Label-free metabolic imaging for sensitive and robust monitoring of anti-CD47 immunotherapy response in triple-negative breast cancer.

BACKGROUND: Immunotherapy is revolutionizing cancer treatment from conventional radiotherapies and chemotherapies to immune checkpoint inhibitors which use patients' immune system to recognize and attack cancer cells. Despite the huge clinical success and vigorous development of immunotherapies, there is a significant unmet need for a robust tool to identify responders to specific immunotherapy. Early and accurate monitoring of immunotherapy response is indispensable for personalized treatment and effective drug development. METHODS: We established a label-free metabolic intravital imaging (LMII) technique to detect two-photon excited autofluorescence signals from two coenzymes, NAD(P)H (reduced nicotinamide adenine dinucleotide (phosphate) hydrogen) and FAD (flavin adenine dinucleotide) as robust imaging markers to monitor metabolic responses to immunotherapy. Murine models of triple-negative breast cancer (TNBC) were established and tested with different therapeutic regimens including anti-cluster of differentiation 47 (CD47) immunotherapy to monitor time-course treatment responses using the developed metabolic imaging technique. RESULTS: We first imaged the mechanisms of the CD47-signal regulatory protein alpha pathway in vivo, which unravels macrophage-mediated antibody-dependent cellular phagocytosis and illustrates the metabolism of TNBC cells and macrophages. We further visualized the autofluorescence of NAD(P)H and FAD and found a significant increase during tumor growth. Following anti-CD47 immunotherapy, the imaging signal was dramatically decreased demonstrating the sensitive monitoring capability of NAD(P)H and FAD imaging for therapeutic response. NAD(P)H and FAD intravital imaging also showed a marked decrease after chemotherapy and radiotherapy. A comparative study with conventional whole-body bioluminescence and fluorescent glucose imaging demonstrated superior sensitivity of metabolic imaging. Flow cytometry validated metabolic imaging results. In vivo immunofluorescent staining revealed the targeting ability of NAD(P)H imaging mainly for tumor cells and a small portion of immune-active cells and that of FAD imaging mainly for immunosuppressive cells such as M2-like tumor-associated macrophages. CONCLUSIONS: Collectively, this study showcases the potential of the LMII technique as a powerful tool to visualize dynamic changes of heterogeneous cell metabolism of cancer cells and immune infiltrates in response to immunotherapy thus providing sensitive and complete monitoring. Leveraged on ability to differentiate cancer cells and immunosuppressive macrophages, the presented imaging approach provides particularly useful imaging biomarkers for emerged innate immune checkpoint inhibitors such as anti-CD47 therapy.

Current Developments of Artificial Intelligence in Digital Pathology and Its Future Clinical Applications in Gastrointestinal Cancers.

The implementation of DP will revolutionize current practice by providing pathologists with additional tools and algorithms to improve workflow. Furthermore, DP will open up opportunities for development of AI-based tools for more precise and reproducible diagnosis through computational pathology. One of the key features of AI is its capability to generate perceptions and recognize patterns beyond the human senses. Thus, the incorporation of AI into DP can reveal additional morphological features and information. At the current rate of AI development and adoption of DP, the interest in computational pathology is expected to rise in tandem. There have already been promising developments related to AI-based solutions in prostate cancer detection; however, in the GI tract, development of more sophisticated algorithms is required to facilitate histological assessment of GI specimens for early and accurate diagnosis. In this review, we aim to provide an overview of the current histological practices in AP laboratories with respect to challenges faced in image preprocessing, present the existing AI-based algorithms, discuss their limitations and present clinical insight with respect to the application of AI in early detection and diagnosis of GI cancer.

Designing a Novel Functional Peptide With Dual Antimicrobial and Anti-inflammatory Activities via in Silico Methods.

As spider venom is composed of various bioactive substances, it can be utilized as a platform for discovering future therapeutics. Host defense peptides are great candidates for developing novel antimicrobial agents due to their multifunctional properties. In this study, novel functional peptides were rationally designed to have dual antibacterial and anti-inflammatory activities with high cytocompatibility. Based on a template sequence from the transcriptome of spider Agelena koreana, a series of via in silico analysis were conducted, incorporating web-based machine learning tools along with the alteration of amino acid residues. Two peptides, Ak-N' and Ak-N'm, were designed and were subjected to functional validation. The peptides inhibited gram-negative and gram-positive bacteria by disrupting the outer and bacterial cytoplasmic membrane. Moreover, the peptides down-regulated the expression of pro-inflammatory mediators, tumor necrosis factor-α, interleukin (IL)-1ß, and IL6. Along with low cytotoxicity, Ak-N'm was shown to interact with macrophage surface receptors, inhibiting both Myeloid differentiation primary response 88-dependent and TIR-domain-containing adapter-inducing interferon-ß-dependent pathways of Toll-like receptor 4 signaling on lipopolysaccharide-stimulated THP-1-derived macrophages. Here, we rationally designed functional peptides based on the suggested in silico strategy, demonstrating new insights for utilizing biological resources as well as developing therapeutic agents with enhanced properties.

Immunotherapy for Glioblastoma: Current State, Challenges, and Future Perspectives.

Glioblastoma is the most lethal intracranial primary malignancy by no optimal treatment option. Cancer immunotherapy has achieved remarkable survival benefits against various advanced tumors, such as melanoma and non-small-cell lung cancer, thus triggering great interest as a new therapeutic strategy for glioblastoma. Moreover, the central nervous system has been rediscovered recently as a region for active immunosurveillance. There are vibrant investigations for successful glioblastoma immunotherapy despite the fact that initial clinical trial results are somewhat disappointing with unique challenges including T-cell dysfunction in the patients. This review will explore the potential of current immunotherapy modalities for glioblastoma treatment, especially focusing on major immune checkpoint inhibitors and the future strategies with novel targets and combo therapies. Immune-related adverse events and clinical challenges in glioblastoma immunotherapy are also summarized. Glioblastoma provides persistent difficulties for immunotherapy with a complex state of patients' immune dysfunction and a variety of constraints in drug delivery to the central nervous system. However, rational design of combinational regimens and new focuses on myeloid cells and novel targets to circumvent current limitations hold promise to advent truly viable immunotherapy for glioblastoma.

TSPO-targeted NIR-fluorescent ultra-small iron oxide nanoparticles for glioblastoma imaging.

The translocator protein 18 kDa (TSPO) is mainly located in outer membrane of mitochondria and results highly expressed in a variety of tumor including breast, colon, prostate, ovarian and brain (such as glioblastoma). Glioblastoma multiforme (GBM) is the most common and lethal type of primary brain tumor. Although GBM patients had currently available therapies, the median survival is <14 months. Complete surgical resection of GBM is critical to improve GBM treatment. In this study, we performed the one-step synthesis of water-dispersible ultra-small iron oxide nanoparticles (USPIONs) and combine them with an imidazopyridine based TSPO ligand and a fluorescent dye. The optical and structural characteristics of TSPO targeted-USPIONs were properly evaluated at each step of preparation demonstrating the high colloidal stability in physiological media and the ability to preserve the relevant optical properties in the NIR region. The cellular uptake in TSPO expressing cells was assessed by confocal microscopy. The TSPO selectivity was confirmed in vivo by competition studies with the TSPO ligand PK 11195. In vivo fluorescence imaging of U87-MG xenograft models were performed to highlight the great potential of the new NIR imaging nanosystem for diagnosis and successful delineation of GBM.

In vivo delineation of glioblastoma by targeting tumor-associated macrophages with near-infrared fluorescent silica coated iron oxide nanoparticles in orthotopic xenografts for surgical guidance.

Glioblastoma multiforme (GBM) is the most aggressive and lethal type of human brain cancer. Surgery is a current gold standard for GBM treatment but the complete surgical resection of GBM is almost impossible due to their diffusive characteristics into surrounded normal brain tissues. There is an urgent need to develop a sensitive imaging tool for accurate delineation of GBM in the operating room to guide surgeons. Here we illustrate the feasibility of using near-infrared fluorescent silica coated iron oxide nanoparticles (NF-SIONs) with high water dispersion capacity and strong fluorescence stability for intraoperative imaging of GBM by targeting tumor-associated macrophages. Abundant macrophage infiltration is a key feature of GBM margins and it is well associated with poor prognosis. We synthesized NF-SIONs of about 37 nm to maximize endocytosis activity for macrophage uptake. The NF-SIONs selectively visualized tumor-associated macrophage populations by in vitro live-cell imaging and in vivo fluorescence imaging. In the orthotopic GBM xenograft models, the NF-SIONs could successfully penetrate blood-brain barrier and delineated tumor burden specifically. Taken together, this study showcased the potential applications in GBM treatment for improved intraoperative staging and more radical surgery as well as dual modality benefit in order to circumvent previous clinical failure.

Facile scalable synthesis of highly monodisperse small silica nanoparticles using alkaline buffer solution and their application for efficient sentinel lymph node mapping.

Cancer nanomedicine involving nanotechnology-based drugs and in vivo imaging agents is an active field of nanoscience that provides new ways of enhancing therapeutic and diagnostic efficacy. Translating cancer nanomedicine mainly comes from rational and scalable design of nanoparticles to achieve versatile properties including specific size because nanomaterials whose properties confer unique advantages can only optimize clinical impact. Here, a facile scalable synthesis of highly monodisperse small silica nanoparticles was developed by screening various alkaline buffer solutions as catalysts. The size of silica nanoparticles ranging from 7 to 30 nm was finely controlled by varying the reaction temperature. Moderate sized silica nanoparticles in the range of 30 to 50 nm and large sized silica nanoparticles (>100 nm) were readily synthesized by in situ adding tetraethylorthosilicate (TEOS) and applying the Stöber method in the reaction solution using small silica nanoparticles as the seeds, respectively. Having shown the ability to precisely synthesize size controlled silica nanoparticles with a process compatible with good manufacturing practices, we performed in vivo fluorescence imaging and immunofluorescence analysis of sentinel lymph nodes (SLNs) with the synthesized nanoparticles having different sizes to investigate the size effect for effective identification of SLNs. The synthesized nanoparticles with a diameter of 12 nm showed effective SLN uptake within 10 min after intradermal injection both in noninvasive and in intraoperative imaging mode and were localized evenly inside the SLN, whereas the 120 nm sized nanoparticles failed to identify the SLN with noninvasive imaging at 10 min post-injection and distributed only in the medulla region not in the superficial cortex of the SLN. Taken together, a new facile scalable synthesis technique to achieve fine size controlling capability from very small silica nanoparticles (7 nm) was developed and it made possible to investigate the optimal size of nanoparticles for efficient SLN mapping which is still controversial.

In vivo detection of macrophage recruitment in hind-limb ischemia using a targeted near-infrared fluorophore.

Macrophages are an essential component of the immune system and have protective and pathogenic functions in various diseases. Imaging of macrophages in vivo could furnish new tools to advance evaluation of disease and therapies. Critical limb ischemia is a disease in which macrophages have considerable pathogenic roles, and are potential targets for cell-based immunotherapy. We sought to develop a new near-infrared fluorescence (NIRF) imaging probe to target macrophages specifically in vivo in various pathological states, including hind-limb ischemia. We rapidly screened the photostable cyanine-based NIRF library against different blood cell lines. The identified monocyte/macrophage-selective hit was tested in vitro in live-cell labeling assay. Non-invasive NIRF imaging was performed with murine models of paw inflammation by lipopolysaccharide challenge and hind-limb ischemia with femoral artery ligation. in vivo macrophage targeting was further evaluated using intravital microscopy with Csf1r-EGFP transgenic mice and immunofluorescent staining with macrophage-specific markers. We discovered MF800, a Macrophage-specific near-infrared Fluorophore, which showed selective live-cell imaging performance in a panel of cell lines and primary human blood samples. MF800 outperforms the clinically-available NIRF contrast agent ICG for in vivo specificity in paw inflammation and hind-limb ischemia models. We observed a marked overlap of MF800-labeled cells and EGFP-expressing macrophages in intravital imaging of Csf1r-EGFP transgenic mice. In the histologic analysis, MF800-positive cells also expressed the macrophage markers CD68 and CD169. NIRF imaging showcased the potential of using MF800 to understand macrophage behavior in vivo, characterize macrophage-associated diseases, and may help in assessing therapeutic responses in the clinic.

Urinary excretion of ß2-microglobulin as a prognostic marker in immunoglobulin A nephropathy.

BACKGROUND/AIMS: ß2-microglobulin (ß2-MG) is freely filtered at the glomerulus and subsequently reabsorbed and catabolized by proximal renal tubular cells. Urinary ß2-MG is an early and sensitive biomarker of acute kidney injury; however, its utility as a biomarker of immunoglobulin A nephropathy (IgAN) is unclear. METHODS: We included urinary ß2-MG levels in the routine laboratory examination of all inpatients with biopsy-proven IgAN at our hospital from 2006 to 2010. We retrospectively analyzed the correlation between ß2-MG levels and clinical parameters as a prognostic biomarker of IgAN. RESULTS: A total of 51 patients (30 males, 21 females; mean age, 33.01 ± 12.73 years) with IgAN were included in this study. Initial demographic, clinical, and laboratory data for all patients are listed. The mean initial estimated glomerular filtration rate and 24-hour urine protein levels were 94.69 ± 34.78 mL/min/1.73 m(2) and 1.28 ± 1.75 g/day, respectively. The mean level of urinary ß2-MG was 1.92 ± 7.38 µg/mg creatinine. There was a significant correlation between initial serum creatinine (iSCr), urine protein creatinine ratio (UPCR), and the level of ß2-MG (r = 0.744, r = 0.667, p < 0.01). There was also a significant correlation between renal function tests and the level of urinary ß2-MG (p < 0.01). Cox regression analysis showed that albumin, ß2-MG, iSCr, and UPCR were significant predictors of disease progression in IgAN. CONCLUSIONS: Urinary ß2-MG levels showed a significant correlation with renal function and proteinuria in IgAN. Thus, we propose that urinary ß2-MG may be an additional prognostic factor in patients with IgAN.

A macrophage-specific fluorescent probe for intraoperative lymph node staging.

Successful identification of nodal metastases in patients with cancer is crucial to prescribe suitable treatment regimens that can improve recurrence-free survival. Although some new imaging technologies for nodal staging have been developed, such as nanoparticle-enhanced MRI and quantum-dot-based fluorescence imaging, sound technologies for intraoperative differentiation of metastatic and inflamed lymph nodes remain lacking. In this study, we illustrate the feasibility of using a macrophage-specific fluorescent probe (MFP) to visualize sentinel lymph nodes during surgery, highlighting abnormalities related to inflammation and tumor infiltration with signal enhancement and reduction methods using this technology. MFP was identified by high-throughput screening of fluorescent small-molecule libraries synthesized with a diversity-oriented approach. It selectively visualized monocyte and macrophage cell populations in vitro, by live-cell imaging and flow cytometry, as well as in vivo, for imaging-guided surgery. Collectively, this study provides preclinical proof of concept for an intraoperative imaging platform to accurately assess lymph node status, eliminating the need for invasive nodal dissections that can contribute to complications of cancer therapy.

Primo vascular system accompanying a blood vessel from tumor tissue and a method to distinguish it from the blood or the lymph system.

A primo vessel was observed in the abdominal cavity in the lung cancer mouse model, and its function as an extra metastatic path was observed. In this work, we found a primo vessel accompanying a blood vessel emanating from a tumor in the skin. We also presented simple and efficient criteria to distinguish a primo vessel from a blood or a lymph vessel and from a nerve. The criteria for using DAPI and Phalloidin will be useful in clinical situations to find and identify the primo vessels among the blood vessels, lymph vessels, or nerves in the tissue surrounding a tumor such as a melanoma or breast cancer.

Evidence for an additional metastatic route: in vivo imaging of cancer cells in the primo-vascular system around tumors and organs.

PURPOSE: Researchers have been studying the mechanisms by which metastasis can be prevented via blocking the hematogenous and the lymphatic routes for a long time now. However, metastasis is still the single most challenging obstacle for successful cancer management. In a new twist that may require some retooling of this established approach, we investigated the hypothesis that tumor metastases can occur via an independent fluid-conducting system called the primo-vascular system. PROCEDURES: The dissemination and growth of near-infrared quantum dot (NIR QD)-electroporated cancer cells in metastatic sites were investigated using in vivo multispectral imaging techniques. RESULTS: Our results show that the NIR QD-labeled cancer cells were able to migrate through not only the blood vascular and lymphatic systems but also the primo-vascular system extending from around the tumor to inside the abdominal cavity. Furthermore, the NIR QD-labeled cancer cells, which had been seeded intraperitoneally, specifically infiltrated the primo-vascular system in the omentum and in the gonadal fat. CONCLUSIONS: These findings strongly suggest that the primo-vascular system may be an additional metastasis route, complementing the lymphatic and hematogenous routes, which facilitate the dissemination and colonization of cancer cells at secondary sites.

Characterization of the primo-vascular system in the abdominal cavity of lung cancer mouse model and its differences from the lymphatic system.

Cancer growth and dissemination have been extensively studied for a long time. Nevertheless, many new observations on anatomy and histopathology of cancer events are still reported such as formation of a vasculogenic-like network inside aggressive tumors. In this research, new kinds of micro-conduits, named primo-vessels, were found inside the abdominal cavity of NCI-H460 lung cancer murine xenograft models. These vascular threads were largely distributed on the surfaces of various organs and were often connected to peritoneal tumor nodules. Histological and immunofluorescent investigations showed that the primo-vessels had characteristic features that were distinctively different from those of similar-looking lymphatic vessels. They had multiple channels surrounded with loose collageneous matrices, which is in contrast to the single-channel structure of other vascular systems. The rod-shaped nuclei aligned longitudinally along the channels were assumed to be the endothelial cells of the primo-vessels, but LYVE-1, a specific marker of lymphatics, was not expressed, which indicates a clear difference from lymphatic endothelial cells. Taken together these findings on and characterization of the novel threadlike vascular structures in cancer models may have important implications for cancer prognosis and for therapy.

Current concepts and future perspectives on surgical optical imaging in cancer.

There are vibrant developments of optical imaging systems and contrast-enhancing methods that are geared to enhancing surgical vision and the outcome of surgical procedures. Such optical technologies designed for intraoperative use can offer high integration in the operating room compared to conventional radiological modalities adapted to intraoperative applications. Simple fluorescence epi-illumination imaging, in particular, appears attractive but may lead to inaccurate observations due to the complex nature of photon-tissue interaction. Of importance therefore are emerging methods that account for the background optical property variation in tissues and can offer accurate, quantitative imaging that eliminates the appearance of false negatives or positives. In parallel, other nonfluorescent optical imaging methods are summarized and overall progress in surgical optical imaging applications is outlined. Key future directions that have the potential to shift the paradigm of surgical health care are also discussed.

Bonghan ducts as possible pathways for cancer metastasis.

OBJECTIVE: The present study has been designed to find a possible new route for the metastasis of cancer cells on the fascia surrounding tumor tissue using a novel technique of trypan blue staining. MATERIALS AND METHODS: Tumor tissues were grown in the skin of nude mice after sub-cutaneous inoculation with human lung cancer cells. Trypan blue was recently identified as a dye with specificity for Bonghan ducts (BHDs) and not other tissues, such as blood or lymph vessels or nerves. RESULTS: We demonstrate that the trypan blue staining technique allows the first visualization of BHDs which are connected to tumor tissues. CONCLUSION: Since BHDs are known to make up a circulatory system corresponding to acupuncture meridians or collaterals, we propose that, in addition to the currently known blood or lymph vessels, BHDs on tumor tissue fascia may be a novel pathway for metastasis.

Comparative analysis of cell surface proteins in chronic and acute leukemia cell lines.

This study was designed to identify the cell surface protein markers that can differentiate between chronic myeloid leukemia (CML) and acute promyelocytic leukemia cells (APL). The differentially expressed plasma membrane proteins were analyzed between CML cell line (K562) and APL cell line (NB4) using the comparative proteomic approach. The cell membrane proteins were enriched by labeling with a membrane-impermeable biotinylation reagent, sulfo-NHS-SS-Biotin, and subjected to liquid chromatography tandem mass spectrometry (LC-MS/MS). By comparative proteomic analysis of K562 and NB4 cells, we identified 25 membrane and 14 membrane-associated proteins. The result of LC-MS/MS combined with chemical tagging method was validated by confirming the expression and localization of one of the differentially expressed plasma membrane proteins, CD43, by FACS and confocal microscopy. Our results indicate that CD43 could be a potential candidate for differentiating CML from APL.

Electron microscopic study of novel threadlike structures on the surfaces of mammalian organs.

The ultrastructures of novel threadlike structures (NTSs) and corpuscles on the surfaces of internal organs of rats were investigated using electron microscopy. The samples were studied in situ by using a stereomicroscope and were taken for further morphological analysis. Scanning electron microscope (SEM) images revealed a bundle structure of threadlike tissue, which was composed of several 10-micro m-thick subducts. The surfaces of the corpuscles were rather coarse and fenestrated. The corpuscles had cucumber-like shapes with an average length of about 2 mm and a thickness of about 400 micro m. Transmission electron microscope (TEM) images disclosed disordered collagen fibers, which formed the extracellular matrix of the threadlike tissue, and immune-function cells, like macrophages, mast cells, and eosinophils. Sinuses of various diameters, which were thought to be cross-sections of the lumens of the subducts, were observed in the TEM, cryo-SEM and focused-ion-beam SEM images. These SEM images were obtained for the first time to reveal the detailed structure of the NTSs that were only recently discovered.

FHIT protein enhances paclitaxel-induced apoptosis in lung cancer cells.

The fragile histidine triad (FHIT) gene is a frequent target of deletions in lung cancer. Previous studies have shown that FHIT gene transfer into lung cancer cells lacking FHIT expression results in induction of apoptosis. However, the effect of FHIT expression on apoptosis induced by chemotherapeutic agents and its intracellular mechanism is poorly understood. This study was undertaken to elucidate the effect of FHIT expression and the role of Bcl-2-caspase signaling in paclitaxel-induced apoptosis in lung cancer cells. NCI-H358 lung cancer cells, which lack FHIT expression, were stably transfected with plasmid vector containing FLAG-tagged wildtype FHIT. We investigated effects of paclitaxel on apoptosis, activation of caspase system and expression of Bcl-2 family. We next evaluated whether these effects were reversed by blocking FHIT expression using siRNA. Paclitaxel enhanced apoptosis in FHIT-expressing cells compared to that in control vector-transfected cells, and this enhancement was suppressed by siRNA treatment. Activities of caspase-3 and caspase-7, but not of caspase-8, were higher in FHIT-expressing cells than in control vector-transfected cells, and this was reduced by siRNA treatment. When caspase activation was blocked by a pan-caspase inhibitor in FHIT-expressing cells, paclitaxel-induced apoptotic cell death was decreased similar to that in control vector-transfected cells. Bcl-2 and Bcl-xL expressions were down-regulated after paclitaxel treatment in FHIT-expressing cells, whereas Bax and Bad expressions were up-regulated. These were reversed by siRNA treatment. These results indicate that paclitaxel-induced apoptosis enhanced by FHIT expression in lung cancer cells might be associated with modulation of Bcl-2-caspase signaling.