Follicular helper- and peripheral helper-like T cells drive autoimmune disease in human immune system mice.

Human immune system (HIS) mice constructed in various ways are widely used for investigations of human immune responses to pathogens, transplants and immunotherapies. In HIS mice that generate T cells de novo from hematopoietic progenitors, T cell-dependent multisystem autoimmune disease occurs, most rapidly when the human T cells develop in the native NOD.Cg- Prkdc scid Il2rg tm1Wjl (NSG) mouse thymus, where negative selection is abnormal. Disease develops very late when human T cells develop in human fetal thymus grafts, where robust negative selection is observed. We demonstrate here that PD-1 + CD4 + peripheral (Tph) helper-like and follicular (Tfh) helper-like T cells developing in HIS mice can induce autoimmune disease. Tfh-like cells were more prominent in HIS mice with a mouse thymus, in which the highest levels of IgG were detected in plasma, compared to those with a human thymus. While circulating IgG and IgM antibodies were autoreactive to multiple mouse antigens, in vivo depletion of B cells and antibodies did not delay the development of autoimmune disease. Conversely, adoptive transfer of enriched Tfh- or Tph-like cells induced disease and autoimmunity-associated B cell phenotypes in recipient mice containing autologous human APCs without T cells. T cells from mice with a human thymus expanded and induced disease more rapidly than those originating in a murine thymus, implicating HLA-restricted T cell-APC interactions in this process. Since Tfh, Tph, autoantibodies and LIP have all been implicated in various forms of human autoimmune disease, the observations here provide a platform for the further dissection of human autoimmune disease mechanisms and therapies.

The impact of enhanced recovery after surgery on inflammatory indicators and prognosis related to complex appendicitis in children.

Objective: To explore the application effect of enhanced recovery after surgery (ERAS) perioperative plan in the treatment of complex appendicitis in children, and further enrich the implementation plan of ERAS in the field of pediatric surgery. Method: This study selected 122 children who underwent laparoscopic complex appendectomy at Inner Mongolia Maternal and Child Health Hospital and Baotou Fourth Hospital from August 2018 to July 2022, and randomly divided them into a traditional surgery group (TS) and an enhanced recovery surgery group (ERAS). The changes of white blood cell (WBC), hypersensitive C-reactive protein (CRP), pro Calcitonin (PCT) and interleukin 6 (IL-6) before and after surgery were compared. The degree of pain, recovery time of intestinal function, length of hospital stay, hospital costs, postoperative complications and parental satisfaction were compared between the two groups. Result: The WBC and CRP levels in the ERAS group at 6 h after surgery, as well as the IL-6 levels on the 3rd day after surgery, were lower than those in the TS group. Meanwhile, the analgesic effect of ERAS group at 3 h and 6 h after surgery was better than that of TS group. And the ERAS group had a shorter postoperative first exhaust time, fewer overall hospital stays, and lower hospitalization costs. In addition, the ERAS group had high parental satisfaction during hospitalization. There was no statistically significant difference in postoperative complications between the two groups of children. Conclusion: ERAS can promote postoperative recovery of children, reduce surgical stress, save family medical expenses, alleviate the pain of children, and improve parental satisfaction. It is a safe and effective method for treating complex appendicitis in children.

Origin, phenotype and autoimmune potential of T cells in human immune system mice receiving neonatal human thymus tissue.

Robust human immune system (HIS) mice are created using human fetal thymus tissue and hematopoietic stem cells (HSCs). A HIS mouse model using neonatal human thymus tissue and umbilical cord blood (CB) HSCs (NeoHu) was recently described. We improved the model by removing the native murine thymus, which can also generate human T cells, and demonstrated definitively the capacity of human T cells to develop in a grafted neonatal human thymus. Human T cells derived from the neonatal thymus tissue appeared in peripheral blood early post-transplantation and CB-derived T cells appeared later. Naïve T cells were demonstrated in peripheral blood but effector memory and T peripheral helper phenotypes predominated later, in association with development of autoimmunity in some animals. Treatment of thymus grafts with 2-deoxyglucose (2-DG) increased the proportion of stem cells derived from injected HSCs, delayed onset of autoimmune disease, reduced early T cell reconstitution, and reduced effector/memory T cell conversion. Younger neonatal human thymus tissue was associated with improved T cell reconstitution. While the NeoHu model bypasses the need for fetal tissue, it has yet to demonstrate equivalent reconstitution to fetal tissue, though 2-DG can improve results by removing native thymocytes prior to transplantation.

Extraction of tree crown parameters of high-density pure Cunninghamia lanceolata plantations by combining the U-Net model and watershed algorithm.

As one of the important timber species in China, Cunninghamia lanceolata is widely distributed in southern China. The information of tree individuals and crown plays an important role in accurately monitoring forest resources. Therefore, it is particularly significant to accurately grasp such information of individual C. lanceolata tree. For high-canopy closed forest stands, the key to correctly extract such information is whether the crowns of mutual occlusion and adhesion can be accurately segmented. Taking the Fujian Jiangle State-owned Forest Farm as the research area and using the UAV image as the data source, we developed a method to extract crown information of individual tree based on deep learning method and watershed algorithm. Firstly, the deep learning neural network model U-Net was used to segment the coverage area of the canopy of C. lanceolata, and then the traditional image segmentation algorithm was used to segment the individual tree to obtain the number and crown information of individual tree. Under the condition of maintaining the same training set, validation set and test set, the extraction results of the canopy coverage area by the U-Net model and traditional machine learning methods [random forest (RF) and support vector machine (SVM)] were compared. Then, two individual tree segmentation results were compared, one using the marker-controlled watershed algorithm, and the other using the combination of the U-Net model and marker-controlled watershed algorithm. The results showed that the segmentation accuracy (SA), precision, IoU (intersection over union) and F1-score (harmonic mean of precision and recall) of the U-Net model were higher than those of RF and SVM. Compared with RF, the value of those four indicators increased by 4.6%, 14.9%, 7.6% and 0.05, respectively. Compared with SVM, the four indicators increased by 3.3%, 8.5%, 8.1% and 0.05, respectively. In terms of extracting the number of trees, the overall accuracy (OA) of the U-Net model combined with the marker-controlled watershed algorithm was 3.7% higher than that of the marker-controlled watershed algorithm, with the mean absolute error (MAE) being decreased by 3.1%. In terms of extracting crown area and crown width of individual tree, R2 increased by 0.11 and 0.09, mean squared error decreased by 8.49 m2 and 4.27 m, and MAE decreased by 2.93 m2 and 1.72 m, respectively. The combination of deep learning U-Net model and watershed algorithm could overcome the challenges in accurately extracting the number of trees and the crown information of individual tree of high-density pure C. lanceolata plantations. It was an efficient and low-cost method of extracting tree crown parameters, which could provide a basis for developing intelligent forest resource monitoring.

Cholesterol paradox in the community-living old adults: is higher better?

OBJECTIVE: To evaluate the associations of lipid indicators and mortality in Beijing Elderly Comprehensive Health Cohort Study. METHODS: A prospective cohort was conducted based on Beijing Elderly Comprehensive Health Cohort Study with 4499 community older adults. After the baseline survey, the last follow-up was March 31, 2021 with an average 8.13 years of follow-up. Cox proportional hazard model was used to estimate the hazard ratios (HR) with 95% CI for cardiovascular disease (CVD) death and all-cause death in associations with baseline lipid indicators. RESULTS: A total of 4499 participants were recruited, and the mean levels of uric acid, body mass index, systolic blood pressure, diastolic blood pressure, fasting plasma glucose, total cholesterol (TC), triglyceride, and low-density lipoprotein cholesterol (LDL-C) showed an upward trend with the increasing remnant cholesterol (RC) quarters (Ptrend < 0.05), while the downward trend was found in high-density lipoprotein cholesterol (HDL-C). During the total 36,596 person-years follow-up, the CVD mortality and all-cause mortality during an average 8.13 years of follow-up was 3.87% (95% CI: 3.30%-4.43%) and 14.83% (95% CI: 13.79%-15.86%) with 174 CVD death participants and 667 all-cause death participants. After adjusting for confounders, the higher level of TC (HR = 0.854, 95% CI: 0.730-0.997), LDL-C (HR = 0.817, 95% CI: 0.680-0.982) and HDL-C (HR = 0.443, 95% CI: 0.271-0.724) were associated with lower risk of CVD death, and the higher level of HDL-C (HR = 0.637, 95% CI: 0.501-0.810) were associated with lower risk of all-cause death. The higher level of RC (HR = 1.276, 95% CI: 1.010-1.613) increase the risk of CVD death. Compared with the normal lipid group, TC ≥ 6.20 mmol/L group and LDL-C ≥ 4.10 mmol/L group were no longer associated with lower risk of CVD death, while RC ≥ 0.80 mmol/L group was still associated with higher risk of CVD death. In normal lipid group, the higher levels of TC, LDL-C and HDL-C were related with lower CVD death. CONCLUSIONS: In community older adults, higher levels of TC and HDL-C were associated with lower CVD mortality in normal lipid reference range. Higher RC was associated with higher CVD mortality, which may be a better lipid indicator for estimating the CVD death risk in older adults.

T1D patient-derived hematopoietic stem cells are programmed to generate Tph, Tfh, and autoimmunity-associated B cell subsets in human immune system mice.

Interactions between B cells and CD4+ T cells play a central role in the development of Type 1 Diabetes (T1D). Two helper cell subsets, follicular (Tfh) and peripheral (Tph) helper T cells, are increased in patients with T1D but their role in driving B cell autoimmunity is undefined. We used a personalized immune (PI) mouse model to generate human immune systems de novo from hematopoietic stem cells (HSCs) of patients with T1D or from healthy controls (HCs). Both groups developed Tfh and Tph-like cells, and those with T1D-derived immune systems demonstrated increased numbers of Tph-like and Tfh cells compared to HC-derived PI mice. T1D-derived immune systems included increased proportions of unconventional memory CD27-IgD- B cells and reduced proportions of naïve B cells compared to HC PI mice, resembling changes reported for patients with systemic lupus erythematosus. Our findings suggest that T1D HSCs are genetically programmed to produce increased proportions of T cells that promote the development of unconventional, possibly autoreactive memory B cells. PI mice provide an avenue for further understanding of the immune abnormalities that drive autoantibody pathogenesis and T1D.

Mixed xenogeneic porcine chimerism tolerizes human anti-pig natural antibody-producing cells in a humanized mouse model.

BACKGROUND: A major obstacle to the success of organ transplantation from pigs to humans, necessitated by the shortage of human organs, is robust humoral immune rejection by pig-reactive human antibodies. Mixed xenogeneic hematopoietic chimerism induces xenoreactive B cell tolerance in rodents, but whether mixed pig/human chimerism could induce tolerance of human B cells to pig xenoantigens is unknown. METHODS: We investigated this question using a humanized mouse model in which durable mixed (pig-human) xenogeneic chimerism can be established. RESULTS: Human natural anti-pig cytotoxic antibodies, predominantly IgM, are detectable in non-chimeric humanized mouse serum, and pig-reactive antibodies were reduced in mixed chimeric versus non-chimeric humanized mice. This difference required persistent mixed chimerism and was not due to the adsorption of antibodies on pig cells in vivo. Furthermore, human B cells from spleens of mixed chimeric mice produced lower levels of anti-pig antibodies when stimulated in vitro compared with those from non-chimeric mice. CONCLUSIONS: Our findings demonstrate that mixed chimerism reduces human natural antibodies to pig xenoantigens, providing the first in vivo evidence of human B cell tolerance induction by mixed xenogeneic chimerism and supporting further evaluation of this approach for inducing human B cell tolerance to xenografts.

Metastasis and Immune Evasion from Extracellular cGAMP Hydrolysis.

Cytosolic DNA is characteristic of chromosomally unstable metastatic cancer cells, resulting in constitutive activation of the cGAS-STING innate immune pathway. How tumors co-opt inflammatory signaling while evading immune surveillance remains unknown. Here, we show that the ectonucleotidase ENPP1 promotes metastasis by selectively degrading extracellular cGAMP, an immune-stimulatory metabolite whose breakdown products include the immune suppressor adenosine. ENPP1 loss suppresses metastasis, restores tumor immune infiltration, and potentiates response to immune checkpoint blockade in a manner dependent on tumor cGAS and host STING. Conversely, overexpression of wild-type ENPP1, but not an enzymatically weakened mutant, promotes migration and metastasis, in part through the generation of extracellular adenosine, and renders otherwise sensitive tumors completely resistant to immunotherapy. In human cancers, ENPP1 expression correlates with reduced immune cell infiltration, increased metastasis, and resistance to anti-PD-1/PD-L1 treatment. Thus, cGAMP hydrolysis by ENPP1 enables chromosomally unstable tumors to transmute cGAS activation into an immune-suppressive pathway. SIGNIFICANCE: Chromosomal instability promotes metastasis by generating chronic tumor inflammation. ENPP1 facilitates metastasis and enables tumor cells to tolerate inflammation by hydrolyzing the immunotransmitter cGAMP, preventing its transfer from cancer cells to immune cells.This article is highlighted in the In This Issue feature, p. 995.

Negative selection of human T cells recognizing a naturally-expressed tissue-restricted antigen in the human thymus.

During T cell development in mice, thymic negative selection deletes cells with the potential to recognize and react to self-antigens. In human T cell-dependent autoimmune diseases such as Type 1 diabetes, multiple sclerosis, and rheumatoid arthritis, T cells reactive to autoantigens are thought to escape negative selection, traffic to the periphery and attack self-tissues. However, physiological thymic negative selection of autoreactive human T cells has not been previously studied. We now describe a human T-cell receptor-transgenic humanized mouse model that permits the study of autoreactive T-cell development in a human thymus. Our studies demonstrate that thymocytes expressing the autoreactive Clone 5 TCR, which recognizes insulin B:9-23 presented by HLA-DQ8, are efficiently negatively selected at the double and single positive stage in human immune systems derived from HLA-DQ8+ HSCs. In the absence of hematopoietic expression of the HLA restriction element, negative selection of Clone 5 is less efficient and restricted to the single positive stage. To our knowledge, these data provide the first demonstration of negative selection of human T cells recognizing a naturally-expressed tissue-restricted antigen. Intrathymic antigen presenting cells are required to delete less mature thymocytes, while presentation by medullary thymic epithelial cells may be sufficient to delete more mature single positive cells. These observations set the stage for investigation of putative defects in negative selection in human autoimmune diseases.

Reduced positive selection of a human TCR in a swine thymus using a humanized mouse model for xenotolerance induction.

BACKGROUND: Tolerance-inducing approaches to xenotransplantation would be optimal and may be necessary for long-term survival of transplanted pig organs in human patients. The ideal approach would generate donor-specific unresponsiveness to the pig organ without suppressing the patient's normal immune function. Porcine thymus transplantation has shown efficacy in promoting xenotolerance in humanized mice and large animal models. However, murine studies demonstrate that T cells selected in a swine thymus are positively selected only by swine thymic epithelial cells, and therefore, cells expressing human HLA-restricted TCRs may not be selected efficiently in a transplanted pig thymus. This may lead to suboptimal patient immune function. METHODS: To assess human thymocyte selection in a pig thymus, we used a TCR transgenic humanized mouse model to study positive selection of cells expressing the MART1 TCR, a well-characterized human HLA-A2-restricted TCR, in a grafted pig thymus. RESULTS: Positive selection of T cells expressing the MART1 TCR was inefficient in both a non-selecting human HLA-A2- or swine thymus compared with an HLA-A2+ thymus. Additionally, CD8 MART1 TCRbright T cells were detected in the spleens of mice transplanted with HLA-A2+ thymi but were significantly reduced in the spleens of mice transplanted with swine or HLA-A2- thymi. [Correction added on October 15, 2019, after first online publication: The missing superscript values +, -, and bright have been included in the Results section.] CONCLUSIONS: Positive selection of cells expressing a human-restricted TCR in a transplanted pig thymus is inefficient, suggesting that modifications to improve positive selection of cells expressing human-restricted TCRs in a pig thymus may be necessary to support development of a protective human T-cell pool in future patients.

Rapid thymectomy of NSG mice to analyze the role of native and grafted thymi in humanized mice.

We developed a rapid method to remove the native mouse thymus from NSG mice, which allowed us to compare the behavior of human immune cells in the presence or absence of human T cells in human immune system mice. Removing the native mouse thymus is critical for studies of human thymopiesis in grafted thymic tissue in humanized mice.

Author Correction: Plumbagin attenuates cancer cell growth and osteoclast formation in the bone microenvironment of mice.

During re-read of our previously article Plumbagin attenuates cancer cell growth and osteoclast formation in the bone microenvironment of micepublished in Acta Pharmacologica Sinica, we were regretted to point out a mistake shown in Fig. 2a. The representative figure chosen to indicate the inhibitory effect of 4 mg/kg of plumbagin treatment at 1 week against MDA-MB-231SArfp cells localization within bone environment was incorrect due to the mishandling in manuscript preparation. Although this correction does not affect the results and conclusion of the paper, all the authors agree on the correction of our negligence as providing the corrected Fig. 2a presented below. We feel sorry and apologize for all the inconvenience it caused.An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Correction to: Suppressive effects of plumbagin on invasion and migration of breast cancer cells via the inhibition of STAT3 signaling and down-regulation of inflammatory cytokine expressions.

[This corrects the article DOI: 10.4248/BR201304007.].

Dual IFN-γ/hypoxia priming enhances immunosuppression of mesenchymal stromal cells through regulatory proteins and metabolic mechanisms.

The immunosuppressive capacity of human mesenchymal stromal cells (MSCs) renders them promising candidates for treating diverse immune disorders. However, after hundreds of clinical trials, there are still no MSC therapies approved in the United States. MSCs require specific cues to adopt their immunosuppressive phenotype, and yet most clinical trials use cells expanded in basic culture medium and growth conditions. We propose that priming MSCs prior to administration will improve their therapeutic efficacy. Interferon-gamma (IFN-γ) priming are cues common to situations of immune escape that have individually shown promise as MSC priming cues but have not been systematically compared. Using mixed lymphocyte reactions, we show that priming MSCs with either cue alone improves T-cell inhibition. However, combining the two cues results in additive effects and markedly enhances the immunosuppressive phenotype of MSCs. We demonstrate that IFN-γ induces expression of numerous immunosuppressive proteins (IDO, PD-L1, HLA-E, HLA-G), whereas hypoxia switches MSCs to glycolysis, causing rapid glucose consumption and production of T-cell inhibitory lactate levels. Dual IFN-γ/hypoxia primed MSCs display both attributes and have even higher induction of immunosuppressive proteins over IFN-γ priming alone (IDO and HLA-G), which may reflect another benefit of metabolic reconfiguration.

CD150high Bone Marrow Tregs Maintain Hematopoietic Stem Cell Quiescence and Immune Privilege via Adenosine.

A crucial player in immune regulation, FoxP3+ regulatory T cells (Tregs) are drawing attention for their heterogeneity and noncanonical functions. Here, we describe a Treg subpopulation that controls hematopoietic stem cell (HSC) quiescence and engraftment. These Tregs highly expressed an HSC marker, CD150, and localized within the HSC niche in the bone marrow (BM). Specific reduction of BM Tregs achieved by conditional deletion of CXCR4 in Tregs increased HSC numbers in the BM. Adenosine generated via the CD39 cell surface ectoenzyme on niche Tregs protected HSCs from oxidative stress and maintained HSC quiescence. In transplantation settings, niche Tregs prevented allogeneic (allo-) HSC rejection through adenosine and facilitated allo-HSC engraftment. Furthermore, transfer of niche Tregs promoted allo-HSC engraftment to a much greater extent than transfer of other Tregs. These results identify a unique niche-associated Treg subset and adenosine as regulators of HSC quiescence, abundance, and engraftment, further highlighting their therapeutic utility.

MHC Class I Expression by Donor Hematopoietic Stem Cells Is Required to Prevent NK Cell Attack in Allogeneic, but Not Syngeneic Recipient Mice.

NK cells resist engraftment of syngeneic and allogeneic bone marrow (BM) cells lacking major histocompatibility (MHC) class I molecules, suggesting a critical role for donor MHC class I molecules in preventing NK cell attack against donor hematopoietic stem and progenitor cells (HSPCs), and their derivatives. However, using high-resolution in vivo imaging, we demonstrated here that syngeneic MHC class I knockout (KO) donor HSPCs persist with the same survival frequencies as wild-type donor HSPCs. In contrast, syngeneic MHC class I KO differentiated hematopoietic cells and allogeneic MHC class I KO HSPCs were rejected in a manner that was significantly inhibited by NK cell depletion. In vivo time-lapse imaging demonstrated that mice receiving allogeneic MHC class I KO HSPCs showed a significant increase in NK cell motility and proliferation as well as frequencies of NK cell contact with and killing of HSPCs as compared to mice receiving wild-type HSPCs. The data indicate that donor MHC class I molecules are required to prevent NK cell-mediated rejection of syngeneic differentiated cells and allogeneic HSPCs, but not of syngeneic HSPCs.

Rapid Functional Decline of Activated and Memory Graft-versus-Host-Reactive T Cells Encountering Host Antigens in the Absence of Inflammation.

Inflammation in the priming host environment has critical effects on the graft-versus-host (GVH) responses mediated by naive donor T cells. However, it is unclear how a quiescent or inflammatory environment impacts the activity of GVH-reactive primed T and memory cells. We show in this article that GVH-reactive primed donor T cells generated in irradiated recipients had diminished ability compared with naive T cells to increase donor chimerism when transferred to quiescent mixed allogeneic chimeras. GVH-reactive primed T cells showed marked loss of cytotoxic function and activation, and delayed but not decreased proliferation or accumulation in lymphoid tissues when transferred to quiescent mixed chimeras compared with freshly irradiated secondary recipients. Primed CD4 and CD8 T cells provided mutual help to sustain these functions in both subsets. CD8 help for CD4 cells was largely IFN-γ dependent. TLR stimulation after transfer of GVH-reactive primed T cells to mixed chimeras restored their cytotoxic effector function and permitted the generation of more effective T cell memory in association with reduced PD-1 expression on CD4 memory cells. Our data indicate that an inflammatory host environment is required for the maintenance of GVH-reactive primed T cell functions and the generation of memory T cells that can rapidly acquire effector functions. These findings have important implications for graft-versus-host disease and T cell-mediated immunotherapies.

Xenograft tolerance and immune function of human T cells developing in pig thymus xenografts.

Transplantation of xenogeneic thymus tissue allows xenograft tolerance induction in the highly disparate pig-to-mouse model. Fetal swine thymus (SW THY) can support the generation of a diverse human T cell repertoire that is tolerant of the pig in vitro. We demonstrate that SW THY generates all human T cell subsets, including regulatory T cells (Tregs), in similar numbers as fetal human thymus (HU THY) grafts in immunodeficient mice receiving the same human CD34(+) cells. Peripheral T cells are specifically tolerant to the mouse and to the human and porcine donors, with robust responses to nondonor human and pig Ags. Specific tolerance is observed to pig skin grafts sharing the THY donor MHC. SW THY-generated peripheral Tregs show similar function, but include lower percentages of naive-type Tregs compared with HU THY-generated Tregs. Tregs contribute to donor-pig specific tolerance. Peripheral human T cells generated in SW THY exhibit reduced proportions of CD8(+) T cells and reduced lymphopenia-driven proliferation and memory-type conversion, accelerated decay of memory-type cells, and reduced responses to protein Ags. Thus, SW thymus transplantation is a powerful xenotolerance approach for human T cells. However, immune function may be further enhanced by strategies to permit positive selection by autologous HLA molecules.

Plumbagin attenuates cancer cell growth and osteoclast formation in the bone microenvironment of mice.

AIM: To investigate the effects of plumbagin, a naphthoquinone derived from the medicinal plant Plumbago zeylanica, on human breast cancer cell growth and the cancer cell-induced osteolysis in the bone microenvironment of mice. METHODS: Human breast cancer cell subline MDA-MB-231SA with the ability to spread and grow in the bone was tested. The cell proliferation was determined using the CCK-8 assay. Apoptosis was detected with Annexin V/PI double-labeled flow cytometry. Red fluorescent protein-labeled MDA-MB-231SArfp cells were injected into the right tibia of female BALB/c-nu/nu mice. Three days after the inoculation, the mice were injected with plumbagin (2, 4, or 6 mg/kg, ip) 5 times per week for 7 weeks. The growth of the tumor cells was monitored using an in vivo imaging system. After the mice were sacrificed, the hind limbs were removed for radiographic and histological analyses. RESULTS: Plumbagin (2.5-20 µmol/L) concentration-dependently inhibited the cell viability and induced apoptosis of MDA-MB-231SA cells in vitro (the IC50 value of inhibition of cell viability was 14.7 µmol/L). Administration of plumbagin to breast cancer bearing mice delayed the tumor growth by 2-3 weeks and reduced the tumor volume by 44%-74%. The in vivo imaging study showed that plumbagin dose-dependently inhibited MDA-MB-231SArfp cell growth in bone microenvironment. Furthermore, X-ray images and micro-CT study demonstrated that plumbagin reduced bone erosion area and prevented a decrease in bone tissue volume. Histological studies showed that plumbagin dose-dependently inhibited the breast cancer cell growth, enhanced the cell apoptosis and reduced the number of TRAcP-positive osteoclasts. CONCLUSION: Plumbagin inhibits the cell growth and induces apoptosis in human breast cancer cells in mice bone microenvironment, leading to significant reduction in osteolytic lesions caused by the tumor cells.