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Abstract The PD‐1 immune checkpoint‐based therapy has emerged as a promising therapy strategy for treating the malignant brain tumor glioblastoma (GBM). However, patient response varies in clinical trials, mainly due to the tumor heterogeneity and immunological resistance in the tumor microenvironment. To further understand how mechanistically the niche interplay and competition drive anti‐PD‐1 resistance, an in silico model is established to quantitatively describe the biological rationale of critical GBM‐immune interactions, such as tumor growth and apoptosis, T cell activation and cytotoxicity, and tumor‐associated macrophage (TAM) mediated immunosuppression. Such an in silico experimentation and predictive model, based on the in vitro microfluidic chip‐measured end‐point data and patient‐specific immunological characteristics, allows for a comprehensive and dynamic analysis of multiple TAM‐associated immunosuppression mechanisms against the anti‐PD‐1 immunotherapy. The computational model demonstrates that the TAM‐associated immunosuppression varies in severity across different GBM subtypes, which results in distinct tumor responses. The prediction results indicate that a combination therapy by co‐targeting of PD‐1 checkpoint and TAM‐associated CSF‐1R signaling can enhance the immune responses of GBM patients, especially those patients with mesenchymal GBM who are irresponsive to the single anti‐PD‐1 therapy. The development of a patient‐specific in silico–in vitro GBM model will help navigate and personalize immunotherapies for GBM patients. 

Abstract Sorting cells in a single cell per microwell format is of great interest to basic biological studies, biotherapeutics, and biosensing including cell phenotyping. For instance, isolation of individual immune T cells in rectangular microwells has been shown to empower the multiplex cytokine profiling at the single cell level for therapeutic applications. The present study, shows that there is an existing bias in temporal cytokine sensing that originates from random “unpredicted” positions of loaded cells within the rectangular microwells. To eliminate this bias, the isolated cells need to be well‐aligned with each other and relative to the sensing elements. Hence, an approach that utilizes the in situ formation and release of airplugs to localize cells toward the center of the rectangular microwells is reported. The chip includes 2250 microwells (each 500 × 50 × 20 µm³) arranged in nine rows. Results show 20% efficiency in trapping single T cells per microwells, where cells are localized within ± 3% of the center of microwells. The developed platform could provide real‐time dynamic and unbiased multiplex cytokine detection from single T cells for phenotyping and biotherapeutics studies. 

Abstract Macrophages are a predominant immune cell population that drive inflammatory responses and exhibit transitions in phenotype and function during tissue remodeling in disease and repair. Thus, engineering an immunomodulatory biomaterial has significant implications for resolving inflammation. Here, a biomimetic and photoresponsive hyaluronan (HA) hydrogel nanocomposite with tunable 3D extracellular matrix (ECM) adhesion sites for dynamic macrophage immunomodulation is engineered. Photodegradative alkoxylphenacyl‐based polycarbonate (APP) nanocomposites are exploited to permit user‐controlled Arg–Gly–Asp (RGD) adhesive peptide release and conjugation to a HA‐based ECM for real‐time integrin activation of macrophages encapsulated in 3D HA–APP nanocomposite hydrogels. It is demonstrated that photocontrolled 3D ECM–RGD peptide conjugation can activate αvβ3 integrin of macrophages, and periodic αvβ3 integrin activation can enhance anti‐inflammatory M2 macrophage polarization. Altogether, an emerging use of biomimetic, photoresponsive, and bioactive HA–APP nanocomposite hydrogel is highlighted to command 3D cell–ECM interactions for modulating macrophage polarization, which may shed light on cell–ECM interactions in innate immunity and inspire new biomaterial‐based immunomodulatory therapies. 

Abstract Rapid and accurate immune monitoring plays a decisive role in effectively treating immune‐related diseases especially at point‐of‐care, where an immediate decision on treatment is needed upon precise determination of the patient immune status. Derived from the emerging clinical demands, there is an urgent need for a cytokine immunoassay that offers unprecedented sensor performance with high sensitivity, throughput, and multiplexing capability, as well as short turnaround time at low system complexity, manufacturability, and scalability. In this paper, a label‐free, high throughput cytokine immunoassay based on a magnet patterned Fe₃O₄/Au core–shell nanoparticle (FACSNP) sensing array is developed. By exploiting the unique superparamagnetic and plasmonic properties of the core–shell nanomaterials, a facile microarray patterning technique is established that allows the fabrication of a uniform, self‐assembled microarray on a large surface area with remarkable tunability and scalability. The sensing performance of the FACSNP microarray is validated by real‐time detection of four cytokines in complex biological samples, showing high sensitivity (≈20 pg mL⁻¹), selectivity and throughput with excellent statistical accuracy. The developed immunoassay is successfully applied for rapid determination of the functional immunophenotype of leukemia tumor‐associated macrophages, manifesting its potential clinical applications for real‐time immune monitoring, early cancer detection, and therapeutic drug stratification toward personalized medicine. 

B cell acute lymphoblastic leukemia (B-ALL) blasts hijack the bone marrow (BM) microenvironment to form chemoprotective leukemic BM “niches,” facilitating chemoresistance and, ultimately, disease relapse. However, the ability to dissect these evolving, heterogeneous interactions among distinct B-ALL subtypes and their varying BM niches is limited with current in vivo methods. Here, we demonstrated an in vitro organotypic “leukemia-on-a-chip” model to emulate the in vivo B-ALL BM pathology and comparatively studied the spatial and genetic heterogeneity of the BM niche in regulating B-ALL chemotherapy resistance. We revealed the heterogeneous chemoresistance mechanisms across various B-ALL cell lines and patient-derived samples. We showed that the leukemic perivascular, endosteal, and hematopoietic niche-derived factors maintain B-ALL survival and quiescence (e.g., CXCL12 cytokine signal, VCAM-1/OPN adhesive signals, and enhanced downstream leukemia-intrinsic NF-κB pathway). Furthermore, we demonstrated the preclinical use of our model to test niche-cotargeting regimens, which may translate to patient-specific therapy screening and response prediction. 

Programmed cell death protein-1 (PD-1) checkpoint immunotherapy efficacy remains unpredictable in glioblastoma (GBM) patients due to the genetic heterogeneity and immunosuppressive tumor microenvironments. Here, we report a microfluidics-based, patient-specific ‘GBM-on-a-Chip’ microphysiological system to dissect the heterogeneity of immunosuppressive tumor microenvironments and optimize anti-PD-1 immunotherapy for different GBM subtypes. Our clinical and experimental analyses demonstrated that molecularly distinct GBM subtypes have distinct epigenetic and immune signatures that may lead to different immunosuppressive mechanisms. The real-time analysis in GBM-on-a-Chip showed that mesenchymal GBM niche attracted low number of allogeneic CD154+CD8+ T-cells but abundant CD163+ tumor-associated macrophages (TAMs), and expressed elevated PD-1/PD-L1 immune checkpoints and TGF-β1, IL-10, and CSF-1 cytokines compared to proneural GBM. To enhance PD-1 inhibitor nivolumab efficacy, we co-administered a CSF-1R inhibitor BLZ945 to ablate CD163+ M2-TAMs and strengthened CD154+CD8+ T-cell functionality and GBM apoptosis on-chip. Our ex vivo patient-specific GBM-on-a-Chip provides an avenue for a personalized screening of immunotherapies for GBM patients. 

Introduction— In response to external stress, cells alter their morphology, metabolic activity, and functions to mechanically adapt to the dynamic, local environment through cell allostasis. To explore mechanotransduction in cellular allostasis, we applied an integrated micromechanical system that combines an ‘ultrasound tweezers’-based mechanical stressor and a Förster resonance energy transfer (FRET)-based molecular force biosensor, termed “actinin-sstFRET,” to monitor in situ single-cell allostasis in response to transient stimulation in real time. Methods— The ultrasound tweezers utilize 1 Hz, 10-second transient ultrasound pulses to acoustically excite a lipid-encapsulated microbubble, which is bound to the cell membrane, and apply a pico- to nano-Newton range of forces to cells through an RGD-integrin linkage. The actinin-sstFRET molecular sensor, which engages the actin stress fibers in live cells, is used to map real-time actomyosin force dynamics over time. Then, the mechanosensitive behaviors were examined by profiling the dynamics in Ca2+ influx, actomyosin cytoskeleton (CSK) activity, and GTPase RhoA signaling to define a single-cell mechanical allostasis. Results—By subjecting a 1 Hz, 10-second physical stress, single vascular smooth muscle cells (VSMCs) were observed to remodeled themselves in a biphasic mechanical allostatic manner within 30 minutes that caused them to adjust their contractility and actomyosin activities. The cellular machinery that underscores the vital role of CSK equilibrium in cellular mechanical allostasis, includes Ca2+ influx, remodeling of actomyosin CSK and contraction, and GTPase RhoA signaling. Mechanical allostasis was observed to be compromised in VSMCs from patients with type II diabetes mellitus (T2DM), which could potentiate an allostatic maladaptation. Conclusions— By integrating tools that simultaneously permit localized mechanical perturbation and map actomyosin forces, we revealed distinct cellular mechanical allostasis profiles in our micromechanical system. Our findings of cell mechanical allostasis and maladaptation provide the potential for mechanophenotyping cells to reveal their pathogenic contexts and their biophysical mediators that underlie multi-etiological diseases such as diabetes, hypertension, or aging. 

Although many advanced biosensing techniques have been proposed for cytokine profiling, there are no clinically available methods that integrate high-resolution immune cell monitoring and in situ multiplexed cytokine detection together in a biomimetic tissue microenvironment. The primary challenge arises due to the lack of suitable label-free sensing techniques and difficulty for sensor integration. In this work, we demonstrated a novel integration of a localized-surface plasmon resonance (LSPR)-based biosensor with a biomimetic microfluidic ‘adipose-tissue-on-chip’ platform for an in situ label-free, high-throughput and multiplexed cytokine secretion analysis of obese adipose tissue. Using our established adipose-tissue-on-chip platform, we were able to monitor the adipose tissue initiation, differentiation, and maturation and simulate the hallmark formation of crown-like structures (CLSs) during pro-inflammatory stimulation. With integrated antibody-conjugated LSPR barcode sensor arrays, our platform enables simultaneous multiplexed measurements of pro-inflammatory (IL-6 and TNF-α) and anti-inflammatory (IL-10 and IL-4) cytokines secreted by the adipocytes and macrophages. As a result, our adipose-tissue-on-chip platform is capable of identifying stage-specific cytokine secretion profiles from a complex milieu during obesity progression, highlighting its potential as a high-throughput preclinical readout for personalized obesity treatment strategies.