected from cytotoxicity by the action of an inhibitory receptor ("blocker") via a mechanism that involves blocker modulation of CAR surface expression. The blocker is triggered by the product of a polymorphic HLA allele (e.g., HLA-A∗02) deleted in a significant subset of solid tumors via loss of heterozygosity. Moreover, Tmod constructs that target mouse homologs of EGFR or HLA-E for activation, and a mouse-equivalent of HLA-A∗02 for inhibition, protect mice from toxicity caused by the CAR alone. The blocker also controls graft vs. host response in allogeneic T cells in vitro, consistent with the use of Tmod cells for off-the-shelf therapy without additional gene-editing.

A carcinoembryonic antigen-specific cell therapy selectively targets tumor cells with HLA loss of heterozygosity in vitro and in vivo.

The CEACAM5 gene product [carcinoembryonic antigen (CEA)] is an attractive target for colorectal cancer because of its high expression in virtually all colorectal tumors and limited expression in most healthy adult tissues. However, highly active CEA-directed investigational therapeutics have been reported to be toxic, causing severe colitis because CEA is expressed on normal gut epithelial cells. Here, we developed a strategy to address this toxicity problem: the Tmod dual-signal integrator. CEA Tmod cells use two receptors: a chimeric antigen receptor (CAR) activated by CEA and a leukocyte Ig-like receptor 1 (LIR-1)-based inhibitory receptor triggered by human leukocyte antigen (HLA)-A*02. CEA Tmod cells exploit instances of HLA heterozygous gene loss in tumors to protect the patient from on-target, off-tumor toxicity. CEA Tmod cells potently killed CEA-expressing tumor cells in vitro and in vivo. But in contrast to a traditional CEA-specific T cell receptor transgenic T cell, Tmod cells were highly selective for tumor cells even when mixed with HLA-A*02-expressing cells. These data support further development of the CEA Tmod construct as a therapeutic candidate for colorectal cancer.

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.

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.

Edge-localized alteration in pluripotency state of mouse ES cells forming topography-confined layers on designed mesh substrates.

Self-organization of pluripotent stem cells during tissue formation is directed by the adhesion microenvironment, which defines the resulting tissue topography. Although the influence of tissue topography on pluripotency state has been inferred, this aspect of self-organization remains largely unexplored. In this study, to determine the effect of self-organized tissue topography on pluripotency loss, we designed novel island mesh substrates to confine the self-organization process of mouse embryonic stem cells, enabling us to generate isolated cell layers with an island-like topography and overhanging edges. Using immunofluorescence microscopy, we determined that cells at the tissue edge exhibited deformed nuclei associated with low OCT3/4, in contrast with cells nested in the tissue interior which had round-shaped nuclei and exhibited sustained OCT3/4 expression. Interestingly, F-actin and phospho-myosin light chain were visibly enriched at the tissue edge where ERK activation and elevated AP-2γ expression were also found to be localized, as determined using both immunofluorescence microscopy and RT-qPCR analysis. Since actomyosin contractility is known to cause ERK activation, these results suggest that mechanical condition at the tissue edge can contribute to loss of pluripotency leading to differentiation. Thus, our study draws attention to the influence of self-organized tissue topography in stem cell culture and differentiation.

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.

Extensive functional comparisons between chimeric antigen receptors and T cell receptors highlight fundamental similarities.

Though TCRs have been subject to limited engineering in the context of therapeutic design and optimization, they are used largely as found in nature. On the other hand, CARs are artificial, composed of different segments of proteins that function in the immune system. This characteristic raises the possibility of altered response to immune regulatory stimuli. Here we describe a large-scale, systematic comparison of CARs and TCRs across 5 different pMHC targets, with a total of 19 constructs examined in vitro. These functional measurements include CAR- and TCR-mediated activation, proliferation, and cytotoxicity in both acute and chronic settings. Surprisingly, we find no consistent difference between CARs and TCRs as receptor classes with respect to their relative sensitivity to major regulators of T cell activation: PD-L1, CD80/86 and IL-2. Though TCRs often emerge from human blood directly as potent, selective receptors, CARs must be heavily optimized to attain these properties for pMHC targets. Nonetheless, when iteratively improved and compared head to head in functional tests, CARs appear remarkably similar to TCRs with respect to immune modulation.

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.

Author Correction: Warm hole in Pacific Arctic sea ice cover forced mid-latitude Northern Hemisphere cooling during 2017-18.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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.

Modulation of adhesion microenvironment using mesh substrates triggers self-organization and primordial germ cell-like differentiation in mouse ES cells.

The cell adhesion microenvironment plays contributory roles in the induction of self-organized tissue formation and differentiation of pluripotent stem cells (PSCs). However, physical factors emanating from the adhesion microenvironment have been less investigated largely in part due to overreliance on biochemical approaches utilizing cytokines to drive in vitro developmental processes. Here, we report that a mesh culture technique can potentially induce mouse embryonic stem cells (mESCs) to self-organize and differentiate into cells expressing key signatures of primordial germ cells (PGCs) even with pluripotency maintained in the culture medium. Intriguingly, mESCs cultured on mesh substrates consisting of thin (5 µm-wide) strands and considerably large (200 µm-wide) openings which were set suspended in order to minimize the cell-substrate adhesion area, self-organized into cell sheets relying solely on cell-cell interactions to fill the large mesh openings by Day 2, and further into dome-shaped features around Day 6. Characterization using microarray analysis and immunofluorescence microscopy revealed that sheet-forming cells exhibited differential gene expressions related to PGCs as early as Day 2, but not other lineages such as epiblast, primitive endoderm, and trophectoderm, implying that the initial interaction with the mesh microenvironment and subsequent self-organization into cells sheets might have triggered PGC-like differentiation to occur differently from the previously reported pathway via epiblast-like differentiation. Overall, considering that the observed differentiation occurred without addition of known biochemical inducers, this study highlights that bioengineering techniques for modulating the adhesion microenvironment alone can be harnessed to coax PSCs to self-organize and differentiate, in this case, to a PGC-like state.

Warm hole in Pacific Arctic sea ice cover forced mid-latitude Northern Hemisphere cooling during 2017-18.

In North America and Asia, extreme cold weather characterized the winter of 2017-18. At the same time, the Pacific, the Bering Sea, and the Atlantic Arctic regions experienced anomalously low sea ice extent in the early winter. The jet stream dividing cold Arctic air from warm air deviated from normal zonal patterns northward into the ice-free areas north of the Bering Strait. Large southward jet stream pathways formed over Asia and America, allowing cold air to spread into Asia and the southern areas of North America. We hypothesise that the late autumn Bering Strait sea-ice anomaly and Pacific atmospheric rivers were partially responsible for the cold winter. We used data analyses and numerical experiments to test this hypothesis. We propose a positive feedback mechanism between the sea ice anomaly and atmospheric river activity, with anomalous south winds toward the sea ice anomaly potentially leading to more warm water injected by the wind-driven current through the Bering Strait. Our findings suggest that Poleward propagation of the atmospheric rivers made upper air warm, leading to their upgliding, which further heated the overlying air, causing poleward jet meanders. As a part of this response the jet stream meandered southward over Asia and North America, resulting in cold intrusions. We speculate that the positive feedback mechanism observed during the 2017-18 winter could recur in future years when the sea-ice reduction in the Pacific Arctic interacts with enhanced atmospheric river activity.

Spatial Regulation of Mitochondrial Heterogeneity by Stromal Confinement in Micropatterned Tumor Models.

Heterogeneity of mitochondrial activities in cancer cells exists across different disease stages and even in the same patient, with increased mitochondrial activities associated with invasive cancer phenotypes and circulating tumor cells. Here, we use a micropatterned tumor-stromal assay (µTSA) comprised of MCF-7 breast cancer cells and bone marrow stromal cells (BMSCs) as a model to investigate the role of stromal constraints in altering the mitochondrial activities of cancer cells within the tumor microenvironment (TME). Using microdissection and RNA sequencing, we revealed a differentially regulated pattern of gene expression related to mitochondrial activities and metastatic potential at the tumor-stromal interface. Gene expression was confirmed by immunostaining of mitochondrial mass, and live microscopic imaging of mitochondrial membrane potential (ΔΨm) and optical redox ratio. We demonstrated that physical constraints by the stromal cells play a major role in ΔΨm heterogeneity, which was positively associated with nuclear translocation of the YAP/TAZ transcriptional co-activators. Importantly, inhibiting actin polymerization and Rho-associated protein kinase disrupted the differential ΔΨm pattern. In addition, we showed a positive correlation between ΔΨm level and metastatic burden in vivo in mice injected with MDA-MB-231 breast cancer cells. This study supports a new regulatory role for the TME in mitochondrial heterogeneity and metastatic potential.

Evaluating CAR-T Cell Therapy in a Hypoxic 3D Tumor Model.

Despite its revolutionary success in hematological malignancies, chimeric antigen receptor T (CAR-T) cell therapy faces disappointing clinical results in solid tumors. The poor efficacy has been partially attributed to the lack of understanding in how CAR-T cells function in a solid tumor microenvironment. Hypoxia plays a critical role in cancer progression and immune editing, which potentially results in solid tumors escaping immunosurveillance and CAR-T cell-mediated cytotoxicity. Mechanistic studies of CAR-T cell biology in a physiological environment has been limited by the complexity of tumor-immune interactions in clinical and animal models, as well as by a lack of reliable in vitro models. A microdevice platform that recapitulates a 3D tumor section with a gradient of oxygen and integrates fluidic channels surrounding the tumor for CAR-T cell delivery is engineered. The design allows for the evaluation of CAR-T cell cytotoxicity and infiltration in the heterogeneous oxygen landscape of in vivo solid tumors at a previously unachievable scale in vitro.

Fabrication of liposomal doxorubicin exhibiting ultrasensitivity against phospholipase A2 for efficient pulmonary drug delivery to lung cancers.

Phospholipase A2 (PLA2) is expressed in inflammation-related tissue, including cancer tumors. We report that a hybrid liposome composed of phospholipid (DPPC) and PEGylated block-copolymer (Poloxamer 188) can rapidly release an encapsulated hydrophilic drug in the presence of PLA2. DPPC/P188 liposomes released approximately 80% of the encapsulated calcein (a fluorescence marker) within 10min in the presence of 120 mU of PLA2 at 37°C in vitro, whereas several other liposomal compositions used for inhalation therapy did not. DPPC/P188 liposomes were stable in the absence of PLA2 at 37°C after 60min incubation and drug release by PLA2 was dependent on the amount of P188 incorporated into the DPPC liposomes. Drug release from doxorubicin (DOX, anticancer drug)-loaded DPPC/P188 liposomes was facilitated at higher PLA2 concentrations and was dependent on the temperature and the presence of calcium ion, thus partially explaining PLA2-responsive drug release. DOX release from liposomes triggered by PLA2 exhibited the same cytotoxic effects on the A549 lung cancer cell line as did DOX in free solution. These findings suggest that DPPC/P188 liposomes are a promising drug carrier for delivering drug efficiently at PLA2-expressing sites such as inflammatory lung cancer.

A Microdevice Platform Recapitulating Hypoxic Tumor Microenvironments.

Hypoxia plays a central role in cancer progression and resistance to therapy. We have engineered a microdevice platform to recapitulate the intratumor oxygen gradients that drive the heterogeneous hypoxic landscapes in solid tumors. Our design features a "tumor section"-like culture by incorporating a cell layer between two diffusion barriers, where an oxygen gradient is established by cellular metabolism and physical constraints. We confirmed the oxygen gradient by numerical simulation and imaging-based oxygen sensor measurement. We also demonstrated spatially-resolved hypoxic signaling in cancer cells through immunostaining, gene expression assay, and hypoxia-targeted drug treatment. Our platform can accurately generate and control oxygen gradients, eliminates complex microfluidic handling, allows for incorporation of additional tumor components, and is compatible with high-content imaging and high-throughput applications. It is well suited for understanding hypoxia-mediated mechanisms in cancer disease and other biological processes, and discovery of new therapeutics.

A cost-effective micromilling platform for rapid prototyping of microdevices.

Micromilling has great potential in producing microdevices for lab-on-a-chip and organ-on-a-chip applications, but has remained under-utilized due to the high machinery costs and limited accessibility. In this paper, we assessed the machining capabilities of a low-cost 3-D mill in polycarbonate material, which were showcased by the production of microfluidic devices. The study demonstrates that this particular mill is well suited for the fabrication of multi-scale microdevices with feature sizes from micrometers to centimeters.