Lycii Radicis Cortex (LRC) is a medicinal and food homologous plant with various pharmacological activities, including anti-tumor effects. This study explores the anti-tumor effect of LRC on non-small cell lung cancer (NSCLC) and its molecular mechanism using mice bearing Lewis lung carcinoma cells. LRC significantly suppressed the growth of NSCLC. Besides, RNA sequencing of mice tumors and hematoxylin & eosin and immunofluorescence staining revealed that LRC promoted the infiltration of T lymphocytes, specifically GZMB+CD8+T lymphocytes, in tumor tissues. The Gene Set Enrichment Analysis of spleen RNA indicated that LRC up-regulated PD-1-downstream pathways, suggesting that LRC exerted its effects through the PD-L1/PD-1 pathway. Further experiments revealed that LRC interacted with PD-L1, blocking PD-L1/PD-1 binding and thus restoring the T cell killing activity on tumor cells. Together, these results support using LRC as healthy food to improve anti-tumor immunity in patients with NSCLC.

Alzheimer's disease (AD) starts decades before cognitive symptoms develop. Easily accessible and cost-effective biomarkers that accurately reflect AD pathology are essential for both monitoring and therapeutics of AD. Neurofilament light chain (NfL) levels in blood and cerebrospinal fluid are increased in AD more than a decade before the expected onset, thus providing one of the most promising blood biomarkers for monitoring of AD. The clinical practice of employing single-molecule array (Simoa) technology for routine use in patient care is limited by the high costs. Herein, we developed a microarray chip-based high-throughput screening method and screened an attractive self-assembling peptide targeting NfL. Through directly "imprinting" and further analyzing the sequences, morphology, and affinity of the identified self-assembling peptides, the Pep-NfL peptide nanosheet with high binding affinity toward NfL (K-D = 1.39 x 10(-9) mol/L), high specificity, and low cost was characterized. The superior binding ability of Pep-NfL was confirmed in AD mouse models and cell lines. In the clinical setting, the Pep-NfL peptide nanosheets hold great potential for discriminating between patients with AD (P < 0.001, n = 37), mild cognitive impairment (P < 0.05, n = 26), and control groups (n = 30). This work provides a high-throughput, high-sensitivity, and economical system for noninvasive tracking of AD to monitor neurodegeneration at different stages of disease. The obtained Pep-NfL peptide nanosheet may be useful for assessing dynamic changes in plasma NfL concentrations to evaluate disease-modifying therapies as a surrogate end point of neurodegeneration in clinical trials.

Breast cancer has become the leading cause of cancer mortality in women. Although immune checkpoint inhibitors targeting programmed death-1 (PD-1) are promising, it remains unclear whether PD-L1 expression on circulating tumor cells (CTCs) has predictive and prognostic values in predicting and stratifying metastatic breast cancer (MBC) patients who can benefit from anti-PD-1 immunotherapy. Twenty six MBC patients that received anti-PD-1 immunotherapy were enrolled in this study. The peptide-based Pep@MNPs method was used to isolate and enumerate CTCs from 2.0 ml of peripheral venous blood. The expression of PD-L1 on CTCs was evaluated by an established immunoscoring system categorizing into four classes (negative, low, medium, and high). Our data showed that 92.3

Rationale: Breast cancer has become the leading cause of cancer mortality in women. Although immune checkpoint inhibitors targeting programmed death-1 (PD-1) are promising, it remains unclear whether PD-L1 expression on circulating tumor cells (CTCs) has predictive and prognostic values in predict and stratify metastatic breast cancer (MBC) patients who can benefit from anti-PD-1 immunotherapy. Methods: Twenty six MBC patients that received anti-PD-1 immunotherapy were enrolled in this study. The peptide-based Pep@MNPs method was used to isolate and enumerate CTCs from 2.0 ml of peripheral venous blood. The expression of PD-L1 on CTCs was evaluated by an established immunoscoring system categorizing into four classes (negative, low, medium, and high). Results: Our data showed that 92.3% (24/26) of patients had CTCs, 83.3% (20/26) of patients had PD-L1-positive CTCs, and 65.4% (17/26) of patients had PD-L1-high CTCs. We revealed that the clinical benefit rate (CBR) of patients with a cut-off value of ≥ 35% PD-L1-high CTCs (66.6%) was higher than the others (29.4%). We indicated that PD-L1 expression on CTCs from MBC patients treated with anti-PD-1 monotherapy was dynamic. We demonstrated that MBC patients with a cut-off value of ≥ 35% PD-L1-high CTCs had longer PFS (P< 0.05) and OS (P< 0.01) compared with patients with a cut-off value of < 35% PD-L1-high CTCs. Conclusion: Our findings suggested that PD-L1 expression on CTCs could predict the therapeutic response and clinical outcomes, providing a valuable predictive and prognostic biomarker for patients treated with anti-PD-1 immunotherapy. Citation Format: Ying Zhou, Jinmei Zhou, Haoyuan Shi, Zhiyuan Hu, Yanlian Yang, Zefei Jiang, Tao Wang. Prognostic relevance of PD-L1 expression on circulating tumor cells in metastatic breast cancer patients treated with anti-PD-1 immunotherapy [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P5-02-12.

Glioblastoma (GBM) has been regarded as one of the most deadly and challenging cancers to treat with extremely poor prognosis. The limited efficacy of current chemotherapies might be attributed to the presence of glioma stem cells (GSCs) as well as the difficulties in passing through the blood–brain barrier (BBB) and targeting tumor cells. Tumor-derived exosomes are emerging as novel and promising drug delivery systems. However, great concerns regarding the biosafety and BBB penetrability remain to be addressed. Herein, we have developed a simple and feasible strategy to engineer GBM cell-derived exosomes with improved biosafety termed “Exo@TDPs” to deliver the cargos of chemotherapeutic agents temozolomide (TMZ) and doxorubicin (DOX) into GBM tissues. Exo@TDPs decorated with angiopep-2 (Ang-2) and CD133-targeted peptides improve the capacity to penetrate the BBB and target tumor cells. Both in vitro and in vivo studies demonstrate that Exo@TDPs can cross the BBB, target GBM cells, penetrate into deep tumor parenchyma, and release the therapeutic cargos effectively. Synergistic delivery of TMZ and DOX by Exo@TDPs exerts therapeutic effects to suppress the tumor growth and prolong the survival time of orthotopic syngeneic mouse GBM models. These findings suggest that our developed Exo@TDPs loaded with chemotherapeutic drugs may bring new possibilities for the application of tumor cell-derived exosomes for brain tumor treatment.

Alzheimer’s disease (AD) is a complex multifactorial disorder that poses a substantial burden on patients, caregivers, and society. Considering the increased aging population and life expectancy, the incidence of AD will continue to rise in the following decades. However, the molecular pathogenesis of AD remains controversial, superior blood-based biomarker candidates for early diagnosis are still lacking, and effective therapeutics to halt or slow disease progression are urgently needed. As powerful genetic regulators, microRNAs (miRNAs) are receiving increasing attention due to their implications in the initiation, development, and theranostics of various diseases, including AD. In this review, we summarize miRNAs that directly target microtubule-associated protein tau (MAPT), amyloid precursor protein (APP), and β-site APP-cleaving enzyme 1 (BACE1) transcripts and regulate the alternative splicing of tau and APP. We also discuss related kinases, such as glycogen synthase kinase (GSK)-3β, cyclin-dependent kinase 5 (CDK5), and death-associated protein kinase 1 (DAPK1), as well as apolipoprotein E, that are directly targeted by miRNAs to control tau phosphorylation and amyloidogenic APP processing leading to Aβ pathologies. Moreover, there is evidence of miRNA-mediated modulation of inflammation. Furthermore, circulating miRNAs in the serum or plasma of AD patients as noninvasive biomarkers with diagnostic potential are reviewed. In addition, miRNA-based therapeutics optimized with nanocarriers or exosomes as potential options for AD treatment are discussed.

Recently, female breast cancer (BC) has surpassed lung cancer to occupy the first place of the most commonly diagnosed cancer. The unsatisfactory prognosis of endocrine therapy for breast cancer might be attributed to the discordance in estrogen receptor (ER) status between primary tumors and corresponding metastases, as well as temporal and spatial receptor status heterogeneity at point-in-time between biopsy and treatment. The purpose of this study was to evaluate the prognostic and predictive value of ER status in circulating tumor cells (CTCs) in BC patients. We analyzed ER expression on CTCs isolated using the Pep@MNPs method in 2.0 ml of blood samples from 70 patients with BC and 67 female controls. The predictive and prognostic value of ER expression in CTCs and immunohistochemistry results of biopsies for progression-free survival (PFS) and overall survival (OS) of patients in response to therapies were assessed. The detection rate for CTCs was 95.71% (67/70 patients), with a median of 8 CTCs within 2 ml of peripheral venous blood (PVB). A concordance of 76.56% in ER status between CTCs and corresponding primary tumor and 69.23% between CTCs and corresponding metastases was observed. We also found that patients with ER-positive CTCs (CTC ER+ ) had longer PFS and OS than those without ER-positive CTCs (CTC ER- ). Our findings suggested that ER status in CTCs of BC patients may provide valuable predictive and prognostic insights into endocrine therapies, although further evaluation in larger prospective trials is required.

Rationale: Cancer immunotherapy has demonstrated significant antitumor activity in a variety of tumors; however, extensive infiltration of immunosuppressive tumor-associated macrophages (TAMs) in the glioblastoma (GBM) tumor microenvironment (TME) and the existence of the blood-brain barrier (BBB) might lead to failure of the checkpoint blockade therapy. Methods: Herein, we have developed a smart “Trojan horse” BBB-permeable nanocapsule termed “NAcp@CD47” to deliver anti-CD47 antibodies and stimulator of interferon genes (STING) agonists into GBM tissues in a stealth-like manner to reshaped the immune microenvironment by switching the phenotype of microglia and macrophages. Results: Both in vitro and in vivo studies demonstrate that NAcp@CD47 could effectively penetrate the BBB, increase the polarization of M1-phenotype TAMs, help reduce tumor immunosuppression, and inhibit the orthotopic GBM growth by phagocytosis of macrophages and microglia. Conclusions: Our findings indicate that the well-designed NAcp@CD47 not only enhances the phagocytosis of cancer cells but also efficiently enhance antitumor immunogenicity and reverses immune suppression to convert uninflamed “cold” tumors into “hot” tumors.

Background: There are no obvious clinical signs and symptoms in the early stages of Alzheimer’s disease (AD), and most patients usually have mild cognitive impairment (MCI) before diagnosis. Therefore, early diagnosis of AD is very critical. This paper mainly discusses the blood biomarkers of AD patients and uses machine learning methods to study the changes of blood transcriptome during the development of AD and to search for potential blood biomarkers for AD.Methods: Individualized blood mRNA expression data of 711 patients were downloaded from the GEO database, including the control group (CON) (238 patients), MCI (189 patients), and AD (284 patients). Firstly, we analyzed the subcellular localization, protein types and enrichment pathways of the differentially expressed mRNAs in each group, and established an artificial intelligence individualized diagnostic model. Furthermore, the XCell tool was used to analyze the blood mRNA expression data and obtain blood cell composition and quantitative data. Ratio characteristics were established for mRNA and XCell data. Feature engineering operations such as collinearity and importance analysis were performed on all features to obtain the best feature solicitation. Finally, four machine learning algorithms, including linear support vector machine (SVM), Adaboost, random forest and artificial neural network, were used to model the optimal feature combinations and evaluate their classification performance in the test set.Results: Through feature engineering screening, the best feature collection was obtained. Moreover, the artificial intelligence individualized diagnosis model established based on this method achieved a classification accuracy of 91.59% in the test set. The area under curve (AUC) of CON, MCI, and AD were 0.9746, 0.9536, and 0.9807, respectively.Conclusion: The results of cell homeostasis analysis suggested that the homeostasis of Natural killer T cell (NKT) might be related to AD, and the homeostasis of Granulocyte macrophage progenitor (GMP) might be one of the reasons for AD.

Gastric cancer (GC) is a major global cancer burden, and only HER2-targeted therapies have been approved in first line clinical therapy. CLDN18.2 has been regarded as a potential therapeutic target for gastrointestinal tumors, and global clinical trials have been in process. Hence, the precise, efficient, and noninvasive detection of CLDN18.2 expression is important for the effective application of this attractive target. A high similarity of protein sequence between CLDN18.1 and -18.2 made RNA become more suitable for the detection of CLDN18.2 expression. In this study, CLDN18.2 molecular beacon (MB) with a stem-loop hairpin structure was optimized by phosphorothioate and 2'-O-methyl for stability and efficiency. The MB could recognize CLDN18.2 RNA rapidly. Its resolution and selectivity has been verified in several model cells, demonstrating that MB can distinguish CLDN18.2 expression in several model cells. Furthermore, it was applied successfully to the circulating tumor cell (CTC) assay. The concordance in the expression of CLDN18.2 between CTCs and tissue biopsy is 100% (negative: 3 vs 3; positive: 7 vs 7), indicating that CLDN18.2 RNA detection in CTCs based on a MB will be a promising approach for searching potential patients to CLDN 18.2 targeted drug.

Cancer stem cells (CSCs) are a small subset of cells that sit atop the hierarchical ladder in many cancer types. Liver CSCs have been associated with high chemoresistance and recurrence rates in hepatocellular carcinoma (HCC). However, as of yet, no satisfactorily effective liver CSC-targeted treatment is available, which drove us to design and investigate the efficacy of a liposome-based delivery system. Here, we introduce a redox-triggered dual-targeted liposome, CEP-LP@S/D, capable of co-delivering doxorubicin (Dox) and salinomycin (Sal) for the synergistic treatment of liver cancer. This system is based on the association of CD133- and EpCAM-targeted peptides to form Y-shaped CEP ligands that were anchored to the surface of the liposome and allowed the selective targeting of CD133(+)EpCAM(+)liver CSCs. After arriving to the CSCs, the CEP-LP@S/D liposome undergoes endocytosis to the cytoplasm, where a high concentration of glutathione (GSH) breaks its disulfide bonds, thereby degrading the liposome. This then induces a rapid release of Dox and Sal to synergistically inhibit tumor growth. Notably, this effect occurs through Dox-induced apoptosis and concurrent lysosomal iron sequestration by Sal. Interestingly, bothin vitroandin vivostudies indicated that our GSH-responsive co-delivery system not only effectively enhanced CSC targeting but also eliminated the non-CSC faction, thereby exhibiting high antitumor efficacy. We believe that the smart liposome nanocarrier-based co-delivery system is a promising strategy to combat liver cancer, which may also lay the groundwork for more enhanced approaches to target other cancer types as well.

In vivo molecular imaging of tumors targeting a specific cancer cell marker is a promising strategy for cancer diagnosis and imaging guided surgery and therapy. While targeted imaging often relies on antibody-modified probes, peptides can afford targeting probes with small sizes, high penetrating ability, and rapid excretion. Recently, in vivo fluorescence imaging in the second near-infrared window (NIR-II, 1000-1700 nm) shows promise in reaching sub-centimeter depth with microscale resolution. Here, a novel peptide (named CP) conjugated NIR-II fluorescent probe is reported for molecular tumor imaging targeting a tumor stem cell biomarker CD133. The click chemistry derived peptide-dye (CP-IRT dye) probe afforded efficient in vivo tumor targeting in mice with a high tumor-to-normal tissue signal ratio (T/NT > 8). Importantly, the CP-IRT probes are rapidly renal excreted (≈87% excretion within 6 h), in stark contrast to accumulation in the liver for typical antibody-dye probes. Further, with NIR-II emitting CP-IRT probes, urethra of mice can be imaged fluorescently for the first time noninvasively through intact tissue. The NIR-II fluorescent, CD133 targeting imaging probes are potentially useful for human use in the clinic for cancer diagnosis and therapy.

Greatly reduced scattering in the second near-infrared (NIR-II) region (1000-1700 nm) opens up many new exciting avenues of bioimaging research, yet NIR-II fluorescence imaging is mostly implemented by using nontargeted fluorophores or wide-field imaging setups, limiting the signal-to-background ratio and imaging penetration depth due to poor specific binding and out-of-focus signals. A newly developed high-performance NIR-II bioconjugate enables targeted imaging of a specific organ in the living body with high quality. Combined with a home-built NIR-II confocal set-up, the enhanced imaging technique allows 900 µm-deep 3D organ imaging without tissue clearing techniques. Bioconjugation of two hormones to nonoverlapping NIR-II fluorophores facilitates two-color imaging of different receptors, demonstrating unprecedented multicolor live molecular imaging across the NIR-II window. This deep tissue imaging of specific receptors in live animals allows development of noninvasive molecular imaging of multifarious models of normal and neoplastic organs in vivo, beyond the traditional visible to NIR-I range. The developed NIR-II fluorescence microscopy will become a powerful imaging technique for deep tissue imaging without any physical sectioning or clearing treatment of the tissue.

In vivo fluorescence imaging in the near-infrared region between 1500–1700 nm (NIR-IIb window) affords high spatial resolution, deep-tissue penetration, and diminished auto-fluorescence due to the suppressed scattering of long-wavelength photons and large fluorophore Stokes shifts. However, very few NIR-IIb fluorescent probes exist currently. Here, we report the synthesis of a down-conversion luminescent rare-earth nanocrystal with cerium doping (Er/Ce co-doped NaYbF 4 nanocrystal core with an inert NaYF 4 shell). Ce doping is found to suppress the up-conversion pathway while boosting down-conversion by ~9-fold to produce bright 1550 nm luminescence under 980 nm excitation. Optimization of the inert shell coating surrounding the core and hydrophilic surface functionalization minimize the luminescence quenching effect by water. The resulting biocompatible, bright 1550 nm emitting nanoparticles enable fast in vivo imaging of blood vasculature in the mouse brain and hindlimb in the NIR-IIb window with short exposure time of 20 ms for rare-earth based probes.

Yuqiao Wang1, Ying Zhou2, Zhiyuan Hu2,3 1 The Cooper Union for the Advancement of Science and Art, New York, USA; 2 Center for Neuroscience Research, School of Basic Medical Sciences, Fujian Medical University, Fuzhou, Fujian Province, China; 3 CAS Key Laboratory of Standardization and Measurement for Nanotechnology, CAS Center for Excellence in Nanoscience, National Center for Nanoscience and Technology, Beijing, China

Cyclin-dependent kinase 5 (CDK5) regulates important neuronal functions via p35. p35 undergoes cleavage in response to neuronal activity and neurotoxic conditions to release its subunit p25. Although p25 has been implicated in various neurodegenerative diseases, the mechanisms by which p25 mediates neurodegenerative impairment have not been fully elucidated. We aimed to determine the role of p25-mediated neurodegeneration on neurogenesis in an inducible transgenic mouse line overexpressing p25 (p25 TG) in the forebrain. Adult neuronal progenitor cells (NPCs) were labeled with BrdU in vivo, which were significantly increased in numbers in the subventricular zone, the hippocampus, and the cortex of p25 TG mice. Consistently, more mitotic cells were observed in p25 TG mice than in controls, even in the cortex and the CA1, which are not neurogenic regions. BrdU-positive cells were negative for GFAP or γ-H2AX, suggesting that they are not astrocytes or dying cells. Neurospheres derived from the dentate gyrus and the cortex were significantly increased in p25 TG mice and can be differentiated into astrocytes and neurons. However, p25 TG decreased the long-term survival of proliferating NPCs and severely impaired adult neurogenesis. A Transwell co-culture system was used to assess the influence of p25-expressing primary neurons on adult NPCs. Co-culture with p25-expressing neurons downregulated Ki67 expression and upregulated cleaved caspase-3, indicating that the paracrine signaling in cell-cell communication is essential for NPC survival and proliferation. Moreover, increased CDK5 activity impairs Wnt activation. This study demonstrates that hyperactivation of p25 may temporarily enhance NPC proliferation, but impair their long-term survival.

Cyclin-dependent kinase 5 regulates numerous neuronal functions with its activator, p35. Under neurotoxic conditions, p35 undergoes proteolytic cleavage to liberate p25, which has been implicated in various neurodegenerative diseases. Here, we show that p25 is generated following neuronal activity under physiological conditions in a GluN2B- and CaMKIIα-dependent manner. Moreover, we developed a knockin mouse model in which endogenous p35 is replaced with a calpain-resistant mutant p35 (Δp35KI) to prevent p25 generation. The Δp35KI mice exhibit impaired long-term depression and defective memory extinction, likely mediated through persistent GluA1 phosphorylation at Ser845. Finally, crossing the Δp35KI mice with the 5XFAD mouse model of Alzheimer's disease (AD) resulted in an amelioration of β-amyloid (Aβ)-induced synaptic depression and cognitive impairment. Together, these results reveal a physiological role of p25 production in synaptic plasticity and memory and provide new insights into the function of p25 in Aβ-associated neurotoxicity and AD-like pathology.

Memory formation is modulated by pre- and post-synaptic signaling events in neurons. The neuronal protein kinase Cyclin-Dependent Kinase 5 (Cdk5) phosphorylates a variety of synaptic substrates and is implicated in memory formation. It has also been shown to play a role in homeostatic regulation of synaptic plasticity in cultured neurons. Surprisingly, we found that Cdk5 loss of function in hippocampal circuits results in severe impairments in memory formation and retrieval. Moreover, Cdk5 loss of function in the hippocampus disrupts cAMP signaling due to an aberrant increase in phosphodiesterase (PDE) proteins. Dysregulation of cAMP is associated with defective CREB phosphorylation and disrupted composition of synaptic proteins in Cdk5-deficient mice. Rolipram, a PDE4 inhibitor that prevents cAMP depletion, restores synaptic plasticity and memory formation in Cdk5-deficient mice. Collectively, our results demonstrate a critical role for Cdk5 in the regulation of cAMP-mediated hippocampal functions essential for synaptic plasticity and memory formation.

Primary autosomal-recessive microcephaly (MCPH) and Majewski osteodysplastic primordial dwarfism type II (MOPDII) are both genetic diseases that result in decreased brain size at birth. MCPH is thought to arise from alterations in the size of the neural progenitor pool, but the cause of this defect has not been thoroughly explored. We find that one of the genes associated with MCPH, Cdk5rap2, is highly expressed in the neural progenitor pool and that its loss results in a depletion of apical progenitors and increased cell-cycle exit leading to premature neuronal differentiation. We link Cdk5rap2 function to the pericentriolar material protein pericentrin, loss of function of which is associated with MOPDII. Depletion of pericentrin in neural progenitors phenocopies effects of Cdk5rap2 knockdown and results in decreased recruitment of Cdk5rap2 to the centrosome. Our findings uncover a common mechanism, involving aberrations in the neurogenesis program, that may underlie the development of microcephaly in multiple diseases.

Organogenesis is a highly integrated process with a fundamental requirement for precise cell cycle control. Mechanistically, the cell cycle is composed of transitions and thresholds that are controlled by coordinated post-translational modifications. In this study, we describe a novel mechanism controlling the persistence of the transcription factor ATF4 by multisite phosphorylation. Proline-directed phosphorylation acted additively to regulate multiple aspects of ATF4 degradation. Stabilized ATF4 mutants exhibit decreased β-TrCP degron phosphorylation, β-TrCP interaction, and ubiquitination, as well as elicit early G(1) arrest. Expression of stabilized ATF4 also had significant consequences in the developing neocortex. Mutant ATF4 expressing cells exhibited positioning and differentiation defects that were attributed to early G(1) arrest, suggesting that neurogenesis is sensitive to ATF4 dosage. We propose that precise regulation of the ATF4 dosage impacts cell cycle control and impinges on neurogenesis.