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.

Quantitative multiphoton imaging of cell metabolism, stromal fibers, and keratinization enables label-free discrimination of esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) features atypical clinical manifestations and a low 5-year survival rate (< 5% in many developing countries where most of the disease occurs). Precise ESCC detection and grading toward timely and effective intervention are therefore crucial. In this study, we propose a multidimensional, slicing-free, and label-free histopathological evaluation method based on multispectral multiphoton fluorescence lifetime imaging microscopy (MM-FLIM) for precise ESCC identification. To assess the feasibility of this method, comparative imaging on fresh human biopsy specimens of different ESCC grades is performed. By constructing fluorescence spectrum- and lifetime-coded images, ESCC-induced morphological variations are unveiled. Further quantification of cell metabolism and stromal fibers reveals potential indicators for ESCC detection and grading. The specific identification of keratin pearls provides additional support for the early detection of ESCC. These findings demonstrate the viability of using MM-FLIM and the series of derived indicators for histopathological evaluation of ESCC. As there is an increasing interest in developing multiphoton endoscopes and multiphoton FLIM systems for clinical use, the proposed method would probably allow noninvasive, label-free, and multidimensional histological detection and grading of ESCC in the future.

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.

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.

Mouse Strain- and Charge-Dependent Vessel Permeability of Nanoparticles at the Lower Size Limit.

Remarkable advancement has been made in the application of nanoparticles (NPs) for cancer therapy. Although NPs have been favorably delivered into tumors by taking advantage of the enhanced permeation and retention (EPR) effect, several physiological barriers present within tumors tend to restrict the diffusion of NPs. To overcome this, one of the strategies is to design NPs that can reach lower size limits to improve tumor penetration without being rapidly cleared out by the body. Several attempts have been made to achieve this, such as selecting appropriate nanocarriers and modifying surface properties. While many studies focus on the optimal design of NPs, the influence of mouse strains on the effectiveness of NPs remains unknown. Therefore, this study aimed to assess whether the vascular permeability of NPs near the lower size limit differs among mouse strains. We found that the vessel permeability of dextran NPs was size-dependent and dextran NPs with a size below 15 nm exhibited leakage from postcapillary venules in all strains. Most importantly, the leakage rate of 8-nm fluorescein isothiocyanate dextran was significantly higher in the BALB/c mouse strain than in other strains. This strain dependence was not observed in slightly positive TRITC-dextran with comparable sizes. Our results indicate that the influence on mouse strains needs to be taken into account for the evaluation of NPs near the lower size limit.

A universal strategy for the fabrication of single-photon and multiphoton NIR nanoparticles by loading organic dyes into water-soluble polymer nanosponges.

The development of optical organic nanoparticles (NPs) is desirable and widely studied. However, most organic dyes are water-insoluble such that the derivatization and modification of these dyes are difficult. Herein, we demonstrated a simple platform for the fabrication of organic NPs designed with emissive properties by loading ten different organic dyes (molar masses of 479.1-1081.7 g/mol) into water-soluble polymer nanosponges composed of poly(styrene-alt-maleic acid) (PSMA). The result showed a substantial improvement over the loading of commercial dyes (3.7-50% loading) while preventing their spontaneous aggregation in aqueous solutions. This packaging strategy includes our newly synthesized organic dyes (> 85% loading) designed for OPVs (242), DSSCs (YI-1, YI-3, YI-8), and OLEDs (ADF-1-3, and DTDPTID) applications. These low-cytotoxicity organic NPs exhibited tunable fluorescence from visible to near-infrared (NIR) emission for cellular imaging and biological tracking in vivo. Moreover, PSMA NPs loaded with designed NIR-dyes were fabricated, and photodynamic therapy with these dye-loaded PSMA NPs for the photolysis of cancer cells was achieved when coupled with 808 nm laser excitation. Indeed, our work demonstrates a facile approach for increasing the biocompatibility and stability of organic dyes by loading them into water-soluble polymer-based carriers, providing a new perspective of organic optoelectronic materials in biomedical theranostic applications.

Noninvasive assessment of liver function reserve with fluorescent dosimetry of indocyanine green.

Using in vivo multiphoton fluorescent dosimetry, we demonstrate that the clearance dynamics of Indocyanine Green (ICG) in the blood can quickly reveal liver function reserve. In normal rats, the ICG retention rate was below 10% at the 15-minute post-administration; While in the rat with severe hepatocellular carcinoma (HCC), the 15-minute retention rate is over 40% due to poor liver metabolism. With a 785 nm CW laser, the fluorescence dosimeter can evaluate the liver function reserve at a 1/10 clinical dosage of ICG without any blood sampling. In the future, this low-dosage ICG 15-minute retention dosimetry can be applied for the preoperative assessment of hepatectomy or timely perioperative examination.

One step synthesis of efficient red emissive carbon dots and their bovine serum albumin composites with enhanced multi-photon fluorescence for in vivo bioimaging.

Efficient red emissive carbon dots (CDs) in aqueous solutions are very scarce for high performance bioimaging applications. In this work, we report a one-step solvothermal treatment to synthesize pure red emissive CDs (FA-CDs) from citric acid and urea in formic acid without complicated purification procedures. Photoluminescence quantum yield (PLQY) of 43.4% was observed in their dimethyl sulfoxide solutions. High PLQY up to 21.9% in aqueous solutions was achieved in their bovine serum albumin (BSA) composites (FA-CDs@BSA) with significantly enhanced multi-photon fluorescence. The strong surface electron-withdrawing structure of FA-CDs caused by the high content of C = O groups contributes for their pure red emission. Owing to the significantly enhanced single and multi-photon red fluorescence and enlarged particle sizes after composing with BSA, in vivo tumor imaging and two-photon fluorescence imaging of blood vessels in mouse ear have been realized via intravenous injection of FA-CDs@BSA aqueous solutions.

Intravital lipid droplet labeling and imaging reveals the phenotypes and functions of individual macrophages in vivo.

Macrophages play pivotal roles in the maintenance of tissue homeostasis. However, the reactivation of macrophages toward proinflammatory states correlates with a plethora of inflammatory diseases, including atherosclerosis, obesity, neurodegeneration, and bone marrow (BM) failure syndromes. The lack of methods to reveal macrophage phenotype and function in vivo impedes the translational research of these diseases. Here, we found that proinflammatory macrophages accumulate intracellular lipid droplets (LDs) relative to resting or noninflammatory macrophages both in vitro and in vivo, indicating that LD accumulation serves as a structural biomarker for macrophage phenotyping. To realize the staining and imaging of macrophage LDs in vivo, we developed a fluorescent fatty acid analog-loaded poly(lactic-co-glycolic acid) nanoparticle to label macrophages in mice with high efficiency and specificity. Using these novel nanoparticles, we achieved in situ functional identification of single macrophages in BM, liver, lung, and adipose tissues under conditions of acute or chronic inflammation. Moreover, with this intravital imaging platform, we further realized in vivo phenotyping of individual macrophages in the calvarial BM of mice under systemic inflammation. In conclusion, we established an efficient in vivo LD labeling and imaging system for single macrophage phenotyping, which will aid in the development of diagnostics and therapeutic monitoring. Moreover, this method also provides new avenues for the study of lipid trafficking and dynamics in vivo.

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.

Novel zebrafish polycystic kidney disease models reveal functions of the Hippo pathway in renal cystogenesis.

The Hippo signaling pathway is a kinase cascade that plays an important role in organ size control. As the main effectors of the Hippo pathway, transcription coactivators Yap1/Wwtr1 are regulated by the upstream kinase Stk3. Recent studies in mammals have implicated the Hippo pathway in kidney development and kidney diseases. To further illustrate its roles in vertebrate kidney, we generated a series of zebrafish mutants targeting stk3, yap1 and wwtr1 genes. The stk3-/- mutant exhibited edema, formation of glomerular cysts and pronephric tubule dilation during the larval stage. Interestingly, disruption of wwtr1, but not yap1, significantly alleviated the renal phenotypes of the stk3-/- mutant, and overexpression of Wwtr1 with the CMV promoter also induced pronephric phenotypes, similar to those of the stk3-/- mutant, during larval stage. Notably, adult fish with Wwtr1 overexpression developed phenotypes similar to those of human polycystic kidney disease (PKD). Overall, our analyses revealed roles of Stk3 and Wwtr1 in renal cyst formation. Using a pharmacological approach, we further demonstrated that Stk3-deficient zebrafish could serve as a PKD model for drug development.

Functionalizing Collagen with Vessel-Penetrating Two-Photon Phosphorescence Probes: A New In Vivo Strategy to Map Oxygen Concentration in Tumor Microenvironment and Tissue Ischemia.

The encapsulation and/or surface modification can stabilize and protect the phosphorescence bio-probes but impede their intravenous delivery across biological barriers. Here, a new class of biocompatible rhenium (ReI ) diimine carbonyl complexes is developed, which can efficaciously permeate normal vessel walls and then functionalize the extravascular collagen matrixes as in situ oxygen sensor. Without protective agents, ReI -diimine complex already exhibits excellent emission yield (34%, λem   = 583 nm) and large two-photon absorption cross-sections (σ2   = 300 GM @ 800 nm) in water (pH 7.4). After extravasation, remarkably, the collagen-bound probes further enhanced their excitation efficiency by increasing the deoxygenated lifetime from 4.0 to 7.5 µs, paving a way to visualize tumor hypoxia and tissue ischemia in vivo. The post-extravasation functionalization of extracellular matrixes demonstrates a new methodology for biomaterial-empowered phosphorescence sensing and imaging.

Remodeling of Tumor Microenvironment by Tumor-Targeting Nanozymes Enhances Immune Activation of CAR T Cells for Combination Therapy.

Targeting B7-H3 chimeric antigen receptor (CAR) T cells has antitumor potential for therapy of non-small cell lung cancer (NSCLC) in preclinical studies. However, CAR T cell therapy remains a formidable challenge for the treatment of solid tumors due to the heterogeneous and immunosuppressive tumor microenvironment (TME). Nanozymes exhibit merits modulating the immunosuppression of the tumor milieu. Here, a synergetic strategy by combination of nanozymes and CAR T cells in solid tumors is described. This nanozyme with dual photothermal-nanocatalytic properties is endowed to remodel TME by destroying its compact structure. It is found that the B7-H3 CAR T cells infused in mice engrafted with the NSCLC cells have superior antitumor activity after nanozyme ablation of the tumor. Importantly, it is found that the changes altered immune-hostile cancer environment, resulting in enhanced activation and infiltration of B7-H3 CAR T cells. The first evidence that the process of combination nanozyme therapy effectively improves the therapeutic index of CAR T cells is presented. Thus, this study clearly supports that the TME-immunomodulated nanozyme is a promising tool to improve the therapeutic obstacles of CAR T cells against solid tumors.

Low-toxicity FePt nanoparticles for the targeted and enhanced diagnosis of breast tumors using few centimeters deep whole-body photoacoustic imaging.

A considerable amount of early breast tumors grown at a depth over 2 cm in breast tissues. With high near-infrared absorption of iron-platinum (FePt) nanoparticles, we achieved few centimeters deep photoacoustic (PA) imaging for the diagnosis of breast tumors. The imaging depth can extend over 5 cm in chicken breast tissues at the low laser energy density of 20 mJ/cm2 (≤ ANSI safety limit). After anti-VEGFR conjugation and the tail-vein injection, we validated their targeting on tumor sites by the confocal microscopy and PA imaging. Using a home-made whole-body in vivo PA imaging, we found that the nanoparticles were rapidly cleared away from the site of the tumor and majorly metabolized through the liver. These results validated the clinical potential of the FePt nanoparticles in the low-toxicity PA theragnosis of early breast cancer and showed the capacity of our whole-body PA imaging technique on monitoring the dynamic biodistribution of nanoparticles in the living body.

Imaging of macrophage mitochondria dynamics in vivo reveals cellular activation phenotype for diagnosis.

Highly plastic macrophages are pivotal players in the body's homeostasis and pathogenesis. Grasping the molecular or cellular factors that drive and support the macrophage activation will help to develop diagnostics and manipulate their functions in these contexts. However, the lack of in vivo characterization methods to reveal the dynamic activation of macrophages impedes these studies in various disease contexts. Methods: Here, in vitro bone marrow-derived macrophages (BMDMs) and in vivo Matrigel plug were used to evaluate how mitochondria dynamics supports cellular activation and functions. We conducted macrophage repolarization in vitro to track mitochondria dynamics during the shift of activation status. For in vivo diagnosis, a novel MitoTracker-loaded liposome was first developed to label macrophage mitochondria in mice before/after inflammatory stimulation. Results: Based on the typical activation of in vitro BMDMs, we found glycolysis based macrophages have punctate and discrete mitochondria, while OXPHOS active macrophages have elongated and interconnected mitochondria. M1, M2a, M2b, and M2c activated BMDMs showed clustered and differentiable features in mitochondrial morphology. These features also hold for Matrigel plug-recruited macrophages in mice. Furthermore, with the interventions on M2a macrophages in vitro, we demonstrated that mitochondria morphology could be a metabolic index to evaluate macrophage activation status under drug manipulation. Using the MitoTracker-loaded liposomes, we further achieved subcellular imaging of macrophage mitochondria in vivo. Their organization dynamics revealed the dynamic change from anti-inflammatory macrophages to inflammatory ones in vivo under the lipopolysaccharide (LPS) challenge. Conclusion: These results reveal that subcellular imaging of mitochondria organization can characterize the activation status of macrophage in vitro and in vivo at a single-cell level, which is critical for the studies of noninvasive diagnosis and therapeutic drug monitoring.

Prophylactic and therapeutic effects of honey on radiochemotherapy-induced mucositis: a meta-analysis of randomized controlled trials.

PURPOSE: Oral mucositis is a common side effect of radiochemotherapy and may adversely affect the patients' quality of life (QoL). Honey application may reduce the mucositis grade in patients. Here, we conducted a meta-analysis of randomized controlled trials (RCTs) to evaluate the prophylactic and therapeutic effects of honey on radiochemotherapy-induced oral mucositis. METHODS: Publications on RCTs were extracted from the PubMed, Embase, CINAHL, and Cochrane Library databases. The primary outcomes were mucositis grades and pain scores. Secondary outcomes were the recovery time and QoL. The study was registered with PROSPERO (number CRD42018108486). RESULTS: Nineteen RCTs, involving 1276 patients, were reviewed. Honey considerably mitigated oral mucositis in both prophylactic and therapeutic phases. In the prophylactic phase, intolerable mucositis development was significantly prevented in the honey-treated group (RR = 0.18, 95% confidence interval [CI] = 0.09 to 0.41). Patients treated with honey showed significant decrease in pain scores in the first month of treatment (weighted mean difference [WMD] = - 3.25, 95% CI = - 4.41 to - 2.09) and at the end of the treatment (WMD = - 2.32, 95% CI = - 4.47 to - 0.18). CONCLUSION: Honey, which is relatively cheap and easily available, prevented mucositis and effectively mitigate mucositis in patients after radiochemotherapy. Moreover, it significantly reduced the mucositis grade and engendered a fast and painless healing process. Therefore, honey use during and after radiochemotherapy is recommended for mucositis prevention and treatment.

Discovering Macrophage Functions Using In Vivo Optical Imaging Techniques.

Macrophages are an important component of host defense and inflammation and play a pivotal role in immune regulation, tissue remodeling, and metabolic regulation. Since macrophages are ubiquitous in human bodies and have versatile physiological functions, they are involved in virtually every disease, including cancer, diabetes, multiple sclerosis, and atherosclerosis. Molecular biological and histological methods have provided critical information on macrophage biology. However, many in vivo dynamic behaviors of macrophages are poorly understood and yet to be discovered. A better understanding of macrophage functions and dynamics in pathogenesis will open new opportunities for better diagnosis, prognostic assessment, and therapeutic intervention. In this article, we will review the advances in macrophage tracking and analysis with in vivo optical imaging in the context of different diseases. Moreover, this review will cover the challenges and solutions for optical imaging techniques during macrophage intravital imaging.

Fiber-based 1150-nm femtosecond laser source for the minimally invasive harmonic generation microscopy.

Harmonic generation microscopy (HGM) has become one unique tool of optical virtual biopsy for the diagnosis of cancer and the in vivo cytometry of leukocytes. Without labeling, HGM can reveal the submicron features of tissues and cells in vivo. For deep imaging depth and minimal invasiveness, people commonly adopt 1100- to 1300-nm femtosecond laser sources. However, those lasers are typically based on bulky oscillators whose performances are sensitive to environmental conditions. We demonstrate a fiber-based 1150-nm femtosecond laser source, with 6.5-nJ pulse energy, 86-fs pulse width, and 11.25-MHz pulse repetition rate. It was obtained by a bismuth borate or magnesium-doped periodically poled lithium niobate (MgO:PPLN) mediated frequency doubling of the 2300-nm solitons, generated from an excitation of 1550-nm femtosecond pulses on a large mode area photonic crystal fiber. Combined with a home-built laser scanned microscope and a tailor-made frame grabber, we achieve a pulse-per-pixel HGM imaging in vivo at a 30-Hz frame rate. This integrated solution has the potential to be developed as a stable HGM system for routine clinical use.

Infrared-active quadruple contrast FePt nanoparticles for multiple scale molecular imaging.

A single nanomaterial with multiple imaging contrasts and functions is highly desired for multiscale theragnosis. Herein, we demonstrate single 1-1.9 µm infrared-active FePt alloy nanoparticles (FePt NPs) offering unprecedented four-contrast-in-one molecular imaging - computed tomography (CT), magnetic resonance imaging (MRI), photoacoustic (PA) imaging, and high-order multiphoton luminescence (HOMPL) microscopy. The PA response of FePt NPs outperforms that of infrared-active gold nanorods by 3- to 5.6-fold under identical excitation fluence and particle concentrations. HOMPL (680 nm) of an isolated FePt NP renders spatial full-width-at-half-maximum values of 432 nm and 300 nm beyond the optical diffraction limit for 1230-nm and 920-nm excitation, respectively. The in vivo targeting function was successfully visualized using HOMPL, PA imaging, CT, and MRI, thereby validating FePt as a single nanomaterial system covering up to four types (Optical/PA/CT/MRI) of molecular imaging contrast, ranging from the microscopic level to whole-body scale investigation.

Controlled Au-Polymer Nanostructures for Multiphoton Imaging, Prodrug Delivery, and Chemo-Photothermal Therapy Platforms.

We have successfully introduced a proton-induced controlled reaction of HAuCl4 and poly(styrene-alt-maleic acid) (PSMA) sodium salt to prepare triangular and multicore Au@polymer nanoparticles (NPs). The interparticle interactions in the core gave rise to an absorption band at the near-infrared wavelength. The near-infrared optical properties of the resulting Au-polymer nanostructures are highly stable in a physiological environment, which offered strong photo-to-thermal conversion by a moderate continuous-wave 808 nm laser and exhibited multiphoton fluorescence for imaging using a 1230 nm light excitation (femtosecond laser). Exposure of the carboxylate groups at the polymer shell made the surface structure of the Au multicore @polymer NPs directly conjugate Pt(II)-/Pt(IV)-based drugs, which possessed the elimination of the immediate toxicity over the short time and resulted in an anticancer effect after 3 days. A synergistic effect of the chemo-photothermal therapy showed a moderate hyperthermia assistance (<1 W/cm(2)) and better anticancer performance over time compared with the individual treatments. We demonstrated that such PSMA-based methodology not only enables a broad range of chemical material synthesis in the kinetic control to form Au nano-octahedrons and nanotriangles using Br(-)/I(-) ions additives but also could be extended to form Au/Fe3O4@polymer nanocomposites via proton-assisted PSMA self-assembly.