Deep learning unlocks label-free viability assessment of cancer spheroids in microfluidics.

Despite recent advances in cancer treatment, refining therapeutic agents remains a critical task for oncologists. Precise evaluation of drug effectiveness necessitates the use of 3D cell culture instead of traditional 2D monolayers. Microfluidic platforms have enabled high-throughput drug screening with 3D models, but current viability assays for 3D cancer spheroids have limitations in reliability and cytotoxicity. This study introduces a deep learning model for non-destructive, label-free viability estimation based on phase-contrast images, providing a cost-effective, high-throughput solution for continuous spheroid monitoring in microfluidics. Microfluidic technology facilitated the creation of a high-throughput cancer spheroid platform with approximately 12 000 spheroids per chip for drug screening. Validation involved tests with eight conventional chemotherapeutic drugs, revealing a strong correlation between viability assessed via LIVE/DEAD staining and phase-contrast morphology. Extending the model's application to novel compounds and cell lines not in the training dataset yielded promising results, implying the potential for a universal viability estimation model. Experiments with an alternative microscopy setup supported the model's transferability across different laboratories. Using this method, we also tracked the dynamic changes in spheroid viability during the course of drug administration. In summary, this research integrates a robust platform with high-throughput microfluidic cancer spheroid assays and deep learning-based viability estimation, with broad applicability to various cell lines, compounds, and research settings.

Engineered Bio-Based Hydrogels for Cancer Immunotherapy.

Immunotherapy represents a revolutionary paradigm in cancer management, showcasing its potential to impede tumor metastasis and recurrence. Nonetheless, challenges including limited therapeutic efficacy and severe immune-related side effects are frequently encountered, especially in solid tumors. Hydrogels, a class of versatile materials featuring well-hydrated structures widely used in biomedicine, offer a promising platform for encapsulating and releasing small molecule drugs, biomacromolecules, and cells in a controlled manner. Immunomodulatory hydrogels present a unique capability for augmenting immune activation and mitigating systemic toxicity through encapsulation of multiple components and localized administration. Notably, hydrogels based on biopolymers have gained significant interest owing to their biocompatibility, environmental friendliness, and ease of production. This review delves into the recent advances in bio-based hydrogels in cancer immunotherapy and synergistic combinatorial approaches, highlighting their diverse applications. It is anticipated that this review will guide the rational design of hydrogels in the field of cancer immunotherapy, fostering clinical translation and ultimately benefiting patients.

Tumor Microenvironment-Responsive Nanoparticles Amplifying STING Signaling Pathway for Cancer Immunotherapy.

Insufficient activation of the stimulator of interferon genes (STING) signaling pathway and profoundly immunosuppressive microenvironment largely limits the effect of cancer immunotherapy. Herein, tumor microenvironment (TME)-responsive nanoparticles (PMM NPs) are exploited that simultaneously harness STING and Toll-like receptor 4 (TLR4) to augment STING activation via TLR4-mediated nuclear factor-kappa B signaling pathway stimulation, leading to the increased secretion of type I interferons (i.e., 4.0-fold enhancement of IFN-ß) and pro-inflammatory cytokines to promote a specific T cell immune response. Moreover, PMM NPs relieve the immunosuppression of the TME by decreasing the percentage of regulatory T cells, and polarizing M2 macrophages to the M1 type, thus creating an immune-supportive TME to unleash a cascade adaptive immune response. Combined with an anti-PD-1 antibody, synergistic efficacy is achieved in both inflamed colorectal cancer and noninflamed metastatic breast tumor models. Moreover, rechallenging tumor-free animals with homotypic cells induced complete tumor rejection, indicating the generation of systemic antitumor memory. These TME-responsive nanoparticles may open a new avenue to achieve the spatiotemporal orchestration of STING activation, providing a promising clinical candidate for next-generation cancer immunotherapy.

Single-cell morphological and transcriptome analysis unveil inhibitors of polyploid giant breast cancer cells in vitro.

Considerable evidence suggests that breast cancer therapeutic resistance and relapse can be driven by polyploid giant cancer cells (PGCCs). The number of PGCCs increases with the stages of disease and therapeutic stress. Given the importance of PGCCs, it remains challenging to eradicate them. To discover effective anti-PGCC compounds, there is an unmet need to rapidly distinguish compounds that kill non-PGCCs, PGCCs, or both. Here, we establish a single-cell morphological analysis pipeline with a high throughput and great precision to characterize dynamics of individual cells. In this manner, we screen a library to identify promising compounds that inhibit all cancer cells or only PGCCs (e.g., regulators of HDAC, proteasome, and ferroptosis). Additionally, we perform scRNA-Seq to reveal altered cell cycle, metabolism, and ferroptosis sensitivity in breast PGCCs. The combination of single-cell morphological and molecular investigation reveals promising anti-PGCC strategies for breast cancer treatment and other malignancies.

Manganese-Enriched Zinc Peroxide Functional Nanoparticles for Potentiating Cancer Immunotherapy.

Immunotherapies have shown high clinical success, however, the therapeutical efficacy is largely restrained by insufficient immune activation and an immunosuppressive microenvironment. Herein, we report tumor microenvironment (TME)-responsive manganese-enriched zinc peroxide nanoparticles (MONPs) for synergistic cancer immunotherapy by inducing the immunogenic death (ICD) of cancer cells and activating the stimulator of the interferon gene (STING) pathway. MONPs especially disassociate upon exposure to acidic tumor tissue and in situ generate •OH for the ICD effect. Moreover, Mn2+ activated the STING and synergistically induced the secretion of type I interferon and inflammatory cytokines for specific T cell responses. Meanwhile, MONPs relieved the immunosuppression of TME through decreasing Tregs and polarizing M2 macrophages to the M1 type to unleash a cascade adaptive immune response. In combination with the anti-PD-1 antibody, MONPs showed superior efficacy in inhibiting tumor growth and preventing lung metastasis. Our study demonstrates the feasibility of functional nanoparticles to amplify STING innate stimulation, showing a prominent strategy for cancer immunotherapy.

Microfluidic single-cell migration chip reveals insights into the impact of extracellular matrices on cell movement.

Cell migration is a complex process that plays a crucial role in normal physiology and pathologies such as cancer, autoimmune diseases, and mental disorders. Conventional cell migration assays face limitations in tracking a large number of individual migrating cells. To address this challenge, we have developed a high-throughput microfluidic cell migration chip, which seamlessly integrates robotic liquid handling and computer vision to swiftly monitor the movement of 3200 individual cells, providing unparalleled single-cell resolution for discerning distinct behaviors of the fast-moving cell population. This study focuses on the ECM's role in regulating cellular migration, utilizing this cutting-edge microfluidic technology to investigate the impact of ten different ECMs on triple-negative breast cancer cell lines. We found that collagen IV, collagen III, and collagen I coatings were the top enhancers of cell movement. Combining these ECMs increased cell motility, but the effect was sub-additive. Furthermore, we examined 87 compounds and found that while some compounds inhibited migration on all substrates, significantly distinct effects on differently coated substrates were observed, underscoring the importance of considering ECM coating. We also utilized cells expressing a fluorescent actin reporter and observed distinct actin structures in ECM-interacting cells. ScRNA-Seq analysis revealed that ECM coatings induced EMT and enhanced cell migration. Finally, we identified genes that were particularly up-regulated by collagen IV and the selective inhibitors successfully blocked cell migration on collagen IV. Overall, the study provides insights into the impact of various ECMs on cell migration and dynamics of cell movement with implications for developing therapeutic strategies to combat diseases related to cell motility.

Aldose reductase inhibition decelerates optic nerve degeneration by alleviating retinal microglia activation.

As part of the central nervous system (CNS), retinal ganglion cells (RGCs) and their axons are the only neurons in the retina that transmit visual signals from the eye to the brain via the optic nerve (ON). Unfortunately, they do not regenerate upon injury in mammals. In ON trauma, retinal microglia (RMG) become activated, inducing inflammatory responses and resulting in axon degeneration and RGC loss. Since aldose reductase (AR) is an inflammatory response mediator highly expressed in RMG, we investigated if pharmacological inhibition of AR can attenuate ocular inflammation and thereby promote RGC survival and axon regeneration after ON crush (ONC). In vitro, we discovered that Sorbinil, an AR inhibitor, attenuates BV2 microglia activation and migration in the lipopolysaccharide (LPS) and monocyte chemoattractant protein-1 (MCP-1) treatments. In vivo, Sorbinil suppressed ONC-induced Iba1 + microglia/macrophage infiltration in the retina and ON and promoted RGC survival. Moreover, Sorbinil restored RGC function and delayed axon degeneration one week after ONC. RNA sequencing data revealed that Sorbinil protects the retina from ONC-induced degeneration by suppressing inflammatory signaling. In summary, we report the first study demonstrating that AR inhibition transiently protects RGC and axon from degeneration, providing a potential therapeutic strategy for optic neuropathies.

High-Throughput Cellular Heterogeneity Analysis in Cell Migration at the Single-Cell Level.

Cancer cell migration represents an essential step toward metastasis and cancer deaths. However, conventional drug discovery focuses on cytotoxic and growth-inhibiting compounds rather than inhibitors of migration. Drug screening assays generally measure the average response of many cells, masking distinct cell populations that drive metastasis and resist treatments. Here, this work presents a high-throughput microfluidic cell migration platform that coordinates robotic liquid handling and computer vision for rapidly quantifying individual cellular motility. Using this innovative technology, 172 compounds were tested and a surprisingly low correlation between migration and growth inhibition was found. Notably, many compounds were found to inhibit migration of most cells while leaving fast-moving subpopulations unaffected. This work further pinpoints synergistic drug combinations, including Bortezomib and Danirixin, to stop fast-moving cells. To explain the observed cell behaviors, single-cell morphological and molecular analysis were performed. These studies establish a novel technology to identify promising migration inhibitors for cancer treatment and relevant applications.

Engineered Nanoprobes for Immune Activation Monitoring.

The activation of the immune system is critical for cancer immunotherapy and treatments of inflammatory diseases. Non-invasive visualization of immunoactivation is designed to monitor the dynamic nature of the immune response and facilitate the assessment of therapeutic outcomes, which, however, remains challenging. Conventional imaging modalities, such as positron emission tomography, computed tomography, etc., were utilized for imaging immune-related biomarkers. To explore the dynamic immune monitoring, probes with signals correlated to biomarkers of immune activation or prognosis are urgently needed. These emerging molecular probes, which turn on the signal only in the presence of the intended biomarker, can improve the detection specificity. These probes with "turn on" signals enable non-invasive, dynamic, and real-time imaging with high sensitivity and efficiency, showing significance for multifunctionality/multimodality imaging. As a result, more and more innovative engineered nanoprobes combined with diverse imaging modalities were developed to assess the activation of the immune system. In this work, we comprehensively review the recent and emerging advances in engineered nanoprobes for monitoring immune activation in cancer or other immune-mediated inflammatory diseases and discuss the potential in predicting the efficacy following treatments. Research on real-time in vivo immunoimaging is still under exploration, and this review can provide guidance and facilitate the development and application of next-generation imaging technologies.

LANCE: a Label-Free Live Apoptotic and Necrotic Cell Explorer Using Convolutional Neural Network Image Analysis.

Identifying and quantifying cell death is the basis for all cell death research. Current methods for obtaining these quantitative measurements rely on established biomarkers, yet the marker-based approach suffers from limited marker specificity, high cost of reagents, lengthy sample preparation, and fluorescence imaging. Based on the morphological difference, we developed a Live, Apoptotic, and Necrotic Cell Explorer (LANCE) to categorize cell death status in a label-free manner, by incorporating machine learning and image processing. The LANCE workflow includes cropping individual cells from microscopic images having hundreds of cells, formation of an image database of around 5000 events, training and validation of the convolutional neural network models using multiple cell lines, and treatment conditions. With LANCE, we precisely categorized live, apoptotic, and necrotic cells with a high accuracy of 96.3 ± 0.5%. More importantly, the nondestructive label-free LANCE method allows for tracking time dynamics of the cell death process, which enhances the understanding of subtle cell death regulation at the molecular level. Hence, LANCE is a fast, low-cost, and nondestructive label-free method to distinguish cell status, which can be applied to cell death studies as well as many other biomedical applications.

Is it beneficial for patients with pT1-2N1M0 breast cancer to receive postmastectomy radiotherapy? An analysis based on RecurIndex assay.

The benefit of postmastectomy radiotherapy (PMRT) for pT1-2N1M0 breast cancer patients currently remains controversial. This study was conducted to investigate whether pT1-2N1M0 breast cancer patients could benefit from PMRT based on RecurIndex assay. The clinical data of 213 pT1-2N1M0 breast cancer patients were retrospectively analyzed. Through RecurIndex assay, 81 cases were assessed as the low risk, and 132 as the high risk. Compared to low-risk patients, high-risk patients especially those not receiving PMRT had a significantly increased risk of recurrence and metastasis, and worse 7-year local-regional recurrence-free interval (LRFI), distance recurrence-free interval (DRFI) and recurrence-free survival (RFS) rates. PMRT-based subgroup analysis indicated no significant differences between the low-risk patients with and without PMRT in 7-year LRFI, DRFI, RFS and overall survival (OS) rates, but apparent differences were all shown between the high-risk patients with and without PMRT in 7-year LRFI, DRFI, RFS and OS rates. Overall, for pT1-2N1M0 breast cancer patients at low risk of recurrence and metastasis stratified by RecurIndex assay, there may be a phenomenon of no PMRT benefits, while for those at high risk, use of PMRT may produce survival benefits.

The beneficial role of Asian-based RecurIndex test in the prognostic prediction in Chinese male breast cancer patients.

RecurIndex, a multigene profiling assay, can predict the risk of local recurrence and distant metastasis in female breast cancer (FBC), but its role in male breast cancer (MBC) remains unclear. In this study, the clinicopathological data of 43 consecutive MBC patients undergoing surgeries between 2009 and 2018 were retrospectively analysed. Their paraffin-embedded tissue sections were examined by RecurIndex test which comprised 2 models: recurrence index for local recurrence (RI-LR) and recurrence index for distant recurrence (RI-DR). Of 43 patients, there were 26 low-risk and 17 high-risk patients assessed by RI-LR, while 17 low-risk and 26 high-risk patients by RI-DR. For RI-LR, tumor N stage showed statistically significant (P < 0.001) between low- and high-risk patients; for RI-DR, differences were pronounced in tumor grade (P = 0.033), T stage (P = 0.043) and N stage (P = 0.003). In terms of clinical outcomes, the overall survival (OS) of low- and high-risk patients stratified by RI-LR showed no statistically significant differences (P = 0.460), while high-risk patients identified by RI-DR had a significantly worse distant recurrence-free survival (DRFS) (P = 0.035), progression-free survival (PFS) (P = 0.019) and OS (P = 0.044) than low-risk patients. Overall, RI-DR can effectively predict the DRFS, PFS and OS of MBC patients and identify those at low risk of recurrence, which may serve as a potential prognostic tool for MBC.

Single-cell transcriptomics uncovers phenotypic alterations in the monocytes in a Chinese population with chronic cadmium exposure.

BACKGROUND: Cadmium is the most prevalent form of heavy metal contaminant globally and its exposure rises serious health concern. Chronic exposure to cadmium causes immune disturbances. However, few studies have addressed how it affects circulating immune cells, one of the most essential elements for the host defense system, at both population and molecular level. Therefore, this is the first single-cell transcriptomic analysis of the response of the human circulating immune system to plasma cadmium level. METHODS: We conducted a cross-sectional study in Hunan province, which has the highest level of cadmium land contamination in China. A total of 3283 individuals were eligible for analyzing the association between plasma cadmium levels and the monocyte counts and its subgroups. Another 780 individuals were assigned for validation. Thirty propensity-matched individuals without chronic disease from the lowest- and highest-quartile groups according to serum cadmium levels were selected for single-cell RNA sequencing (scRNA-seq) and flow cytometry analyses. Moreover, the monocyte phenotypic alterations in the heavy metal-exposed population were validated with a cecal ligation and puncture sepsis mouse model. RESULTS: From August 2016 to July 2017, we conducted a cross-sectional study to identify phenotypic alterations in peripheral immune cells in cadmium polluted areas in China. Monocyte percentages were negatively associated with plasma cadmium levels in multivariable linear regression analysis. Peripheral blood mononuclear cell scRNA-seq revealed that the CD14+ monocyte subset was dramatically reduced in the highest-quartile cadmium-exposed group. Moreover, we assessed different hallmarks of immune cell dysfunction-such as host defense capability, apoptotic signaling, cellular diversity and malignant gene expression in monocytes. Importantly, cadmium induced phenotypic alterations in the immune system were validated in the cecal ligation and puncture sepsis mouse model, in which chronic exposure to cadmium not only increased the death rate but also decreased monocyte numbers and the ability to clear bacterial infections. CONCLUSION: This transcriptomic analysis provides molecular information about how the most important hallmarks of immune cell dysfunction are affected by plasma cadmium level. The significant phenotypic alterations in monocytes serving as early indicators of increased susceptibility to infectious and malignant diseases.

Paroxetine Attenuates Cardiac Hypertrophy Via Blocking GRK2 and ADRB1 Interaction in Hypertension.

Background ADRB1 (adrenergic receptor beta 1) responds to neuroendocrine stimulations, which have great implications in hypertension. GRK2 (G protein-coupled receptor kinase 2) is an essential regulator for many G protein-coupled receptors and subsequent cell signaling cascades, but its role as a regulator of ADRB1 and associated cardiac hypertrophy in hypertension remains to be elucidated. Methods and Results In this study, we found the expressions of GRK2 and ADRB1 in peripheral blood mononuclear cells were positively associated with blood pressure levels in hypertensive patients and with their expression in heart. In vitro evidence showed a direct interaction in ADRB1 and GRK2 and genetic depletion of GRK2 blocks epinephrine-induced upregulation of hypertrophic and fibrotic genes in cardiomyocytes. Meanwhile, we discovered a selective serotonin reuptake inhibitor paroxetine specifically blockades GRK2 and ADRB1 interaction. In vivo, paroxetine treatment ameliorates hypertension-induced cardiac hypertrophy, dysfunction, and fibrosis in animal models. We found that paroxetine suppressed sympathetic overdrive and increased the adrenergic receptor sensitivity to catecholamines. Paroxetine treatment also blocks epinephrine-induced upregulation of hypertrophic and fibrotic genes as well as ADRB1 internalization in cardiomyocytes. Coadministration of paroxetine further potentiates metoprolol-induced reductions in blood pressure and heart rate, further attenuating cardiac hypertrophy in spontaneously hypertensive rats. Furthermore, in patients with hypertension accompanied with depression, we observed that cardiac remodeling was less severe in those with paroxetine treatment compared with those with other types of anti-depressive agents. Conclusions Paroxetine promotes ADRB1 sensitivity and attenuates cardiac hypertrophy partially via blocking GRK2-mediated ADRB1 activation and internalization in the context of hypertension.

Duration of dual antiplatelet therapy after percutaneous coronary intervention with drug-eluting stent: systematic review and network meta-analysis.

OBJECTIVE: To evaluate the efficacy and safety of standard term (12 months) or long term (>12 months) dual antiplatelet therapy (DAPT) versus short term (<6 months) DAPT after percutaneous coronary intervention (PCI) with drug-eluting stent (DES). DESIGN: Systematic review and network meta-analysis. DATA SOURCES: Relevant studies published between June 1983 and April 2018 from Medline, Embase, Cochrane Library for clinical trials, PubMed, Web of Science, ClinicalTrials.gov, and Clinicaltrialsregister.eu. REVIEW METHODS: Randomised controlled trials comparing two of the three durations of DAPT (short term, standard term, and long term) after PCI with DES were included. The primary study outcomes were cardiac or non-cardiac death, all cause mortality, myocardial infarction, stent thrombosis, and all bleeding events. RESULTS: 17 studies (n=46 864) were included. Compared with short term DAPT, network meta-analysis showed that long term DAPT resulted in higher rates of major bleeding (odds ratio 1.78, 95% confidence interval 1.27 to 2.49) and non-cardiac death (1.63, 1.03 to 2.59); standard term DAPT was associated with higher rates of any bleeding (1.39, 1.01 to 1.92). No noticeable difference was observed in other primary endpoints. The sensitivity analysis revealed that the risks of non-cardiac death and bleeding were further increased for ≥18 months of DAPT compared with short term or standard term DAPT. In the subgroup analysis, long term DAPT led to higher all cause mortality than short term DAPT in patients implanted with newer-generation DES (1.99, 1.04 to 3.81); short term DAPT presented similar efficacy and safety to standard term DAPT with acute coronary syndrome (ACS) presentation and newer-generation DES placement. The heterogeneity of pooled trials was low, providing more confidence in the interpretation of results. CONCLUSIONS: In patients with all clinical presentations, compared with short term DAPT (clopidogrel), long term DAPT led to higher rates of major bleeding and non-cardiac death, and standard term DAPT was associated with an increased risk of any bleeding. For patients with ACS, short term DAPT presented similar efficacy and safety with standard term DAPT. For patients implanted with newer-generation DES, long term DAPT resulted in more all cause mortality than short term DAPT. Although the optimal duration of DAPT should take personal ischaemic and bleeding risks into account, this study suggested short term DAPT could be considered for most patients after PCI with DES, combining evidence from both direct and indirect comparisons. SYSTEMATIC REVIEW REGISTRATION: PROSPERO CRD42018099519.

Investigation into attitudes toward epilepsy among non-/neurological doctors and nurses in southern China.

PURPOSE: To assess attitudes concerning epilepsy among non-/neurological medical personnel from basic-level hospitals in southern China and identify significant predictive factors for future stigma reduction interventions. METHOD: The Chinese Public Attitudes Toward Epilepsy (CPATE) scale was administered to 184 neurological and 264 non-neurological medical staff members from basic-level hospitals in southern China. Positively stated items in the PATE scale were reversely scored; in this case, a higher score would indicate a more negative attitude. RESULTS: The mean score of each item among the neurological group was statistically lower than that of the non-neurological group. The mean scores in both the general and personal domains of the CPATE scale were significantly lower among the doctors as compared to nurses. CONCLUSIONS: This was the first study to evaluate the attitudes toward epilepsy among medical personnel with different characteristics in basic-level hospitals in China. Health education can be a new model for stigma-reducing public interventions in China. Future strategies should first focus on improving access to correct knowledge about epilepsy among non-neurological medical staffs. This would be a vital breakthrough point to improve attitudes toward epilepsy in the whole society to help ease the disease burden on people with epilepsy, their families and the community in the years to come.