Probing the glioma micro-environment: analysis using biopsy in combination with ultra-fast cyclic immunolabeling.

The interaction between gliomas and the immune system is poorly understood and thus hindering development of effective immunotherapies for glioma patients. The immune response is highly variable during tumor development, and affected by therapies such as surgery, radiation, and chemotherapy. Currently, analysis of these local changes is difficult due to poor accessibility of the tumor and high-morbidity of sampling. In this study, we developed a model for repeat-biopsy in mice to study these local immunological changes over time. Using fine needle biopsy we were able to safely and repeatedly collect cells from intracranial tumors in mice. Ultra-fast cycling technology (FAST) was used for multi-cycle immunofluorescence of retrieved cells, and provided insights in the changing immune response over time. The combination of these techniques can be utilized to study changes in the immune response in glioma or other intracranial diseases over time, and in response to treatment within the same animal.

Hybrid LNP Prime Dendritic Cells for Nucleotide Delivery.

The efficient activation of professional antigen-presenting cells-such as dendritic cells (DC)-in tumors and lymph nodes is critical for the design of next-generation cancer vaccines and may be able to provide anti-tumor effects by itself through immune stimulation. The challenge is to stimulate these cells without causing excessive toxicity. It is hypothesized that a multi-pronged combinatorial approach to DC stimulation would allow dose reductions of innate immune receptor-stimulating TLR3 agonists while enhancing drug efficacy. Here, a hybrid lipid nanoparticle (LNP) platform is developed and tested for double-stranded RNA (polyinosinic:polycytidylic acid for TLR3 agonism) and immune modulator (L-CANDI) delivery. This study shows that the ≈120 nm hybrid nanoparticles-in-nanoparticles effectively eradicate tumors by themselves and generate long-lasting, durable anti-tumor immunity in mouse models.

Dual Immunostimulatory Pathway Agonism through a Synthetic Nanocarrier Triggers Robust Anti-Tumor Immunity in Murine Glioblastoma.

Myeloid cells are abundant, create a highly immunosuppressive environment in glioblastoma (GBM), and thus contribute to poor immunotherapy responses. Based on the hypothesis that small molecules can be used to stimulate myeloid cells to elicit anti-tumor effector functions, a synthetic nanoparticle approach is developed to deliver dual NF-kB pathway-inducing agents into these cells via systemic administration. Synthetic, cyclodextrin-adjuvant nanoconstructs (CANDI) with high affinity for tumor-associated myeloid cells are dually loaded with a TLR7 and 8 (Toll-like receptor, 7 and 8) agonist (R848) and a cIAP (cellular inhibitor of apoptosis protein) inhibitor (LCL-161) to dually activate these myeloid cells. Here CANDI is shown to: i) readily enter the GBM tumor microenvironment (TME) and accumulate at high concentrations, ii) is taken up by tumor-associated myeloid cells, iii) potently synergize payloads compared to monotherapy, iv) activate myeloid cells, v) fosters a "hot" TME with high levels of T effector cells, and vi) controls the growth of murine GBM as mono- and combination therapies with anti-PD1. Multi-pathway targeted myeloid stimulation via the CANDI platform can efficiently drive anti-tumor immunity in GBM.

Deep Learning Pipeline for Automated Cell Profiling from Cyclic Imaging.

Recent advances in microscopy allow scientists to generate vast amounts of biological data from a single biopsy sample. Cyclic fluorescence microscopy, in particular, enables multiple targets to be detected simultaneously. This, in turn, has deepened our understanding of tissue composition, cell-to-cell interactions, and cell signaling. Unfortunately, analysis of these datasets can be time-prohibitive due to the sheer volume of data. In this paper, we present CycloNET, a computational pipeline tailored for analyzing raw fluorescent images obtained through cyclic immunofluorescence. The automated pipeline pre-processes raw image files, quickly corrects for translation errors between imaging cycles, and leverages a pre-trained neural network to segment individual cells and generate single-cell molecular profiles. We applied CycloNET to a dataset of 22 human samples from head and neck squamous cell carcinoma patients and trained a neural network to segment immune cells. CycloNET efficiently processed a large-scale dataset (17 fields of view per cycle and 13 staining cycles per specimen) in 10 minutes, delivering insights at the single-cell resolution and facilitating the identification of rare immune cell clusters. We expect that this rapid pipeline will serve as a powerful tool to understand complex biological systems at the cellular level, with the potential to facilitate breakthroughs in areas such as developmental biology, disease pathology, and personalized medicine.

TNIK Inhibition Has Dual Synergistic Effects on Tumor and Associated Immune Cells.

Treatment with checkpoint inhibitors can be extraordinarily effective in a fraction of patients, particularly those whose tumors are pre-infiltrated by T cells. In others, efficacy is considerably lower, which has led to interest in developing strategies for sensitization to immunotherapy. Using various colorectal cancer mouse models, it is shown that the use of Traf2 and Nck-interacting protein kinase inhibitors (TNIKi) unexpectedly increases tumor infiltration by PD-1+ CD8+ T cells, thus contributing to tumor control. This appears to happen by two independent mechanisms, by inducing immunogenic cell death and separately by directly activating CD8. The use of TNIKi achieves complete tumor control in 50% of mice when combined with checkpoint inhibitor targeting PD-1. These findings reveal immunogenic properties of TNIKi and indicate that the proportion of colorectal cancers responding to checkpoint therapy can be increased by combining it with immunogenic kinase inhibitors.

Multiplexed single-cell analysis of FNA allows accurate diagnosis of salivary gland tumors.

Diagnosing salivary gland tumors (SGTs) through fine-needle aspiration (FNA) biopsies is challenging due to the overlapping cytomorphologic features between benign and malignant tumors. The authors developed an innovative, multiplexed cycling technology for the rapid analyses of single cells obtained from FNA that can facilitate the molecular analyses and diagnosis of SGTs. Antibodies against 29 protein markers associated with 7 SGT subtypes were validated and chemically modified via custom linker-bio-orthogonal probes (FAST). Single-cell homogenates and FNA samples were profiled by FAST cyclic imaging and computational analysis. A prediction model was generated using a training set of 151,926 cells from primary SGTs (N = 26) and validated on a separate cohort (N = 30). Companion biomarker testing, such as neurotrophic tyrosine receptor kinase (NTRK), was also assessed with the FAST technology. The FAST molecular diagnostic assay was able to distinguish between benign and malignant SGTs with an accuracy of 0.86 for single-cell homogenate samples and 0.88 for FNA samples. Profiling of multiple markers as compared to a single marker increased the diagnostic accuracy (0.82 as compared to 0.65-0.74, respectively), independent of the cell number sampled. NTRK expression was also assessed by the FAST assay, highlighting the potential therapeutic application of this technology. Application of the novel multiplexed single-cell technology facilitates rapid biomarker testing from FNA samples at low cost. The customizable and modular FAST-FNA approach has relevance to multiple pathologies and organ systems where cytologic samples are often scarce and/or indeterminate resulting in improved diagnostic workflows and timely therapeutic clinical decision-making.

Hydrogel Stamping for Rapid, Multiplexed, Point-of-Care Immunostaining of Cells and Tissues.

In the era of precision oncology, multicolor fluorescence imaging has become a core technology for multiplexed molecular analysis of cellular and tissue specimens. However, conventional solution-based staining is labor-intensive and time-consuming and requires considerable expertise to yield optimal results, which creates difficulties for employing this technology in resource-limited settings. Here, we report a new immunostaining method based on hydrogel stamping, which is simple, fast, easy to use, and reproducible. We showed that a hydrophilic hydrogel stamp could effectively transfer fluorescent antibodies to targets and withdraw an excess solution when the reaction is completed, obviating the need for extra washing. This unique property allows for quality immunostaining in 5 min for cells using one-eighth of antibody consumption compared to the conventional solution-based method. Furthermore, we implemented fluorescence quenching and immunocycling with hydrogel staining for multiplexed analysis of 9 protein markers at a single cell level. Finally, we applied the immunocycling method to human breast cancer tissue samples and showed quality immunostaining over a large area (∼2 cm2) in 30 min for molecular subtyping of breast cancer. The hydrogel immunostaining could open new opportunities for rapid, automated, and multiplexed profiling in compact point-of-care systems for molecular cancer diagnosis.

Cellular point-of-care diagnostics using an inexpensive layer-stack microfluidic device.

Cellular analyses are increasingly used to diagnose diseases at point-of-care and global healthcare settings. Some analyses are simple as they rely on chromogenic stains (blood counts, malaria) but others often require higher multiplexing to define and quantitate cell populations (cancer diagnosis, immunoprofiling). Simplifying the latter with inexpensive solutions represents a current bottleneck in designing start-end pipelines. Based on the hypothesis that novel film adhesives could be used to create inexpensive disposable devices, we tested a number of different designs and materials, to rapidly perform 12-15 channel single-cell imaging. Using an optimized passive pumping layer-stack microfluidic (PLASMIC) device (<1 $ in supplies) we show that rapid, inexpensive cellular analysis is feasible.

Integrated Analytical System for Clinical Single-Cell Analysis.

High-dimensional analyses of cancers can potentially be used to better define cancer subtypes, analyze the complex tumor microenvironment, and perform cancer cell pathway analyses for drug trials. Unfortunately, integrated systems that allow such analyses in serial fine needle aspirates within a day or at point-of-care currently do not exist. To achieve this, an integrated immunofluorescence single-cell analyzer (i2SCAN) for deep profiling of directly harvested cells is developed. By combining a novel cellular imaging system, highly cyclable bioorthogonal FAST antibody panels, and integrated computational analysis, it is shown that same-day analysis is possible in thousands of harvested cells. It is demonstrated that the i2SCAN approach allows comprehensive analysis of breast cancer samples obtained by fine needle aspiration or core tissues. The method is a rapid, robust, and low-cost solution to high-dimensional analysis of scant clinical specimens.

A rapid assay provides on-site quantification of tetrahydrocannabinol in oral fluid.

Tetrahydrocannabinol (THC), the primary psychoactive ingredient of cannabis, impairs cognitive and motor function in a concentration-dependent fashion. Drug testing is commonly performed for employment and law enforcement purposes; however, available tests produce low-sensitive binary results (lateral flow assays) or have long turnaround (gas chromatography­mass spectrometry). To enable on-site THC quantification in minutes, we developed a rapid assay for oral THC analysis called EPOCH (express probe for on-site cannabis inhalation). EPOCH features distinctive sensor design such as a radial membrane and transmission optics, all contained in a compact cartridge. This integrated approach permitted assay completion within 5 min with a detection limit of 0.17 ng/ml THC, which is below the regulatory guideline (1 ng/ml). As a proof of concept for field testing, we applied EPOCH to assess oral fluid samples from cannabis users (n = 43) and controls (n = 43). EPOCH detected oral THC in all specimens from cannabis smokers (median concentration, 478 ng/ml) and THC-infused food consumers. Longitudinal monitoring showed a fast drop in THC concentrations within the first 6 hours of cannabis smoking (half-life, 1.4 hours).

Rapid Serial Immunoprofiling of the Tumor Immune Microenvironment by Fine Needle Sampling.

PURPOSE: There is increasing effort to discover and integrate predictive and/or prognostic biomarkers into treatment algorithms. While tissue-based methods can reveal tumor-immune cell compositions at a single time point, we propose that single-cell sampling via fine needle aspiration (FNA) can facilitate serial assessment of the tumor immune microenvironment (TME) with a favorable risk-benefit profile. EXPERIMENTAL DESIGN: Primary antibodies directed against 20 murine and 25 human markers of interest were chemically modified via a custom linker-bio-orthogonal quencher (FAST) probe. A FAST-FNA cyclic imaging and analysis pipeline were developed to derive quantitative response scores. Single cells were harvested via FNA and characterized phenotypically and functionally both in preclinical and human samples using the newly developed FAST-FNA assay. RESULTS: FAST-FNA samples analyzed manually versus the newly developed deep learning-assisted pipeline gave highly concordant results. Subsequently, an agreement analysis showed that FAST and flow cytometry of surgically resected tumors were positively correlated with an R2 = 0.97 in preclinical samples and an R2 = 0.86 in human samples with the detection of the relevant tumor and immune biomarkers of interest. Finally, the feasibility of applying this minimally invasive approach to analyze the TME during immunotherapy was assessed in patients with cancer revealing local antitumor immune programs. CONCLUSIONS: The FAST-FNA is an innovative technology that combines bio-orthogonal chemistry coupled with a computational analysis pipeline for the comprehensive profiling of single cells obtained through FNA. This is the first demonstration that the complex and rapidly evolving TME during treatment can be accurately and serially measured by simple FNA.

The effect of continuity of care on medical costs in patients with chronic shoulder pain.

Unnecessary surgery could be prevented through continuity of care (COC). The present study aimed to investigate the relationships between COC, surgery and cost associated with chronic shoulder pain. We used the Health Insurance Review and Assessment Service national patient sample (HIRA-NPS) in 2017. A total of 1717 patients were included. Bice-Boxerman Continuity of Care Index was used as the indicator for measuring the COC. Occurrence of surgery, associated costs, and direct medical costs were analysed. Logistic regression, a two-part model with recycled predictions and generalized linear model with gamma distribution were used. The majority of patients were 40-65 years old (high COC: 68.4%; low COC: 64.4%). The odds ratio (OR) for surgery was 0.41 in the high-COC group compared to the low COC group (95% CI, 0.20 to 0.84). Direct medical cost was 14.09% (95% CI, 8.12% to 19.66%) and 58.00% lower in surgery cost (95% CI, 57.95 to 58.05) in the high-COC group. Interaction with COC and shoulder impingement syndrome was significant lower in direct medical cost (15.05% [95% CI, 1.81% to 26.51%]). High COC was associated with low medical cost in patients diagnosed with chronic shoulder pain.

Radio frequency heating and material processing using carbon susceptors.

Carbon nanomaterials have been shown to rapidly evolve heat in response to electromagnetic fields. Initial studies focused on the use of microwaves, but more recently, it was discovered that carbon nanomaterial systems heat in response to electric fields in the radio frequency range (RF, 1-200 MHz). This is an exciting development because this range of radio frequencies is safe and versatile compared to microwaves. Additional RF susceptor materials include other carbonaceous materials such as carbon black, graphite, graphene oxide, laser-induced graphene, and carbon fibers. Such conductive fillers can be dispersed in matrices such as polymer or ceramics; these composites heat rapidly when stimulated by electromagnetic waves. These findings are valuable for materials processing, where volumetric and/or targeted heating are needed, such as curing composites, bonding multi-material surfaces, additive manufacturing, chemical reactions, actuation, and medical ablation. By changing the loading of these conductive RF susceptors in the embedding medium, material properties can be customized to achieve different heating rates, with possible other benefits in thermo-mechanical properties. Compared to traditional heating and processing methods, RF heating provides faster heating rates with lower infrastructure requirements and better energy efficiency; non-contact RF applicators or capacitors can be used for out-of-oven processing, allowing for distributed manufacturing.

CytoPAN-Portable cellular analyses for rapid point-of-care cancer diagnosis.

Rapid, automated, point-of-care cellular diagnosis of cancer remains difficult in remote settings due to lack of specialists and medical infrastructure. To address the need for same-day diagnosis, we developed an automated image cytometry system (CytoPAN) that allows rapid breast cancer diagnosis of scant cellular specimens obtained by fine needle aspiration (FNA) of palpable mass lesions. The system is devoid of moving parts for stable operations, harnesses optimized antibody kits for multiplexed analysis, and offers a user-friendly interface with automated analysis for rapid diagnoses. Through extensive optimization and validation using cell lines and mouse models, we established breast cancer diagnosis and receptor subtyping in 1 hour using as few as 50 harvested cells. In a prospective patient cohort study (n = 68), we showed that the diagnostic accuracy was 100% for cancer detection and the receptor subtyping accuracy was 96% for human epidermal growth factor receptor 2 and 93% for hormonal receptors (ER/PR), two key biomarkers associated with breast cancer. A combination of FNA and CytoPAN offers faster, less invasive cancer diagnoses than the current standard (core biopsy and histopathology). This approach should enable the ability to more rapidly diagnose breast cancer in global and remote settings.

Ultra-fast Cycling for Multiplexed Cellular Fluorescence Imaging.

Rapid analysis of single and scant cell populations is essential in modern diagnostics, yet existing methods are often limited and slow. Herein, we describe an ultra-fast, highly efficient cycling method for the analysis of single cells based on unique linkers for tetrazine (Tz)/trans-cyclooctene (TCO)-mediated quenching. Surprisingly, the quenching reaction rates were more than 3 orders of magnitude faster (t1/2 <1 s) than predicted. This allowed multi-cycle staining and immune cell profiling within an hour, leveraging the accelerated kinetics to open new diagnostic possibilities for rapid cellular analyses.

Ultra-fast cycling for multiplexed cellular fluorescence imaging.

Rapid analysis of single and scant cell populations is essential in modern diagnostics, yet existing methods are often limited and slow. Here we describe an ultra-fast, highly efficient cycling method for the analysis of single cells based on unique linkers for tetrazine (Tz) / trans-cyclooctene (TCO) mediated quenching. Surprisingly, the quenching reaction rates were more than 3 orders of magnitude faster (t1/2 < 1 sec) than predicted. This allowed multi-cycle staining and immune cell profiling within an hour, leveraging the accelerated kinetics to open new diagnostic possibilities for rapid cellular analyses.

Multichannel digital heteronuclear magnetic resonance biosensor.

Low-field, mobile NMR systems are increasingly used across diverse fields, including medical diagnostics, food quality control, and forensics. The throughput and functionality of these systems, however, are limited due to their conventional single-channel detection: one NMR probe exclusively uses an NMR console at any given time. Under this design, multi-channel detection could only be accomplished by either serially accessing individual probes or stacking up multiple copies of NMR electronics; this approach still retains limitations such as long assay times and increased system complexity. Here we present a new scalable architecture, HERMES (hetero-nuclear resonance multichannel electronic system), for versatile, high-throughput NMR analyses. HERMES exploits the concept of software-defined radio by virtualizing NMR electronics in the digital domain. This strategy i) creates multiple NMR consoles without adding extra hardware; ii) acquires signals from multiple NMR channels in parallel; and iii) operates in wide frequency ranges. All of these functions could be realized on-demand in a single compact device. We interfaced HERMES with an array of NMR probes; the combined system simultaneously measured NMR relaxation from multiple samples and resolved spectra of hetero-nuclear spins (1H, 19F, 13C). For potential diagnostic uses, we applied the system to detect dengue fever and molecularly profile cancer cells through multi-channel protein assays. HERMES holds promise as a powerful analytical tool that enables rapid, reconfigurable, and parallel detection.

Age-related tumor growth in mice is related to integrin α 4 in CD8+ T cells.

Cancer incidence increases with age, but paradoxically, cancers have been found to grow more quickly in young mice compared with aged ones. The cause of differential tumor growth has been debated and, over time, attributed to faster tumor cell proliferation, decreased tumor cell apoptosis, and/or increased angiogenesis in young animals. Despite major advances in our understanding of tumor immunity over the past 2 decades, little attention has been paid to comparing immune cell populations in young and aged mice. Using mouse colon adenocarcinoma model MC38 implanted in young and mature mice, we show that age substantially influences the number of tumor-infiltrating cytotoxic CD8+ T cells, which control cancer progression. The different tumor growth pace in young and mature mice was abrogated in RAG1null mice, which lack mature T and B lymphocytes, and upon selective depletion of endogenous CD8+ cells. Transcriptome analysis further indicated that young mice have decreased levels of the Itga4 gene (CD49d, VLA-4) in tumor-infiltrating lymphocytes when compared with mature mice. Hypothesizing that VLA-4 can have a tumor-protective effect, we depleted the protein, which resulted in accelerated tumor growth in mature mice. These observations may explain the paradoxical growth rates observed in murine cancers, point to the central role of VLA-4 in controlling tumor growth, and open new venues to therapeutic manipulation.

Arg1 expression defines immunosuppressive subsets of tumor-associated macrophages.

Tumor-associated macrophages (TAM) have attracted attention as they can modulate key cancer-related activities, yet TAM represent a heterogenous group of cells that remain incompletely characterized. In growing tumors, TAM are often referred to as M2-like macrophages, which are cells that display immunosuppressive and tumorigenic functions and express the enzyme arginase 1 (Arg1). Methods: Here we combined high resolution intravital imaging with single cell RNA seq to uncover the topography and molecular profiles of immunosuppressive macrophages in mice. We further assessed how immunotherapeutic interventions impact these cells directly in vivo. Results: We show that: i) Arg1+ macrophages are more abundant in tumors compared to other organs; ii) there exist two morphologically distinct subsets of Arg1 TAM defined by previously unknown markers (Gbp2b, Bst1, Sgk1, Pmepa1, Ms4a7); iii) anti-Programmed Cell Death-1 (aPD-1) therapy decreases the number of Arg1+ TAM while increasing Arg1- TAM; iv) accordingly, pharmacological inhibition of arginase 1 does not synergize with aPD-1 therapy. Conclusion: Overall, this research shows how powerful complementary single cell analytical approaches can be used to improve our understanding of drug action in vivo.

Stem cell aging: mechanisms, regulators and therapeutic opportunities.

Aging tissues experience a progressive decline in homeostatic and regenerative capacities, which has been attributed to degenerative changes in tissue-specific stem cells, stem cell niches and systemic cues that regulate stem cell activity. Understanding the molecular pathways involved in this age-dependent deterioration of stem cell function will be critical for developing new therapies for diseases of aging that target the specific causes of age-related functional decline. Here we explore key molecular pathways that are commonly perturbed as tissues and stem cells age and degenerate. We further consider experimental evidence both supporting and refuting the notion that modulation of these pathways per se can reverse aging phenotypes. Finally, we ask whether stem cell aging establishes an epigenetic 'memory' that is indelibly written or one that can be reset.