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Chimeric antigen receptor (CAR) T cell therapy has shown significant efficacy for hematological malignancies, however, it needs to be further optimized. Recently, the lipid nanoparticle (LNP)-mRNA delivery system as a nonviral gene transfer vector has gained rapid progress in CAR-T cell therapy. The claudin-6 (CLDN6) mRNA is delivered to antigen presenting cells (APCs) through LNP system, thereby enhancing the function of CLDN6 CAR-T cells for the clearance of solid tumor cells. For treatment of acute cardiac injury, the fibroblast activation protein (FAP) CAR mRNA can be delivered to T cells through LNP system for the in vivo production of FAP CAR-T cells, thereby blocking the process of myocardial fibrosis. The LNP-mRNA delivery system has advantages including having no integration in host genome, inexpensiveness, low toxicity and modifiability; on the other hand, it has certain disadvantages such as limited cell persistence caused by transient protein expression and limitations in preparation techniques. This article reviews the research advance in LNP-mRNA in vivo delivery system and its application in CAR-T cell therapy.

Clonal hematopoiesis (CH) is a clonal expansion of single hematopoietic stem cells (HSC) with germline or somatic mutations accumulated in specific genes, which usually leads to clonal dominance via multiple biological pathways and increases the risk of developing hematological malignancies. With the advancement of Next Generation Sequencing (NGS) technology, CH can be widely detected in healthy individuals, many patients complicated with bone marrow failure (BMF), or even in those with myeloproliferative neoplasm (MPN) and therapy-related MDS/AML (t-MDS/AML). Recently, several proof-of-concept studies showed that it is likely to discriminate the CH with high risk developing malignancy through predictive models many years before its onset. Additionally, it has been shown that the CH-related premalignant clones can be abated or eradicated through chemical or genetic measures in animal models, thereby providing an insightful idea for preventing leukemogenesis. However, in despite of these advancements, the clinical implication and early diagnosis of CH for predicting hematological malignancies remain to be more investigated. In this review, we will first review the brief history, risk factors and molecular mechanisms of CH. Then, we will discuss the involved biological process of CH in bone marrow failure diseases, MPN and t-MDS/AML, pointing to its clinical implication. Finally, we will discuss the advancement of early detection of CH at premalignant stage for predicting hematological malignancies, and express perspectives and challenges for early intervention of CH in the future.

The diagnostic, monitoring and therapeutic options for cancers currently remain limited. These limitations represent a large threat to human health. Adaptive immunity, which is dependent on diverse repertoires of B cell receptors (BCRs) and T cell receptors (TCRs), plays a critical role in the anti-tumor immune response. Modulation and surveillance of adaptive immunity has become a powerful weapon to combat cancers. Recently, the high-throughput sequencing of immune repertoire (HTS-IR) technology, which provides a robust tool for deep sequencing repertoires of BCRs or TCRs, has been applied in the development of tumor biomarkers and immunotherapeutics for cancers. This review will first provide an overview of the advancement of HTS-IR technology at the population-cell and single-cell levels. It will then provide a current summary of the applications of HTS-IR technology in the diagnosis and monitoring of minimal residual disease (MRD), focusing on immune reconstitution after the treatment of allogeneic hematopoietic stem cell transplantation (allo-HSCT) in B/T-cell malignancies, and the precise detection of tumor-infiltrating lymphocytes (TILs) in non-B/T-cell malignancies. Finally, current advances of HTS-IR technology in cancer immunotherapeutic applications, such as therapeutic antibodies, CAR-T cell based-adoptive immunotherapies, and neoantigen-specific TCR-T cell-based adoptive immunotherapies, will be introduced.

Intratumor heterogeneity of tumor clones and an immunosuppressive microenvironment in cancer ecosystems contribute to inherent difficulties for tumor treatment. Recently, chimeric antigen receptor (CAR) T-cell therapy has been successfully applied in the treatment of B-cell malignancies, underscoring its great potential in antitumor therapy. However, functional challenges of CAR-T cell therapy, especially in solid tumors, remain. Here, we describe cancer-immunity phenotypes from a clonal-stromal-immune perspective and elucidate mechanisms of T-cell exhaustion that contribute to tumor immune evasion. Then we assess the functional challenges of CAR-T cell therapy, including cell trafficking and infiltration, targeted-recognition and killing of tumor cells, T-cell proliferation and persistence, immunosuppressive microenvironment and self-control regulation. Finally, we delineate tumor precision informatics and advancements in engineered CAR-T cells to counteract inherent challenges of the CAR-T cell therapy, either alone or in combination with traditional therapeutics, and highlight the therapeutic potential of this approach in future tumor precision treatment.

Context: Inflammatory disease is a big threat to human health. Leukocyte chemotactic migration is required for efficient inflammatory response. Inhibition of leukocyte chemotactic migration to the inflammatory site has been shown to provide therapeutic targets for treating inflammatory diseases. Objective: Our study was designed to discover effective and safe compounds that can inhibit leukocyte chemotactic migration, thus providing possible novel therapeutic strategy for treating inflammatory diseases. Materials and methods: In this study, we used transgenic zebrafish model (Tg:zlyz-EGFP line) to visualize the process of leukocyte chemotactic migration. Then, we used this model to screen the hit compound and evaluate its biological activity on leukocyte chemotactic migration. Furthermore, western blot analysis was performed to evaluate the effect of the hit compound on the AKT or ERK-mediated pathway, which plays an important role in leukocyte chemotactic migration. Results: In this study, using zebrafish-based chemical screening, we identified that the hit compound meisoindigo (25 μM, 50 μM, 75 μM) can significantly inhibit zebrafish leukocyte chemotactic migration in a dose-dependent manner (p = 0.01, p = 0.0006, p

Ubiquitin-specific protease 10 (USP10), a novel deubiquitinating enzyme, is associated with androgen receptor transcriptional activity and pathological processes of tumor. However, information between USP10 and the adrenal gland is limited. In particular, the role of USP10 in adrenal tumors has not been elucidated yet. This study aims to investigate the expression of USP10 in the human normal adrenal gland and various adrenal tumors. Tissue samples were obtained from 30 adrenocortical adenomas, nine adrenocortical adenocarcinomas, and 20 pheochromocytomas following laparoscopic surgery. Twenty normal adrenal glands were obtained from kidney surgical resection conducted due to renal cell carcinomas. USP10 expression was investigated on protein levels using immunohistochemistry and on mRNA levels using bioinformatics analysis in the Gene Expression Omnibus (GEO) Datasets. In the 20 cases of normal adrenal glands analyzed, USP10 protein was constantly expressed in situ in the cortex of the adrenal glands, but in the medulla of the gland, only the sustentacular cells were detected positive. In adrenal tumors, detectable levels of USP10 protein were found in 100 % (30/30) adrenocortical adenomas, 88.89 % (8/9) adrenocortical carcinomas, and 10 % (2/20) pheochromocytomas. Bioinformatics analysis did not show a significant difference in USP10 messenger RNA (mRNA) expression between adrenal tumors and normal adrenal gland tissues. A positive USP10 immunoreaction can be useful in distinguishing adrenal cortical tumors from pheochromocytoma.

Clonal hematopoiesis (CH) is characterized by clonal expansion of hematopoietic stem/progenitor cells with the capability of multilineage differentiation and can be detected through the biomarker of genetic mutations. Bone marrow failure (BMF) diseases including idiopathic cytopenia of undetermined significance (ICUS), aplastic anemia (AA), paroxysmal nocturnal hemoglobinuria (PNH) and myelodysplasia syndrome (MDS) are reported to partially coexist with CH. However, till now, the CH and genetic mutations in acquired BMF diseases are still not very clear. Therefore, we designed a panel that contains more than 500 candidate genes to perform a targeted sequencing of bone marrow cells isolated from 48 BMF patients, taking their mucosal cells as controls. In total, 75 somatic mutations and two germline mutations (PTCH1 c.1177G>A, CHEK2 c.1240C>T) were profiled in 30 patients, including 5 ICUS (also termed CCUS (clonal cytopenia of undetermined significance)), 14 AA, 3 PNH and 7 MDS patients. 67.53% missense mutations, 10.39% nonsense mutations, 10.39% frameshift mutations, 1.30% splicing mutations, 7.79% cds-del mutations, 2.60% CNV (copy number variations) can be found in our sequenced mutations. In comparison, the genetic mutation burden is relatively lower in AA and ICUS but higher in PNH and MDS, suggesting that the extent of CH possibly correlated with disease progression. Genetic mutations in our assay also implicated insightful views in the pathogenesis of BMF diseases. We can functionally divide genetic mutations into several types such as signaling transduction (21.6%), transcriptional regulation (19.6%), epigenetic regulation (15.5%), cell cycle regulation (7.2%), DNA repair (3.1%), cohesion complex (2.1%), immunity (3.1%), RNA splicing (4.1%) and others (3.1%). Specifically, we found a serial of novel genetic mutations. For example, the mutations in extracellular membrane receptors such as Notch (Notch1 c.4759A>C , Notch2 c.4819C>T, Notch3 c.3592A>C), CXCR4(c.598C>T), IGF1(c.341C>T), MPL(c.611C>T) suggest bone marrow environment is possibly associated with BMF diseases. In detail, Notch, IGF1, CXCR4 and MPL-mediated signaling are critical for HSC niche regulation, suggesting niche signals serve as a promoting factor for acquired BMF pathogenesis, which is consistent with previous reports. Also, we found the immunity-related mutations in HLA-A(c.750_751delGG) and CD58(c.475T>G), which are closely related with specific T cell recognition, implicated that clonal hematopoiesis and evolution to malignancies can be partially attributed to immune evasion possibly. The mutation ERBB4 (c.3424C>T), a membrane receptor which binds to and is activated by nerve cell released neuregulins, implicated that nerve fiber might involve in BMF. Additionally, we also found novel mutations in epigenetic factors such as KDM5C (c.265delA) and CHD3 (cds-del c.222GCC [7>6]) in PNH and MDS patients. Interestingly, LRP1B (c.13771G>A) that plays an important role in lipid metabolism was found in PNH patient firstly. These gene mutations provide clues for potential biomarkers for BMF diagnosis and prognosis and need more functional investigations. To systemically observe the clonal hematopoiesis at individual patient-level, we delineated the mutational composition in 30 individual BMF patients. We found that 56.7% patients have more than two mutations (eg. MDS 85.71%, PNH 100%, CCUS 60% and AA 35.7%), suggesting that multiple gene mutations-driven clonal expansion constitutes a complex mutational composition and their interaction possibly influences disease progression. At individual patient-level, mutational composition better promotes our understanding on BMF pathogenesis and clinical diagnosis. For example, an MDS patient bearing several mutations in ASXL (non-sense mutation, c.3187C>T, VAF: 18.2%), DNMT3A (missense mutation, c.2644C>T, VAF: 3.01%) and RUNX1 (missense, c.602G>A, VAF: 2.08%) suggested a complex pathological process and poor prognosis. Therefore, mutational composition structure can be revealed in individual acquired BMF patients, promoting precise diagnosis and prognosis. In conclusion, our study not only found a serial of novel genetic mutations that need further investigations, but also provided potential clues for the identification of biomarkers in acquired BMF diseases. Figure Disclosures No relevant conflicts of interest to declare.

Cellular heterogeneity is a fundamental characteristic of many cancers. A lack of cellular homogeneity contributes to difficulty in designing targeted oncological therapies. Therefore, the development of novel methods to determine and characterize oncologic cellular heterogeneity is a critical next step in the development of novel cancer therapies. Single-cell sequencing (SCS) technology has been recently employed for analyzing the genetic polymorphisms of individual cells at the genome-wide level. SCS requires (1) precise isolation of the single cell of interest; (2) isolation and amplification of genetic material; and (3) descriptive analysis of genomic, transcriptomic, and epigenomic data. In addition to targeted analysis of single cells isolated from tumor biopsies, SCS technology may be applied to circulating tumor cells, which may aid in predicting tumor progression and metastasis. In this paper, we provide an overview of SCS technology and review the current literature on the potential application of SCS to clinical oncology and research.