Mesenchymal Stromal Cells Increase the Natural Killer Resistance of Circulating Tumor Cells via Intercellular Signaling of cGAS-STING-IFNß-HLA.

Circulating tumor cells (CTCs) shed from primary tumors must overcome the cytotoxicity of immune cells, particularly natural killer (NK) cells, to cause metastasis. The tumor microenvironment (TME) protects tumor cells from the cytotoxicity of immune cells, which is partially executed by cancer-associated mesenchymal stromal cells (MSCs). However, the mechanisms by which MSCs influence the NK resistance of CTCs remain poorly understood. This study demonstrates that MSCs enhance the NK resistance of cancer cells in a gap junction-dependent manner, thereby promoting the survival and metastatic seeding of CTCs in immunocompromised mice. Tumor cells crosstalk with MSCs through an intercellular cGAS-cGAMP-STING signaling loop, leading to increased production of interferon-ß (IFNß) by MSCs. IFNß reversely enhances the type I IFN (IFN-I) signaling in tumor cells and hence the expression of human leukocyte antigen class I (HLA-I) on the cell surface, protecting the tumor cells from NK cytotoxicity. Disruption of this loop reverses NK sensitivity in tumor cells and decreases tumor metastasis. Moreover, there are positive correlations between IFN-I signaling, HLA-I expression, and NK tolerance in human tumor samples. Thus, the NK-resistant signaling loop between tumor cells and MSCs may serve as a novel therapeutic target.

NK cell exhaustion in the tumor microenvironment.

Natural killer (NK) cells kill mutant cells through death receptors and cytotoxic granules, playing an essential role in controlling cancer progression. However, in the tumor microenvironment (TME), NK cells frequently exhibit an exhausted status, which impairs their immunosurveillance function and contributes to tumor immune evasion. Emerging studies are ongoing to reveal the properties and mechanisms of NK cell exhaustion in the TME. In this review, we will briefly introduce the maturation, localization, homeostasis, and cytotoxicity of NK cells. We will then summarize the current understanding of the main mechanisms underlying NK cell exhaustion in the TME in four aspects: dysregulation of inhibitory and activating signaling, tumor cell-derived factors, immunosuppressive cells, and metabolism and exhaustion. We will also discuss the therapeutic approaches currently being developed to reverse NK cell exhaustion and enhance NK cell cytotoxicity in the TME.

Coculture of cancer cells with platelets increases their survival and metastasis by activating the TGFß/Smad/PAI-1 and PI3K/AKT pathways.

When cancer cells enter the bloodstream, they can interact with platelets to acquire stronger survival and metastatic abilities. To elucidate the underlying mechanisms, we cocultured metastatic melanoma and triple-negative breast cancer cells with species-homologous platelets. We found that cocultured cancer cells displayed higher viabilities in circulation, stronger capacities for cell migration, invasion, and colony formation in vitro, and more tumorigenesis and metastasis in mice. RNA sequencing analysis revealed that the level of serpin family E member 1 (SERPINE1) was significantly upregulated in cocultured cancer cells. Knockdown of SERPINE1 reversed the coculture-elevated survival and metastatic phenotypes of cancer cells. Mechanistic studies indicated that coculture with platelets activated the TGFß/Smad pathway to induce SERPINE1 expression in cancer cells, which encodes plasminogen activator inhibitor 1 (PAI-1). PAI-1 then activated PI3K to increase the phosphorylation of AKTThr308 and Bad to elevate Bcl-2, which enhanced cell survival in circulation. Moreover, higher levels of PAI-1 were detected in metastatic tumors from melanoma and triple-negative breast cancer patients than in normal tissues, and high levels of PAI-1 were associated with a shorter overall survival time and worse disease progression in breast cancer. PAI-1 may act as a potential biomarker for detecting and treating metastatic tumor cells.

Navigating chimeric antigen receptor-engineered natural killer cells as drug carriers via three-dimensional mapping of the tumor microenvironment.

Chimeric antigen receptor (CAR)-modified natural killer (NK) cells are recognized as promising immunotherapeutic agents for cancer treatment. However, the efficacy and trafficking of CAR-NK cells in solid tumors are hindered by the complex barriers present in the tumor microenvironment (TME). We have developed a novel strategy that utilizes living CAR-NK cells as carriers to deliver anticancer drugs specifically to the tumor site. We also introduce a time-lapse method for evaluating the efficacy and tumor specificity of CAR-NK cells using a two-photon microscope in live mouse models and three-dimensional (3D) tissue slide cultures. Our results demonstrate that CAR-NK cells exhibit enhanced antitumor immunity when combined with photosensitive chemicals in both in vitro and in vivo tumor models. Additionally, we have successfully visualized the trafficking, infiltration, and accumulation of drug-loaded CAR-NK cells in deeply situated TME using non-invasive intravital two-photon microscopy. Our findings highlight that tumor infiltration of CAR-NK cells can be intravitally monitored through the two-photon microscope approach. In conclusion, our study demonstrates the successful integration of CAR-NK cells as drug carriers and paves the way for combined cellular and small-molecule therapies in cancer treatment. Furthermore, our 3D platform offers a valuable tool for assessing the behavior of CAR cells within solid tumors, facilitating the development and optimization of immunotherapeutic strategies with clinical imaging approaches.

The Interaction between Macrophages and Triple-negative Breast Cancer Cells Induces ROS-Mediated Interleukin 1α Expression to Enhance Tumorigenesis and Metastasis.

Triple-negative breast cancer (TNBC) has higher mortality than non-TNBC because of its stronger metastatic capacity. Increasing studies reported that TNBC tumors had more macrophage infiltration than non-TNBC tumors, which promoted the metastasis of TNBC cells. However, how TNBC cells become more malignant after interacting with macrophages is less reported. In this study, it is observed that when TNBC cells are co-cultured with macrophages, they display higher viability and stronger metastatic ability than non-TNBC cells. Mechanistic studies reveal that TNBC cells acquired these abilities via interactions with macrophages in three phases. First, within 12 h of co-culture with macrophages, some TNBC cells have significantly elevated levels of reactive oxygen species (ROS), which upregulate interleukin 1α (IL1α) expression in ERK1/2-c-Jun- and NF-κB-dependent manners at 24-48 h. Second, the secreted IL1α bound to IL1R1 activates the ERK1/2-ZEB1-VIM pathway which increases metastasis. Third, IL1α/IL1R1 facilitates its own synthesis and induces the expression of IL1ß and IL8 at 72-96 h through the MKK4-JNK-c-Jun and NF-κB signaling pathways. Moreover, a higher level of IL1α is positively correlated with more macrophage infiltration and shorter overall survival in breast cancer patients. Thus, reducing ROS elevation or downregulating IL1α expression can serve as new strategies to decrease metastasis of TNBC.

Shear Stress Drives the Cleavage Activation of Protease-Activated Receptor 2 by PRSS3/Mesotrypsin to Promote Invasion and Metastasis of Circulating Lung Cancer Cells.

When circulating tumor cells (CTCs) travel in circulation, they can be killed by detachment-induced anoikis and fluidic shear stress (SS)-mediated apoptosis. Circulatory treatment, which can make CTCs detached but also generate SS, can increase metastasis of cancer cells. To identify SS-specific mechanosensors without detachment impacts, a microfluidic circulatory system is used to generate arteriosus SS and compare transcriptome profiles of circulating lung cancer cells with suspended cells. Half of the cancer cells can survive SS damage and show higher invasion ability. Mesotrypsin (PRSS3), protease-activated receptor 2 (PAR2), and the subunit of activating protein 1, Fos-related antigen 1 (FOSL1), are upregulated by SS, and their high expression is responsible for promoting invasion and metastasis. SS triggers PRSS3 to cleave the N-terminal inhibitory domain of PAR2 within 2 h. As a G protein-coupled receptor, PAR2 further activates the Gαi protein to turn on the Src-ERK/p38/JNK-FRA1/cJUN axis to promote the expression of epithelial-mesenchymal transition markers, and also PRSS3, which facilitates metastasis. Enriched PRSS3, PAR2, and FOSL1 in human tumor samples and their correlations with worse outcomes reveal their clinical significance. PAR2 may serve as an SS-specific mechanosensor cleavable by PRSS3 in circulation, which provides new insights for targeting metastasis-initiating CTCs.

Fluorescence intensity and lifetime imaging of lipofuscin-like autofluorescence for label-free predicting clinical drug response in cancer.

Conventional techniques for in vitro cancer drug screening require labor-intensive formalin fixation, paraffin embedding, and dye staining of tumor tissues at fixed endpoints. This way of assessment discards the valuable pharmacodynamic information in live cells over time. Here, we found endogenous lipofuscin-like autofluorescence acutely accumulated in the cell death process. Its unique red autofluorescence could report the apoptosis without labeling and continuously monitor the treatment responses in 3D tumor-culture models. Lifetime imaging of lipofuscin-like red autofluorescence could further distinguish necrosis from apoptosis of cells. Moreover, this endogenous fluorescent marker could visualize the apoptosis in live zebrafish embryos during development. Overall, this study validates that lipofuscin-like autofluorophore is a generic cell death marker. Its characteristic autofluorescence could label-free predict the efficacy of anti-cancer drugs in organoids or animal models.

Preclinical safety evaluation and tracing of human mesenchymal stromal cell spheroids following intravenous injection into cynomolgus monkeys.

We have previously demonstrated that mesenchymal stromal/stem cells (MSCs) in spheroids (MSCsp) tolerate ambient and hypoxic conditions for a prolonged time. Local administration of MSCsp, but not dissociated MSCs (MSCdiss), promotes wound healing and relieves multiple sclerosis and osteoarthritis in mice and monkeys. These findings indicate an advantage of MSCsp over MSCdiss in sustaining cell viability and efficacy following transplantation, which, however, does not appear to apply to intravenous (i.v.) injection for the principal concern that MSCsp might cause embolism in small blood vessels of the host, leading to sudden death. Here, we addressed this concern by injecting human MSCsp (∼450 µm) or MSCdiss i.v. into cynomolgus monkeys. Surprisingly, no deaths occurred until sacrifice at day 21 or 60 post injection, and no remarkable physiological changes were found in the animals following the i.v. injection. The big diameters of large blood vessels in monkeys, compared to small animals like mice, may allow sufficient time for MSCsp to dissociate into single cells so they can pass through small vessels without causing embolism. Retention of MSCsp was lower in the lungs but higher in the blood than retention of MSCdiss at 1 h post injection and both disappeared at day 21. In vitro, MSCsp tolerated fluidic shear stress with higher survival than MSCdiss. Thus, i.v. injection of MSCsp into nonhuman primates is feasible, safe, and probably associated with better survival, less lung entrapment and higher efficacy than administration of MSCdiss.

Apoptosis-Sensing Xenograft Zebrafish Tumor Model for Anticancer Evaluation of Redox-Responsive Cross-Linked Pluronic Micelles.

A suitable animal model for preclinical screening and evaluation in vivo could vastly increase the efficiency and success rate of nanomedicine development. Compared with rodents, the transparency of the zebrafish model offers unique advantages of real-time and high-resolution imaging of the whole body and cellular levels in vivo. In this research, we established an apoptosis-sensing xenograft zebrafish tumor model to evaluate the anti-cancer effects of redox-responsive cross-linked Pluronic polymeric micelles (CPPMs) visually and accurately. First, doxorubicin (Dox)-loaded CPPMs were fabricated and characterized with glutathione (GSH)-responsive drug release. Then, the B16F10 xenograft zebrafish tumor model was established to mimic the tumor microenvironment with angiogenesis and high GSH generation for redox-responsive tumor-targeting evaluation in vivo. The high GSH generation was first verified in the xenograft zebrafish tumor model. Compared with ordinary Pluronic polymeric micelles, Dox CPPMs had a much higher accumulation in zebrafish tumor sites. Finally, the apoptosis-sensing B16F10-C3 xenograft zebrafish tumor model was established for visual, rapid, effective, and noninvasive assessment of anti-cancer effects at the cellular level in vivo. The Dox CPPMs significantly inhibited the proliferation of cancer cells and induced apoptosis in the B16F10-C3 xenograft zebrafish tumor model. Therefore, the redox-responsive cross-linked Pluronic micelles showed effective anti-cancer therapy in the xenograft zebrafish tumor model. This xenograft zebrafish tumor model is available for rapid screening and assessment of anti-cancer effects in preclinical studies.

Visualization of natural killer cell-mediated killing of cancer cells at single-cell resolution in live zebrafish.

Tumor immunotherapy has been an important advancement in cancer treatment in recent years. Compared with T cell-based therapy, natural killer (NK) cell-based therapy does not require human leukocyte antigen matching and has fewer side effects; thus, NK cell therapy has gradually attracted the attention of researchers and clinicians. Reliable and effective animal models are essential for evaluating the effects of NK cell therapy. NK cells kill cancer cells mainly through apoptosis. In this study, we first established a 3D coculture model using fluorescence resonance energy transfer (FRET)-based lung or breast cancer cells and tdTomato-labeled NK cells. We observed that cancer cells changed from green to blue when undergoing apoptosis induced by red NK cells. We then coinjected these green cancer cells with red NK cells into zebrafish to visualize the interaction between them and the killing process of NK cells against cancer cells in real-time and at single-cell resolution in circulation. Using this model, we found that NK cells can quickly kill cancer cells in zebrafish circulation in 40 min and the caspase-3 can be activated in 5-10 min. This FRET-based zebrafish tumor model can serve as a powerful in vivo tool that can facilitate the development of NK cell-based therapy. More importantly, cancer cells from cancer patients can be labeled with our apoptotic biosensor and then transplanted into zebrafish to evaluate the sensitivity of the cancer cells to NK cells to help clinicians make treatment plans that can benefit patients.

The interactions between integrin α5ß1 of liver cancer cells and fibronectin of fibroblasts promote tumor growth and angiogenesis.

Hepatocellular carcinoma (HCC) progression is closely related to pathological fibrosis, which involves heterotypic intercellular interactions (HIIs) between liver cancer cells and fibroblasts. Here, we studied them in a direct coculture model, and identified fibronectin from fibroblasts and integrin-α5ß1 from liver cancer cells as the primary responsible molecules utilizing CRISPR/Cas9 gene-editing technology. Coculture led to the formation of 3D multilayer microstructures, and obvious fibronectin remodeling was caused by upregulated integrin-α5ß1, which greatly promoted cell growth in 3D microstructures. Integrin-α5 was more sensitive and specific than integrin-ß1 in this process. Subsequent mechanistic exploration revealed the activation of integrin-Src-FAK, AKT and ERK signaling pathways. Importantly, the growth-promoting effect of HIIs was verified in a xenograft tumor model, in which more blood vessels were observed in bigger tumors derived from the coculture group than that derived from monocultured groups. Hence, we conducted triculture by introducing human umbilical vein endothelial cells, which aligned to and differentiated along multilayer microstructures in an integrin-α5ß1 dependent manner. Furthermore, fibronectin, integrin-α5, and integrin-ß1 were upregulated in 52 HCC tumors, and fibronectin was related to microvascular invasion. Our findings identify fibronectin, integrin-α5, and integrin-ß1 as tumor microenvironment-related targets and provide a basis for combination targeted therapeutic strategies for future HCC treatment.

Applications of Microfluidics and Organ-on-a-Chip in Cancer Research.

Taking the life of nearly 10 million people annually, cancer has become one of the major causes of mortality worldwide and a hot topic for researchers to find innovative approaches to demystify the disease and drug development. Having its root lying in microelectronics, microfluidics seems to hold great potential to explore our limited knowledge in the field of oncology. It offers numerous advantages such as a low sample volume, minimal cost, parallelization, and portability and has been advanced in the field of molecular biology and chemical synthesis. The platform has been proved to be valuable in cancer research, especially for diagnostics and prognosis purposes and has been successfully employed in recent years. Organ-on-a-chip, a biomimetic microfluidic platform, simulating the complexity of a human organ, has emerged as a breakthrough in cancer research as it provides a dynamic platform to simulate tumor growth and progression in a chip. This paper aims at giving an overview of microfluidics and organ-on-a-chip technology incorporating their historical development, physics of fluid flow and application in oncology. The current applications of microfluidics and organ-on-a-chip in the field of cancer research have been copiously discussed integrating the major application areas such as the isolation of CTCs, studying the cancer cell phenotype as well as metastasis, replicating TME in organ-on-a-chip and drug development. This technology's significance and limitations are also addressed, giving readers a comprehensive picture of the ability of the microfluidic platform to advance the field of oncology.

Recipients of COVID-19 vaccines face challenges of SARS-CoV-2 variants.

The coronavirus disease 19 (COVID-19) has been rampant since 2019, severely affecting global public health, and causing 5.75 million deaths worldwide. So far, many vaccines have been developed to prevent the infection of SARS-CoV-2 virus. However, the emergence of new variants may threat vaccine recipients as they might evade immunological surveillance that depends on the using of anti-SARS-CoV-2 antibody to neutralize the viral particles. Recent studies have found that recipients who received two doses of vaccination plus an additional booster shoot were able to quickly elevate neutralization response and immune response against wild-type SARS-CoV-2 virus and some initially appeared viral variants. In this review, we assessed the real-world effectiveness of different COVID-19 vaccines by population studies and neutralization assays and compared neutralization responses of booster vaccines in vitro. Finally, as the efficacy of COVID-19 vaccine is expected to decline over time, continued vaccination should be considered to achieve a long-term immune protection against coronavirus.

Chemo-Phototherapy with Carfilzomib-Encapsulated TiN Nanoshells Suppressing Tumor Growth and Lymphatic Metastasis.

The design of nanomedicine for cancer therapy, especially the treatment of tumor metastasis has received great attention. Proteasome inhibition is accepted as a new strategy for cancer therapy. Despite being a big breakthrough in multiple myeloma therapy, carfilzomib (CFZ), a second-in-class proteasome inhibitor is still unsatisfactory for solid tumor and metastasis therapy. In this study, hollow titanium nitride (TiN) nanoshells are synthesized as a drug carrier of CFZ. The TiN nanoshells have a high loading capacity of CFZ, and their intrinsic inhibitory effect on autophagy synergistically enhances the activity of CFZ. Due to an excellent photothermal conversion efficiency in the second near-infrared (NIR-II) region, TiN nanoshell-based photothermal therapy further induces a synergistic anticancer effect. In vivo study demonstrates that TiN nanoshells readily drain into the lymph nodes, which are responsible for tumor lymphatic metastasis. The CFZ-loaded TiN nanoshell-based chemo-photothermal therapy combined with surgery offers a remarkable therapeutic outcome in greatly inhibiting further metastatic spread of cancer cells. These findings suggest that TiN nanoshells act as an efficient carrier of CFZ for realizing enhanced outcomes for proteasome inhibitor-based cancer therapy, and this work also presents a "combined chemo-phototherapy assisted surgery" strategy, promising for future cancer treatment.

Acetyltanshinone IIA reduces the synthesis of cell cycle-related proteins by degrading p70S6K and subsequently inhibits drug-resistant lung cancer cell growth.

Targeted therapies using tyrosine kinase inhibitors (TKIs) against epidermal growth factor receptor (EGFR) have improved the outcomes of patients with non-small cell lung cancer (NSCLC). However, due to genetic mutations of EGFR or activation of other oncogenic pathways, cancer cells can develop resistance to TKIs, resulting in usually temporary and reversible therapeutic effects. Therefore, new anticancer agents are urgently needed to treat drug-resistant NSCLC. In this study, we found that acetyltanshinone IIA (ATA) displayed much stronger potency than erlotinib in inhibiting the growth of drug-resistant NSCLC cells and their-derived xenograft tumors. Our analyses revealed that ATA achieved this effect by the following mechanisms. First, ATA could bind p70S6K at its ATP-binding pocket to prevent phosphorylation, and second by increasing the ubiquitination of p70S6K to cause its degradation. Since phosphorylation of S6 ribosome protein (S6RP) by p70S6K can induce protein synthesis at the ribosome, the dramatic reduction of p70S6K after ATA treatment led to great reductions of new protein synthesis on several cell cycle-related proteins including cyclin D3, aurora kinase A, polo-like kinase, cyclin B1, survivin; and reduced the levels of EGFR and MET. In addition, ATA treatment increased the levels of p53 and p21 proteins, which blocked cell cycle progression in the G1/S phase. Taken together, as ATA can effectively block multiple signaling pathways essential for protein synthesis and cell proliferation, ATA can potentially be developed into a multi-target anti-cancer agent to treat TKI-resistant NSCLC.

High Expression of G6PD Increases Doxorubicin Resistance in Triple Negative Breast Cancer Cells by Maintaining GSH Level.

Resistance to doxorubicin (DOX) remains a big challenge to breast cancer treatment especially for triple negative breast cancer (TNBC). Our previous study revealed that the antioxidant system plays an important role in conferring metastasis derived DOX resistance. In this study, we used two-dimensional difference gel electrophoresis (2D-DIGE) proteomics to compare the expression profiles of two generations of TNBC cell lines which have increased metastatic ability in nude mice and exhibited resistance to DOX. Through careful analyses, one antioxidant protein: glucose-6-phosphate dehydrogenase (G6PD) was identified with 3.2-fold higher level in metastatic/DOX-resistant 231-M1 than its parental 231-C3 cells. Analyses of clinical data showed that TNBC patients with higher G6PD levels exhibited lower overall survival than patients with lower G6PD level. Reducing G6PD expression by siRNA or inhibiting its activity with dehydroepiandrosterone (DHEA) significantly increased DOX's cytotoxicity in both cell lines. Importantly, inhibiting G6PD's activity with DHEA dramatically increased the apoptotic rate of 1.25 µM DOX from 2% to 54%. Our results suggest that high level of G6PD can help TNBC to resist DOX-induced oxidative stress. Thus, inhibiting G6PD shall be a good strategy to treat DOX-resistant TNBC.

Accelerating precision anti-cancer therapy by time-lapse and label-free 3D tumor slice culture platform.

The feasibility of personalized medicine for cancer treatment is largely hampered by costly, labor-intensive and time-consuming models for drug discovery. Herein, establishing new pre-clinical models to tackle these issues for personalized medicine is urgently demanded. Methods: We established a three-dimensional tumor slice culture (3D-TSC) platform incorporating label-free techniques for time-course experiments to predict anti-cancer drug efficacy and validated the 3D-TSC model by multiphoton fluorescence microscopy, RNA sequence analysis, histochemical and histological analysis. Results: Using time-lapse imaging of the apoptotic reporter sensor C3 (C3), we performed cell-based high-throughput drug screening and shortlisted high-efficacy drugs to screen murine and human 3D-TSCs, which validate effective candidates within 7 days of surgery. Histological and RNA sequence analyses demonstrated that 3D-TSCs accurately preserved immune components of the original tumor, which enables the successful achievement of immune checkpoint blockade assays with antibodies against PD-1 and/or PD-L1. Label-free multiphoton fluorescence imaging revealed that 3D-TSCs exhibit lipofuscin autofluorescence features in the time-course monitoring of drug response and efficacy. Conclusion: This technology accelerates precision anti-cancer therapy by providing a cheap, fast, and easy platform for anti-cancer drug discovery.

Developing effective siRNAs to reduce the expression of key viral genes of COVID-19.

The COVID-19 pandemic has been raging worldwide for more than a year. Many efforts have been made to create vaccines and develop new antiviral drugs to cope with the disease. Here, we propose the application of short interfering RNAs (siRNAs) to degrade the viral genome, thus reducing viral infection. By introducing the concept of the probability of binding efficiency (PBE) and combining the secondary structures of RNA molecules, we designed 11 siRNAs that target the consensus regions of three key viral genes: the spike (S), nucleocapsid (N) and membrane (M) genes of SARS-CoV-2. The silencing efficiencies of the siRNAs were determined in human lung and endothelial cells overexpressing these viral genes. The results suggested that most of the siRNAs could significantly reduce the expression of the viral genes with inhibition rates above 50% in 24 hours. This work not only provides a strategy for designing potentially effective siRNAs against target genes but also validates several potent siRNAs that can be used in the clinical development of preventative medication for COVID-19 in the future.

Nerve growth factor receptor increases the tumor growth and metastatic potential of triple-negative breast cancer cells.

Cellular heterogeneity and the lack of metastatic biomarkers limit the diagnosis of and development of therapies for metastatic triple-negative breast cancer (TNBC). Thus, development of new clinically relevant markers is urgently needed. By using RNA-seq analysis, we found that nerve growth factor receptor (NGFR) was highly expressed in metastatic lung clones of MDA-MB-231 cells. This high level of NGFR expression was necessary for TNBC cells to grow into tumor spheres under nonadhesive conditions, resist anoikis, promote primary tumor growth and increase metastasis in mice. NGFR was also expressed at a high level in a greater number of TNBC patients (45%) than non-TNBC patients (23%), enriched in higher grade tumors, and negatively correlated with the overall survival of TNBC patients. Mechanistic analysis indicated that NGFR exerted its prometastatic effects by binding with neurotrophic receptor tyrosine kinase 3 (TrkC) mainly through a ligand-independent manner, which activated the MEK-ERK1-ZEB1 and PI3K-AKT signaling pathways, increased the level of fibronectin, and decreased the expression of PUMA. Notably, we observed that NGFR expression in TrkC-positive metastatic clones reduced cellular sensitivity to anti-Trk therapy. Moreover, WNT family member 5a (WNT5A) and TrkC activated NGFR transcription in a ZEB1-dependent manner. Taken together, this study identified NGFR as a novel driver for transforming TNBC into higher grade metastatic tumors. Our findings provide the basis for the future development of NGFR as a diagnostic and prognostic marker for determining the metastatic potential of TNBC and as a therapeutic target for treating TNBC patients.

Fluorescence resonance energy transfer-based sensor zebrafish for detecting toxic agents with single-cell sensitivity.

Zebrafish are widely used for detecting toxic agents because of their unique advantages. The conventional zebrafish-based tests use lethal rates and morphological changes as criteria to evaluate the toxicity. To increase the sensitivity of using zebrafish to detect toxic agents, a fluorescence resonance energy transfer-based apoptotic biosensor was introduced into zebrafish genome to generate transgenic sensor zebrafish. Seven chemicals including heavy metals, nanomaterials and DNA-damaging agents were used to treat the sensor zebrafish to determine the sensitivity of the sensor zebrafish. The results showed that sensor zebrafish can detect the toxicity of the tested agents with single-cell sensitivity. Using the sensor zebrafish, we found that, at 100 nM, heavy metal cadmium (Cd) induced apoptosis of zebrafish cells, while no obvious morphological or behavioral changes were observed from the sensor zebrafish. Even at 44.5 nM (the maximum allowable concentration in drinking water), Cd induced a significant increase of apoptosis in sensor zebrafish. ZnO nanoparticles caused apoptosis in sensor zebrafish at a very low concentration of 100 ng/mL. DNA-damaging agents induced the apoptosis of many cells in sensor zebrafish. The sensor zebrafish are much more sensitive than the conventional zebrafish-based tests and can serve as a powerful tool for detecting toxic agents.