Homogeneous,Low‐volume,Efficient, and Sensitive Quantitation of Circulating Exosomal PD‐L1 for Cancer Diagnosis and Immunotherapy Response Prediction

Immunotherapy has revolutionized cancer treatment, but its efficacy is severely hindered by the lack of effective predictors. Herein, we developed a homogeneous, low‐volume, efficient, and sensitive exosomal programmed death‐ligand 1 (PD‐L1, a type of transmembrane protein) quantitation method for cancer diagnosis and immunotherapy response prediction (HOLMES‐Exo_PD‐L1). The method combines a newly evolved aptamer that efficiently binds to PD‐L1 with less hindrance by antigen glycosylation than antibody, and homogeneous thermophoresis with a rapid binding kinetic. As a result, HOLMES‐Exo_PD‐L1 is higher in sensitivity, more rapid in reaction time, and easier to operate than existing enzyme‐linked immunosorbent assay (ELISA)‐based methods. As a consequence of an outstanding improvement of sensitivity, the level of circulating exosomal PD‐L1 detected by HOLMES‐Exo_PD‐L1 can effectively distinguish cancer patients from healthy volunteers, and for the first time was found to correlate positively with the metastasis of adenocarcinoma. Overall, HOLMES‐Exo_PD‐L1 brings a fresh approach to exosomal PD‐L1 quantitation, offering unprecedented potential for early cancer diagnosis and immunotherapy response prediction.     

A novel peptide-based probe 99mTc-PEG6-RD-PDP2 for the molecular imaging of tumor PD-L2 expression

Tumor-related PD-L2 expression is associated with the clinical efficacy of PD-1/PD-L1 blockade therapy. PD-L2-specific imaging can help selecting patients for appropriate immunotherapy. In this study, a PD-L2-targeting peptide (PDP2) was screened by the one-bead one-compound combinatorial library approach. Using the retro-inverso d-peptide of PDP2 (RD-PDP2) and PEGylation strategies, we developed a novel Tc-99m-labeled PD-L2-targeting peptide as a SPECT tracer (^99mTc-PEG₆-RD-PDP2) for imaging of tumor PD-L2 expression. The radiolabeling yield of ^99mTc-PEG₆-RD-PDP2 was greater than 95% by the standard HYNIC/tricine/TPPTS labeling procedure. ^99mTc-PEG₆-RD-PDP2 displayed high PD-L2-binding specificity both in vitro and in vivo. SPECT/CT imaging with ^99mTc-PEG₆-RD-PDP2 showed that the A549-PD-L2 tumors were clearly visualized, whereas the signals in PD-L2-negative A549 tumors were much lower. In vivo blocking study suggested that the tumor uptake of ^99mTc-PEG₆-RD-PDP2 was PD-L2 specifically mediated. ^99mTc-PEG₆-RD-PDP2 is a promising SPECT probe for the non-invasive imaging of tumor PD-L2 expression and has a great potential in guiding the anti-PD-1 or anti-PD-L1 immunotherapy of cancer.     

Discovery of Novel Small-Molecule Inhibitors of PD-1/PD-L1 Interaction via Structural Simplification Strategy

Blockade of the programmed cell death 1 (PD-1)/programmed cell death-ligand 1 (PD-L1) interaction is currently the focus in the field of cancer immunotherapy, and so far, several monoclonal antibodies (mAbs) have achieved encouraging outcomes in cancer treatment. Despite this achievement, mAbs-based therapies are struggling with limitations including poor tissue and tumor penetration, long half-life time, poor oral bioavailability, and expensive production costs, which prompted a shift towards the development of the small-molecule inhibitors of PD-1/PD-L1 pathways. Even though many small-molecule inhibitors targeting PD-1/PD-L1 interaction have been reported, their development lags behind the corresponding mAb, partly due to the challenges of developing drug-like small molecules. Herein, we report the discovery of a series of novel inhibitors targeting PD-1/PD-L1 interaction via structural simplification strategy by using BMS-1058 as a starting point. Among them, compound A9 stands out as the most promising candidate with excellent PD-L1 inhibitory activity (IC₅₀ = 0.93 nM, LE = 0.43) and high binding affinity to hPD-L1 (K_D = 3.64 nM, LE = 0.40). Furthermore, A9 can significantly promote the production of IFN-γ in a dose-dependent manner by rescuing PD-L1 mediated T-cell inhibition in Hep3B/OS-8/hPD-L1 and CD3-positive T cells co-culture assay. Taken together, these results suggest that A9 is a promising inhibitor of PD-1/PD-L1 interaction and is worthy for further study.     

The role of exosomal lncRNAs in cancer biology and clinical management

Exosomes play a vital role in cell–cell communication within the cancer microenvironment. Exosomal long noncoding RNAs (lncRNAs) are important regulators in cancer development and are involved in multiple processes, including cancer cell proliferation, angiogenesis, metastasis, drug resistance, and immunomodulation. Changes in the levels of exosomal lncRNAs often appear with the occurrence and development of cancer. Therefore, exosomal lncRNAs can be used as biomarkers for cancer diagnosis and prognosis. Exosomal lncRNAs can also indicate the treatment response of patients receiving chemotherapy. Moreover, exosomal lncRNAs are potential therapeutic targets for cancer treatment. In this review, we summarize the role of exosomal lncRNAs in cancer biology as well as in clinical management. A more comprehensive and in-depth understanding of the role of exosomal lncRNAs in cancer may help us better understand the mechanism of cancer development and clinically manage cancer patients.Subject terms: Cancer, Molecular biology     

A Novel d-Peptide Identified by Mirror-Image Phage Display Blocks TIGIT/PVR for Cancer Immunotherapy

The low response rate and adaptive resistance of PD-1/PD-L1 blockade demands the studies on novel therapeutic targets for cancer immunotherapy. We discovered that a novel immune checkpoint TIGIT expressed higher than PD-1 in many tumors especially anti-PD-1 resistant tumors. Here, mirror-image phage display bio-panning was performed using the d -enantiomer of TIGIT synthesized by hydrazide-based native chemical ligation. d -peptide ^DTBP-3 was identified, which could occupy the binding interface and effectively block the interaction of TIGIT with its ligand PVR. ^DTBP-3 showed proteolytic resistance, tumor tissue penetrating ability, and significant tumor suppressing effects in a CD8⁺ T cell dependent manner. More importantly, ^DTBP-3 could inhibit tumor growth and metastasis in anti-PD-1 resistant tumor model. This is the first d -peptide targeting TIGIT, which could serve as a potential candidate for cancer immunotherapy.     

Rapid Detection of Exosomal MicroRNAs Using Virus‐Mimicking Fusogenic Vesicles

Exosomal microRNAs (miRNAs) are important biomarkers for clinical diagnosis and disease treatment monitoring. However, most approaches for exosomal miRNA detection are time‐consuming, laborious, and expensive. Herein, we report a virus‐mimicking fusogenic vesicle (Vir‐FV) that enables rapid, efficient, and high‐throughput detection of exosomal miRNAs within 2 h. Fusogenic proteins on Vir‐FVs can specifically target the sialic‐acid‐containing receptors on exosomes, inducing efficient fusion of Vir‐FVs and exosomes. Upon vesicle content mixing, the molecular beacons encapsulated in Vir‐FVs specifically hybridize with the target miRNAs in the exosomes, generating fluorescence. Combined with flow cytometry, the Vir‐FVs can not only detect exosomal miRNAs but also distinguish tumor exosomes from normal exosomes by sensing the tumor‐related miRNAs, paving the way towards the rapid and efficient detection of exosomal miRNAs for diagnosis and prognosis prediction of diseases.     

A dendritic cell-like biomimetic nanoparticle enhances T cell activation for breast cancer immunotherapy

Cancer immunotherapy has remarkably improved the therapeutic effect of melanoma and non-small cell lung cancer in the clinic. Nevertheless, it showed disappointing clinical outcomes for treating immunosuppressive tumors, wherein aggressive T cells are rather limited in tumor sites. Therefore, regulating the behavior of T cells in tumor sites to increase their attack ability for suppressing the immunosuppressive tumor is highly desirable. Inspiringly, we designed a dendritic cell-like biomimetic nanoparticle (DMSNs³@HA) to regulate the behavior of T cells for improving the immunotherapy effect against immunosuppressive tumors. In this work, anti-CD3 and anti-CD28 were responsible for mimicking dendritic cells to activate T cells, and anti-PD-1 for blocking the pathway of PD-1/PD-L1 to break the immune “brake”, which synergistically regulated the behavior of T cells to attack cancer cells. Experimental results indicated that DMSNs³@HA can effectively activate T cells and improve their immune response to significantly inhibit the growth of breast cancer. Moreover, it also proved that T cell activation combining immune checkpoint blocking induced the “1 + 1 >2” immunotherapy effect against immunosuppressive tumors. We expect that this strategy will provide new insights into tumor immunotherapy by modulating T cell behavior.

A dendritic cell-like biomimetic nanoparticle has been designed to regulate the behavior of T cells for improving the immunotherapy effect against immunosuppressive tumors.     

Virtual Screening and In Vitro Evaluation of PD-1 Dimer Stabilizers for Uncoupling PD-1/PD-L1 Interaction from Natural Products

Genetic mutations accumulated overtime could generate many growth and survival advantages for cancer cells, but these mutations also mark cancer cells as targets to be eliminated by the immune system. To evade immune surveillance, cancer cells adopted different intrinsic molecules to suppress immune response. PD-L1 is frequently overexpressed in many cancer cells, and its engagement with PD-1 on T cells diminishes the extent of cytotoxicity from the immune system. To resume immunity for fighting cancer, several therapeutic antibodies disrupting the PD-1/PD-L1 interaction have been introduced in clinical practice. However, their immunogenicity, low tissue penetrance, and high production costs rendered these antibodies beneficial to only a limited number of patients. PD-L1 dimer formation shields the interaction interface for PD-1 binding; hence, screening for small molecule compounds stabilizing the PD-L1 dimer may make immune therapy more effective and widely affordable. In the current study, 111 candidates were selected from over 180,000 natural compound structures through virtual screening, contact fingerprint analysis, and pharmacological property prediction. Twenty-two representative candidates were further evaluated in vitro. Two compounds were found capable of inhibiting the PD-1/PD-L1 interaction and promoting PD-L1 dimer formation. Further structure optimization and clinical development of these lead inhibitors will eventually lead to more effective and affordable immunotherapeutic drugs for cancer patients.     

Acid-activatible micelleplex delivering siRNA-PD-L1 for improved cancer immunotherapy of CDK4/6 inhibition

Cyclin-dependent kinases 4 and 6 inhibitors(CDK4/6i) have been demonstrated to trigger antitumor immunity for tumor regression. However, the therapeutic performance of CDK4/6i-meadiated cancer immunotherapy was impaired by the immunosuppressive tumor microenvironment(ITM) due to overexpression of programmed death ligand 1(PD-L1) on the surface of cancer cell membrane. To improve the immunotherapeutic performance of CDK4/6i, we herein developed endosomal acidactivatable micelleplex for si RNA delivery and PD-L1 knockdown in the tumor cells in vitro and in vivo. We further demonstrated that the combination of PD-L1 knockdown and CDK4/6 inhibition facilitated intratumoral infiltration of cytotoxic T lymphocytes(CTLs), and elicited protective immune response and efficiently suppressed tumor growth in vivo. This study revealed the importance of molecular design of the micelleplex for highly efficient si RNA delivery, which might provide a novel insight for RNAi-based cancer immunotherapy.     

Multispecific Platinum(IV) Complex Deters Breast Cancer via Interposing Inflammation and Immunosuppression as an Inhibitor of COX-2 and PD-L1

Breast cancer (BC) is one of the most common malignancies in women and often accompanied by inflammatory processes. Cyclooxygenase-2 (COX-2) plays a vital role in the progression of BC, correlating with the expression of programmed death-ligand 1 (PD-L1). Overexpression of PD-L1 contributes to the immune escape of cancer cells, and its blockade would stimulate anticancer immunity. Two multispecific platinum(IV) complexes DNP and NP were prepared using non-steroidal antiinflammatory drug naproxen (NPX) as axial ligand(s) to inhibit the BC cells. DNP exhibited high cytotoxicity and antiinflammatory properties superior over NP, cisplatin and NPX; moreover, it displayed potent antitumor activity and almost no general toxicity in mice bearing triple-negative breast cancer (TNBC). Mechanistic studies revealed that DNP could downregulate the expression of COX-2 and PD-L1 in vitro and vivo, inhibit the secretion of prostaglandin, reduce the expression of BC-associated protein BRD4 and phosphorylation of extracellular signal-regulated kinases 1/2 (Erk1/2), and block the oncogene c-Myc in BC cells. These findings demonstrate that DNP is capable of intervening in inflammatory, immune, and metastatic processes of BC, thus presenting a new mechanism of action for anticancer platinum(IV) complexes. The multispecificity offers a special superiority for DNP to treat TNBC by combining chemotherapy and immunotherapy in one molecule.     

Sodium bicarbonate,an inorganic salt and a potential active agent for cancer therapy

Cancer is a serious threat to humans due to its high mortality. The efforts to fully understand cancer and to fight against it have never been stopped. The traditional therapies, such as surgery, radiotherapy and chemotherapy, are useful but cannot meet the increasing demands of patients. As such, novel approaches against cancer are urgently required. It has been found that the acidic tumor microenvironment plays important roles in promoting the cancer progression. In recent years, sodium bicarbonate (NaHCO₃), a simple inorganic salt, has been found to be able to reverse the pH of tumor microenvironment and inhibit the invasion, metastasis, immune evasion, drug resistance and hypoxia of tumor cells. Thus, NaHCO₃-based therapy is a potential approach for the treatment of cancer, and the related studies have been increasingly reported. Herein, we aim to provide a comprehensive understanding of the acidic tumor microenvironment and summarize the applications and mechanisms of NaHCO₃ in cancer therapy. The combination of NaHCO₃ with chemotherapy, immunotherapy or nanoparticles systems is discussed. In addition, the concerns of NaHCO₃ in clinical use and the potential ways to use NaHCO₃ for cancer therapy are also discussed.     

A microfluidic surface-enhanced Raman scattering (SERS) sensor for microRNA in extracellular vesicles with nucleic acid-tyramine cascade amplification

Exosomal microRNA (miRNA) is an ideal candidate of noninvasive biomarker for the early diagnosis of cancer. Sensitive and accurate sensing of abnormal exosomal miRNA plays essential role for clinical promotion due to its close correlation with tumor proliferation and progression. Herein, a microfluidic surface-enhanced Raman scattering (SERS) sensor was proposed for an on-line detection of exosomal miRNA based on rolling circle amplification (RCA) and tyramine signal amplification (TSA) strategy. The microfluidic chip consists of a magnetic enrichment chamber, a serpentine fluidic mixer and a plasmonic SERS substrate functionalized with capture probes. The released miRNA activates the capture probe, triggers RCA reaction, and generates a large number of single-stranded DNA products to drive the catalysis of nanotags deposition via TSA, producing numerous “hot spots” to enhance the SERS signals. In merit of the microfluidics chip and nucleic acid-tyramine cascade amplification, the developed SERS sensor significantly improves the sensitivity for the exosomal miRNA assay, resulting in a limit of detection (LOD) as low as 1 pmol/L and can be successfully applied in the analysis of exosomes secreted from breast tumor cells, which demonstrates the potential utility in practical applications.     

A Tandem-Locked Fluorescent NETosis Reporter for the Prognosis Assessment of Cancer Immunotherapy

NETosis, the peculiar type of neutrophil death, plays important roles in pro-tumorigenic functions and inhibits cancer immunotherapy. Non-invasive real-time imaging is thus imperative for prognosis of cancer immunotherapy yet remains challenging. Herein, we report a T andem-locked N ETosis R eporter 1 (TNR₁) that activates fluorescence signals only in the presence of both neutrophil elastase (NE) and cathepsin G (CTSG) for the specific imaging of NETosis. In the aspect of molecular design, the sequence of biomarker-specific tandem peptide blocks can largely affect the detection specificity towards NETosis. In live cell imaging, the tandem-locked design allows TNR₁ to differentiate NETosis from neutrophil activation, while single-locked reporters fail to do so. The near-infrared signals from activated TNR₁ in tumor from living mice were consistent with the intratumoral NETosis levels from histological results. Moreover, the near-infrared signals from activated TNR₁ negatively correlated with tumor inhibition effect after immunotherapy, thereby providing prognosis for cancer immunotherapy. Thus, our study not only demonstrates the first sensitive optical reporter for noninvasive monitoring of NETosis levels and evaluation of cancer immunotherapeutic efficacy in tumor-bearing living mice, but also proposes a generic approach for tandem-locked probe design.     

A Measurement Technique for Organic Nitrates and Halocarbons in Ambient Air

A cryogenic preconcentration/high-resolution gas chromatographic technique has been developed for the rapid, simultaneous quantitation of C₁–C₄ organic nitrates and halocarbons in ambient air. Whole-air samples are collected in Tedlar^TM bags by an evacuated-chamber method. Samples were stable in 0.010-cm-thick bags for 24 h if they were immediately stored in a freezer at −25°C. Analytes in a 50-cm³ air sample were efficiently preconcentrated on fused-silica beads at −180°C and thermally desorbed at 30°C. High-resolution gas chromatography with a cross-linked polydimethylsiloxane fused-silica capillary column and an electron-capture detector were used for separation and quantitation of the analytes. An analysis time of about 12 min was facilitated by sample cryofocusing at −180°C and oven temperature programming. Recoveries of the analytes by the evacuated-chamber method were better than 95%. The sensitivity of the technique for sample volumes of 50 cm³ is in the sub-parts-per-trillion by volume (ppt[v]) range for many of the analytes, with an average precision of about ±5% for analytes at levels of about 10 ppt(v).     

An Activatable NIR‐II Nanoprobe for In Vivo Early Real‐Time Diagnosis of Traumatic Brain Injury

Traumatic brain injury (TBI) is one of the most dangerous acute diseases resulting in high morbidity and mortality. Current methods remain limited with respect to early diagnosis and real‐time feedback on the pathological process. Herein, a targeted activatable fluorescent nanoprobe (V&A@Ag₂S) in the second near‐infrared window (NIR‐II) is presented for in vivo optical imaging of TBI. Initially, the fluorescence of V&A@Ag₂S is turned off owing to energy transfer from Ag₂S to the A1094 chromophore. Upon intravenous injection, V&A@Ag₂S quickly accumulates in the inflamed vascular endothelium of TBI based on VCAM1‐mediated endocytosis, after which the nanoprobe achieves rapid recovery of the NIR‐II fluorescence of Ag₂S quantum dots (QDs) owing to the bleaching of A1094 by the prodromal biomarker of TBI, peroxynitrite (ONOO^?). The nanoprobe offers high specificity, rapid response, and high sensitivity toward ONOO^?, providing a convenient approach for in vivo early real‐time assessment of TBI.     

A Novel Method for the Quantitation of Warfarin and its Metabolites in Plasma

Abstract

A procedure for the rapid, quantitative recovery of warfarin and its metabolites (diastereoisomeric alcohol, 4′-, 6-, 7-, 8-benzylic-hydroxywarfarin and dehydrowarfarin) from plasma with Sep-Pak C₁₈ cartridges has been developed. A solution of warfarin and its metabolites in plasma was acidified with NH₄OAc buffer (pH 4.85), adsorbed on the Sep-Pak C₁₈ resin, washed free of polar constituents and eluted with methanol. Dilution of the eluate with buffer followed by gradient high performance liquid chromatography permitted accurate quantitation of the desired compounds when detected at 313 nm. The recovery of warfarin and each metabolite was greater than 95% over an investigated range of 0.5–10.0 μg/ml of plasma and the limit of quantitation by the assay was 0.1 μg/ml of plasma. For more rapid quantitation of warfarin, without simultaneous analysis of metabolites, the chromatographic parameters were modified so that elution of warfarin occurred within 13 minutes, and the sensitivity of the assay increased to 0.03 μg of warfarin/ml of plasma. The quantitative recovery of warfarin and its metabolites coupled with the chromatographic versatility of the method make it ideally suited for either detailed pharmacokinetic studies or routine plasma analysis of warfarin.     

Quantitative analysis of calcium and fluorine by high-sensitivity low-energy ion scattering: Calcium fluoride

Low-energy ion scattering (LEIS) probes the atomic composition of the outer surface. Well-defined reference samples are used for the quantitation. For elements like fluorine and calcium, it is not easy to find suitable, clean, and homogeneous references, since fluorine is a gas and calcium is a very reactive metal. In contrast to surface analytic techniques such as XPS, the extreme surface sensitivity of LEIS makes it difficult to use stable compounds like CaF₂ as reference, since these compounds are not homogeneous at the atomic scale. With LEIS, CaF₂ is not expected to show an atomic ratio F/Ca = 2.0. Thus, before CaF₂ can be used as reference, its atomic surface concentrations have to be determined. Here, 3-keV He⁺ scattering by a LiF(001) single crystal, an evaporated layer of Ca, and a Cu foil are used as basic references. High-purity CaF₂ is available in two forms: a single crystal and a powder. For a practical reference, powders are preferred, since if bulk impurities segregate to the surface, they will be dispersed over a large surface area. It is found that both CaF₂ (111) and powder have similar F/Ca atomic ratios. This confirms the F termination for both samples. For the powder, the F and Ca signals are reduced by 0.77 ± 0.03 in comparison with those for the single crystal. The atomic sensitivity factors and relative sensitivity factors have been determined for F, Ca, and Cu.     

Rapid and sensitive lateral flow immunoassay method for determining alpha fetoprotein in serum using europium (III) chelate microparticles-based lateral flow test strips

Alpha-fetoprotein (AFP), a primary marker for many diseases including various cancers, is important in clinical tumor diagnosis and antenatal screening. Most immunoassays provide high sensitivity and accuracy for determining AFP, but they are expensive, often complex, time-consuming procedures. A simple and rapid point-of-care system that integrates Eu (III) chelate microparticles with lateral flow immunoassay (LFIA) has been developed to determine AFP in serum with an assay time of 15 min. The approach is based on a sandwich immunoassay performed on lateral flow test strips. A fluorescence strip reader was used to measure the fluorescence peak heights of the test line (H_T) and the control line (H_C); the H_T/H_C ratio was used for quantitation. The Eu (III) chelate microparticles-based LFIA assay exhibited a wide linear range (1.0–1000 IU mL⁻¹) for AFP with a low limit of detection (0.1 IU mL⁻¹) based on 5ul of serum. Satisfactory specificity and accuracy were demonstrated and the intra- and inter-assay coefficients of variation (CV) for AFP were both <10%. Furthermore, in the analysis of human serum samples, excellent correlation (n = 284, r = 0.9860, p < 0.0001) was obtained between the proposed method and a commercially available CLIA kit. Results indicated that the Eu (III) chelate microparticles-based LFIA system provided a rapid, sensitive and reliable method for determining AFP in serum, indicating that it would be suitable for development in point-of-care testing.