CAR-redirected natural killer T cells demonstrate superior antitumor activity to CAR-T cells through multimodal CD1d-dependent mechanisms.

Human natural killer T (NKT) cells have been proposed as a promising cell platform for chimeric antigen receptor (CAR) therapy in solid tumors. Here we generated murine CAR-NKT cells and compared them with CAR-T cells in immune-competent mice. Both CAR-NKT cells and CAR-T cells showed similar antitumor effects in vitro, but CAR-NKT cells showed superior antitumor activity in vivo via CD1d-dependent immune responses in the tumor microenvironment. Specifically, we show that CAR-NKT cells eliminate CD1d-expressing M2-like macrophages. In addition, CAR-NKT cells promote epitope spreading and activation of endogenous T cell responses against tumor-associated neoantigens. Finally, we observed that CAR-NKT cells can co-express PD1 and TIM3 and show an exhaustion phenotype in a model of high tumor burden. PD1 blockade as well as vaccination augmented the antitumor activity of CAR-NKT cells. In summary, our results demonstrate the multimodal function of CAR-NKT cells in solid tumors, further supporting the rationale for developing CAR-NKT therapies in the clinic.

DNA-Engineered Degradable Invisibility Cloaking for Tumor-Targeting Nanoparticles.

Nanoparticle (NP) delivery systems have been actively exploited for cancer therapy and vaccine development. Nevertheless, the major obstacle to targeted delivery lies in the substantial liver sequestration of NPs. Here we report a DNA-engineered approach to circumvent liver phagocytosis for enhanced tumor-targeted delivery of nanoagents in vivo. We find that a monolayer of DNA molecules on the NP can preferentially adsorb a dysopsonin protein in the serum to induce functionally invisibility to livers; whereas the tumor-specific uptake is triggered by the subsequent degradation of the DNA shell in vivo. The degradation rate of DNA shells is readily tunable by the length of coated DNA molecules. This DNA-engineered invisibility cloaking (DEIC) is potentially generic as manifested in both Ag2S quantum dot- and nanoliposome-based tumor-targeted delivery in mice. Near-infrared-II imaging reveals a high tumor-to-liver ratio of up to ∼5.1, approximately 18-fold higher than those with conventional nanomaterials. This approach may provide a universal strategy for high-efficiency targeted delivery of theranostic agents in vivo.

ADAMTS16 Suppression by MicroRNA-25 Leads to Oncogenic Properties in Renal Cell Carcinoma.

INTRODUCTION: Increasing evidence indicates that microRNAs (miRNAs) play a crucial role in modulating tumor growth. This study is centered on investigating the contribution of miR-25 to the progression of Renal Cell Carcinoma (RCC). METHODS: The investigators examined the expression levels of miR-25 and ADAMTS16 in RCC samples and cell lines. The association between miR-25 and ADAMTS16 was validated via a luciferase reporter assay. Cell viability, apoptosis, migration, and invasion were evaluated utilizing CCK-8 and flow cytometry techniques, while the expression levels of ADAMTS16, ß-catenin, GSK-3ß, and p-GSK-3ß were assessed through western blot analysis. RESULTS: The investigation revealed elevated expression levels of miR-25 in RCC tissues. Subsequently, ADAMTS16 was identified as a target of miR-25. Increased miR-25 levels were associated with decreased expression of ADAMTS16, resulting in enhanced cell viability and diminished apoptosis. Conversely, inhibition of miR-25 led to decreased cell viability, proliferation, and migration. Additionally, the researchers observed that miR-25 triggered the phosphorylation of GSK-3ß and ß-catenin while leaving the total GSK-3ß level unaffected. CONCLUSION: This study suggests that miR-25 regulates the expression of ADAMTS16 through the Wnt/ß-catenin signaling pathway, providing new insights into the cause and potential treatment of RCC.

Primary and Orthotopic Murine Models of Nasopharyngeal Carcinoma Reveal Molecular Mechanisms Underlying its Malignant Progression.

Nasopharyngeal carcinoma (NPC), a squamous cell carcinoma originating in the nasopharynx, is a leading malignancy in south China and other south and east Asia areas. It is frequently associated with Epstein-Barr virus (EBV) infection, while there are also some NPC patients without EBV infection. Here, it is shown that the EBV+ (EBV positive) and EBV- (EBV negative) NPCs contain both shared and distinct genetic abnormalities, among the latter are increased mutations in TP53. To investigate the functional roles of NPC-associated genetic alterations, primary, orthotopic, and genetically defined NPC models were developed in mice, a key tool missed in the field. These models, initiated with gene-edited organoids of normal nasopharyngeal epithelium, faithfully recapitulated the pathological features of human disease. With these models, it is found that Trp53 and Cdkn2a deficiency are crucial for NPC initiation and progression. And latent membrane protein1 (LMP1), an EBV-coding oncoprotein, significantly promoted the distal metastasis. Further, loss of TGFBR2, which is frequently disrupted both in EBV- and EBV+ NPCs, dramatically accelerated the progression and lung metastasis of NPC probably by altering tumor microenvironment. Taken together, this work establishes a platform to dissect the genetic mechanisms underlying NPC pathogenesis and might be of value for future translational studies.

Refining chimeric antigen receptors via barcoded protein domain combination pooled screening.

Chimeric antigen receptor (CAR)-T cells represent a promising frontier in cancer immunotherapy. However, the current process for developing new CAR constructs is time consuming and inefficient. To address this challenge and expedite the evaluation and comparison of full-length CAR designs, we have devised a novel cloning strategy. This strategy involves the sequential assembly of individual CAR domains using blunt ligation, with each domain being assigned a unique DNA barcode. Applying this method, we successfully generated 360 CAR constructs that specifically target clinically validated tumor antigens CD19 and GD2. By quantifying changes in barcode frequencies through next-generation sequencing, we characterize CARs that best mediate proliferation and expansion of transduced T cells. The screening revealed a crucial role for the hinge domain in CAR functionality, with CD8a and IgG4 hinges having opposite effects in the surface expression, cytokine production, and antitumor activity in CD19- versus GD2-based CARs. Importantly, we discovered two novel CD19-CAR architectures containing the IgG4 hinge domain that mediate superior in vivo antitumor activity compared with the construct used in Kymriah, a U.S. Food and Drug Administration (FDA)-approved therapy. This novel screening approach represents a major advance in CAR engineering, enabling accelerated development of cell-based cancer immunotherapies.

Anti-GD2 CAR-NKT cells in relapsed or refractory neuroblastoma: updated phase 1 trial interim results.

Vα24-invariant natural killer T cells (NKTs) have anti-tumor properties that can be enhanced by chimeric antigen receptors (CARs). Here we report updated interim results from the first-in-human phase 1 evaluation of autologous NKTs co-expressing a GD2-specific CAR with interleukin 15 (IL15) (GD2-CAR.15) in 12 children with neuroblastoma (NB). The primary objectives were safety and determination of maximum tolerated dose (MTD). The anti-tumor activity of GD2-CAR.15 NKTs was assessed as a secondary objective. Immune response evaluation was an additional objective. No dose-limiting toxicities occurred; one patient experienced grade 2 cytokine release syndrome that was resolved by tocilizumab. The MTD was not reached. The objective response rate was 25% (3/12), including two partial responses and one complete response. The frequency of CD62L+NKTs in products correlated with CAR-NKT expansion in patients and was higher in responders (n = 5; objective response or stable disease with reduction in tumor burden) than non-responders (n = 7). BTG1 (BTG anti-proliferation factor 1) expression was upregulated in peripheral GD2-CAR.15 NKTs and is a key driver of hyporesponsiveness in exhausted NKT and T cells. GD2-CAR.15 NKTs with BTG1 knockdown eliminated metastatic NB in a mouse model. We conclude that GD2-CAR.15 NKTs are safe and can mediate objective responses in patients with NB. Additionally, their anti-tumor activity may be enhanced by targeting BTG1. ClinicalTrials.gov registration: NCT03294954 .

Interaction between tumor cell TNFR2 and monocyte membrane-bound TNF-α triggers tumorigenic inflammation in neuroblastoma.

BACKGROUND: Tumor progression and resistance to therapy in children with neuroblastoma (NB), a common childhood cancer, are often associated with infiltration of monocytes and macrophages that produce inflammatory cytokines. However, the mechanism by which tumor-supportive inflammation is initiated and propagated remains unknown. Here, we describe a novel protumorigenic circuit between NB cells and monocytes that is triggered and sustained by tumor necrosis factor alpha (TNF-α). METHODS: We used NB knockouts (KOs) of TNF-α and TNFRSF1A mRNA (TNFR1)/TNFRSF1B mRNA (TNFR2) and TNF-α protease inbitor (TAPI), a drug that modulates TNF-α isoform expression, to assess the role of each component in monocyte-associated protumorigenic inflammation. Additionally, we employed NB-monocyte cocultures and treated these with clinical-grade etanercept, an Fc-TNFR2 fusion protein, to neutralize signaling by both membrane-bound (m) and soluble (s)TNF-α isoforms. Further, we treated NOD/SCID/IL2Rγ(null) mice carrying subcutaneous NB/human monocyte xenografts with etanercept and evaluated the impact on tumor growth and angiogenesis. Gene set enrichment analysis (GSEA) was used to determine whether TNF-α signaling correlates with clinical outcomes in patients with NB. RESULTS: We found that NB expression of TNFR2 and monocyte membrane-bound tumor necrosis factor alpha is required for monocyte activation and interleukin (IL)-6 production, while NB TNFR1 and monocyte soluble TNF-α are required for NB nuclear factor kappa B subunit 1 (NF-κB) activation. Treatment of NB-monocyte cocultures with clinical-grade etanercept completely abrogated release of IL-6, granulocyte colony-stimulating factor (G-CSF), IL-1α, and IL-1ß and eliminated monocyte-induced enhancement of NB cell proliferation in vitro. Furthermore, etanercept treatment inhibited tumor growth, ablated tumor angiogenesis, and suppressed oncogenic signaling in mice with subcutaneous NB/human monocyte xenografts. Finally, GSEA revealed significant enrichment for TNF-α signaling in patients with NB that relapsed. CONCLUSIONS: We have described a novel mechanism of tumor-promoting inflammation in NB that is strongly associated with patient outcome and could be targeted with therapy.

DNA framework carriers with asymmetric hydrophobic drug patterns for enhanced cellular cytotoxicity.

We devise a class of amphiphilic drug complexes by programming hydrophobic drug patterns (HDPs) on DNA frameworks. We investigate the effect of HDPs on cellular uptake efficiency and drug potency. We achieve enhanced cytotoxicity against tumor cells by using an asymmetric HDP.

LEF1 Drives a Central Memory Program and Supports Antitumor Activity of Natural Killer T Cells.

Vα24-invariant natural killer T cells (NKT) possess innate antitumor properties that can be exploited for cancer immunotherapy. We have shown previously that the CD62L+ central memory-like subset of these cells drives the in vivo antitumor activity of NKTs, but molecular mediators of NKT central memory differentiation remain unknown. Here, we demonstrate that relative to CD62L- cells, CD62L+ NKTs express a higher level of the gene encoding the Wnt/ß-catenin transcription factor lymphoid enhancer binding factor 1 (LEF1) and maintain active Wnt/ß-catenin signaling. CRISPR/Cas9-mediated LEF1 knockout reduced CD62L+ frequency after antigenic stimulation, whereas Wnt/ß-catenin activator Wnt3a ligand increased CD62L+ frequency. LEF1 overexpression promoted NKT expansion and limited exhaustion following serial tumor challenge and was sufficient to induce a central memory-like transcriptional program in NKTs. In mice, NKTs expressing a GD2-specific chimeric-antigen receptor (CAR) with LEF1 demonstrated superior control of neuroblastoma xenograft tumors compared with control CAR-NKTs. These results identify LEF1 as a transcriptional activator of the NKT central memory program and advance development of NKT cell-based immunotherapy. See related Spotlight by Van Kaer, p. 144.

ERK Inhibitor Ulixertinib Inhibits High-Risk Neuroblastoma Growth In Vitro and In Vivo.

Neuroblastoma (NB) is a pediatric tumor of the peripheral nervous system. Approximately 80% of relapsed NB show RAS-MAPK pathway mutations that activate ERK, resulting in the promotion of cell proliferation and drug resistance. Ulixertinib, a first-in-class ERK-specific inhibitor, has shown promising antitumor activity in phase 1 clinical trials for advanced solid tumors. Here, we show that ulixertinib significantly and dose-dependently inhibits cell proliferation and colony formation in different NB cell lines, including PDX cells. Transcriptomic analysis revealed that ulixertinib extensively inhibits different oncogenic and neuronal developmental pathways, including EGFR, VEGF, WNT, MAPK, NGF, and NTRK1. The proteomic analysis further revealed that ulixertinib inhibits the cell cycle and promotes apoptosis in NB cells. Additionally, ulixertinib treatment significantly sensitized NB cells to the conventional chemotherapeutic agent doxorubicin. Furthermore, ulixertinib potently inhibited NB tumor growth and prolonged the overall survival of the treated mice in two different NB mice models. Our preclinical study demonstrates that ulixertinib, either as a single agent or in combination with current therapies, is a novel and practical therapeutic approach for NB.

Identifying a confused cell identity for esophageal squamous cell carcinoma.

The cell identity of malignant cells and how they acquire it are fundamental for our understanding of cancer. Here, we report that esophageal squamous cell carcinoma (ESCC) cells display molecular features equally similar but distinct to all three types of normal esophageal epithelial cells, which we term as confused cell identity (CCI). CCI is an independent prognostic marker associated with poor prognosis in ESCC. Further, we identify tropomyosin 4 (TPM4) as a critical CCI gene that promotes the aggressiveness of ESCC in vitro and in vivo. And TPM4 creates CCI through activating the Jak/STAT-SOX2 pathway. Thus, our study suggests an unrecognized feature of ESCC cells, which might be of value for clinic prognosis and potential interference.

Artificial Intelligence for Detecting and Delineating Margins of Early ESCC Under WLI Endoscopy.

INTRODUCTION: Conventional white light imaging (WLI) endoscopy is the most common screening technique used for detecting early esophageal squamous cell carcinoma (ESCC). Nevertheless, it is difficult to detect and delineate margins of early ESCC using WLI endoscopy. This study aimed to develop an artificial intelligence (AI) model to detect and delineate margins of early ESCC under WLI endoscopy. METHODS: A total of 13,083 WLI images from 1,239 patients were used to train and test the AI model. To evaluate the detection performance of the model, 1,479 images and 563 images were used as internal and external validation data sets, respectively. For assessing the delineation performance of the model, 1,114 images and 211 images were used as internal and external validation data sets, respectively. In addition, 216 images were used to compare the delineation performance between the model and endoscopists. RESULTS: The model showed an accuracy of 85.7% and 84.5% in detecting lesions in internal and external validation, respectively. For delineating margins, the model achieved an accuracy of 93.4% and 95.7% in the internal and external validation, respectively, under an overlap ratio of 0.60. The accuracy of the model, senior endoscopists, and expert endoscopists in delineating margins were 98.1%, 78.6%, and 95.3%, respectively. The proposed model achieved similar delineating performance compared with that of expert endoscopists but superior to senior endoscopists. DISCUSSION: We successfully developed an AI model, which can be used to accurately detect early ESCC and delineate the margins of the lesions under WLI endoscopy.

Directing Multivalent Aptamer-Receptor Binding on the Cell Surface with Programmable Atom-Like Nanoparticles.

Multivalent interactions of biomolecules play pivotal roles in physiological and pathological settings. Whereas the directionality of the interactions is crucial, the state-of-the-art synthetic multivalent ligand-receptor systems generally lack programmable approaches for orthogonal directionality. Here, we report the design of programmable atom-like nanoparticles (aptPANs) to direct multivalent aptamer-receptor binding on the cell interface. The positions of the aptamer motifs can be prescribed on tetrahedral DNA frameworks to realize atom-like orthogonal valence and direction, enabling the construction of multivalent molecules with fixed aptamer copy numbers but different directionality. These directional-yet-flexible aptPAN molecules exhibit the adaptability to the receptor distribution on cell surfaces. We demonstrate the high-affinity tumor cell binding with a linear aptPAN oligomer (≈13-fold improved compared to free aptamers), which leads to ≈50 % suppression of cell growth.

Endoscopic transcecal appendectomy: a new endotherapy for appendiceal orifice lesions.

BACKGROUND: Endoscopic transcecal appendectomy (ETA) has been reported as a minimally invasive alternative procedure for lesions involving the appendiceal orifice. The aim of this case series study was to evaluate the feasibility, safety, and effectiveness of ETA for lesions at the appendiceal orifice. METHODS: This retrospective study included consecutive patients with appendiceal orifice lesions who underwent ETA between December 2018 and March 2021. The primary outcome was technical success. The secondary outcomes included postoperative adverse events, postoperative hospital stay, and recurrence. RESULTS: 13 patients with appendiceal orifice lesions underwent ETA during the study period. The median lesion size was 20 mm (range 8-50). Lesions morphologies were polypoid lesions (n = 5), laterally spreading tumors (n = 4), and submucosal lesions (n = 4). Technical success with complete resection was achieved in all 13 cases. There were no postoperative bleeding, perforation, or intra-abdominal abscess. The median length of hospital stay after ETA was 8 days (range 6-18). There was no tumor recurrence during a median follow-up of 17 months (range 1-28). CONCLUSIONS: ETA is feasible, safe, and effective for complete resection of appendiceal orifice lesions. Larger, multicenter, prospective studies are needed to further assess this technique.

Artificial intelligence for detecting superficial esophageal squamous cell carcinoma under multiple endoscopic imaging modalities: A multicenter study.

BACKGROUND AND AIM: Diagnosis of esophageal squamous cell carcinoma (ESCC) is complicated and requires substantial expertise and experience. This study aimed to develop an artificial intelligence (AI) system for detecting superficial ESCC under multiple endoscopic imaging modalities. METHODS: Endoscopic images were retrospectively collected from West China Hospital, Sichuan University as a training dataset and an independent internal validation dataset. Images from other four hospitals were used as an external validation dataset. The AI system was compared with 11 experienced endoscopists. Furthermore, videos were collected to assess the performance of the AI system. RESULTS: A total of 53 933 images from 2621 patients and 142 videos from 19 patients were used to develop and validate the AI system. In the internal and external validation datasets, the performance of the AI system under all or different endoscopic imaging modalities was satisfactory, with sensitivity of 92.5-99.7%, specificity of 78.5-89.0%, and area under the receiver operating characteristic curves of 0.906-0.989. The AI system achieved comparable performance with experienced endoscopists. Regarding superficial ESCC confined to the epithelium, the AI system was more sensitive than experienced endoscopists on white-light imaging (90.8% vs 82.5%, P = 0.022). Moreover, the AI system exhibited good performance in videos, with sensitivity of 89.5-100% and specificity of 73.7-89.5%. CONCLUSIONS: We developed an AI system that showed comparable performance with experienced endoscopists in detecting superficial ESCC under multiple endoscopic imaging modalities and might provide valuable support for inexperienced endoscopists, despite requiring further evaluation.

A Nanoradiomics Approach for Differentiation of Tumors Based on Tumor-Associated Macrophage Burden.

Objective: Tumor-associated macrophages (TAMs) within the tumor immune microenvironment (TiME) of solid tumors play an important role in treatment resistance and disease recurrence. The purpose of this study was to investigate if nanoradiomics (radiomic analysis of nanoparticle contrast-enhanced images) can differentiate tumors based on TAM burden. Materials and Methods: In vivo studies were performed in transgenic mouse models of neuroblastoma with low (N = 11) and high (N = 10) tumor-associated macrophage (TAM) burden. Animals underwent delayed nanoparticle contrast-enhanced CT (n-CECT) imaging at 4 days after intravenous administration of liposomal-iodine agent (1.1 g/kg). CT imaging-derived conventional tumor metrics (tumor volume and CT attenuation) were computed for segmented tumor CT datasets. Nanoradiomic analysis was performed using a PyRadiomics workflow implemented in the quantitative image feature pipeline (QIFP) server containing 900 radiomic features (RFs). RF selection was performed under supervised machine learning using a nonparametric neighborhood component method. A 5-fold validation was performed using a set of linear and nonlinear classifiers for group separation. Statistical analysis was performed using the Kruskal-Wallis test. Results: N-CECT imaging demonstrated heterogeneous patterns of signal enhancement in low and high TAM tumors. CT imaging-derived conventional tumor metrics showed no significant differences (p > 0.05) in tumor volume between low and high TAM tumors. Tumor CT attenuation was not significantly different (p > 0.05) between low and high TAM tumors. Machine learning-augmented nanoradiomic analysis revealed two RFs that differentiated (p < 0.002) low TAM and high TAM tumors. The RFs were used to build a linear classifier that demonstrated very high accuracy and further confirmed by 5-fold cross-validation. Conclusions: Imaging-derived conventional tumor metrics were unable to differentiate tumors with varying TAM burden; however, nanoradiomic analysis revealed texture differences and enabled differentiation of low and high TAM tumors.

Poly-Adenine-Based Spherical Nucleic Acids for Efficient Live-Cell MicroRNA Capture.

Direct delivery of exogenous non-coding nucleic acids into living cells has attracted intense interest in biological applications. However, the cell entry efficiency and target capture ability remain to be improved. Herein, we report a method for compartmenting the nucleic acids on the surface of poly-adenine-based spherical nucleic acids (polyA-SNAs) for efficient capture of oncogenic microRNAs (miRNAs) in living cells. We find that polyA-SNAs exhibit high cell entry efficiency, which is insensitive to the configuration of the anti-miRNA sequences. By programming the length of polyAs, we precisely engineered the spatial configuration of the anti-miRNA sequences in polyA-SNAs. Compartmentalized polyA-SNAs bind to miRNAs with improved capture ability as compared to densely compacted SNAs. We further demonstrate that polyA-SNAs serve as high-efficacy miRNA sponges for capturing oncogenic miRNAs both in living cells and in mice. The efficient inhibition of miRNAs results in significant suppression of tumor growth.