Comparison of the Medium-term Outcomes of Anterior Lumbar Discectomy and Fusion with Minimally Invasive Transforaminal Lumbar Interbody Fusion: A Retrospective Cohort Study.

OBJECTIVE: Lumbar degenerative diseases (LDDs) with huge herniation in the left lateral recess or central canal present challenges for oblique lateral lumbar interbody fusion (OLIF) or endoscope-assisted OLIF procedures. Currently, minimally invasive transforaminal lumbar interbody fusion (MIS-TLIF) is the primary approach for this issue. This study aims to provide a standardized technical description of the anterior lumbar discectomy and fusion (ALDF) and evaluate the medium-term clinical effectiveness of both ALDF and MIS-TLIF techniques. METHODS: A retrospective review was performed on LDDs who underwent ALDF and MIS-TLIF surgery from January 2018 to January 2020. The evaluation encompassed various clinical outcomes, such as the visual analogue scale (VAS) scores for back pain and leg pain (VAS-back, VAS-leg), the Oswestry disability index (ODI), the 36-item short-form health survey mental component summary (SF-36 MCS), and the physical component summary (SF-36 PCS). Additionally, radiological parameters, including disc height (DH), segmental disk angle (SDA), lumbar lordosis (LL), and cross-sectional area (CSA), were assessed. Data including radiculopathy, estimated blood loss, operation time, time of getting out of bed, fusion rate, and complications were recorded. Student's independent samples t test and Pearson's chi-square test were used to compare the differences between groups. RESULTS: In total, 47 patients were treated by ALDF and 48 patients were treated by MIS-TLIF. The ALDF group exhibited statistically significant lower estimated blood loss and earlier time of getting out of bed compared to the MIS-TLIF group (p < 0.05). The ALDF group demonstrated lower VAS-back scores and a higher remission rate of low back pain 3 years after the surgery (p < 0.05). During the entire follow-up period, the ALDF group exhibited higher increases in DH and SDA compared to the MIS-TLIF group (p < 0.05). At 6 months, the fusion rate in the ALDF group was significantly higher than in the MIS-TLIF group (p < 0.05). The comparison revealed no statistically significant differences in complication rates between the two groups (p > 0.05). CONCLUSION: The ALDF could be considered as a viable surgical alternative for the treatment of LDDs that necessitate ventral neural direct decompression. ALDF exhibited favorable medium-term outcomes in patients with LDDs, displaying advantages in facilitating expedited recovery, enhancing radiographic outcomes, and elevating the remission rate of low back pain. Although ALDF presents slightly higher complication rates compared to MIS-TLIF, it does not adversely affect clinical outcomes.

Endoscopic Arthroplasty via Mini-open Direct Anterior Approach Improves Postoperative Complications and Acetabular Components of Total Hip Arthroplasty in Obese Patients.

To overcome the high-risk complications and poor alignment of acetabular components in obese patients associated with direct anterior approach (DAA) for total hip arthroplasty (THA), we innovated an endoscopic arthroplasty via mini-open direct anterior approach technique (Endo-DAA). The purpose of this study was to compare the clinical and radiographic outcomes in obese patients subjected to THA between Endo-DAA, Bikini DAA, and conventional DAA. In this retrospective controlled study, a total of 360 consecutive primary THA on obese patients (body mass index greater than 28 kg/m2) via Endo-DAA, Bikini DAA, and conventional DAA performed from October 2017 to October 2022 by different surgeons and in a single center were included. Assessments including perioperative parameters, clinical outcomes, complications, and radiologic measurements were retrieved from patients before the surgery, perioperative period and the latest follow-up. A total of 360 consecutive THA (Endo-DAA = 108, Bikini DAA = 116, Conventional DAA = 136) with complete follow-up data were analyzed. Compared to Bikini DAA or conventional DAA, Endo-DAA significantly shortened the length of incision (5.46 ± 0.53), the duration of operation (64.47 ± 12.38), and postoperative hospital stay (2.15 ± 0.89). Endo-DAA significantly reduces wound related complications compared with conventional DAA. Besides, Endo-DAA achieved a significantly better alignment of acetabular components compared to Bikini DAA or conventional DAA. Furthermore, Endo-DAA improved postoperative pain at the activity at 24 h postoperatively and early functional scores. The Endo-DAA THA technique provides better short-term clinical and radiographic results in obese patients with a low rate of postoperative complications compared to Bikini DAA or conventional DAA.

Prostate cancer-induced endothelial-to-osteoblast transition generates an immunosuppressive bone tumor microenvironment.

Immune checkpoint therapy has limited efficacy for patients with bone metastatic castrate-resistant prostate cancer (bmCRPC). In this study, we revealed a novel mechanism that may account for the relative resistance of bmCRPC to immune checkpoint therapy. We found that prostate cancer (PCa)-induced bone via endothelial-to-osteoblast (EC-to-OSB) transition causes an ingress of M2-like macrophages, leading to an immunosuppressive bone tumor microenvironment (bone-TME). Analysis of a bmCRPC RNA-seq dataset revealed shorter overall survival in patients with an M2-high versus M2-low signature. Immunohistochemical (IHC) analysis showed CD206 + M2-like macrophages were enriched in bmCRPC specimens compared with primary tumors or lymph node metastasis. In osteogenic PCa xenografts, CD206 + macrophages were enriched adjacent to tumor-induced bone. FACS analysis showed an increase in CD206 + cells in osteogenic tumors compared to non-osteogenic tumors. Genetic or pharmacological inhibition of the EC-to-OSB transition reduced aberrant bone and M2-like macrophages in osteogenic tumors. RNAseq analysis of tumor-associated macrophages from osteogenic (bone-TAMs) versus non-osteogenic (ctrl-TAMs) tumors showed high expression of an M2-like gene signature, canonical and non-canonical Wnt pathways, and a decrease in an M1-like gene signature. Isolated bone-TAMs suppressed T-cell proliferation while ctrl-TAMs did not. Mechanistically, EC-OSB hybrid cells produced paracrine factors, including Wnts, CXCL14 and LOX, which induced M2 polarization and recruited M2-like TAMs to bone-TME. Our study thus links the unique EC-to-OSB transition as an "upstream" event that drives "downstream" immunosuppression in the bone-TME. These studies suggest that therapeutic strategies that inhibit PCa-induced EC-to-OSB transition may reverse immunosuppression to promote immunotherapeutic outcomes in bmCRPC. Significance: The insight that prostate cancer-induced bone generates an immunosuppressive bone tumor microenvironment offers a strategy to improve responses to immunotherapy approaches in patients with bone metastatic castrate-resistant prostate cancer.

Effect of intravenous low-dose norepinephrine on blood loss in non-tourniquet total knee arthroplasty under general anesthesia: a randomized, double-blind, controlled, single-center trial.

OBJECTIVE: This prospective trial aimed to evaluate the effects of low-dose intravenous norepinephrine (NE) on intraoperative blood loss and bleeding from osteotomy sites during non-tourniquet total knee arthroplasty (TKA) under general anesthesia. METHODS: A total of 120 patients who underwent TKA between December 2020 and May 2022 were enrolled and randomly assigned to the intravenous low-dose NE Group (NE Group) or the control group (C Group). During surgery, NE Group received 0.05-0.1 µg/(kg min) of NE intravenously to raise and maintain the patient's mean arterial pressure (MAP). C Group received the same dose of saline as placebo. Intraoperative blood loss, bleeding score at osteotomy sites, Δlactate levels (Lac), postoperative complications, and transfusion rate during hospitalization were compared between groups. RESULTS: Intraoperative and osteotomy blood loss was significantly lower in the NE Group than in the C Group (P < 0.001). No significant difference was observed in ΔLac between groups (P > 0.05). There was no significant difference in complications between the groups 3 days after surgery (P > 0.05). In addition, there was no significant difference in blood transfusion rates between the two groups during hospitalization (P > 0.05). CONCLUSION: In non-tourniquet TKA under general anesthesia, low-dose intravenous NE safely and effectively reduced intraoperative blood loss and provided a satisfactory osteotomy site while maintaining a higher MAP.

Photoluminescence of Cesium-Doped Sodium Iodide Films Irradiated by UV LED.

Alkali metal halides have long been used as scintillators for applications as sensors and detectors. Usually, a small amount of impurities are added to these inorganic materials to improve their luminescence efficiencies. We investigate the structures and luminescent properties of un-doped sodium iodide (NaI) and cesium-doped NaI (NaI:Cs) films deposited by thermal vacuum evaporation. Instead of using the toxic element thallium (Tl), we introduced cesium dopant into NaI. This is the first study for the NaI:Cs film excited by UV LED's ultraviolet C (273 nm, 4.54 eV). The luminescence spectra show two main peaks at 3.05 and 4.32/3.955 eV (for fused silica/B270 substrate), originating from the intrinsic defects and/or activator excited states and the intrinsic self-trapped excitons (STEs), respectively. In general, both Cs-doping and post-annealing processes enhance the luminescence performance of NaI films.

High FAM111B expression predicts aggressive clinicopathologic features and poor prognosis in ovarian cancer.

BACKGROUNDS: Ovarian cancer (OC) is the second most common gynecological tumor with the highest mortality rate worldwide. High FAM111B expression has been reported as a predictor of poor prognosis in other cancers, but its correlation with OC has not been reported. METHODS: Immunohistochemistry of tissue microarrays was performed to detect FAM111B expression levels in 141 OC patient tissues. The prognostic value of FAM111B was determined by Kaplan-Meier survival analysis, and correlations between FAM111B expression and clinicopathologic features were investigated by the Clu-square test. The significance of FAM111B expression was verified bioinformatically using the Gene Expression Omnibus database. Protein-protein interaction were performed to explore downstream mechanisms of FAM111B in OC. RESULTS: Among 141 OC patients, FAM111B was positively expressed in 87.23%, 58.16%, and 87.94%; and highly expressed in 8.51%, 17.02%, and 19.86%, as evaluated by cytoplasmic, nuclear, and combined cytoplasmic/nuclear staining. FAM111B expression was positively correlated with the expression of tumor protein markers KI67, EGFR, and PDL-1. Patients with high FAM111B expression had aggressive clinicopathologic features and shorter overall survival (P value 0.0428, 0.0050, 0.0029) and progression-free survival (P value 0.0251, 0.012, 0.0596) compared to the low FAM111B expression group for cytoplasmic, nuclear, and combined cytoplasmic/nuclear groups, respectively. These results were verified using patient data from the Gene Expression Omnibus. Seventeen genes co-expressed with FAM111B were primarily involved in "negative regulation of histone modification", "hippo signaling" and "inner ear receptor cell differentiation". CONCLUSIONS: High FAM111B expression may serve as a novel prognostic predictor and molecular therapeutic target for OC.

Association between serum 25-hydroxyvitamin D and osteoarthritis: A national population-based analysis of NHANES 2001-2018.

Background: Previous studies have not provided a consensus on the effect of serum 25-hydroxyvitamin D [25(OH)D] on osteoarthritis (OA). We aimed to evaluate the association using a large, nationally representative sample. Methods: The cross-sectional data were obtained from the 2001 to 2018 National Health and Nutrition Examination Survey (NHANES). Individuals aged ≥40 years who had information of serum 25(OH)D, self-report OA, and related covariates were included. Multivariable logistic regression analysis was employed to assess the association between serum 25(OH)D and osteoarthritis. Results: Among the 21,334 participants included (weighted mean age, 56.9 years; 48.5% men), the proportion of participants with high serum 25(OH)D concentrations (≥100 nmol/L) increased significantly from 4.2% in 2001-2006 to 18.8% in 2013-2018. Higher serum 25(OH)D levels were associated with more osteoarthritis prevalence in fully adjusted model (odd ratio [OR] 1.25 [95% CI: 1.10, 1.43] for the 50-75 nmol/L group; OR 1.62 [95% CI: 1.42, 1.85] for the 75-100 nmol/L group; OR 1.91 [95% CI: 1.59, 2.30] for the ≥100 nmol/L group; with <50 nmol/L group as the reference) (p < 0.001 for trend). The association was consistent across several sensitivity analyses, including propensity score methods and excluding participants who had received vitamin D supplement. In subgroup analysis, the OR for the association increased significantly with body mass index (BMI) (BMI < 25 kg/m2, 1.01 [95% CI: 1.04, 1.08]; BMI 25-30 kg/m2, 1.05 [95% CI: 1.01, 1.08]; BMI ≥ 30 kg/m2, 1.10 [95% CI: 1.06, 1.13]; p = 0.004 for interaction). Conclusion: There was a positive correlation between serum 25(OH)D and osteoarthritis with a possible modification by BMI. Our finding raises concerns about the potential adverse effects of high serum 25(OH)D on osteoarthritis, particularly among obese individuals. More well-designed studies are still needed to validate our findings in future.

Alendronate conjugate for targeted delivery to bone-forming prostate cancer.

Bone is the primary metastasis site for lethal prostate cancer, often resulting in poor prognosis, crippling pain, and diminished functioning that drastically reduce both quality of life and survivability Uniquely, prostate cancer bone metastasis induces aberrant bone overgrowth, due to an increase of osteoblasts induced by tumor-secreted bone morphogenetic protein 4 (BMP4). Conjugating drugs to substances that target the tumor-induced bone area within the metastatic tumor foci would be a promising strategy for drug delivery. To develop such a strategy, we conjugated a near infrared (NIR) fluorescent probe, the dye Cy5.5, to serve as a surrogate for drugs, with alendronate, which targets bone. Characterization, such as infrared spectroscopy, confirmed the synthesis of the Cy5.5-ALN conjugate. The maximum absorbance of free Cy5.5, which was at 675 nm, did not change upon conjugation. Alendronate targeted the bone component hydroxyapatite in a dose-dependent manner up to 2.5 µM, with a maximum of 85% of Cy5.5-ALN bound to hydroxyapatite, while free Cy5.5 alone had 6% binding. In in vitro cell binding studies, Cy5.5-ALN bound specifically with mineralized bone matrix of differentiated MC3T3-E1 cells or 2H11 endothelial cells that were induced to become osteoblasts through endothelial-to-osteoblast transition, the underlying mechanism of prostate-cancer-induced bone formation. Neither Cy5.5-ALN nor free Cy5.5 bound to undifferentiated MC3T3-E1 or 2H11 cells. Bone-targeting efficiency studies in non-tumor-bearing mice revealed accumulation over time in the spine, jaw, knees, and paws injected with Cy5.5-ALN, and quantification showed higher accumulation in femurs than in muscle at up to 28 days, while the free Cy5.5 dye was observed circulating without preferential accumulation and decreased over time. There was a linear relationship with fluorescence when the injected concentration of Cy5.5-ALN was between 0.313 and 1.25 nmol/27 g of mouse, as quantified in mouse femurs both in vivo and ex vivo. Ex vivo evaluation of bone-targeting efficiency in nude mice was 3 times higher for bone-forming C4-2b-BMP4 tumors compared to non-bone-forming C4-2b tumors (p-value <0.001). Fluorescence microscopy imaging of the tumors showed that Cy5.5-ALN co-localized with the bone matrix surrounding tumor-induced bone, but not with the viable tumor cells. Together, these results suggest that a drug-ALN conjugate is a promising approach for targeted delivery of drug to the tumor-induced bone area in the metastatic foci of prostate cancer.

Endothelial-to-osteoblast transition in normal mouse bone development.

Metastatic prostate cancer (PCa) in bone induces bone-forming lesions. We have previously shown that PCa-induced bone originates from endothelial cells (ECs) that have undergone EC-to-osteoblast (OSB) transition. Here, we investigated whether EC-to-OSB transition also occurs during normal bone formation. We developed an EC and OSB dual-color reporter mouse (DRM) model that marks EC-OSB hybrid cells with red and green fluorescent proteins. We observed EC-to-OSB transition (RFP and GFP co-expression) in both endochondral and intramembranous bone formation during embryonic development and in adults. Co-expression was confirmed in cells isolated from DRM. Bone marrow- and lung-derived ECs underwent transition to OSBs and mineralization in osteogenic medium. RNA-sequencing revealed GATA family transcription factors were upregulated in EC-OSB hybrid cells and knockdown of GATA3 inhibited BMP4-induced mineralization. Our findings support that EC-to-OSB transition occurs during normal bone development and suggest a new paradigm regarding the endothelial origin of OSBs.

Inhibiting KCNMA1-AS1 promotes osteogenic differentiation of HBMSCs via miR-1303/cochlin axis.

OBJECTIVE: Osteoporosis is a progressive systemic skeletal disorder. Multiple profiling studies have contributed to characterizing biomarkers and therapeutic targets for osteoporosis. However, due to the limitation of the platform of miRNA sequencing, only a part of miRNA can be sequenced based on one platform. MATERIALS AND METHODS: In this study, we performed miRNA sequencing in osteoporosis bone samples based on a novel platform Illumina Hiseq 2500. Bioinformatics analysis was performed to construct osteoporosis-related competing endogenous RNA (ceRNA) networks. Gene interference and osteogenic induction were used to examine the effect of identified ceRNA networks on osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (HBMSCs). RESULTS: miR-1303 was lowly expressed, while cochlin (COCH) and KCNMA1-AS1 were highly expressed in the osteoporosis subjects. COCH knockdown improved the osteogenic differentiation of HBMSCs. Meanwhile, COCH inhibition compensated for the suppression of osteogenic differentiation of HBMSCs by miR-1303 knockdown. Further, KCNMA1-AS1 knockdown promoted osteogenic differentiation of HBMSCs through downregulating COCH by sponging miR-1303. CONCLUSIONS: Our findings suggest that the KCNMA1-AS1/miR-1303/COCH axis is a promising biomarker and therapeutic target for osteoporosis.

High Sensitivity SERS Substrate of a Few Nanometers Single-Layer Silver Thickness Fabricated by DC Magnetron Sputtering Technology.

Surface-enhanced Raman spectroscopy (SERS) is commonly used for super-selective analysis through nanostructured silver layers in the environment, food quality, biomedicine, and materials science. To fabricate a high-sensitivity but a more accessible device of SERS, DC magnetron sputtering technology was used to realize high sensitivity, low cost, a stable deposition rate, and rapid mass production. This study investigated various thicknesses of a silver film ranging from 3.0 to 12.1 nm by field emission scanning electron microscope, X-ray diffraction, and X-ray photoelectron spectroscopy. In the rhodamine 6G (R6G) testing irradiated by a He-Ne laser beam, the analytical enhancement factor (AEF) of 9.35 × 108, the limit of detection (LOD) of 10-8 M, and the relative standard deviation (RSD) of 1.61% were better than the other SERS substrates fabricated by the same DC sputtering process because the results showed that the 6 nm thickness silver layer had the highest sensitivity, stability, and lifetime. The paraquat and acetylcholine analytes were further investigated and high sensitivity was also achievable. The proposed SERS samples were evaluated and stored in a low humidity environment for up to forty weeks, and no spectrum attenuation could be detected. Soon, the proposed technology to fabricate high sensitivity, repeatability, and robust SERS substrate will be an optimized process technology in multiple applications.

Inhibiting uptake of extracellular vesicles derived from senescent bone marrow mesenchymal stem cells by muscle satellite cells attenuates sarcopenia.

Objective: Osteoporosis is associated with senescence of bone marrow mesenchymal stem cells (BMSCs). Extracellular vesicles derived from senescent BMSCs (BMSC-EVs) could be uptaken by muscle satellite cells (SCs). We hypothesized that inhibiting the uptake of harmful BMSC-EVs by SCs could prevent patients with osteoporosis complicated with sarcopenia. Methods: Bioinformatics analysis was used to analyze senescent SCs. Myogenic potential of SCs was measured using myogenesis assay and immunofluorescence while muscle atrophy was measured using histological evaluation. And the interaction of cluster of differentiation (CD) 81 and the membrane proteins of SCs was verified using biotin pulldown assay.. CD81-specific siRNA (si-CD81) was used to knockdown CD81 and anti-CD81 antibody (anti-CD81 Ab) was used to block CD81. Results: Differentially expressed genes in senescent SCs were enriched in muscle cell differentiation. The myogenic potential of senescent SCs was significantly decreased. Senescent BMSC-EVs impaired myogenesis of SCs. CD81 on the surface of BMSC-EVs could bind to membrane proteins of SCs. Both knockdown of CD81 and blocking CD81 prevented the uptake of senescent BMSC-EVs by SCs, thus relieving harmful effects of senescent BMSC-EVs on muscle atrophy. Conclusion: Blocking CD81 on the surface of senescent BMSC-EVs attenuates sarcopenia in aged mice, which could be useful for prevention of sarcopenia in patients with osteoporosis in clinical practice. Translational potential of this article: Inhibiting uptake of extracellular vesicles derived from senescent bone marrow mesenchymal stem cells by muscle satellite cells can prevent muscle atrophy in aged mice and has potential for application in treating sarcopenia.

Retinoic Acid Receptor Activation Reduces Metastatic Prostate Cancer Bone Lesions by Blocking the Endothelial-to-Osteoblast Transition.

Metastatic prostate cancer in the bone induces bone-forming lesions that contribute to progression and therapy resistance. Prostate cancer-induced bone formation originates from endothelial cells (EC) that have undergone endothelial-to-osteoblast (EC-to-OSB) transition in response to tumor-secreted BMP4. Current strategies targeting prostate cancer-induced bone formation are lacking. Here, we show that activation of retinoic acid receptor (RAR) inhibits EC-to-OSB transition and reduces prostate cancer-induced bone formation. Treatment with palovarotene, an RARγ agonist being tested for heterotopic ossification in fibrodysplasia ossificans progressiva, inhibited EC-to-OSB transition and osteoblast mineralization in vitro and decreased tumor-induced bone formation and tumor growth in several osteogenic prostate cancer models, and similar effects were observed with the pan-RAR agonist all-trans-retinoic acid (ATRA). Knockdown of RARα, ß, or γ isoforms in ECs blocked BMP4-induced EC-to-OSB transition and osteoblast mineralization, indicating a role for all three isoforms in prostate cancer-induced bone formation. Furthermore, treatment with palovarotene or ATRA reduced plasma Tenascin C, a factor secreted from EC-OSB cells, which may be used to monitor treatment response. Mechanistically, BMP4-activated pSmad1 formed a complex with RAR in the nucleus of ECs to activate EC-to-OSB transition. RAR activation by palovarotene or ATRA caused pSmad1 degradation by recruiting the E3-ubiquitin ligase Smad ubiquitination regulatory factor1 (Smurf1) to the nuclear pSmad1/RARγ complex, thus blocking EC-to-OSB transition. Collectively, these findings suggest that palovarotene can be repurposed to target prostate cancer-induced bone formation to improve clinical outcomes for patients with bone metastasis. SIGNIFICANCE: This study provides mechanistic insights into how RAR agonists suppress prostate cancer-induced bone formation and offers a rationale for developing RAR agonists for prostate cancer bone metastasis therapy. See related commentary by Bhowmick and Bhowmick, p. 2975.

Prostate tumor-induced stromal reprogramming generates Tenascin C that promotes prostate cancer metastasis through YAP/TAZ inhibition.

Metastatic prostate cancer (PCa) in bone induces bone-forming lesions that enhance PCa progression. How tumor-induced bone formation enhances PCa progression is not known. We have previously shown that PCa-induced bone originates from endothelial cells (ECs) that have undergone endothelial-to-osteoblast (EC-to-OSB) transition by tumor-secreted bone morphogenetic protein 4 (BMP4). Here, we show that EC-to-OSB transition leads to changes in the tumor microenvironment that increases the metastatic potential of PCa cells. We found that conditioned medium (CM) from EC-OSB hybrid cells increases the migration, invasion, and survival of PC3-mm2 and C4-2B4 PCa cells. Quantitative mass spectrometry (Isobaric Tags for Relative and Absolute Quantitation) identified Tenascin C (TNC) as one of the major proteins secreted from EC-OSB hybrid cells. TNC expression in tumor-induced OSBs was confirmed by immunohistochemistry of MDA PCa-118b xenograft and human bone metastasis specimens. Mechanistically, BMP4 increases TNC expression in EC-OSB cells through the Smad1-Notch/Hey1 pathway. How TNC promotes PCa metastasis was next interrogated by in vitro and in vivo studies. In vitro studies showed that a TNC-neutralizing antibody inhibits EC-OSB-CM-mediated PCa cell migration and survival. TNC knockdown decreased, while the addition of recombinant TNC or TNC overexpression increased migration and anchorage-independent growth of PC3 or C4-2b cells. When injected orthotopically, PC3-mm2-shTNC clones decreased metastasis to bone, while C4-2b-TNC-overexpressing cells increased metastasis to lymph nodes. TNC enhances PCa cell migration through α5ß1 integrin-mediated YAP/TAZ inhibition. These studies elucidate that tumor-induced stromal reprogramming generates TNC that enhances PCa metastasis and suggest that TNC may be a target for PCa therapy.

Statins reduce castration-induced bone marrow adiposity and prostate cancer progression in bone.

A fraction of patients undergoing androgen deprivation therapy (ADT) for advanced prostate cancer (PCa) will develop recurrent castrate-resistant PCa (CRPC) in bone. Strategies to prevent CRPC relapse in bone are lacking. Here we show that the cholesterol-lowering drugs statins decrease castration-induced bone marrow adiposity in the tumor microenvironment and reduce PCa progression in bone. Using primary bone marrow stromal cells (BMSC) and M2-10B4 cells, we showed that ADT increases bone marrow adiposity by enhancing BMSC-to-adipocyte transition in vitro. Knockdown of androgen receptor abrogated BMSC-to-adipocyte transition, suggesting an androgen receptor-dependent event. RNAseq analysis showed that androgens reduce the secretion of adipocyte hormones/cytokines including leptin during BMSC-to-adipocyte transition. Treatment of PCa C4-2b, C4-2B4, and PC3 cells with leptin led to an increase in cell cycle progression and nuclear Stat3. RNAseq analysis also showed that androgens inhibit cholesterol biosynthesis pathway, raising the possibility that inhibiting cholesterol biosynthesis may decrease BMSC-to-adipocyte transition. Indeed, statins decreased BMSC-to-adipocyte transition in vitro and castration-induced bone marrow adiposity in vivo. Statin pre-treatment reduced 22RV1 PCa progression in bone after ADT. Our findings with statin may provide one of the mechanisms to the clinical correlations that statin use in patients undergoing ADT seems to delay progression to "lethal" PCa.

Multiple pathways coordinating reprogramming of endothelial cells into osteoblasts by BMP4.

Cell type transition occurs during normal development and under pathological conditions. In prostate cancer bone metastasis, prostate cancer-secreted BMP4 induces endothelial cell-to-osteoblast (EC-to-OSB) transition. Such tumor-induced stromal reprogramming supports prostate cancer progression. We delineate signaling pathways mediating EC-to-OSB transition using EC lines 2H11 and SVR. We found that BMP4-activated pSmad1-Notch-Hey1 pathway inhibits EC migration and tube formation. BMP4-activated GSK3ß-ßcatenin-Slug pathway stimulates Osx expression. In addition, pSmad1-regulated Dlx2 converges with the Smad1 and ß-catenin pathways to stimulate osteocalcin expression. By co-expressing Osx, Dlx2, Slug and Hey1, we were able to achieve EC-to-OSB transition, leading to bone matrix mineralization in the absence of BMP4. In human prostate cancer bone metastasis specimens and MDA-PCa-118b and C4-2b-BMP4 osteogenic xenografts, immunohistochemical analysis showed that ß-catenin and pSmad1 are detected in activated osteoblasts rimming the tumor-induced bone. Our results elucidated the pathways and key molecules coordinating prostate cancer-induced stromal programming and provide potential targets for therapeutic intervention.

Combinatorial effect of radium-223 and irreversible electroporation on prostate cancer bone metastasis in mice.

BACKGROUND: Metastatic prostate cancer in bone is difficult to treat as the tumor cells are relatively resistant to hormonal or chemotherapies when compared to primary prostate cancer. Irreversible electroporation (IRE) is a minimally invasive ablation procedure that has potential applications in the management of prostate cancer in bone. However, a common limitation of IRE is tumor recurrence, which arises from incomplete ablation that allows remaining cancer cells to proliferate. In this study, we combined IRE with radium-223 (Ra-223), a bone-seeking radionuclide that emits short track length alpha particles and thus is associated with reduced damage to the bone marrow and evaluated the impact of the combination treatment on bone-forming prostate cancer tumors. METHODS: The antitumor activity of IRE and Ra-223 as single agents and in combination was tested in vitro against three bone morphogenetic protein 4 (BMP4)-expressing prostate cancer cell lines (C4-2B-BMP4, Myc-CaP-BMP4, and TRAMP-C2-BMP4). Similar evaluation was performed in vivo using a bone-forming C4-2B-BMP4 tumor model in nude mice. RESULTS: IRE and Ra-223 as monotherapy inhibited prostate cancer cell proliferation in vitro, and their combination resulted in significant reduction in cell viability compared to monotherapy. In vivo evaluation revealed that IRE with single-dose administration of Ra-233, compared to IRE alone, reduced the rate of tumor recurrence by 40% following initial apparent complete ablation and decreased the rate of proliferation of incompletely ablated tumor as quantified in Ki-67 staining (53.58 ± 16.0% for IRE vs. 20.12 ± 1.63%; for IRE plus Ra-223; p = 0.004). Histological analysis qualitatively showed the enhanced killing of tumor cells adjacent to bone by Ra-223 compared to those treated with IRE alone. CONCLUSION: IRE in combination with Ra-223, which enhanced the destruction of cancer cells that are adjacent to bone, resulted in reduction of tumor recurrence through improved clearance of proliferative cells in the tumor region.

Radium-223 Treatment Increases Immune Checkpoint Expression in Extracellular Vesicles from the Metastatic Prostate Cancer Bone Microenvironment.

PURPOSE: Radium-223 prolongs survival in a fraction of men with bone metastatic prostate cancer (PCa). However, there are no markers for monitoring response and resistance to Radium-223 treatment. Exosomes are mediators of intercellular communication and may reflect response of the bone microenvironment to Radium-223 treatment. We performed molecular profiling of exosomes and compared the molecular profile in patients with favorable and unfavorable overall survival. EXPERIMENTAL DESIGN: We performed exosomal transcriptome analysis in plasma derived from our preclinical models (MDA-PCa 118b tumors, TRAMP-C2/BMP4 PCa) and from the plasma of 25 patients (paired baseline and end of treatment) treated with Radium-223. All samples were run in duplicate, and array data analyzed with fold changes +2 to -2 and P < 0.05. RESULTS: We utilized the preclinical models to establish that genes derived from the tumor and the tumor-associated bone microenvironment (bTME) are differentially enriched in plasma exosomes upon Radium-223 treatment. The mouse transcriptome analysis revealed changes in bone-related and DNA damage repair-related pathways. Similar findings were observed in plasma-derived exosomes from patients treated with Radium-223 detected changes. In addition, exosomal transcripts detected immune-suppressors (e.g., PD-L1) that were associated with shorter survival to Radium-223. Treatment of the Myc-CaP mouse model with a combination of Radium-223 and immune checkpoint therapy (ICT) resulted in greater efficacy than monotherapy. CONCLUSIONS: These clinical and coclinical analyses showed that RNA profiling of plasma exosomes may be used for monitoring the bTME in response to treatment and that ICT may be used to increase the efficacy of Radium-223.

Cabozantinib Reverses Renal Cell Carcinoma-mediated Osteoblast Inhibition in Three-dimensional Coculture In Vitro and Reduces Bone Osteolysis In Vivo.

Renal cell carcinoma bone metastases (RCCBM) are typically osteolytic. We previously showed that BIGH3 (beta Ig-h3/TGFBI), secreted by 786-O renal cell carcinoma, plays a role in osteolytic bone lesion in RCCBM through inhibition of osteoblast (OSB) differentiation. To study this interaction, we employed three-dimensional (3D) hydrogels to coculture bone-derived 786-O (Bo-786) renal cell carcinoma cells with MC3T3-E1 pre-OSBs. Culturing pre-OSBs in the 3D hydrogels preserved their ability to differentiate into mature OSB; however, this process was decreased when pre-OSBs were cocultured with Bo-786 cells. Knockdown of BIGH3 in Bo-786 cells recovered OSB differentiation. Furthermore, treatment with bone morphogenetic protein 4, which stimulates OSB differentiation, or cabozantinib (CBZ), which inhibits VEGFR1 and MET tyrosine kinase activities, also increased OSB differentiation in the coculture. CBZ also inhibited pre-osteoclast RAW264.7 cell differentiation. Using RCCBM mouse models, we showed that CBZ inhibited Bo-786 tumor growth in bone. CBZ treatment also increased bone volume and OSB number, and decreased osteoclast number and blood vessel density. When tested in SN12PM6 renal cell carcinoma cells that have been transduced to overexpress BIGH3, CBZ also inhibited SN12PM6 tumor growth in bone. These observations suggest that enhancing OSB differentiation could be one of the therapeutic strategies for treating RCCBM that exhibit OSB inhibition characteristics, and that this 3D coculture system is an effective tool for screening osteoanabolic agents for further in vivo studies.

Bone secreted factors induce cellular quiescence in prostate cancer cells.

Disseminated tumor cells (DTCs) undergo a dormant state in the distant metastatic site(s) before becoming overt metastatic diseases. In prostate cancer (PCa), bone metastasis can occur years after prostatectomy, suggesting that bone may provide dormancy-inducing factors. To search for these factors, we prepared conditioned media (CM) from calvariae. Using live-cell imaging, we found that Calvarial-CM treatment increased cellular quiescence in C4-2B4 PCa cells. Mass spectrometry analysis of Calvarial-CM identified 132 secreted factors. Western blot and ELISA analyses confirmed the presence of several factors, including DKK3, BMP1, neogenin and vasorin in the Calvarial-CM. qRT-PCR analysis of total calvariae versus isolated osteoblasts showed that DKK3, BMP1, vasorin and neogenin are mainly expressed by osteoblasts, while MIA, LECT1, NGAL and PEDF are expressed by other calvarial cells. Recombinant human DKK3, BMP1, vasorin, neogenin, MIA and NGAL treatment increased cellular quiescence in both C4-2b and C4-2B4 PCa cells. Mechanistically, DKK3, vasorin and neogenin, but not BMP1, increased dormancy through activating the p38MAPK signaling pathway. Consistently, DKK3, vasorin and neogenin failed to induce dormancy in cells expressing dominant-negative p38αMAPK while BMP1 remained active, suggesting that BMP1 uses an alternative dormancy signaling pathway. Thus, bone secretes multiple dormancy-inducing factors that employ distinct signaling pathways to induce DTC dormancy in bone.