Journal of Pharmaceutical Analysis

Preface for Special Issue: Single-Cell and Spatially Resolved Omics

2023, 13(7): 689-690. doi: 10.1016/j.jpha.2023.07.005

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Single-cell RNA-sequencing and subcellular spatial transcriptomics facilitate the translation of liver microphysiological systems for regulatory application

Dan Li, Zhou Fang, Qiang Shi, Nicholas Zhang, Binsheng Gong, Weida Tong, Ahmet F. Coskun, Joshua Xu

2023, 13(7): 691-693. doi: 10.1016/j.jpha.2023.06.013

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Dissecting the brain with spatially resolved multi-omics

Yijia Fangma, Mengting Liu, Jie Liao, Zhong Chen, Yanrong Zheng

2023, 13(7): 694-710. doi: 10.1016/j.jpha.2023.04.003

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Recent studies have highlighted spatially resolved multi-omics technologies, including spatial genomics, transcriptomics, proteomics, and metabolomics, as powerful tools to decipher the spatial heterogeneity of the brain. Here, we focus on two major approaches in spatial transcriptomics (next-generation sequencing-based technologies and image-based technologies), and mass spectrometry imaging technologies used in spatial proteomics and spatial metabolomics. Furthermore, we discuss their applications in neuroscience, including building the brain atlas, uncovering gene expression patterns of neurons for special behaviors, deciphering the molecular basis of neuronal communication, and providing a more comprehensive explanation of the molecular mechanisms underlying central nervous system disorders. However, further efforts are still needed toward the integrative application of multi-omics technologies, including the real-time spatial multi-omics analysis in living cells, the detailed gene profile in a whole-brain view, and the combination of functional verification.

New discoveries in the field of metabolism by applying single-cell and spatial omics

Baocai Xie, Dengfeng Gao, Biqiang Zhou, Shi Chen, Lianrong Wang

2023, 13(7): 711-725. doi: 10.1016/j.jpha.2023.06.002

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Single-cell multi-Omics (SCM-Omics) and spatial multi-Omics (SM-Omics) technologies provide state-of-the-art methods for exploring the composition and function of cell types in tissues/organs. Since its emergence in 2009, single-cell RNA sequencing (scRNA-seq) has yielded many groundbreaking new discoveries. The combination of this method with the emergence and development of SM-Omics techniques has been a pioneering strategy in neuroscience, developmental biology, and cancer research, especially for assessing tumor heterogeneity and T-cell infiltration. In recent years, the application of these methods in the study of metabolic diseases has also increased. The emerging SCM-Omics and SM-Omics approaches allow the molecular and spatial analysis of cells to explore regulatory states and determine cell fate, and thus provide promising tools for unraveling heterogeneous metabolic processes and making them amenable to intervention. Here, we review the evolution of SCM-Omics and SM-Omics technologies, and describe the progress in the application of SCM-Omics and SM-Omics in metabolism-related diseases, including obesity, diabetes, nonalcoholic fatty liver disease (NAFLD) and cardiovascular disease (CVD). We also conclude that the application of SCM-Omics and SM-Omics approaches can help resolve the molecular mechanisms underlying the pathogenesis of metabolic diseases in the body and facilitate therapeutic measures for metabolism-related diseases. This review concludes with an overview of the current status of this emerging field and the outlook for its future.

Single-cell analyses reveal cannabidiol rewires tumor microenvironment via inhibiting alternative activation of macrophage and synergizes with anti-PD-1 in colon cancer

Xiaofan Sun, Lisha Zhou, Yi Wang, Guoliang Deng, Xinran Cao, Bowen Ke, Xiaoqi Wu, Yanhong Gu, Haibo Cheng, Qiang Xu, Qianming Du, Hongqi Chen, Yang Sun

2023, 13(7): 726-744. doi: 10.1016/j.jpha.2023.04.013

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Colorectal tumors often create an immunosuppressive microenvironment that prevents them from responding to immunotherapy. Cannabidiol (CBD) is a non-psychoactive natural active ingredient from the cannabis plant that has various pharmacological effects, including neuroprotective, antiemetic, anti-inflammatory, and antineoplastic activities. This study aimed to elucidate the specific anticancer mechanism of CBD by single-cell RNA sequencing (scRNA-seq) and single-cell ATAC sequencing (scATAC-seq) technologies. Here, we report that CBD inhibits colorectal cancer progression by modulating the suppressive tumor microenvironment (TME). Our single-cell transcriptome and ATAC sequencing results showed that CBD suppressed M2-like macrophages and promoted M1-like macrophages in tumors both in strength and quantity. Furthermore, CBD significantly enhanced the interaction between M1-like macrophages and tumor cells and restored the intrinsic anti-tumor properties of macrophages, thereby preventing tumor progression. Mechanistically, CBD altered the metabolic pattern of macrophages and related anti-tumor signaling pathways. We found that CBD inhibited the alternative activation of macrophages and shifted the metabolic process from oxidative phosphorylation and fatty acid oxidation to glycolysis by inhibiting the phosphatidylinositol 3-kinase-protein kinase B signaling pathway and related downstream target genes. Furthermore, CBD-mediated macrophage plasticity enhanced the response to anti-programmed cell death protein-1 (PD-1) immunotherapy in xenografted mice. Taken together, we provide new insights into the anti-tumor effects of CBD.

Cux1⁺ proliferative basal cells promote epidermal hyperplasia in chronic dry skin disease identified by single-cell RNA transcriptomics

Minhua Huang, Ning Hua, Siyi Zhuang, Qiuyuan Fang, Jiangming Shang, Zhen Wang, Xiaohua Tao, Jianguo Niu, Xiangyao Li, Peilin Yu, Wei Yang

2023, 13(7): 745-759. doi: 10.1016/j.jpha.2023.04.004

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Pathological dry skin is a disturbing and intractable healthcare burden, characterized by epithelial hyperplasia and severe itch. Atopic dermatitis (AD) and psoriasis models with complications of dry skin have been studied using single-cell RNA sequencing (scRNA-seq). However, scRNA-seq analysis of the dry skin mouse model (acetone/ether/water (AEW)-treated model) is still lacking. Here, we used scRNA-seq and in situ hybridization to identify a novel proliferative basal cell (PBC) state that exclusively expresses transcription factor CUT-like homeobox 1 (Cux1). Further in vitro study demonstrated that Cux1 is vital for keratinocyte proliferation by regulating a series of cyclin-dependent kinases (CDKs) and cyclins. Clinically, Cux1⁺ PBCs were increased in patients with psoriasis, suggesting that Cux1⁺ PBCs play an important part in epidermal hyperplasia. This study presents a systematic knowledge of the transcriptomic changes in a chronic dry skin mouse model, as well as a potential therapeutic target against dry skin-related dermatoses.

Single-cell analysis of cellular heterogeneity and interactions in the ischemia-reperfusion injured mouse intestine

Lianhong Yin, Meng Gao, Lina Xu, Yan Qi, Lan Han, Jinyong Peng

2023, 13(7): 760-775. doi: 10.1016/j.jpha.2023.02.002

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Nine major cell populations among 46,716 cells were identified in mouse intestinal ischemia‒reperfusion (II/R) injury by single-cell RNA sequencing. For enterocyte cells, 11 subclusters were found, in which enterocyte cluster 1 (EC1), enterocyte cluster 3 (EC3), and enterocyte cluster 8 (EC8) were newly discovered cells in ischemia 45 min/reperfusion 720 min (I 45 min/R 720 min) group. EC1 and EC3 played roles in digestion and absorption, and EC8 played a role in cell junctions. For TA cells, after ischemia 45 min/reperfusion 90 min (I 45 min/R 90 min), many TA cells at the stage of proliferation were identified. For Paneth cells, Paneth cluster 3 was observed in the resting state of normal jejunum. After I 45 min/R 90 min, three new subsets were found, in which Paneth cluster 1 had good antigen presentation activity. The main functions of goblet cells were to synthesize and secrete mucus, and a novel subcluster (goblet cluster 5) with highly proliferative ability was discovered in I 45 min/R 90 min group. As a major part of immune system, the changes in T cells with important roles were clarified. Notably, enterocyte cells secreted Guca2b to interact with Gucy2c receptor on the membranes of stem cells, TA cells, Paneth cells, and goblet cells to elicit intercellular communication. One marker known as glutathione S-transferase mu 3 (GSTM3) affected intestinal mucosal barrier function by adjusting mitogen-activated protein kinases (MAPK) signaling during II/R injury. The data on the heterogeneity of intestinal cells, cellular communication and the mechanism of GSTM3 provide a cellular basis for treating II/R injury.

Discovering metabolic vulnerability using spatially resolved metabolomics for antitumor small molecule-drug conjugates development as a precise cancer therapy strategy

Xiangyi Wang, Jin Zhang, Kailu Zheng, Qianqian Du, Guocai Wang, Jianpeng Huang, Yanhe Zhou, Yan Li, Hongtao Jin, Jiuming He

2023, 13(7): 776-787. doi: 10.1016/j.jpha.2023.02.010

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Against tumor-dependent metabolic vulnerability is an attractive strategy for tumor-targeted therapy. However, metabolic inhibitors are limited by the drug resistance of cancerous cells due to their metabolic plasticity and heterogeneity. Herein, choline metabolism was discovered by spatially resolved metabolomics analysis as metabolic vulnerability which is highly active in different cancer types, and a choline-modified strategy for small molecule-drug conjugates (SMDCs) design was developed to fool tumor cells into indiscriminately taking in choline-modified chemotherapy drugs for targeted cancer therapy, instead of directly inhibiting choline metabolism. As a proof-of-concept, choline-modified SMDCs were designed, screened, and investigated for their druggability in vitro and in vivo. This strategy improved tumor targeting, preserved tumor inhibition and reduced toxicity of paclitaxel, through targeted drug delivery to tumor by highly expressed choline transporters, and site-specific release by carboxylesterase. This study expands the strategy of targeting metabolic vulnerability and provides new ideas of developing SMDCs for precise cancer therapy.

The role of signaling crosstalk of microglia in hippocampus on progression of ageing and Alzheimer's disease

He Li, Tianyuan Ye, Xingyang Liu, Rui Guo, Xiuzhao Yang, Yangyi Li, Dongmei Qi, Yihua Wei, Yifan Zhu, Lei Wen, Xiaorui Cheng

2023, 13(7): 788-805. doi: 10.1016/j.jpha.2023.05.008

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Based on single-cell sequencing of the hippocampi of 5× familiar Alzheimer's disease (5× FAD) and wild type mice at 2-, 12-, and 24-month of age, we found an increased percentage of microglia in aging and Alzheimer's disease (AD) mice. Blood brain barrier injury may also have contributed to this increase. Immune regulation by microglia plays a major role in the progression of aging and AD, according to the functions of 41 intersecting differentially expressed genes in microglia. Signaling crosstalk between C−C motif chemokine ligand (CCL) and major histocompatibility complex-1 bridges intercellular communication in the hippocampus during aging and AD. The amyloid precursor protein (APP) and colony stimulating factor (CSF) signals drive 5× FAD to deviate from aging track to AD occurrence among intercellular communication in hippocampus. Microglia are involved in the progression of aging and AD can be divided into 10 functional types. The strength of the interaction among microglial subtypes weakened with aging, and the CCL and CSF signaling pathways were the fundamental bridge of communication among microglial subtypes.

A Scd1-mediated metabolic alteration participates in liver responses to low-dose bavachin

Pan Shen, Zhi-Jie Bai, Lei Zhou, Ning-Ning Wang, Zhe-Xin Ni, De-Zhi Sun, Cong-Shu Huang, Yang-Yi Hu, Cheng-Rong Xiao, Wei Zhou, Bo-Li Zhang, Yue Gao

2023, 13(7): 806-816. doi: 10.1016/j.jpha.2023.03.010

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Hepatotoxicity induced by bioactive constituents in traditional Chinese medicines or herbs, such as bavachin (BV) in Fructus Psoraleae, has a prolonged latency to overt drug-induced liver injury in the clinic. Several studies have described BV-induced liver damage and underlying toxicity mechanisms, but little attention has been paid to the deciphering of organisms or cellular responses to BV at no-observed-adverse-effect level, and the underlying molecular mechanisms and specific indicators are also lacking during the asymptomatic phase, making it much harder for early recognition of hepatotoxicity. Here, we treated mice with BV for 7 days and did not detect any abnormalities in biochemical tests, but found subtle steatosis in BV-treated hepatocytes. We then profiled the gene expression of hepatocytes and non-parenchymal cells at single-cell resolution and discovered three types of hepatocyte subsets in the BV-treated liver. Among these, the hepa3 subtype suffered from a vast alteration in lipid metabolism, which was characterized by enhanced expression of apolipoproteins, carboxylesterases, and stearoyl-CoA desaturase 1 (Scd1). In particular, increased Scd1 promoted monounsaturated fatty acids (MUFAs) synthesis and was considered to be related to BV-induced steatosis and polyunsaturated fatty acids (PUFAs) generation, which participates in the initiation of ferroptosis. Additionally, we demonstrated that multiple intrinsic transcription factors, including Srebf1 and Hnf4a, and extrinsic signals from niche cells may regulate the above-mentioned molecular events in BV-treated hepatocytes. Collectively, our study deciphered the features of hepatocytes in response to BV insult, decoded the underlying molecular mechanisms, and suggested that Scd1 could be a hub molecule for the prediction of hepatotoxicity at an early stage.

Single-cell transcriptome analysis reveals the regulatory effects of artesunate on splenic immune cells in polymicrobial sepsis

Jiayun Chen, Xueling He, Yunmeng Bai, Jing Liu, Yin Kwan Wong, Lulin Xie, Qian Zhang, Piao Luo, Peng Gao, Liwei Gu, Qiuyan Guo, Guangqing Cheng, Chen Wang, Jigang Wang

2023, 13(7): 817-829. doi: 10.1016/j.jpha.2023.02.006

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Sepsis is characterized by a severe and life-threatening host immune response to polymicrobial infection accompanied by organ dysfunction. Studies on the therapeutic effect and mechanism of immunomodulatory drugs on the sepsis-induced hyperinflammatory or immunosuppression states of various immune cells remain limited. This study aimed to investigate the protective effects and underlying mechanism of artesunate (ART) on the splenic microenvironment of cecal ligation and puncture-induced sepsis model mice using single-cell RNA sequencing (scRNA-seq) and experimental validations. The scRNA-seq analysis revealed that ART inhibited the activation of pro-inflammatory macrophages recruited during sepsis. ART could restore neutrophils’ chemotaxis and immune function in the septic spleen. It inhibited the activation of T regulatory cells but promoted the cytotoxic function of natural killer cells during sepsis. ART also promoted the differentiation and activity of splenic B cells in mice with sepsis. These results indicated that ART could alleviate the inflammatory and/or immunosuppressive states of various immune cells involved in sepsis to balance the immune homeostasis within the host. Overall, this study provided a comprehensive investigation of the regulatory effect of ART on the splenic microenvironment in sepsis, thus contributing to the application of ART as adjunctive therapy for the clinical treatment of sepsis.

2023 Vol. 13, No. 7