The most common site of metastasis in breast cancer is the bone, where the balance between osteoclast-mediated bone resorption and osteoblast-mediated bone formation is disrupted. This imbalance causes osteolytic bone metastasis in breast cancer, which leads to bone pain, pathological fractures, spinal cord compression, and other skeletal-related events (SREs). These complications reduce patients' quality of life significantly and have a profound impact on prognosis. In this review, we begin by providing a brief overview of the epidemiology of bone metastasis in breast cancer, including current diagnostic tools, treatment approaches, and existing challenges. Then, we will introduce the pathophysiology of breast cancer bone metastasis (BCBM) and the animal models involved in the study of BCBM. We then come to the focus of this paper: a discussion of several biomarkers that have the potential to provide predictive and prognostic value in the context of BCBM—some of which may be particularly compatible with more comprehensive liquid biopsies. Beyond that, we briefly explore the potential of new technologies such as single-cell sequencing and organoid models, which will improve our understanding of tumor heterogeneity and aid in the development of improved biomarkers. The emerging biomarkers discussed hold promise for future clinical application, aiding in the prevention of BCBM, improving the prognosis of patients, and guiding the implementation of personalized medicine. Breast cancer has surpassed lung cancer as the most common cancer worldwide, with an estimated 2.3 million new cases 11.7%) in 2020. It is also the fifth leading cause of cancer-related deaths globally, with 685,000 deaths (6.9%) in 2020 [1.7% in 2Bone metastasis in breast cancer is challenging to treat and can lead to complications including bone pain, pathological fractures, hypercalcemia, and spinal cord compression, which are collectively known as skeletal-related events (SREs). SREs significantly impact patients' quality of life and reduce survival rates , 5. It iHere, we summarize the epidemiology of BCBM and the current approaches to diagnosis and treatment. We will also discuss the existing views on the underlying mechanisms of BCBM and the relevant research models. But our primary focus will be on summarizing the studies aimed at discovering potential biomarkers for BCBM.There is a considerable body of research on the screening of high-risk populations for BCBM. Studies have identified certain molecular subtypes of breast cancer that are significant risk factors for bone metastasis. Of the main molecular subtypes of breast cancer, Luminal A (LUMA), Luminal B , HER2-positive, basal-like, and triple-negative breast cancer (TNBC), Luminal A and Luminal B subtypes have a significantly higher risk of bone metastasis than the other molecular subtypes , 8. LympDiagnostic guidelines for BCBM recommend timely imaging evaluation and biopsy in cases of suspected bone metastasis based on symptoms such as bone pain, pathological fractures, elevated alkaline phosphatase levels, or hypercalcemia . CommonlConsidering the specificity, sensitivity, cost-effectiveness, and accessibility of various diagnostic methods, imaging evaluation for bone metastasis screening is recommended for patients with stage 3 or higher breast cancer (except for T3N1M0 patients) and those experiencing bone pain, pathological fractures, hypercalcemia, and spinal cord compression or other SREs. NCCN guidelines further recommended tumor markers in the guidelines include alkaline phosphatase, blood calcium, CA15-3, and CA27.29 . Therefore, there is an urgent need in the clinical setting for the development of new biomarkers for BCBM, which would facilitate prevention and prediction by clinicians.When dealing with BCBM, a condition characterized as an end-stage systemic malignant disease, it is imperative to adopt treatment algorithms that primarily focus on systemic therapy. The fundamental treatment modalities include endocrine therapy, chemotherapy, and anti-HER2 therapy. In contemporary clinical practice, new targeted therapies and immunotherapies are also being integrated. It is crucial to customize these therapeutic approaches to cater to the unique needs of individual patients.The selection of the most appropriate treatment should take into consideration a multitude of factors, including but not limited to 1) Patients' General Health: The overall health and condition of the patients. 2) Intrinsic Molecular Subtypes: The specific molecular characteristics of the tumor. 3) Prior Therapies and Their Side Effects: The patient's history of previous treatments  and any associated toxicities. 4) Menopausal Status: Whether the patient is premenopausal or postmenopausal. 5) Bone Pain Control: The requirement for managing bone pain. 6) Socioeconomic and Psychological Factors: The social and psychological aspects impacting the patient's well-being.In addition to systemic treatments, the use of bone-targeted agents such as bisphosphonates and denosumab can effectively control the development of skeletal-related events (SREs). For localized management of bone metastases, surgical intervention and radiotherapy are proven and valuable methods. Moreover, it is imperative to provide comprehensive pain management and supportive care to patients. These supportive measures significantly enhance the patients' quality of life  is expressed at higher levels in luminal-type tumors compared to TNBC, and it significantly activates the NF-κB signaling pathway, upregulating IL-1B and promoting osteolytic metastasis [Our current understanding is that the “seeds” of certain breast cancers, which have inherent characteristics that favor bone metastasis, are provided “fertile soil” for metastasis by a shift from a normal bone microenvironment to a pre-metastatic microenvironment . Cells otastasis . A recentastasis . These stastasis . Bone matastasis .After breast cancer cells disseminate into the circulatory system, they may settle with the help of mobilized hematopoietic stem cells (HSCs) in the perivascular microenvironment and enter a dormant state before clinically detectable overt bone metastasis occurs . EndotheBreast cancer cells interact with the bone microenvironment, resulting in osteolytic lesions, promoting proliferation, and preparing for metastasis to other organs. They regulate the bone microenvironment through multiple mechanisms. Breast cancer cells expressing E-cadherin form heterotypic adhesive connections by binding to N-cadherin on osteoblasts. These adhesive connections promote the activation of the Akt/mTOR signaling pathway in cancer cells, stimulating their proliferation and leading to the formation of overt bone metastasis . The rol2+ that comes as a result of cancer-cell promoted bone resorption serves to further improve the survival and proliferation of cancer cells [The release of TGF-β, IGF, and Caer cells . TGF-β der cells . In the er cells . Tumor-der cells , 50. Theer cells , 52. Theer cells . TGFβ1 der cells . Howeverer cells . In summThe events that unfold following breast cancer bone metastasis are of significant concern. As reported by RE Coleman, a study involving 367 female patients with bone metastasis revealed that 228 of them subsequently experienced extra-osseous metastasis . Notably+ T cells via Siglec15, thereby promoting multi-organ metastasis in the late stages of breast cancer bone metastasis [Furthermore, recent investigations suggest that immune regulation also plays a role in secondary metastasis. Apoptotic bodies originating from osteoclasts have been found to suppress the function of naive CD8tastasis .Knowledge of the mechanisms of BCBM and the development of useful biomarkers are dependent on research with the appropriate animal models. In vivo models offer greater advantages over in vitro work in demonstrating the interactions between cancer cells and the microenvironment, as well as simulating complex metastatic processes. The ideal animal model would provide a human-like environment for studying the mechanisms of BCBM and help uncover deeper insights into the process . SeveralIntracardiac injection has been widely used in bone metastasis research . InjectiAlternatively, cancer cells can be injected through the iliac artery, which specifically delivers cancer cells to hind limb bones, avoiding the spread to other organs and preventing premature death and allowing the bone metastatic lesions to develop fully . This teResearchers have recently developed a caudal artery injection protocol that selectively delivers a large number of cancer cells to the bone marrow of the hind limbs. Compared to iliac artery injection, this method is easier to implement and causes less trauma to the animal , 65. ThiIntraosseous injection is the most direct method of confining cancer cells to the bone and reducing the possibility of metastasis to other organs , 69. HowThe above-mentioned circulation injection models offer unique advantages, but they bypass the earliest stages of metastasis. They cannot be used to study the interaction between tumor cells with bone tropism and the extracellular matrix at the primary site. The orthotopic injection is a more suitable model, in which breast cancer cells are injected into the mammary fat pad of mice, where they develop into primary tumors and subsequently generate bone metastases. This model allows for the complete process of metastasis to occur. The cell lines used in this spontaneous metastasis model are 4T1.2 and 4T1.13, which are sublines of the 4T cell lines and have been shown to have a high propensity to metastasize to lymph nodes, lungs, and bones , 72. ComThough the aforementioned models have significantly advanced knowledge of BCBM, researchers aim to establish models that better reflect the conditions in human body. Humanized models provide tumor cells with microenvironments more similar to the human body, allowing for a closer approximation of tumor progression in patients. Some humanized models are relevant to the study of the interaction between cancer cells and the human bone microenvironment. A recently invented model involves implanting tissue from the femoral heads of patients undergoing hip replacement surgery under the skin of immunodeficient mice with subsequent orthotopic injection of cancer cells. The graft exhibits metabolic activity, bone remodeling and can undergo bone metastasis. This model demonstrates spontaneous metastasis of injected breast cancer to the transplanted bone and may be used for studying the driving factors of metastasis and the bone microenvironment of metastasis research  Table 2.Table 2AIn summary, the continuous development of BCBM models provides the tools needed to further elucidate the mechanisms of BCBM while moving the world of bench research ever closer to the goal of clinical utility. It aids in the exploration of new predictive and prognostic biomarkers and provides a reliable platform for the development of potential therapeutic targets.Using appropriate models and research techniques, researchers have explored potential biomarkers for BCBM, including various proteins, genes, and circulating cells. These biomarkers can be broadly classified into two categories: prognostic biomarkers and predictive biomarkers. Prognostic biomarkers provide information about recurrence and may be associated with progression-free survival (PFS) in metastatic disease patients. Predictive biomarkers can predict a patient's response to treatment or be used for monitoring during treatment to determine if the patient is benefiting from the therapy .Prognostic biomarkers provide information about recurrence and may be associated with progression-free survival in metastatic disease patients .Several studies have investigated the value of protein molecules in predicting the occurrence of bone metastasis.Nuclear p21-activated kinase 4 (nPAK4) can promote BCBM by inhibiting leukemia inhibitory factor receptor (LIFR). LIFR plays an important role in BCBM. It activates STAT3 to promote the dormancy of breast cancer cells and triggers the Hippo kinase cascade, leading to the functional inactivation of yes-associated protein (YAP), thus inhibiting bone metastasis , 77. PAKMacrophage-capping protein (CAPG) and PDZ domain–containing protein GIPC1 (GIPC1) are also involved in BCBM. CAPG, an actin-binding protein, may enhance tumor invasion by altering cytoskeletal dynamics . It has Using similar approach, Westbrook et al. discovered that the expression level of dedicator of cytokinesis protein 4 (DOCK4) was higher in the bone metastatic variant of human breast cancer cell line MDA-MB-231 compared to the parental non-bone metastatic cells. They further validated this finding through western blotting, confirming the potential of DOCK4 as a biomarker. In clinical validation, the correlation between high DOCK4 expression and invasive tumors was confirmed in a tissue microarray containing 345 samples. Subsequent research using samples from 689 patients in the AZURE trial found that adjusted Cox regression analysis showed that high DOCK4 expression was associated with a higher risk of bone metastasis occurrence . DOCK4 mRANKL is a factor regulating bone remodeling . It alsoAnother bone remodeling regulator is prolactin (PRL). It promotes the secretion of osteoclastogenic factor sonic hedgehog (SHH) by breast cancer cells, thereby activating the Hedgehog pathway in osteoclasts and promoting bone resorption . The HH . discovered that the expression level of IL-1B protein was increased in bone metastatic cells compared to the primary breast cancer cells. Subsequently, in a tissue microarray containing 150 cases of stage II and III primary breast tumors, IL-1B was measured using immunohistochemistry. The median follow-up period was 84 months. The study found a significant correlation between IL-1B expression and bone metastasis [IL-1B also plays an important role in bone metastasis. It promotes EMT in breast cancer cells and regulates osteolysis, promoting cancer cell proliferation , 93. IL-tastasis .A recent study found that the specific expression of SCUBE2 in luminal-type breast cancer is associated with shortened bone metastasis-free survival. By analyzing the expression of secreted SCUBE2 protein in the serum of patients from the Qilu cohort, Wu et al. observed an upregulation of SCUBE2 expression in bone metastasis patients (n = 13) compared to patients without distant recurrence (n = 23), confirming the prognostic value of SCUBE2 in luminal-type breast cancer patients. Mechanically, regulated by ER signaling in luminal-type breast cancer, SCUBE2 acts in an autocrine manner on cancer cells, leading to Hedgehog signal activation, osteoblast differentiation, and immune suppression-mediated bone metastasis .. They measured the serum levels of P1NP, CTX, IL-6, and osteocalcin in the blood of 164 pre-treatment stage I-III breast cancer patients to explore the relationship between these markers and bone metastasis and overall survival. They found that in stage I–III breast cancer patients, a serum P1NP level ≥ 75 ng/mL indicated a higher risk of bone metastasis and shorter overall survival [Bone turnover markers also provide prognostic information about bone metastasis. They can be divided into bone formation markers and bone resorption markers. Bone metastasis disrupts the balance between bone formation and bone resorption, leading to changes in the levels of these markers . Procollsurvival   secreted by ER+ breast cancer cells can recruit preosteoclast cells, creating a microenvironment enriched with preosteoclast cells, which facilitates miRNA-19a regulation of bone resorption . Wu et aA study conducted by Adam Artigues reported the dysregulation of miRNA-30b-5p in breast cancer metastasis patients. This is another biomarker that can be detected in blood. The researchers first measured the levels of miRNA-30b-5p in tissue samples and then confirmed its potential as a biomarker in liquid biopsy using peripheral blood samples. They found that miRNA-30b-5p was a valuable non-invasive diagnostic biomarker. The expression level of miRNA-30b-5p in bone metastases was significantly higher than in other metastatic sites. However, it should be noted that although miRNA-30b-5p was found to have predictive value for distant metastasis, with 63 bone metastasis patients included in the cohort, 30 of them had metastasis in other sites. Therefore, further research is needed to determine whether miRNA-30b-5p can specifically predict bone metastasis .miRNA-16 and miRNA-378 have the potential to be used in liquid biopsy as well. Mechanically, miRNA-16 may enhance osteoclast function and bone destruction caused by BCBM by increasing the expression of NFATc1, RANKL, IL-1β, PTHrP, and other factors. The specific mechanism of miRNA-16's action requires further investigation . miR-378miRNA-124 has been found to inhibit BCBM by regulating the bone microenvironment. In vitro experiments have shown that miRNA-124 downregulates cytokines that stimulate osteoclastogenesis, inhibits the survival and differentiation of osteoclast precursors, and thus suppresses bone resorption . FurthermiRNA-429, an RNA whose high expression is also associated with a good prognosis, plays a role in BCBM microenvironment regulation. In vitro experiments have shown that miRNA-429 downregulates CrkL and MMP-9, two important molecules that promote bone metastasis, and promotes osteoblast differentiation. In animal models, miRNA-429 regulates BCBM microenvironment through similar mechanism . Using ILncRNA FGF14-AS2 is another RNA that can inhibit breast cancer metastasis . In the It has been reported that a panel of non-coding RNAs may have prognostic potential. eRNAs are short non-coding RNAs transcribed from enhancer sites . Li analMAF gene at 16q23 mediate bone metastasis in breast cancer. The transcription factor MAF acts on the PTHrP P1 promoter, thereby promoting bone metastasis. Using immunohistochemistry to detect MAF protein levels and FISH to detect 16q23 gains, Pavlovic et al. found that patients with this copy number variation in 334 primary breast cancers had a significant risk of bone metastasis, while no correlation was found with metastasis in other sites [Research of BCBM has identified several genes and mRNAs as biomarkers. Copy number alterations of er sites .. used RT-qPCR to detect ZNF217 mRNA expression in 113 breast cancer samples and found that high expression of ZNF217 is associated with a higher risk of isolated bone metastasis in ER+ patients, but the level of ZNF217 cannot predict metastasis in other subtypes of breast cancer [High expression of zinc-finger protein 217 (ZNF217) in MDA-MB-231 breast cancer cells is associated with EMT and certain gene dysregulation . These gt cancer .Enhancer of zeste homolog 2 (EZH2), as a transcriptional co-repressor of RNA Pol II, increases the expression of integrin β1, leading to focal adhesion kinase (FAK) activation and activation of the TGFβ/Smad2 pathway, promoting bone metastasis . EZH2 haSavci-Heijink et al. identified 15 genes  associated with the development of bone metastasis in 157 cases of primary breast cancer. This gene signature showed good predictive value for both ER-positive and ER-negative tumors. In a training set and a validation set containing 376 breast tumors, they identified bone metastasis in over 80% of cases. Among these 15 genes, three upregulated genes  were associated with EMT in tumors, while the other 12 genes were downregulated in bone metastasis . AdditioAnother combination of five genes  has been reported to have prognostic significance. ROC analysis demonstrated that this gene signature had a predictive accuracy of over 90% for bone metastasis in breast and lung cancer patients and could be detected using liquid biopsy. Mechanically, HSP90, PTK2, and VEGF regulate cancer cell invasion, proliferation, and metastasis, while IL3 and SPP1 regulate osteogenesis .A study conducted by Cosphiadi et al. included 92 patients with advanced breast cancer, of which 46 patients had bone metastasis. Comparing the 17 patients who only had bone metastasis with non-bone metastasis patients, the researchers identified 17 significantly altered genes. They further confirmed the genes associated with an increased risk of bone metastasis, namely ESR1, PGR, BCL2, REPS2, NAT1, GATA3, ANXA9, C9orf116. The combination of these eight genes had an AUC of 0.928, demonstrating better predictive ability than individual genes .IL-6 gene signature was identified by Mp1nichal Rajski et al. Researchers first investigated the impact of cell–cell interactions on overall gene expression in a co-culture experiment using breast cancer cells and osteoblasts. Co-culturing significantly increased the expression of IL-6. Subsequently, these results were applied to 295 early breast cancer specimens from the Netherlands Cancer Institute to evaluate the clinical relevance of the in vitro experiment. The researchers confirmed an IL-6 gene signature composed of 72 genes, which was associated with shorter time to bone metastasis in patients with high expression of this gene signature .A study analyzed the expression of TFF1 and verified its positive correlation with tumor relapse to bone using SAM analysis and quantitative RT-PCR. They used PAM analysis to identify a 31-gene signature predicting bone metastasis with 100% sensitivity and 50% specificity .By comparing gene expression between different sublines of human breast cancer cell line MDA-MB-122, researchers identified four highly overexpressed genes in the bone metastasis group: IL11, CTGF, CXCR4, and MMP-1. They further demonstrated that the expression of these factors was further enhanced by the pro-metastatic cytokine TGFβ, providing evidence for these genes as potential diagnostic markers and therapeutic targets .Woelfle et al. reported significant differences in gene expression between bone metastasis-positive and -negative patients' primary tumors based on cDNA microarray expression analysis. The differentially expressed genes were involved in extracellular matrix remodeling, adhesion, and cytoskeletal plasticity. They then identified a 73-gene signature capable of identifying lymph node-negative patients with or without bone marrow micrometastases. Immunohistochemical analysis of a test set containing 83 primary breast tumor samples, from patients with or without tumor cells in the bone marrow, confirmed that patients with decreased expression of CK8, CK18, or CK19 had an increased incidence of bone marrow micrometastases .FOXC1 mRNA expression and metastasis. Higher FOXC1 expression was positively correlated with brain metastasis (P = 0.02) but negatively correlated with bone metastasis (P = 0.0002) [Ray investigated the prognostic value of FOXC1 gene signature in basal-like breast cancer metastasis. They found that ectopic overexpression of FOXC1 increased breast cancer cell proliferation, migration, and invasion. They constructed a FOXC1 gene signature composed of 30 genes that could predict overall survival and brain metastasis occurrence in patients. Subsequent analysis of a dataset containing 286 samples explored the relationship between  0.0002) .The tumor suppressor gene Raf kinase inhibitory protein (RKIP) inhibits downstream genes involved in BCBM  through miRNA let-7. Using an independent dataset of breast cancer patients, researchers found that patients with high expression of downstream RKIP pathway metastasis genes and low expression of RKIP had a significantly increased risk of metastasis .The function of BMP has been discussed previously. It is also found to compose a prognostic gene signature along with other genes. Research based on the construction of a weighted co-expression network of differentially expressed genes (DEGs) related to EMT in metastatic breast cancer identified hub genes such as FERMT2, ITGA5, ITGB1, MCAM, CEMIP, HGF, TGFBR1, and F2RL. Differential expression of BMP2, BMPR2, and GREM1 was found in two datasets of BCBM. Based on the results of bioinformatics analysis from this study, it is speculated that BMP-2 may regulate immune infiltration processes in breast cancer tissue through the PI3K/Akt signaling pathway, thereby affecting cancer prognosis .As has been discussed earlier, breast cancer cells secrete MSP, which acts on the RON receptor and promotes osteoclast activation . Welm etUsing microarray datasets from GSE2034, GSE2603, GSE12276, and NKI295, Li constructed a Gene Dependency Network and selected 51 genes associated with breast cancer metastasis. Survival analysis using training and testing sets showed that the identified gene signature was associated with shorter bone metastasis-free survival in high-risk cancer patients, confirming the prognostic value of this gene panel .Fan used GSE124647 as the training set and GSE16446, GSE45255, GSE14020 as the validation sets to explore the differential expression levels of key genes between breast cancer non-bone metastasis and bone metastasis groups. Potential prognostic-related genes were analyzed, validated, and a columnar line graph (GESBN) model based on gene expression features was constructed to predict the likelihood of bone metastasis in breast cancer patients. Patients were sorted based on risk scores from the training set and divided into high-risk and low-risk groups for bone metastasis. The GESBN model constructed using GAJ1, SLC24A3, ITGBL1, and SLC44A1 indicated a poorer overall survival (OS) for high-risk individuals. The GESBN model constructed using GJA1, IGFBP6, MDFI, TGFBI, ANXA2, and SLC24A3 indicated a poorer PFS for high-risk individuals. Two hub genes, SLC44A1 and MDFIBC, were also selected and may serve as therapeutic targets for BCBM .Although these gene signatures cover many genes, the overlap between them is relatively small. Possible reasons for this phenomenon include different sample types (breast cancer cell lines or tissues), choice of experimental methods, and selection of patient types . HoweverTFF1 mRNA and bone metastasis. Thus, the specific gene expression of CTCs can be used in bone metastasis prediction [Circulating tumor cells (CTCs) are tumor cells that detach from the tumor and circulate in the bloodstream. Circulating tumor cells can be collected and used for liquid biopsy assays . In patiediction . Furtherediction . These sOsteocalcin is a marker of late osteoblast differentiation. Circulating osteocalcin-positive cells (cOC) are small mononuclear cells expressing osteocalcin in peripheral blood mononuclear cells. Researchers have identified the optimal cutoff value for cOC as 0.069% based on the ROC curve. Patients with high cOC levels have significantly shorter bone metastasis-free survival compared to those with low cOC levels, indicating that cOC can predict early bone metastasis. However, cOC levels do not increase in advanced bone metastasis patients. Animal models have also shown a significant increase in cOC during early bone metastasis, which gradually decreases with tumor progression. Researchers have also found that benign fractures do not affect the elevation of cOC related to metastasis, which is an advantage of using cOC as a predictor of bone metastasis .. found that downregulation of RAI2 is associated with early bone metastasis in ERα-positive breast tumors, suggesting its potential role in inhibiting early hematogenous dissemination of tumor cells to the bone marrow [In addition to the mentioned biomarkers, there are other factors that may have prognostic significance. Tavazoie et al. compared primary tumors resected from 11 patients with lung, bone, or brain metastases to tumors resected from 9 patients without metastatic recurrence. They found that patients with low expression of miRNA-335 and miRNA-126 in the primary tumor were more likely to develop metastasis . Additioe marrow . Subseque marrow . WhetherPredictive biomarkers can predict a patient's response to treatment or be used for monitoring during treatment to determine if the patient is benefiting from the therapy .CAPG and GIPC1 have been confirmed to have prognostic significance by Westbrook et al. In the same study, they also demonstrated the predictive role of these two molecules. Patients with high expression of CAPG and GIPC1 treated with zoledronic acid showed a tenfold reduction in the risk of bone metastasis compared to the control group (P = 0.008). Therefore, detecting CAPG and GIPC1 can help identify patients who would benefit from zoledronic acid treatment .In a previously mentioned AZURE clinical trial, high levels of DOCK4 were found to be associated with bone metastasis in the control group but not statistically correlated in the group treated with zoledronic acid. This indicates that the use of zoledronic acid can effectively reduce the risk of bone metastasis in patients with high expression of DOCK4 .Urinary N-terminal telopeptide (uNTX) is a marker reflecting bone resorption . A retroThe prognostic value of MAF has been discussed earlier. Subsequent studies have found that MAF may be used to guide treatment for BCBM. Using FISH to detect MAF in primary tumor samples, Robert Coleman defined copy number >  = 2.5 as MAF+ve and studied a cohort of 1769 patients, including those from the AZURE trial. Kaplan–Meier survival curve analysis showed that MAF-ve tumor patients benefited from zoledronic acid, while in premenopausal women with MAF+ve tumors, zoledronic acid treatment led to poorer overall survival (OS) . This reThese biomarkers not only have predictive and prognostic value but also participate in the pathophysiology of BCBM Table . Their rA subset of the aforementioned biomarkers have been evaluated for use in liquid biopsies. The technique of liquid biopsy involves real-time combined analysis of circulating tumor cells, circulating tumor DNA, extracellular vesicles, RNA, proteins, and other substances derived from primary or metastatic tumor lesions in blood or other body fluids, providing information about the tumor . It offeWithin the scope of liquid biopsy, the development of novel assays to detect biomarkers within exomes is particularly promising. Exosomes are a subset of extracellular vesicles that mediate cell-to-cell communication by transporting molecular cargoes such as proteins, mRNA, miRNA, and lipids from donor cells to recipient cells, thereby participating in physiological and pathological processes. Exosomes offer several advantages in liquid biopsy. First, they are enriched in body fluids and easily accessible. Second, due to their lipid bilayer structure, exosomes are stable, which allows them to maintain stability during circulation in the body, sample transportation, and storage, making them crucial for the development of biomarkers using retrospective samples , 156.Numerous studies have enriched and purified exosomes through ultracentrifugation and confirmed their role in the process of BCBM and their potential as biomarkers. As mentioned earlier, exosomal miRNA-21 promotes the formation of osteolytic metastatic lesions in breast cancer, and exosomal mRNA of miRNA-19a, HSP90AA1, SPP1, IL3, VEGFA, and PTK2 genes have also been validated as biomarkers , 98, 120Liquid biopsy offers unique advantages compared to tissue biopsy. Tumor cells are heterogeneous, and cancer cells undergo molecular changes because of fluctuations in microenvironment and therapeutic interventions. Whereas tissue biopsies are limited in their ability to be representative of subpopulations of tumor cells and to reflect real-time development of the tumor, liquid biopsies are not . The majTechnological advancements are making nucleic acids increasingly relevant as the targets of screening and diagnosis. A multistage disease as complex and heterogenous as BCBM offers a particular set of challenges that must be addressed by a deliberate, well planned sequencing protocol. The continuous evolution of heterogeneous tumor cell populations resulting in the emergence of treatment-resistant clones, which can cause drug responses in the primary site and the distant metastatic sites to be very different . Traditi. conducted a study employing single-cell RNA sequencing to delve into the cellular components within the microenvironment. Their research uncovered that SCUBE2, expressed by MCF7 cells, plays a pivotal role in inducing the enrichment of a specific osteoblastic subpopulation within metastatic lesions. This induction, in turn, promotes osteoblast differentiation [. explored various cell subpopulations within the tumor microenvironment of bone metastasis. Their research pinpointed the existence of the MSC_MARCKSL1 subpopulation, situated in the early stages of differentiation and characterized by its significant differentiation potential. These findings suggest a potential involvement of this subpopulation in the regulation of bone metastasis [Wu et alntiation . In a setastasis .Sequencing techniques can also provide important insight into how breast to bone metastasis influences further metastasis to other sites . The ori. conducted single-cell RNA sequencing on T cells isolated from human breast cancer tissues. This endeavor revealed the remarkable heterogeneity within the T cell population and culminated in the development of a CD8+ TRM gene signature, derived from the sequencing data. This signature serves as a valuable prognostic biomarker, specifically tailored for early TNBC patients [Single-cell sequencing plays a pivotal role in advancing the development of biomarkers, particularly in breast cancer research. In pertinent studies, breast cancer cells and immune cell populations of distinct molecular subtypes have been systematically classified, leading to the identification of unique subgroups that may hold relevance for prognosis and the understanding of drug resistance . For inspatients . Moreovepatients .., who utilized this technology to uncover the presence of immune-inhibitory immature myeloid cells (IMCs) within breast cancer tumors that displayed resistance to anti-Her2 treatment and the CDK4/6 inhibitor Palbociclib. To surmount this resistance, Wang's research demonstrated the effectiveness of a combined approach. It involved the use of tyrosine kinase inhibitors that targeted IMCs in conjunction with immune checkpoint inhibitors. This innovative combination therapy successfully overcame resistance, highlighting the considerable potential of single-cell sequencing in monitoring the evolution of tumors under the influence of drug interventions. It offers valuable insights into devising more effective treatment strategies [Single-cell sequencing not only holds promise for predicting patient prognosis but also plays a vital role in predicting drug efficacy. An illustrative example of this is the work of Wang et alrategies .Knowledge of the mechanisms behind BCBM is only as good as the in vitro and in vivo experimental models used. Development of more suitable BCBM models is critical to both the development of better screening biomarkers and therapeutic drugs. One of the most promising avenues in the study of BCBM is the use of organoids in precision medicine. Organoids are three-dimensional models containing multiple cell types that simulate the characteristics of tumors in the body and can be used to study the biological properties of tumor cells in detail . They ofOrganoids represent intricate three-dimensional structures that closely mimic the architecture and functionality of organs. They originate from stem cells or organ-specific progenitor cells and are generated through in vitro self-organization processes. When compared to other tumor models, organoids offer conditions that more faithfully replicate the characteristics of the original tumor. This is especially advantageous in the in-depth study of human cancers, including breast cancer. To illustrate, when cultured in conventional tissue culture bottles, there exists a substantial disparity, exceeding 17 million times, between the stiffness of healthy mammary glands and the surrounding cell layer. In contrast, the elastic modulus of Matrigel, a common substance used in organoid culture, is relatively like that of mammary glands. This aligns organoid cultures more closely with the natural tissue environment. Furthermore, in standard two-dimensional (2D) cell cultures, there is a gradual loss of heterogeneity, whereas the tumor environment maintains a high degree of heterogeneity. In contrast, organoids encompass various cell types and can simulate some of the essential functions of organs. As a result, they are exceptionally well-suited for research into the tumor microenvironment (TME) and offer a more comprehensive and physiologically relevant platform for such investigations .The extracellular matrix (ECM) plays critical roles in driving cancer phenotype, disease progression and therapeutic response in vivo. 3D engineered matrices with tunable biochemical and mechanical properties are poised to answer previously untestable hypotheses surrounding mechanisms of these important cancer–matrix interactions . DhimoleOrganoids serve as invaluable tools that offer histological insights into cancer development, enhance our understanding of the carcinogenic process, and open avenues for the discovery of novel treatment approaches. Notably, organoid models have found application in the investigation of bone metastasis in breast cancer. In some studies, researchers have identified a correlation between elevated expression of Sclerostin (SOST) and the occurrence of bone metastasis in breast cancer, as well as a poorer prognosis for breast cancer patients. Silencing SOST expression has been shown to significantly diminish the capacity of SCP2 cells. Moreover, it has been revealed that the interaction between SOST and STAT3 can potentiate the TGF-β/KRAS signaling pathway, consequently promoting tumor growth and bone metastasis. Remarkably, the administration of S6, a leading candidate drug, has demonstrated a notable capacity to inhibit the growth of breast cancer organoids. Additionally, it has exhibited efficacy in mitigating bone metastasis in mouse models. These findings underscore the potential therapeutic value of targeting SOST and its associated pathways in addressing breast cancer and its metastatic spread to the bone . Recent While organoids hold great promise as invaluable models, their practical implementation faces certain limitations. Firstly, despite their ability to offer more realistic 3D models for cancer research, creating an organoid model that faithfully mirrors the heterogeneity of a patient's tumor and its tumor microenvironment (TME) remains a significant challenge. This challenge arises due to the absence of critical components such as stroma, immune cells, blood vessels, and microbiota within organoids. Additionally, the time required for culturing organoids derived from breast cancer patients poses a significant hurdle. The process can be time-consuming, and the efficiency of organoid derivation and reliable in vitro expansion is often characterized by unpredictability and low success rates. These limitations underscore the need for continued research and development efforts to refine organoid models and enhance their capacity to faithfully recapitulate the complexity of tumors and their microenvironments , 171.Bone metastasis is a serious complication that occurs in advanced breast cancer, impairing patients' quality of life and reducing their lifespan. Existing detection methods often struggle to identify early-stage bone metastasis, and many patients are only diagnosed when symptoms arise .In this context, many studies have attempted to explore biomarkers that can predict the occurrence of bone metastasis, enabling the selection of high-risk patients for subsequent follow-up and treatment. Among them, biomarkers compatible with liquid biopsy offer unique advantages over tissue biopsy since measurements can be repeated and assess the risk of bone metastasis multiple times. Future research endeavors in the realm of liquid biopsy are poised to unlock the full potential of these biomarkers for longitudinal monitoring during disease progression and therapeutic interventions. Such studies hold significant promise, particularly in the context of assessing the risk of bone metastasis in breast cancer patients, enabling precise patient stratification, and facilitating informed drug selection. An illustrative example of the potential of longitudinal liquid biopsy comes from the retrospective study conducted by Chin et al., involving 33 breast cancer patients. In this study, serial analysis of circulating tumor DNA (ctDNA) was employed to monitor the status of bone metastatic lesions and to detect acquired genetic alterations linked to drug resistance. The outcomes of this investigation highlight the clinical feasibility and utility of serial ctDNA analysis in tracking tumor responses to CDK4/6 inhibitors. Consequently, this study provides compelling evidence supporting the practical application of longitudinal liquid biopsy in clinical settings . These fOur understanding of the predicting power of the biomarkers discussed is currently limited. Some of the biomarkers mentioned are just beginning to show promise in preclinical testing, whereas others have already demonstrated some degree of utility. Without the use of scaled up clinical trials in large patient populations, it is exceedingly difficult, if not impossible, to determine which biomarkers will be robust enough to predict BCBM. More substantial validation studies are in our future. With continued research in particular patient populations, our understanding of which biomarkers will be clinically useful in which situations will improve.Concurrently, sequencing techniques will continue to become more sophisticated and will make the goal of precision medicine more attainable. Research in the basic sciences and improved models will also undoubtedly advance the discovery of novel biomarkers and therapeutic compounds while providing valuable insight into how to best use them.., who developed CIBERSORT, a tool that employs machine learning and deconvolution algorithms to estimate the composition and abundance of immune cells within mixed cell populations. Building upon this advancement, Guo et al. successfully integrated results from both single-cell sequencing and bulk sequencing. This integration enabled them to delve into T-cell heterogeneity in TNBC and construct a prognostic risk model specifically related to T-cells in TNBC. The integration of these data sources holds significant promise for gaining a comprehensive understanding of tumor biology and the immune microenvironment. This approach has the potential to advance the development of precise prognostic models and personalized therapeutic strategies in breast cancer research and beyond [Despite the significant strides made in cancer research and biomarker development facilitated by single-cell sequencing, there are challenges that need to be addressed before the widespread adoption of this promising technology. One such challenge is the limitation imposed by the sheer number of cells within a solid tumor tissue, which often surpasses the capacity for sequencing offered by single-cell sequencing. Additionally, the high costs associated with this technology pose a barrier to its widespread application , 178. Fud beyond . This unAnother noteworthy limitation is the loss of spatial information . HoweverThe added benefit of discovering and developing novel biomarkers is that most of them also play a role in cancer development and can be potential targets for treatment. For example, RANKL has significance as a biomarker and is also the target of denosumab, an approved for the treatment of bone metastasis . Other bTremendous strides have been made in unraveling the intricate mechanisms underlying breast cancer bone metastasis. Yet, certain processes, especially those related to secondary metastasis following initial bone metastasis, still lack comprehensive characterization. While existing research has shed light on some aspects, such as the role of the bone metastatic microenvironment in promoting secondary metastasis, there remains a need for deeper understanding. Current studies have pointed to specific mechanisms, such as cancer cell-autonomous secretion of EZH2 and immune suppression mediated by osteoclast-derived apoptotic bodies, both of which can be promising targets for therapy , 58. TheIn conclusion, the future trajectory of research in breast cancer bone metastasis should be guided by several key objectives: 1) Mechanistic Understanding: Continued efforts should be dedicated to deepening our understanding of the intricate mechanisms that drive breast cancer bone metastasis. This knowledge will pave the way for more targeted and effective treatments. 2) Biomarker Development: The development of highly accurate and reliable biomarkers is crucial. These biomarkers have the potential to aid in early diagnosis, risk assessment, and treatment stratification for breast cancer patients with bone metastasis. 3) Clinical Validation: It is imperative to subject these biomarkers to extensive large-scale clinical validation to ensure their robustness and reliability. This step is essential before their potential implementation in routine clinical practice. 4) Therapeutic Targets: Exploring whether these biomarkers can also serve as therapeutic targets is a promising avenue of investigation. Targeted therapies based on these biomarkers may offer more effective and personalized treatment options.By pursuing these objectives, the development of new technologies and research models will undoubtedly advance the study of novel biomarkers, with the potential for clinical application in the near future, ultimately assisting in the prevention of BCBM and improving the prognosis of patients with BCBM.


Involvement in caregiving and tailored support services may reduce the risk of mental health symptoms for mothers after their preterm infant’s neonatal intensive care unit (NICU) discharge. We aimed to compare Family-Centered Care (FCC) with mobile-enhanced Family-Integrated Care (mFICare) on post-discharge maternal mental health symptoms.This quasi-experimental study enrolled preterm infant (≤ 33 weeks)/parent dyads from three NICUs into sequential cohorts: FCC or mFICare. We analyzed post-discharge symptoms of perinatal post-traumatic stress disorder (PTSD) and depression using intention-to-treat and per protocol approaches.178 mothers  completed measures. We found no main effect of group assignment. We found an interaction between group and stress, indicating fewer PTSD and depression symptoms among mothers who had higher NICU-related stress and received mFICare, compared with mothers who had high stress and received FCC . Per protocol analyses of mFICare components suggested fewer PTSD and depression symptoms among mothers who had higher NICU stress scores and participated in clinical team rounds and/or group classes, compared with mothers who had high stress and did not participate in rounds or classes.Overall, post-discharge maternal mental health symptoms did not differ between the mFICare and FCC groups. However, for mothers with high levels of stress during the NICU stay, mFICare was associated with fewer post-discharge PTSD and depression symptoms. Parents provide essential caregiving support for preterm infants admitted to neonatal intensive care units (NICU) to promote infant growth and development, including breastfeeding, skin-to-skin contact, developmentally supportive care, positive sensory stimulation, pain management and massage . AlthougFamily-Integrated Care (FICare) is a parent-partnered NICU care delivery model that provides well-defined yet flexible protocols for ensuring meaningful participation of parents in all aspects of NICU caregiving, from an individual infant’s bedside to the hospital boardroom. FICare has four main pillars: NICU environment, NICU team education and support, parent education, and parent support , 5. MobiThe United States (US) healthcare and social contexts are different than in other countries and these differences may affect NICU implementation of FCC and FICare and parental uptake of supportive services. For example, most countries provide more generous perinatal leave benefits and more generous extended leave benefits if a child is critically ill than the US , thus enThis analysis of pre-defined secondary aims was part of a larger quasi-experimental, time-lagged non-randomized intervention trial of the mFICare intervention compared with FCC  [Parents/primary caregivers of infants born ≤ 33 weeks gestation were invited to participate. Participants were excluded if: (1) the parent was not English literate, < 18 years of age, or had no smart phone or tablet access; or (2) the infant had a life-threatening congenital anomaly or was receiving palliative care. Parents received up to $50 in gift cards for completion of study surveys. The study was approved by the institutional review board at each site and written informed consent was obtained from all participants [Parents of current and former NICU patients and NICU healthcare professionals were extensively involved in the design of the trial and the adaptation of the FICare intervention to the local settings. Parents also co-designed and pilot-tested a mobile app for parents. Details of the FCC and mFICare interventions are provided elsewhere . BrieflyThe first cohort of enrolled parents received usual FCC. Then recruitment was paused for mFICare training of NICU staff and parent mentors. Once approximately 80% of each NICU’s staff received training, the second cohort of parent/infant dyads was enrolled and received mFICare.The primary outcomes for this analysis were maternal symptoms of perinatal PTSD and depression measured at least three months after the infant’s NICU discharge. The Perinatal PTSD Questionnaire (PPQ)  was usedMaternal depression was assessed by self-report using the Edinburgh Postnatal Depression Scale (EPDS) . The EPDWe examined four potential moderators of the intervention effect on PTSD or depression symptoms: GA at birth ; Infant age at enrollment ; Infant discharge on respiratory or feeding devices or monitoring equipment ; and Parent stress . The selected moderators have been shown in prior studies to influence PTSD or depression in mothers after their infant’s discharge from the NICU , 26. PerAdditional sample characteristics were obtained from the infant medical record and parents surveys. Parents’ perception of the family-centeredness of NICU care they received was assessed near the time of discharge with the Digi Family-Centered Care-Parent Version (DigiFCC-P); scores range 1–7 with high scores indicating high perceived quality of FCC .Analyses were performed using R v4.1  and StatWe tested for associations between PPQ and EPDS scores (log-transformed) and intervention group using a linear regression model. We first tested for main effects of the intervention. We adjusted for additional covariates using a hybrid approach, forcing in site, and then using a backwards stepwise regression to identify demographic and clinical covariates that contributed p < .1 to the final model.We then tested for moderating effects on the intervention with four factors: infant gestational age at birth, infant chronological age at study enrollment (log transformed), whether the infant was discharged on a respiratory or feeding device, and NICU-related stress level at study enrollment (log-transformed), controlling for additional covariates as described above. Finally, for factors that had an interaction with the intervention group (denoted intervention moderators) with p < .05, we conducted an exploratory per protocol analysis testing for an interaction between the moderator and each of the following mFICare intervention components over the course of the NICU hospitalization: whether the parent had a mentor; participated in at least 2 clinical rounds; attended at least 1 parent class; or logged into the We3health™ app at least 4 times. We report results meeting a nominal p < .05, as none reached Bonferroni-corrected significance.Of the 237 mothers enrolled in the study between April, 2017 and June, 2020, 178  completed PPQ and EPDS measures a mean of 4.2 (SD 1.9) months after their infant’s NICU discharge and were included in this analysis . However, we found a nominally significant interaction association between PSS:NICU scores and intervention group for PPQ . For mothers who experienced high levels of NICU-related stress, participating at least twice in clinical rounds was associated with fewer PTSD symptoms after discharge. However, for mothers who experienced low levels of NICU-related stress, there was no apparent association. This differential association  and fewer symptoms of depression  after discharge than similarly stressed mothers who did not attend parent classes. However, for mothers who had low levels of stress, there was no apparent association  3032-Maternal Mental Health Conditions Education, Early Diagnosis, and Treatment Act , requireOur study had several limitations in design and implementation, including selection bias because of the non-random design, unmeasured participation of families in the usual FCC group and incomplete uptake of the mFICare intervention by all mothers in that group. Selection bias may have also affected recruitment. We note that 59% of eligible FCC participants enrolled and 71% of mFICare participants enrolled; nevertheless, there were few differences in characteristics of the two groups. Timing of the intervention may be important and affected by enrollment later in the admission. Other limitations include mFICare intervention curtailment by the COVID-19 pandemic, further reducing intervention dose for some participants, and the exclusion of non-English-speaking families, limiting generalizability. Strengths of the study include increased generalizability of the findings because of the racially and ethnically diverse participant families as well as populations served by the participating NICUs and use of mobile app technology to increase intervention access.In conclusion, the mFICare model was not associated with fewer mental health symptoms for mothers than the FCC model overall, but it was associated with fewer mental health symptoms for those experiencing higher levels of stress during the NICU stay. Further research on mFICare is urgently needed to address the prevalent and persistent mental health symptoms experienced by parents of preterm infants.


CRISPR/Cas-9 mediated genome editing has recently emerged as a potential and innovative technology in therapeutic development and biomedical research. Several recent studies have been performed to understand gene modification techniques in obtaining effectiveex vivo results. Generally, the disease targets for gene correction will be in specific organs, so understanding the complete potential of genomic treatment methods is essential. From such a perspective, the present review revealed the significant importanceof the CRISPR/ Cas9 delivery system. Both the promising gene-editing delivery systems, such as synthetic  and viral vector systems are discussed in this review. In addition, this paper attempted to summarize the tissue-specific and organ-specificmRNA delivery systems that provide possible research information for future researchers. Further, the major challenges of the CRISPR/Cas9 system, such as off-target delivery, immunogenicity, and limited packaging, were also elucidated. Accordingly, this reviewillustrated a wide range of clinical applications associated with the efficient delivery of CRISPR/ Cas9 gene-editing. Moreover, this article emphasizes the role of the CRISPR/Cas9 system in treating Intra Cerebral haemorrhage (ICH), thereby suggesting futureresearchers to adopt more clinical trials on this breakthrough delivery system. The pervasiveness of biomedical research constantly aims at developing an efficient treatment for genetic diseases. CRISPR, which is the natural component of the immune system of bacteria, stands for Clustered Regularly Interspaced Short Palindromic Repeats(CRISPR) , 2. It iThe mechanism of the CRISPR/Cas system is categorized as type I to VI, in which type I system utilizes cas3, which is a protein containing both helicase domain and DNase for degrading the targeted DNA. Similarly, the type II system uses Cas9, Cas 2 and Cas 1associated with the use of Csn2 or Cas4 to break the nucleotides . SubsequCr RNA (CRISPR RNA)Trace RNA(transactivation RNA)Cas9 protein The Cas9 possesses DNA-cleavage domains like HNH (His/Asn/His) and Ruv C (Recombination UV C) that degrades the ds DNA site situated three base pairs upstream of PAM sequences in the targeted DNA. Here Ruv C domain cleaves the opposite strands of ds DNA and theHNH domain cleaves the complementary crRNA strand. Due to this, the DNA is repaired in-vivo by homology-directed repair termed HDR or non-homologous end joining termed (NHEJ), which are considered error-prone . Here thIt performs a vital role in recognizing and matching the targeted DNA and comprises of a sequence for guiding Cas9 RNP to form R loop at the specific locus. Such R-loop formation activates RuvC and HNH in cleaving the non-targetedand the target DNA strands.It binds to Cas9 protein and crRNA to form a complex. Further, gRNA is developed as a chimeric molecule containing crRNA and tracrRNA preceded by 18 to 20 nucleotide spacer sequence that is complementary to the target located near to PAM. The PAM plays avital role in Cas9 mediated DNA degradation. CRISPR system follows the below specified main steps for gene editing Expression of Cas9 proteinGeneration of gRNA consisting 20 nucleotide (complement to gene of interest) Recognition of NGG PAM site situated adjacent to 3ʹ endWith the guidance of sgRNA, Cas9 explores the target in the genome and generates blunt end double stranded base-pairing at 3bp upstream of the PAM region.The delivery strategies of CRISPR components comprising g RNA and Cas nuclease into the target cells is through the following formulations .Here two plasmids are present, in which one encodes a protein and the other encodes a gRNA or a single plasmid that encodes both the components.Cas protein with mRNA and g RNA as an in-vitro transcribed component.gRNA-RNP complex and Cas Protein; Synthetic gRNA component and Cas Protein; The following table 1 provides the comparison of features between different delivery modesThe viral vectors remain to be promising system in in-vivo CRISPR/Cas9delivery. Because of their huge cellular uptake and editing effectiveness, the viral vectors are extensively used for gene editing. Recently the most widely used viral vectors arelentiviral vectors, adeno associated viruses, adenoviral vectors [The progression of synthetic vectors enrolled non-viral delivery systems with a transformative impact on the gene editing field to drive the treatment applications. They possess a flexible packaging capability and hence it could accommodate huge nucleicacids and protein cargos like Cas9 mRNA, sg RNA, donor DNA and RNP . By combApart from viral delivery, there exists a considerable interest in the development of non-viral vectors that could perform gene editing in the intracranial region of the brain.Intra-cerebral haemorrhage (ICH) is considered a crucial neurological disease associated with high mortality and morbidity of the entire stroke subtypes. The risk factors associated with ICH comprise hypertension, alcohol use, usage of oral anticoagulants andcigarette smoking. There exist no proven efficient surgical or medical treatments available for ICH. However, cerebellar haemorrhage decompression has been widely accepted as a potential lifesaver. More experimental evidence depicted that the blood-brainbarrier (BBB) is one of the key aspects of brain injury and ICH. Raised level of BBB permeability causes vascular-derived oedema after ICH. Treatment methods against BBB disruption attenuate secondary brain damage after ICH. Thus the existing treatmentstrategies against BBB disruption would help attenuate secondary brain damage after ICH. The role of Frizzled 7 signal to preserve BBB post-ICH in the CD 1 mouse model was investigated by . ActivatThe defects in multiple-cell signal pathways cause vascular integrity disruptions that necessitate the optimized regulation of cell adhesive complexes. Such defects were associated with the progression of ICH. Genetic evidence for ICH is observed in humans andalso the signaling of adaptor proteins plays a vital process in cell signaling pathways. The study  is invesThe presented review also observed that C-C chemokine receptor type 1 (CCR1) and CCL 5, its associated ligand which were responsible for the progression of ICH. These compounds actively participate in the pathogenicity of neuro inflammatory diseases. TheCCL/CCR1 signaling in the permeability of blood brain barrier after ICH remains completely unrevealed. Motivated by the above description , 250 CD1Similarly, integration of CRISPR/CAS 9 with the desired single-cell electroporation method for gene editing in the single neurons in-vivo was performed by . This st First, the prevailing research has not explored the process of underlying endogenous marker expression after ICH briefly . After the induction of ICH, all kinds of neuro-protective systems comprising neurons, pericytes, and astrocytes contributing to the injury were not completely elucidated. The impacts of the signalling process on the hematoma resolution and angiogenesis after inducing ICH were also not fully investigated. The general studies did not provide adequate information about the leakage of substances with smaller molecular weight, and hence the extent of leakage could not be determined successfully.Due to lack of carriers for systematic delivering of RNP, several therapeutic targets could not be accessed. They reported generalized methods for engineering the altered lipid nanoparticles to efficiently deliver RNPs to the cells. Following this process,tissue editing comprising brain, muscle, lungs and liver has been accomplished. The intravenous injection enables tissue specific mRNA delivery into the lungs of mouse. A high carrier potential has been leveraged for developing organ specific cancer mo thelungs and livers of mice. The authors  also repDespite the effectiveness of CRISPR/Cas 9, this section describes the potential limitations of the investigated system.There exists an inherent danger, such that it is developed to cleave the targeted genomic site, but it would cleave the other sites and leading to off-target mutations . General Despite the occurrence of off target mutations by CRISPR/Cas9 system, it is found to be very rare, even single cell mutations among the potentially targeted billion of cells leads to more feared complications. Recently there is no strong evidence to prove the safety of the clinical applications of CRISPR/Cas 9. To provide such information, various unbiased methods for gene scanning for billions to millions of cells were adopted, but too went in vain due toconstrained cultured cells or test tubes in vitro . The researchers determined that the gene editing with Cas 9 protein could be proved effectively at in-vivo conditions,since questions arise regarding the long term safety in human tissues. So optimization in terms ofclinical trials seems to be complex .Meanwhile the first CRISPR/Cas9 therapy were performed in sick patients who are supposed to face multiple risks.Although if CRISPR Cas 9 could create a DNA break at a high efficiency site by introducing a particular alteration, efficiency at HDR site is found to be minimum. Further for proliferating active HDR cells, it is complex to ensure that cells utilize HDR to repair the DNA breaks . In addition, since NHEJ is observed to be active in all the cell, large number of cells with NHEJ mediated disruption of target gene were found .This cause high problem during the correction of a gene that causes disease.There are clinical trials in using CRISPR/cas9 targeting CD19 to remove TRAC and HLA-I to cure Non-Hodgkin lymphoma and acute lymphocytic leukemia , 27,28,CRISPR/Cas9 can reduce the resilience of tumours against these molecular targeted drugs or inhibitors by editing genes related to EGFR, ALK, and KRAS, which would support lung cancer patients. The CRISPR/Cas9 technology evaluates the gene targeted by drugsused in chemotherapy and identifies new pathways to reduce or eliminate resistance to chemotherapy in lung cancer. Also, the CRISPR/Cas9 technology used to edit proto-oncogenes or tumour-suppressor genes provides a great opportunity in lung cancer treatment. CRISPR/Inducible CRISPR Cas System has also to be tailored and understood deeply for adopting extensive application in clinical scenario. This paper suggested that RNA editing and epigenetic regulation is much required apart from prevailing gene editing Thepresent review offered new in-depth insights about the viral and non-viral based CRISPR based systems to promote safe and precise genome editing techniques. Since the number of pathological and physiological signals like pH, redox and ATP could beleveragedfor developing various types of gene editing tools, the presented article will functions as a new gateway to such future researchers. The overall challenges provided in this paper will alarm the researchers thereby helps to avoid pre-existing mistakes.


In the past decade, extracellular vesicles (EVs) have emerged as key regulators of bone development, homeostasis and repair. EV-based therapies have the potential to circumnavigate key issues hindering the translation of cell-based therapies including functional tissue engraftment, uncontrolled differentiation and immunogenicity issues. Due to EVs’ innate biocompatibility, low immunogenicity, and high physiochemical stability, these naturally-derived nanoparticles have garnered growing interest as potential acellular nanoscale therapeutics for a variety of diseases. Our increasing knowledge of the roles these cell-derived nanoparticles play, has made them an exciting focus in the development of novel pro-regenerative therapies for bone repair. Although these nano-sized vesicles have shown promise, their clinical translation is hindered due to several challenges in the EV supply chain, ultimately impacting therapeutic efficacy and yield. From the biochemical and biophysical stimulation of parental cells to the transition to scalable manufacture or maximising vesicles therapeutic response in vivo, a multitude of techniques have been employed to improve the clinical efficacy of EVs. This review explores state of the art bioengineering strategies to promote the therapeutic utility of vesicles beyond their native capacity, thus maximising the clinical potential of these pro-regenerative nanoscale therapeutics for bone repair. There is an urgent clinical need for effective therapeutic strategies to treat damaged bone caused by traumatic injury, tumour resection or osteoporosis (OP) , 2. ApprThere has been an increasing body of evidence demonstrating the trophic effects of cell-secreted bioactive products in regulating cellular communication , 10. Of EVs are defined as nanoscale lipid particles that contain a diverse biological cargo of nucleic acids, proteins and bioactive molecules –15. ThesWithin the bone context, several studies have reported the intrinsic role of EVs in regulating bone homeostasis by mediating intercellular communication , 27. MorDespite the growing promise of vesicles as acellular tools to stimulate bone repair, their clinical adoption has been hindered due to issues including the selection of an appropriate cell source, EVs inherent therapeutic efficacy, scalable manufacture and their clinical administration . In thisThe bone microenvironment consists of a diverse subset of cell types including mesenchymal stem/stromal cells (MSCs), osteoblast, osteoclast, osteocytes, macrophages, endothelial cells (ECs), and many more within the marrow, which synergistically regulate bone homeostasis , 36. DueMSCs are an attractive cell population used in bone augmentation due to their ease of procurement from numerous tissues and their ability to differentiate into multiple lineages , 38. WitVesicles derived from MSCs have also been reported to regulate angiogenesis, a key process involved in bone fracture healing. For instance, Lu et al. demonstrated the role of MSC-EVs in promoting both angiogenesis and osteogenesis in vitro and in vivo . EVs isoMSCs-EVs also exhibit similar immunoregulatory properties to their parental cells during different stages of osteogenesis. For example, Nakao et al. reported that treating human gingiva-derived MSCs with TNF-α, resulted in the production of EVs that enhanced the M2 polarisation of macrophages and inhibited periodontal bone loss . Wei et Extensive research has indicated MSCs derived from different tissue origins exhibit differential capacity to promote bone regeneration , 55. As Osteoblasts are derived from bone marrow MSCs and are responsible for the synthesis and mineralisation of the bone matrix Fig.  62, 63], 6362, 6Interestingly, Uenaka et al. recently reported vesicles derived from mature osteoblasts inhibited bone formation and enhanced osteoclastogenesis in vivo . EVs werAdditionally, there is evidence indicating the role of osteoblast-derived EVs in regulating angiogenesis. Tang et al. reported that EVs derived from 21 day osteogenically cultured preosteoblast promoted EC angiogenesis via EV associated MMP activation of the VEGF/Erk1/2 signalling pathway . CollectOsteoclasts are multinucleated cells generated from hematopoietic precursors including blood/bone marrow monocytes and macrophages Fig.  71]. Th. Th71]. Osteoclast-derived EVs have been reported to promote osteoblast differentiation. For example, Chen et al. demonstrated that osteoclast EVs were able to promote the osteogenic differentiation of the mouse preosteoblast KusaO cell line . ContrasHuynh et al. reported that EVs secreted from precursor cells promoted osteoclastogenesis, whilst mature osteoclast-EVs inhibited osteoclast differentiation . Mature Osteocytes are terminally differentiated cells that are embedded within the mineralised matrix of bone Fig.  77]. Th. Th77]. Angiogenesis is an essential process involved in normal fracture healing, with defective blood vessel development associated with poor outcomes , 82. EC-Jia et al. showed during the process of distraction osteogenesis, EVs secreted from ECs were capable of accelerating bone regeneration in rats. Specifically they found that the miR-126 enriched EVs stimulated angiogenesis by targeting the Raf/ERK signalling pathway . MoreoveMacrophages are critical components of the immune response and have been shown to contribute to the regulation of MSC and osteoblast function during bone repair Fig.  85–87].–87.85–87Studies have showed that macrophages are able to stimulate MSCs osteogenic differentiation through EV signalling . Kang etTaken together, these studies emphasise the diverse roles of EVs within the bone microenvironment in regulating skeletal tissue homeostasis and repair Table .As cells are highly sensitive to the environment they reside in, researchers have harnessed biochemical and biophysical cues to augment the cells phenotype and ultimately the therapeutic efficacy of the EVs they secreted for bone regeneration.Whilst there has been growing evidence reporting the regenerative capacity of EVs derived from numerous cell sources, the inherent therapeutic potency of these vesicles is still a factor hindering clinical translation . As EVs Increasing studies have shown the influence of cell culture parameters in modulating the phenotype of parental cells, ultimately augmenting the therapeutic potency of their EVs. For example, Davies et al. reported the impact of culture media composition on modulating the efficacy of secreted EVs . The autOverall, these studies indicate the importance of refining the EV parental cell culture parameters in order to procure vesicles with optimum pro-regenerative capacity Fig. A.Manipulation of a cell’s genetic makeup is one of the most well-established approaches to augment cell function. Thus, it is not surprising that genetic engineering has been increasingly adopted as a strategy to improve EVs utility for bone repair  Fig. 2)2). SeverBesides the loading of nucleic acids, the overexpression of pro-regenerative proteins has also been conducted –105. ForAlthough there is growing evidence demonstrating the promise of engineering EVs through genetic modifications, concerns of transduction efficiency remain. The intensive cost and time associated with this approach may also hinders its clinical utility . MoreoveIn recent years, epigenetic regulation has garnered increasing attention as a critical process in controlling cell fate , 108. EpDue to the growing influence of epigenetic regulation on cell fate, an increasing number of studies have explored this approach to modulate EV therapeutic efficacy , 118. ReDuring the process of bone healing, hypoxia is a critical environmental stimulus regulating tissue regeneration. In vivo oxygen concentrations range from 2 to 8%, whilst in vitro, cells are exposed to concentrations of 21% . Several2) or hypoxic conditions (1% O2) [For instance, Liu et al. investigated the impact of hypoxia on the therapeutic efficacy of MSC-EVs on bone fracture healing. MSCs-derived from human umbilical cord tissue were subjected to either normoxic (21% O (1% O2) . The fin (1% O2) . In thisTo date, EVs are commonly procured from cells cultured on 2D planar tissue culture plastic, which does not replicate the complex physiological environment within the body , 126. HeTitanium alloys have been widely used in numerous orthopaedic applications due to their biocompatibility, mechanical properties and osteoinductivity , 132. DuWithin the regenerative medicine field, growing research has investigated harnessing 3D culture platforms to more closely replicate the cells native environment. Several studies have shown that cells in 3D culture exhibit enhanced osteogenic capacity when compared to cells on 2D surfaces –140. Of Although the use of hydrogel systems for the production of pro-osteogenic EVs have shown promise, the sourcing of naturally-derived biomaterials could suffer from batch and lot inconsistencies, which will hinder the reproducible manufacture of EVs from these 3D systems , 144. MoMechanical stimulation has garnered increased attention as an approach to improve the osteogenic capacity of cells for bone augmentation strategies , 150. Re3O4 nanoparticle composite hydrogel and subjected the construct to 20% magnetic-induced strain [Interestingly, studies have investigated the impact of mechanical stimulation on the secretion of pro-regenerative EVs when cultured in a 3D microenvironment. For instance, Yu et al. cultured hPDLSCs within a collagen/Fed strain . The autDespite the promising osteoinductive capacity of EVs for bone repair, it is expected that large doses of vesicles are required to achieve regenerative effects clinically for both systemic and local EV administration . This neThere has been extensive research harnessing vesicles derived from primary cells. However, issues with cell sourcing, limited window of expansion, heterogeneity, donor and lot variations will hinder the production of therapeutically effective EVs at clinically relevant quantities , 155. ThAnother approach to generating EV producer cells is the use of induced pluripotent stem cells (iPSCs). These cells were first generated in 2006 by the transduction of four transcription factors, Oct3/4, Sox2, c-Myc, and Klf4 into adult fibroblast cells . These iAnother key factor affecting the production of therapeutically-relevant EVs is the use of clinical-grade medium Fig. . CurrentThe acquisition of pro-regenerative EVs at large scales has the potential to transform the treatment of debilitating bone-associated disorders such as OP. The systemic administration of vesicles for OP has shown promise , 166, hoBioreactor platforms have been commonly utilised for the large-scale production of clinical-grade cells for therapeutic applications , 168. AsMicrocarriers have been extensively employed for the manufacture of clinical-grade cells in conjugation with bioreactors systems , 175. DuThus, it is critical to assess the impact of the entire EV supply chain on the scalable and reproducible production of clinically effective vesicles for bone augmentation strategies.The demonstration of EVs pro-osteogenic capacity in vitro has led researchers to investigate their potential use for the treatment of systematic skeletal diseases such as OP. Although EV administration via intravenous injection for the treatment of OP has shown promise , the nanOne approach exploits the affinity of anti-resorptive drugs to bind to the mineral phase of bone tissue Fig. . For insIn recent years, peptides have attracted increasing attention as targeting ligands due to their low immunogenicity, low cost and high specificity . Cui et Several studies have investigated the use of aptamers, single-stranded RNA or DNA that are capable of folding into 3D structures and exhibit high affinity to specific targets , 190. AsAlthough, there have been increasing investigations using vesicles for the repair of systematic skeletal diseases such as OP , 192, thFor the repair of critical-sized bone defects, it is expected that a large quantity of EVs will be required to facilitate effective tissue regeneration. Due to issues with the administration of EVs via systemic routes and the manufacture of vesicles at large quantities, researchers have investigated locally delivering these vesicles directly into the bone defect in conjunction with an appropriate biomaterial –196. ThiAs EVs exhibit a plethora of different physiochemical properties , 198, thThe porosity of a biomaterial influences several factors such as cellular infiltration, proliferation, differentiation and nutrient/waste transport , 200. AdAlthough this approach has shown promise, biomaterials with rapid degradation rates may have a detrimental impact on the stabilisation of the defect, a critical aspect for the repair of load-bearing tissues , 205. MoAnother approach to modulate the release of EVs from biomaterials is electrostatic interactions. Studies have reported that vesicles exhibit an overall negative surface charge, thus harnessing biomaterials that possess positively charged groups provides an approach for EV immobilisation . For exaThe synthetic nanosilicate Laponite (LAP) has been reported to exhibit a broad affinity to bioactive molecules due to their positive rim charge and negative surface charge , 211. MoEVs have been reported to bind to native extracellular components found within the bone matrix, thus providing a biomimetic platform to facilitate the local delivery of pro-regenerative vesicles for bone regeneration Fig.  146, 21. For exaIn a similar approach, researchers have developed an injectable ECM-mimetic hydrogel utilising chitosan and collagen to facilitate EV controlled release for bone repair . ChitosaThere is a growing body of evidence highlighting EVs as a promising acellular but biological approach for the treatment of bone disorders. Although these nanoparticles have shown their potential, from reviewing the current state of the literature, it is clear there are several challenges hindering the clinical translation of vesicles for bone repair Fig. .To facilitate the translation of pro-regenerative vesicles to the clinic, it is essential to elucidate EV composition to determine the “active drug substance(s)” involved in mediating the vesicles’ biological activity. Although increasing efforts in recent years have employed multi-omic approaches to identify key bioactive factors , the majority of research has procured EVs from cells cultured on 2D tissue culture plastic, a highly artificial environment , 220. ReAs EVs have been shown to consist of multiple different biological constituents, they may be able to effect bone regeneration through several pathways, overcoming one of the limitations of current clinically available growth factors . FurtherTo date, there is no consensus on a standardised approach to manufacture pro-regenerative EVs at scale. As it is expected that a large quantity of EVs will be required for the treatment of bone disorders, the use of bioreactor systems for EV manufacture is a logical step toward translation. As the scalable manufacture employing bioreactor systems is cost intensive, it is critical to use GMP-compatible materials and processes early in research and development to facilitate the translation of EV-based products. Moreover, it is key to confirm whether the transition to scalable manufacture adversely impacts the therapeutic potency of EVs when produced at large quantities. This transition to large scale production will ultimately be beneficial to the sustainable manufacture of these pro-regenerative vesicles. An in-depth overview of critical challenges facing the broader EV field for the large scale production of EVs can be found in the “massivEVs” ISEV report . In addiAs highlighted in our review, there has been extensive research harnessing novel bioengineering strategies to promote the therapeutic efficacy of EVs for the treatment of bone disorders. Although their promise has been demonstrated, it is critical to ensure the robustness, reproducibility and scalability of these approaches in order to facilitate the translation of these smart nanoscale therapeutics to the clinic.In addition to the scalable manufacture of these nanoparticles, there is currently a lack of standardised quality assured functional assays to ensure appropriate batch and lot consistency. It is important to develop GMP appropriate functional in vitro assays that offer enhanced reproducibility, high-quality quantification and avoids confounding biological processes. The Minimal Information for Studies of Extracellular Vesicles from The International Society for Extracellular Vesicles provides guidelines for EV manufacture , howeverThere has been increasing research investigating the local delivery of EVs within biomaterials systems, to enhance their bioavailability and maximise their therapeutic response. Although promising results have been observed, it is crucial to determine the impact of the biomaterial on facilitating EV-induced bone formation following administration. Moreover, optimisation of the vesicles dosing regimen in vivo is critical to maximise the nanoparticles therapeutic response, in addition to informing appropriate manufacturing batch requirements for certain clinical inclinations.Finally, the standardisation of storage methods is a critical step towards the translation of EVs . Thus, iWhile great developments have been made in elucidating the role of EVs in regulating bone remodelling, there are still several unmet challenges hindering the translation of clinically-viable vesicles for bone regeneration. This has propelled intensive investigations into novel bioengineering approaches to improve EVs’ therapeutic efficacy for bone repair. In this review, we highlight strategies harnessing biochemical/biophysical stimulation, GMP-compatible scalable production and maximising in vivo efficacy. We trust that continued progress in these areas will streamline the transition of promising bench-side results to clinically-viable smart nanoscale therapies for the effective treatment of bone disorders.


Herpes zoster is characterized by the presence of unilateral, localized vesicular rash in a dermatomal distribution. Although in most cases herpes zoster affects a single dermatome, multidermatomal involvement may occur in rare cases.In this case report, we describe an immunocompetent patient with multidermatomal herpes zoster involving the cranial nerve (CN) V3 and C2 territories with simultaneous vestibulocochlear deficits. We demonstrate the results of temporal bone magnetic resonance imaging and discuss the pathophysiological mechanism of the patient’s characteristic clinical manifestation.The diagnosis of multidermatomal herpes zoster involving CN V3 and C2 territories with simultaneous vestibulocochlear deficit was made. Vestibular function test and pure tone audiometry revealed ipsilateral vestibular impairment and hearing loss.The patient was admitted for treatment with systemic steroids and antiviral agents.After treatment, vertigo and hearing loss improved, and the skin lesions healed well without scarrings.Considering that the afferent sensory fibers of the CN V and C2 converge in the trigeminocervical nucleus, herpes zoster involving contiguous dermatomes of CN V3 and C2 in the patient may be explained by neural-to-neural spread. And considering that temporal bone magnetic resonance imaging revealed enhancement of the dura of the internal auditory canal without enhancement of CN VII or VIII, the vestibulocochlear deficits in the patient may be caused by inflammation of the inner ear transmitted via the cerebrospinal fluid. Herpesviridae. VZV infection is species-specific to humans. Herpes zoster infection is caused by reactivation of VZV in the cell bodies of neurons. Initial infection with VZV is called varicella or chickenpox and usually occurs in childhood; after the initial infection, a latent infection is established in the cranial nerve ganglia or sensory dorsal root. Under some conditions, the dormant VZV reactivates and migrates along the nerve axons to the skin, causing vesicular eruptions that show a localized dermatomal distribution. Herpes zoster generally manifests as painful vesicular eruptions along the unilateral distribution of the dermatome, most commonly affecting a single thoracic truncal dermatome. Less commonly, involvement of the cervical dermatome or cranial nerves (CNs), especially CN V and CN VII, has been reported.–3 Herpes zoster oticus (HZO) refers to VZV reactivation within CN VII, and is characterized by an erythematous vesicular rash in the external ear and otalgia. When accompanied by ipsilesional facial weakness, a diagnosis of Ramsay Hunt syndrome can be made. In HZO, vestibulocochlear symptoms such as hearing loss, tinnitus, and vertigo are often present, and the pathological mechanism of vestibulocochlear deficit has been suggested to be directional spread of VZV to CN VIII from CN VII or CN VIII via the cerebrospinal fluid.–8Varicella zoster virus (VZV), also known as human herpesvirus 3, is a double-stranded DNA virus belonging to the family Here, we present a 35-year-old woman with herpes zoster in multiple adjacent dermatomes involving CN V3 and C2, concomitant with peripheral-type vertigo and hearing loss on the ipsilateral side.http://links.lww.com/MD-CASES/A23). The direction of nystagmus did not change when the head position was changed. No abnormal findings were observed on neurological examinations such as the cerebellar function test and lower cranial nerve examination. Facial weakness was not observed on either side. A bithermal caloric test showed right unilateral weakness with a canal paresis value of 78% , and written informed consent was obtained from the patient for publication of this case report details.This case presentation raises 2 interesting issues that are worth studying. First, herpes zoster involving multiple adjacent dermatomes is very uncommon in young, healthy, immunocompetent patients. Second, except in cases of Ramsay Hunt syndrome, the concomitant vestibulocochlear deficit on the ipsilateral side without evidence of CN VII involvement is also very unusual in individuals with herpes zoster. In some patients, herpes zoster extends beyond a single dermatome, and this multidermatomal involvement often occurs in the presence of underlying immunocompromised conditions such as human immunodeficiency virus infection, hematological malignancy and chemotherapy. Multidermatomal herpes zoster involving the trigeminal nerve,–12 or cervical or thoracic dermatomes has rarely been reported in immunocompetent patients. The exact mechanism underlying multidermatomal herpes zoster has not been clearly elucidated. Yoon et al reported a case of left Ramsay Hunt syndrome with extensive dissemination of vesicular eruptions to the bilateral scalp, posterior neck, shoulder, upper arm, and posterior upper neck in an immunocompetent elderly patient. The authors assumed that the widespread dissemination of herpes zoster infection may occur through interconnections between ganglia or due to viremia of VZV. Gomez and Chernev reported a 95-year-old patient with disseminated multidermatomal herpes zoster involving the left CN VI-III, the bilateral trunk and bilateral extremities and presented a review of 28 immunocompetent patients with disseminated herpes zoster previously reported in the literature. The results showed that the initial presentation of vesicular rash most frequently occurred in dermatomes innervated by the cranial nerves (42%), followed by the thoracic (27%), lumbosacral (19%), and cervical (15%) dermatomes. Extracutaneous manifestations were reported in twelve cases (43%); among theses, mucosal lesions, central nervous system lesions, cranial or peripheral nerve paresis, Ramsay Hunt syndrome, herpes ophthalmicus, and visceral involvement were observed in 4, 2, 2, 2, 1, and 1 cases, respectively. The authors speculated that widespread dissemination of herpes zoster may be caused by VZV viremia, and this was supported by the previous finding that VZV DNA can be amplified from the serum of immunocompetent patients with localized herpes zoster. Our patient presented with characteristic skin findings of herpes zoster involving contiguous dermatomes of CN V3 and C2. It has been reported that the afferent sensory fibers of the CN V and C2 converge in the trigeminocervical nucleus, and VZV spread between these nerves may occur at this point. Another plausible explanation would be spreading of the virus from skin to skin or from peripheral nerve to peripheral nerve, considering that the dermatomal territories show abundant overlap in head and neck region.Herpes zoster generally develops unilaterally in a single dermatome. The thoracic dermatomes are most commonly affected, followed by the cervical and trigeminal dermatomes.,19,20 we suspected herpes zoster oticus as a cause of the vestibulocochlear deficits in this patient. In our previous studies, vestibulocochlear deficits were observed in more than half of patients with Ramsay Hunt syndrome, and postcontrast temporal bone MRI in these patients exhibited enhancement of inner ear structures and of the dura of the internal auditory canal as well as of the facial nerve and the external auditory canal.–8,21,22 However, our patient did not show facial weakness or enhancement of the facial nerve or the external auditory canal, suggesting that the vestibulocochlear deficits were not accompanied by Ramsay Hunt syndrome or herpes zoster oticus. Concomitant vestibulocochlear deficits and ipsilateral multidermatomal herpes zoster involving the CN V3 and C2 territories in the absence of Ramsay Hunt syndrome has not previously been reported in the literature, and the mechanism of this clinical manifestation is not clear. Although anastomosis between the auriculotemporal branch of CN V3 and the temporofacial division of CN VII has been demonstrated, there is no anastomosis between CN V3 and CN VIII. Thus, in our patient, the possibility of VZV spread via neural anastomoses or interganglionic transmission may be low. When VZV infection extends to the cerebrospinal fluid, central nervous system infections such as encephalitis, encephalomyelitis or meningitis have been reported.–25 Eskiizmir et al described an immunocompetent 19-year-old man with Ramsay Hunt syndrome and simultaneous VZV encephalitis. Previously healthy patients with disseminated cutaneous zoster and aseptic meningitis have also been reported.,25 Considering that no evidence of meningitis was found in our patient and that temporal bone MRI revealed enhancement of the dura of the internal auditory canal without enhancement of CN VII or VIII, the vestibulocochlear deficits in our patient may be caused by inflammation of the inner ear transmitted via the cerebrospinal fluid.Another noteworthy manifestation in our patient was comorbid hearing loss and vertigo due to ipsilateral vestibulocochlear deficit. Because Ramsay Hunt syndrome can be associated with herpes zoster involving CN V or C2, as reported in previous studies,To our knowledge, this is the first case report of multidermatomal herpes zoster involving the CN V3 and C2 territories with simultaneous vestibulocochlear deficits. The mechanism of herpes zoster involving contiguous dermatomes of CN V3 and C2 may be neural-to-neural spread, and that of vestibulocochlear deficit may be viral inflammation of the inner ear transmitted via cerebrospinal fluid.Conceptualization: Chang-Hee Kim.Data curation: Taesik Jung, ChanEui Hong, Jung Eun Shin, Chang-Hee Kim.Formal analysis: Taesik Jung, Jung Eun Shin, Chang-Hee Kim.Funding acquisition: Chang-Hee Kim.Investigation: Taesik Jung, ChanEui Hong, Jung Eun Shin, Chang-Hee Kim.Methodology: Taesik Jung, ChanEui Hong, Chang-Hee Kim.Supervision: Chang-Hee Kim.Visualization: Jung Eun Shin.Writing – original draft: Taesik Jung, Chang-Hee Kim.Writing – review & editing: Chang-Hee Kim.