We determined the effect of β2-AR agonists, alone or in combination with corticosteroids, on fibrocyte function.Patients with severe asthma have increased airway remodelling and elevated numbers of circulating fibrocytes with enhanced myofibroblastic differentiation capacity, despite being treated with high doses of corticosteroids, and long acting β2-AR agonist, salmeterol, in the presence or absence of the corticosteroid dexamethasone. The number of fibrocytes (collagen I+/CD45+ cells) and differentiating fibrocytes (α-smooth muscle actin+ cells), and the expression of CC chemokine receptor 7 and of β2-AR were determined using flow cytometry. The role of cyclic adenosine monophosphate (cAMP) was elucidated using the cAMP analogue 8-bromoadenosine 3′,5′-cyclic monophosphate (8-Br-cAMP) and the phosphodiesterase type IV (PDE4) inhibitor, rolipram.Non-adherent non-T cells from peripheral blood mononuclear cells isolated from healthy subjects and patients with non-severe or severe asthma were treated with the β2-AR expression and were relatively insensitive to salmeterol but not to 8-Br-cAMP or rolipram. Dexamethasone increased β2-AR expression and enhanced the inhibitory effect of salmeterol on severe asthma fibrocyte differentiation.Salmeterol reduced the proliferation, myofibroblastic differentiation and CCR7 expression of fibrocytes from healthy subjects and non-severe asthma patients. Fibrocytes from severe asthma patients had a lower baseline surface βFibrocytes from patients with severe asthma are relatively insensitive to the inhibitory effects of salmeterol, an effect which is reversed by combination with corticosteroids.The online version of this article (10.1186/s12931-017-0678-7) contains supplementary material, which is available to authorized users. The CC 2-AR activation induces the production of 3′,5′-cyclic adenosine monophosphate (cAMP) by adenylyl cyclase leading to airway smooth muscle relaxation [2-AR agonists also exert other effects on cellular function such as inhibition of proliferation, α–SMA expression and collagen synthesis in pulmonary fibroblasts [2-AR agonists [The use of LABAs in conjunction with inhaled corticosteroids results in further improvement of symptoms and lung function . β2-AR alaxation . β2-AR aroblasts , 12. Coragonists .2-AR activation has differential effects on the function of fibrocytes from severe asthma patients compared to healthy subjects and non-severe asthma patients. We determined the effect of the β2-AR agonist, salmeterol, in the presence or absence of the corticosteroid dexamethasone on the number, differentiation potential, and CCR7 and β2-AR expression in fibrocytes from patients with non-severe and severe asthma and healthy subjects. The involvement of cAMP in the downstream signalling was investigated using the phosphodiesterase (PDE) 4 inhibitor rolipram and the cAMP analogue 8-bromoadenosine 3′,5′-cyclic monophosphate (8-Br-cAMP) on fibrocytes in cultured non-adherent non-T (NANT) cells from severe asthmatic subjects.We hypothesised that βPatients with severe and non-severe asthma and healthy subjects were recruited as previously described , accordi+ MicroBeads according to the manufacturer’s instructions . The NANT cells were then incubated in Iscove’s Modified Dulbecco’s Medium  supplemented with 30% FBS (Sigma-Aldrich) and 1% BSA in a humidified incubator, at 37∘C with 5% CO2, for 3 days in the presence or absence of treatments. NANT cells were counted on a haemocytometer after Trypan blue staining.NANT cells were isolated as previously described . Briefly+/CD45+), by flow cytometry [+) cells, by staining permeabilised NANT cells with 0.125 μg of PE-conjugated mouse anti-human α–SMA antibody . Cells incubated with isotype-matched antibodies were used as negative controls.Fibrocytes were identified in the NANT cell population, as cells positive for collagen I and CD45  and 0.015 μg of anti-human CD45 antibody for 30 min, followed by a PE–conjugated goat IgG anti-rabbit polyclonal secondary antibody  0.3 μg for 30 min, permeablised and then stained with FITC-conjugated anti-human collagen I antibody 0.5 μg for 30 min.To evaluate the expression of β2-AR in the whole cell, cells were incubated with anti-human CD45 antibody, permeablised, then stained with 1 μg of unconjugated rabbit IgG anti-human β2-AR and 0.5 μg of FITC-conjugated anti-human collagen I antibody for 30 min, followed by 0.5 μ of PE–conjugated goat IgG anti-rabbit polyclonal secondary antibody g for 30 min.To evaluate the expression of β−/PI− were considered live cells, Annexin V+/PI− as early apoptotic and annexin V+/PI+ as late apoptotic cells.NANT cell apoptosis was determined by Annexin V and propidium iodide (PI) staining using the FITC Annexin V/Dead cell apoptosis kit  according to the manufacturer’s instructions. Briefly, following treatment, NANT cells were washed in PBS, incubated with FITC-conjugated annexin V and PI and analysed by flow cytometry. Annexin Vp < 0.05 was considered as statistically significant.Data are presented as mean ± standard error of the mean (SEM). Statistical analysis was carried out using the GraphPad Prism v.5 software package . Intra-group comparisons of more than two conditions were carried out using the Friedman test followed by Dunn’s post-hoc test and pairwise comparisons using a paired t-test. Inter-group comparisons were carried out using the Mann-Whitney test. × 103; 15% ± 2.2) compared to non-severe asthma . For experiments, 1 × 106 NANT cells per well were seeded at day 0. After three days in culture, there was a concomitant increase in the percentage of fibrocytes within each group  the total number of NANT cells were not significantly different between groups, however there were higher percentages of fibrocytes in severe asthma (51 ± 11 −9 - 10−7 M) for 3 days led to a decrease in the number of fibrocytes  and differentiating fibrocytes  from healthy subjects and non-severe asthma patients. Salmeterol did not modulate the percentage of apoptotic NANT cells  led to a concentration-dependent decrease in the number of fibrocytes , which inhibits PDE4-dependent cAMP hydrolysis, alone and in combination with salmeterol reduced fibrocyte number and differentiation  for 3 days led to a reduction in the percentage of fibrocytes positive for surface β2-AR  or the MFI ratio, on the surface  in combination with dexamethasone (10−7 M). For all groups there was an increase in the total the number fibrocytes at day 3 as described above. Although dexamethasone or salmeterol alone had no effect on fibrocyte number and differentiation, the number of differentiating fibrocytes  to cAMP leading to activation of PKA and EPAC-dependent pathways [2-AR activation on the number and differentiation of fibrocytes. These findings are in line with studies showing reduction in proliferation, myofibroblastic differentiation and chemotaxis of fibroblasts by cAMP [2-AR expression or activation.We also show that fibrocytes from severe asthma are less sensitive to the inhibitory effects of salmeterol suggesting that LABAs may be unable to reduce fibrocyte numbers and myofibroblastic differentiation in the airways of patients with severe asthma due to defective βpathways . Increas by cAMP –18. Sign2-AR expression, whilst they have similar whole cell β2-AR expression levels, compared to fibrocytes from healthy subjects. Moreover, salmeterol failed to reduce the surface β2-AR expression in fibrocytes from patients with severe asthma, possibly due to the already low β2-AR surface density. These findings indicate a possible defect in the membrane localisation but not the overall expression of the β2-AR in severe asthma fibrocytes. As this occurs in the absence of salmeterol in vitro, it is possible that this is due to chronic exposure to systemic LABAs or endogenous circulating catecholamines and inflammatory mediators in vivo [2-AR activation triggers its uncoupling from adenylyl cyclase, internalisation of the uncoupled receptors, and increased degradation of internalized receptors, leading to desensitisation [2-AR are subsequently resensitised by recycling back to the cell membrane to interact with agonists [2-AR resensitisation has been shown to cause reduced baseline surface expression and attenuated agonist-induced activation in ASM cells of subjects with fatal asthma [We have demonstrated that severe asthmatic fibrocytes show reduced surface β in vivo , 20. Protisation . Under nagonists . Defectil asthma .2-AR agonist effects by increasing β2-AR density through up-regulation of transcription [2-AR MFI ratio, indicating increased surface receptor density, in response to dexamethasone and salmeterol in severe asthma fibrocytes. Nonetheless, the superior effect of salmeterol and dexamethasone combination may also be a result of potentiation of glucocorticoid receptor (GR) function by salmeterol, as β2-AR agonists are known to induce GR nuclear translocation and modulate phosphorylation of GR, in PBMCs, lung fibroblasts and airway smooth muscle cells [2-AR agonist, formoterol, has been shown to inhibit transforming growth factor (TGF)–β1-induced collagen I synthesis, whilst either drug alone had no effect [2-AR agonists and corticosteroids may be more potent in controlling airway remodelling in severe asthma, than either drug alone.The inhibitory effect of salmeterol on severe asthma fibrocyte differentiation was restored by co-incubation with the corticosteroid, dexamethasone. However, salmeterol and dexamethasone did not modulate the total number of fibrocytes from severe asthma patients in culture and this may be a result of the increased capacity of fibrocytes from patients with severe asthma to survive, as we have previously demonstrated . Resistacription . In agrele cells , 26, 27.o effect . Moreoveo effect . Thus, t2-AR agonist, indacaterol, and the PDE4 inhibitor, roflumilast, inhibits the expression of endothelin-1, connective tissue growth factor and α-SMA, and the secretion of fibronectin from normal human lung fibroblasts, more strongly than with either drug alone [2-AR desensitisation in ASM cells [We showed that rolipram, a first generation selective PDE4 inhibitor , enhanceug alone . SignifiSM cells .2-AR expression and consequently insensitivity to the inhibitory effects of β2-AR agonists. These findings, in conjunction with our previous findings showing corticosteroid insensitivity in severe asthma fibrocytes, highlight a possible mechanism underlining the accumulation of fibrocytes in the circulation and lung tissue of severe asthma patients [2-AR agonists with corticosteroids are more effective in suppressing fibrocyte number and differentiation than each drug alone highlighting the importance of combination therapy in the treatment of remodelling in severe asthma.In this study we have demonstrated that fibrocytes from patients with severe asthma show reduced surface βpatients , 8. MoreAdditional file 1:Supplementary Materials and Methods. (DOCX 19 kb)Additional file 2: Figure S3.+/CD45+; A) and differentiating fibrocytes  within the NANT cell population, were determined in the NANT cells from healthy subjects (n = 8–9) and patients with non-severe (n = 7) or severe asthma (n = 7–9) at day 0 (0 d) or after 3 days (3 d) in the absence or presence of salmeterol (10−9- 10−7 M). Data points represent mean ± SEM. * p < 0.05 and ** p < 0.01 versus vehicle-treated cells for each group. ###p < 0.001 versus healthy group and $$$p < 0.001 versus non-severe asthma group. (PDF 478 kb)Number of fibrocytes and differentiating fibrocytes in NANT cells after culture in the absence or presence of salmeterol. The number of fibrocytes . The percentage of Col I+/α-SMA+ (B) and CD45+/ α-SMA+ cells (D) was determined by flow cytometry. (PDF 112 kb)α–smooth muscle actin-positive cells also express both collagen I and CD45. NANT cells from one healthy subject were harvested after 3 days in culture and stained with antibodies for collagen I (Col I), α- smooth muscle actin (SMA) and CD45 or their respective IgG isotype controls.  were treated with salmeterol (10−8 M) for 3 days and the percentage of live, and early and late apoptotic NANT cells was determined. Bars represent mean ± SEM. (PDF 173 kb)Effect of salmeterol on NANT cell apoptosis. NANT cells from healthy subjects  in the presence or absence of ICI-118,551 (10−5 M) for 3 days. The number of fibrocytes  and differentiating fibrocytes  was determined. Bars represent mean ± SEM.* p < 0.05 and ** p < 0.01. (PDF 69 kb)Effect of ICI-118,551 on salmeterol-mediated reduction in fibrocyte number and differentiation. NANT cells from healthy subjects (n = 6) were treated with salmeterol (10


The mesopores in the CaSiO3 microspheres contributed to the drug delivery property, and the SrFe12O19 particles improved photothermal therapy (PTT) conversion efficacy. With the irradiation of NIR laser, doxorubicin (DOX) was rapidly released from the MCSC/DOX scaffolds. In vitro and in vivo tests demonstrated that the MCSC scaffolds possessed the excellent anti-tumor efficacy via the synergetic effect of DOX drug release and hyperthermia ablation. Moreover, BMP-2/Smad/Runx2 pathway was involved in the MCSC scaffolds promoted proliferation and osteogenic differentiation of human bone marrow stromal cells (hBMSCs). Taken together, the MCSC scaffolds have the ability to promote osteogenesis and enhance synergetic photothermal-chemotherapy against osteosarcoma, indicating MCSC scaffolds may have great application potential for bone tumor-related defects.The development of multifunctional biomaterials to repair bone defects after neoplasm removal and inhibit tumor recurrence remained huge clinical challenges. Here, we demonstrate a kind of innovative and multifunctional magnetic mesoporous calcium sillicate/chitosan (MCSC) porous scaffolds, made of M-type ferrite particles (SrFe The conventional therapeutic strategies for bone tumors include surgical intervention and chemo/radiotherapy, but these approaches often fail to eradicate residual malignant cells, which confer the potential for recurrence3. Additionally, bone defect affects the quality of life in patients receiving surgical resection; chemo/radiotherapy may cause side effects and drug resistance4. Previous studies suggested that residual tumor cells could be effectively killed by controlled drug delivery system mediated photothermal therapy (PTT)6. Local drug delivery systems could facilitate the release of anti-cancer drugs at designated sites with higher local drug concentrations, and minimize the cytotoxicity to normal cells7. Mesoporous CaSiO3 has been widely used for both controlled drug delivery systems and bone repair applications due to good biocompatibility, drug loading efficiency and sustained drug release performance8. The chemotherapeutic drugs loaded-mesoporous CaSiO3 scaffolds may combine bone regenerative abilities with anti-tumor properties. Nevertheless, multifunctional biomaterials with optimal anti-tumor and bone regeneration properties are rarely reported.Bone metastasis has been commonly observed in malignant tumors, notably for patients with breast cancer, lung cancer or kidney cancer11. Conventional photothermal agents mainly include gold nanomaterials13, copper nanomaterials14, carbonnano materials15, near infrared (NIR) dyes17 and magnetic ironoxide nanoparticles19, in which these regimens show good NIR absorption property. Compared with the conventional photothermal agents, the magnetic iron oxide particles exhibited higher NIR absorbance, higher photothermal-conversion efficiency, better thermal conductivity and cytocompatibility19. The NIR irradiation could elevate local temperatures of photothermal particles up to 42~50 °C, thus facilitating tumor hyperthermia ablation20. Moreover, the photothermal treatment can trigger the rapid release of chemotherapeutic drugs from the scaffolds21, and promote cell membrane permeability of drug incorporation22. Therefore, it could be inferred that the photothermal agents could synergize with chemotherapy to drive potent anti-tumor responses for malignant cells.PTT has been shown to be an effective, non-invasive and low cytotoxicity strategy to kill tumor cells3, bioglass (BG), poly (methyl methacrylate) (PMMA) and chitosan (CS), possess desirable osteoconductivity, but their osteoinductivity is insufficient24. Previous study reported that the use of static magnetic fields (SMF) could stimulate osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (hBMSCs) in vitro and initiate early bone formation in vivo as indicated by the upregulation of osteogenic markers, such as alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2), collagen1a1 (COL1a1), osteocalcin (OCN), osteonectin (ON), osteopontin (OPN), and osterix (OSX)25. In addition, magnetic nanoparticles loaded biopolymer scaffolds promoted osteoblastic cells adhesion and differentiation and bone formation in vivo26. However, the current magnetic iron oxide nanoparticles (such as Fe3O4) exhibit too weak magnetism to represent remarkable osteogenesis effect. Therefore, the scaffold incorporated with M-type ferrite particles may overcome these drawbacks, and may be considered a promising biomatrix for bone engineering.The commonly utilized bone repair materials, including hydroxyapatite (HA), CaSiO28. SrFe12O19 is one of the M-type ferrite materials showing strong intrinsic magnetic potential29. Here, we introduced a novel multifunctional magnetic mesoporous calcium sillicate/chitosan porous scaffold, which is made of SrFe12O19, CaSiO3 and CS, possessing both potent anti-tumor and osteogenesis regeneration properties  and bone tissue were obtained from four donors who gave their written informed consent.2/g and 0.41 cm3/g, respectively . The emission scanning electron microscopy (SEM) analysis revealed that the sizes of microspheres were around 200 nm Fig. . Additioely Fig. .Figure 23 microspheres and iron particles were uniformly distributed within and/or on CS films. The calcium (Ca) was derived from the CaSiO3 microspheres  scaffolds were assessed. The MCSC scaffolds with the ferrite/calcium silicate mass ratio at 1:7 and 1:3 were named as MCSC 1:7 and MCSC 1:3, respectively. The three-dimensional interconnected macroporous structure of CSC Fig. 1, MCSC 1res Fig. 1–3, and O19 Fig. 2–3.Figur3 was non-crystalline and CS was semi-crystalline kind of material, therefore, only a broad peak at 2θ = 22° was detected for the CSC porous scaffolds  was 61.66 emu/g and 919 Oe, respectively  were greater than the MCSC 1:7 scaffolds (6.10 emu/g), indicating the ratio of ferrite particles in the scaffolds was positively related to magnetization  assay. As the CaSiOlds Fig. . After tlds Fig. . Then, fely Fig. . After tely Fig. . The sation Fig. . Moreoveion Fig. . The higCompared with CSC scaffolds, the MCSC scaffolds possessed better photothermal conversion efficiency Fig. . With thin vitro and in vivo. After incubation with MCSC 1:7 and MCSC 1:3 scaffolds for 24 h, MG-63 cells exhibited high cell viability indicating that both MCSC 1:7 and MCSC 1:3 scaffolds had good biocompatibility  and heat shock proteins38 as well as decreasing concentrations of metabolites associated with cell proliferation and tumor growth39. Further studies are needed to explore the mechanism of PTT synergy with MCSC scaffolds in inhibiting tumor cell proliferation.In order to eradicate the residual tumor cells to avoid tumor recurrence, we employed the strategies of drug delivery system and photothermal therapy. It was noted that NIR promoted the DOX release from MCSC scaffolds and the cumulative release ratio is 79.3% at 24 h, indicating anti-tumor drugs could be rapidly released locally to combat malignant cells and could potentially minimize the side effects caused by chemo-regimens. This phenomenon could possibly due to accelerated Brownian movement and the accelerated degradation of the chitosan matrix due to the increased temperatures in the particle surrounding, as suggested by a previous studylds Fig. . In vivo40. In this study, we incorporated SrFe12O19 ferrite particles into the CSC scaffolds, resulting in the formation of MCSC porous scaffolds. COL1, OCN, Runx2 and ALP are essential for osteogenesis and their upregulation is a positive indicator for osteogenesis42. Interestingly, the MCSC scaffolds significantly augmented the expression of these indicators for hBMSCs, suggesting MCSC scaffolds have a potential in promoting bone formation. In vivo analysis revealed that the markers for bone formation such as BV/TV and BMD were increased. Micro-CT scan, Van Gieson’s picrofuchsin staining and calcein fluorescence assay further proved that hBMSCs loaded MCSC scaffolds could effectively promote osteogenesis. The role of BMP/Smad signaling pathway in bone formation has been widely acknowledged in bone formation44 and Runx2 has been reported to the downstream mediator for Smad1/5 45. The present study revealed that the expression of BMP, Smad1/5 and Runx2 was upregulated in hBMSCs loaded MCSC scaffolds. These data indicate that BMP/Smad/Runx2 signaling was, at least in part, involved in the magnetic scaffolds promoted osteogenic differentiation in hBMSCs. Furthermore, we observed that the higher ratio of ferrite particles, the higher expression of osteogenic proteins and bone formation. It was reported that composites containing SrFe12O19 can potentially better facilitate bone formation46, which was in line with this study. Therefore, we could reasonably infer that M-type ferrite particles, at least SrFe12O19, had the potential to contribute to osteogenesis, and more studies are warranted to unveil the underlying mechanism.The CSC could support the adhesion, spreading and proliferation of hBMSCs Fig. , but itsin vitro and in vivo. Nevertheless, more studies are needed to evaluate the efficacy and application of MCSC in clinics for patients with bone defects due to bone tumor resection.This study suggested that the MCSC scaffolds could load anti-tumor drugs and synergy with PTT to combat tumor cells. Additionally, MCSC scaffolds could enhance the proliferation of hBMSCs and promote bone formation. These results strongly suggest that MCSC scaffolds are bi-functional and may have a potential role in patients with surgical resection of malignant bone tumors treatment. In summary, we have derived pre-clinical data supporting a role for MCSC scaffolds in harbouring potent anti-tumor and bone regeneration capability both 3·H2O, 3.20 ml) were dissolved in deionized water followed by stirring for 30 min. Under vigorous agitation, tetraethyl orthosilicate  and 9.73 g calcium nitrate tetrahydrate were successively dissolved in the above solution, and were further aged for 20 h. The precipitates  were washed by deionized water, dried at 80 °C and calcined at 550 °C for 4 h.Mesoporous calcium silicate microspheres were prepared by using organic template method. In brief, cetyltrimethyl ammonium bromide  and ammonium hydroxide (NH12O19) particles with approximately 0.5 μm were obtained from Hengdian Group DMEGC Magnetics Co., Ltd. The ferrite powders (0.25 or 0.125 g) and mesoporous calcium silicate microspheres (0.75 or 0.875 g) were added into CS solutions . The mixtures were injected in the cylindrical mold with a diameter of 15.6 mm and height of 19.0 mm. Under external strong magnetic fields, the samples were frozen at −20 °C in a deep freezer . The solidified mixtures were freeze-dried at −35 to −50 °C for 48 h by a in a freeze-drying machine . The MCSC scaffolds were neutralized by a NaOH solution (10.0 wt%) for 24 h, and then washed with deionized water. Finally, the MCSC scaffolds were magnetized by a magnetization machine , resulting in the same magnetic domain directions and high magnetic fields. The mesoporous calcium silicate/CS (CSC) scaffolds which served as control group were fabricated under the same condition without ferrite powders. The MCSC scaffolds with the ferrite/calcium silicate mass ratio at 1:7 and 1:3 were named as MCSC 1:7 and MCSC 1:3, respectively.M-type ferrite  were characterized by field-emission scanning electron microscopy (FESEM) with energy-dispersive spertrometry . The porous structures of mesoporous calcium silicate microspheres were characterized by transmission electron microscopy . N2 adsorption-desorption isotherms of mesoporous BG microspheres of mesoporous microspheres were determined by automatic surface area and porosity analyzer  at 77 K. The crystalline structure of the scaffolds was determined by X-ray power diffraction , and the functional groups were analyzed by Fourier transform infrared . The magnetic properties of magnetic scaffolds were analyzed by vibrating sample magnetometer .A DOX solution with a concentration of 3.75 mg/l was prepared by dissolving DOX in distilled water. The DOX loading were carried out by the injection of 200 μl DOX solution in scaffolds, followed by freeze-drying in a freeze-drier machine . The CSC, MCSC 1:3 and MCSC 1:7 porous scaffolds loaded with DOX were denoted as CSC/DOX, MCSC 1:3/DOX and MCSC 1:7/DOX, respectively.2HPO4·12H2O and 0.8894 g NaH2PO4·2H2O into 1000 mL distilled water. A NIR laser was fixed 15 cm higher than the center of the scaffolds. Under the irradiation of NIR , the temperatures of the media with the scaffolds were detected by thermocouple thermometer . The NIR-light-triggered drug release tests of MCSC 1:3/DOX and MCSC 1:7/DOX scaffolds were performed by the immersion of DOX loaded scaffold in 5 ml PBS solution under orbital shaking of 80 rpm. In brief, the probe of NIR laser  was fixed 15 cm higher from the center of the scaffolds. At given time intervals, the scaffolds were irradiated by NIR laser for 6 min. Then 1.0 ml DOX-release medium was extracted and supplemented with 1.0 ml fresh PBS. The DOX concentrations of the DOX-release medium were characterized by fluorescence spectrophotometer  using a xenon lamp as excitation source (λ = 495 nm). The in vitro drug release of the MCSC 1:7/DOX or MCSC 1:3/DOX scaffolds without NIR irradiation was performed with similar procedures.A PBS solution with pH value of 7.4 was prepared by adding 5.8032 g Na2 at 37 °C and subject for anti-tumor analyses.The human MG-63 and MNNG osteosarcoma cell line was obtained from ATCC, cultured with high glucose DMEM (Gibco) containing 10% FBS (Gibco) and 1/100 penicillin-streptomycin (Gibco). All cells were kept in humidified atmosphere containing 5% CO6 MG-63 cells were incubated with MCSC 1:7, MCSC 1:7/DOX, MCSC 1:3 or MCSC 1:3/DOX scaffolds for 24 h, then cells were irradiated by NIR laser with a power density of  for 3 min or 6 min and cultured for another 24 h. Cells were without irradiation were served as control. Then cell viability was evaluated by cell counting kit-8  according to manufacturer’s instructions.A number of 1 ×10in vitro analyses was 15 mm × 2 mm and the amount of the DOX was 150 ng.As for irradiation twice, the MG-63 cells were first irradiated by NIR for 6 min post 24 h treatment of MCSC 1:7, MCSC 1:7/DOX, MCSC 1:3 or MCSC 1:3/DOX scaffolds, cultured for another 24 h and cells were irradiated the second time for 6 min. The size of MCSC scaffolds for all 6 MG-63 cells were seeded in six-well-plate, 24 h later, cells were incubated with MCSC scaffolds, MCSC/DOX scaffolds for another 24 h with or without 6 min’s NIR irradiation . The live and dead cells were analyzed according to manufacturer’s instruction  and observed under an inverted fluorescence microscope .A number of 2×106 MNNG cells were seeded in a six well plate and cultured for 24 h. Then lentivirus containing EGFP at a multiplicity of infection (MOI) of 150 plaque-forming units (PFU)  were incubated with MNNG cells. The next day, the culture medium was replaced with fresh medium and the fluorescence density was evaluated under inverted fluorescence microscope .Approximately 1 × 1033. In brief, the osteosarcoma MNNG (5 × 106) cells with or without EGFR transfection were subcutaneously inoculated into the left flank of nude mice . When tumors were palpable, the mice were randomly divided into MCSC 1:3 scaffolds, MCSC 1:3/DOX scaffolds with laser irradiation, MCSC 1:3 scaffolds and MCSC 1:3/DOX scaffolds without irradiation groups (n = 5/group). A skin incision was made at the edge of the tumor and then the scaffold  was implanted.The xenograft mouse model bearing osteosarcoma cells was constructed as previously described2) for 6 min post scaffolds implantation. Meanwhile, the temperature in the tumor was monitored by real-time IR thermal imaging system. The first NIR treatment time was regarded as day 0. From day 0, tumor sizes were measured with calipers and their volumes were calculated by the following the formula: (length × width2)/2. V0 is the initial tumor volume-scaffold volume at day 0. The relative tumor size = V/V0. Whole-body fluorescent imaging was performed at day 0 and day 12. The mice were sacrificed at the 12th day, and their tumors were immersed in 4% neutral buffered formalin for 24 h, then the tissues were embedded in paraffin and cut to 5 μM sections. Afterwards, the sections were dehydrated with gradient alcohol and xylene, then they were subjected for hematoxylin-eosin (H&E) staining according to manufacturer’s instructions .For laser irradiation, the mice were exposed to the NIR laser  was measured according to the formula, TCNR = the number of necrotic cells/the total cell number × 100%. The size of MCSC scaffolds for all 47. hBMSCs were cultured in α-MEM , supplemented with 10% fetal bovine serum  and 1/100 penicillin-streptomycin (Gibco), cells were maintained in humidified atmosphere containing 5% CO2 at 37 °C and hBMSCs at 3–5 passages were used for bone regeneration analysis.hBMSCs were harvested from healthy donors as previously described4 hBMSCs at its third passage were seeded in a 24-well culture plate and incubated scaffolds. To evaluate the cell attachment and morphology in CSC, MCSC 1:7 and MCSC 1:3 scaffolds at 1, 3 and 7 days, the scaffolds were fixed in 2.5% glutaraldehyde for 20 min, washed with PBS for three times, dehydrated in a graded ethanol and hexamethyldisilizane. Then dehydrated cellular scaffolds were coated with gold and observed by SEM .A number of 1.0 × 10−ΔΔCt method and actin was used as the reference gene. The primers for RT-PCR are listed in Table After hBMSCs were treated with the scaffolds for 14 days, then total RNA was extracted using the TRIzol reagent . cDNA was reverse-transcribed using SuperScript First-Strand Synthesis System . The expression of ALP, Runx2, OCN and COL1 was measured by RT-PCR using FastStart Universal SYBR Green Master  in a ViiA 7 Real-Time PCR System . The procedure for this reaction is 95 °C, 5 min; 34 cycles for 95 °C, 5 min and 60 °C, 40 s; and 72 °C, 5 min. The data was analyzed by 2The hBMSCs were treated with scaffolds for 14 days, then the Cells were lysed in lysis buffer supplemented with protease and phosphatase inhibitor . Protein concentration was assessed by BCA . Equal amount of protein (20 μg/lane) was separated by 8–12% SDS-PAGE. Then protein was transferred to PVDF membrane , the non-specific signals were blocked with 5% (w/v) skim-milk at room temperature for 1 h. Afterwards, primary antibodies against GAPDH , BMP-2, p-Smad1/5  and Runx2  were incubated overnight at 4 °C. Horseradish peroxidase (HRP)-conjugated polyclonal goat secondary antibodies were then incubated for 2 h at room temperature. The protein bands were detected by electrochemiluminescence (ECL) method . GAPDH was served as internal reference.Twenty Sprague-Dawley male rats (300–350 g) obtained from Silaike  were used to establish the bilateral critical-sized calvarial-defect rat model (n = 5/group). Two parietal defects were created , then either CSC, MCSC 1:7 and MCSC 1:3 scaffolds were implanted into the defect and the incisions were closed. A polychrome sequential fluorescent labeling method was performed to observe bone formation and mineralization on rats at week 12 post-treatment. At 2, 4 and 6 weeks after implantation, fluorochromes  were injected (i.p.) to rats. At 12 weeks post- implantation, the rats were sacrificed and micro-CT . Three dimention (3-D) images were reconstructed using the 3-D Creator software. Besides bone volume to total bone volume (BV/TV) and local bone mineral densities (BMD).After dehydration in ascending concentrations of alcohol the undecalcified specimens were embedded in poly-methylmethacrylate and 150 mm thick sections in the orientation of the sagittal surface were obtained using a microtome . The sections were observed for fluorescent labeling using a confocal microscope . The excitation/emission wavelengths of the chelating fluorochromes used were 405/560–590 nm , 543/580–670 nm  and 488/500–550 nm . Histological analyses were carried out by Van Gieson’s picrofuchsin staining. With a computer-based image analysis system , the area of new bone was quantified as a percentage of the total bone defect area.All statistical analyses were performed using SPSS 20.0 . All the data were expressed as means ± standard deviation (SD) and were analyzed using one-way ANOVA with a post hoc test. A two-tailed p-value < 0.05 was considered statistically significant.


Glioblastoma (GBM) is a rapidly progressive brain cancer that exploits the neural microenvironment, and particularly blood vessels, for selective growth and survival. Anti-angiogenic agents such as the vascular endothelial growth factor-A (VEGF-A) blocking antibody bevacizumab yield short-term benefits to patients due to blood vessel regression and stabilization of vascular permeability. However, tumor recurrence is common, and this is associated with acquired resistance to bevacizumab. The mechanisms that drive acquired resistance and tumor recurrence in response to anti-angiogenic therapy remain largely unknown. Here, we report that Neuropilin-1 (Nrp1) regulates GBM growth and invasion by balancing tumor cell responses to VEGF-A and transforming growth factor βs (TGFβs). Nrp1 is expressed in GBM cells where it promotes TGFβ receptor internalization and signaling via Smad transcription factors. GBM that recur after bevacizumab treatment show down-regulation of Nrp1 expression, indicating that altering the balance between VEGF-A and TGFβ signaling is one mechanism that promotes resistance to anti-angiogenic agents. Collectively, these data reveal that Nrp1 plays a critical role in balancing responsiveness to VEGF-A versus TGFβ to regulate GBM growth, progression, and recurrence after anti-vascular therapy. GBM is a malignant brain cancer that is distinguished from lower grade tumors, in part, by the development of hallmark angiogenesis pathologies, including florid microvascular cell proliferation and breakdown of the intratumoral blood-brain barrier . GBM celThe TGFβ signaling pathway is a major driver of GBM pathogenesis . For exaNrp1 is a co-receptor for multiple secreted factors such as semaphorins , VEGF-A In this study, we analyze the roles of Nrp1 in GBM cell growth and recurrence after anti-angiogenic therapy via activation of TGFβ signaling. Nrp1 expression increases during tumor progression from low-grade to GBM, and is expressed in GBM cell lines and patient samples. Nrp1 acts as a co-receptor with TGFβR2 to enhance TGFβ receptor signaling via Smad3. Additionally, we show that Nrp1 expression is diminished in GBM cells in tumors that acquire resistance to bevacizumab. Hence, understanding how Nrp1-dependent TGFβ signaling drives GBM development and progression will help in identifying new therapeutic targets to counter the deadly growth and invasive behaviors in GBM.Approval for the use of human specimens was obtained from the Institutional Review Board (IRB) at the University of Texas MD Anderson Cancer Center. The IRB waived the requirement for informed consent for previously collected residual tissues from surgical procedures stripped of unique patient identifiers according to the Declaration of Helsinki guidelines. Archived human GBM samples collected before and after bevacizumab treatment were obtained through a protocol approved by the IRB at the University of California San Francisco. All animal procedures and experiments conducted in this study were reviewed and approved by the University of Texas MD Anderson Cancer Center Institutional Animal Care and Use Committee (IACUC).LN229 and U87 GBM cell lines, as well as HEK 293T cells, were purchased from the American Type Culture Collection . GBM cell lines were cultured in DMEM  supplemented with 10% fetal bovine serum  and antibiotics. Nrp1-dpendent LN229 cell proliferation was quantified by counting adherent cells grown in complete media every 24 hours for four consecutive days. Primary human GBM cells from patient samples were cultured in the following growth media: DMEM-F12 (Mediatech), 20 ng/ml EGF and bFGF (Gibco), B27 supplement (Life Technologies) and one unit per ml penicillin-streptomycin (Gibco). After 7 to 10 days spheroids were passaged by trypsin treatment and mechanical disruption using a 1 ml syringe and a 23-gauge needle, and dissociated cells were re-plated in fresh growth media. Nrp1-dpendent GSC growth and survival was quantified using the Alamar Blue viability reagent (Promega). HUVECs and complete growth media were purchased from ScienCell, Inc.http://www.betastasis.com/). The TCGA-based coincidental expression analysis for TGFBR2 and NRP1 in GBM samples was performed using Project Betastasis.Approval for the use of human specimens was obtained from the Institutional Review Board (IRB) at the University of Texas MD Anderson Cancer Center. The IRB waived the requirement for informed consent for previously collected residual tissues from surgical procedures stripped of unique patient identifiers according to the Declaration of Helsinki guidelines. Archived human GBM samples collected before and after bevacizumab treatment were obtained through a protocol approved by the IRB at the University of California San Francisco. TCGA database was queried to compare the expression of Nrp1 between low-grade tumor samples and GBM subtypes. The TCGA database was accessed online through Project Betastasis -approved protocols. The MD Anderson Cancer Center IACUC approved all animal experiments conducted in this study. The IACUC has adopted guidelines to instruct investigators and others working with laboratory animals on the potential for commonly performed procedures to cause pain/stress. NIH Guidelines, PHS Policy, and USDA Regulations require the assessment of the potential for pain or stress in association with any procedure performed on a laboratory animal. Use of animals in research is minimized if in vitro research applications are possible.5 LN229 GBM cells expressing GFP as well as Nrp1 shRNAs (n = 10 mice) or non-targeting control shRNAs (n = 10 mice) as described previously [5 GSC6-27 cells expressing Nrp1 shRNAs (n = 5 mice) or non-targeting control shRNAs (n = 5 mice) were injected intracranially. Mice were anesthetized by intraperitoneal injection of a mixture of Ketamine/ Xylazine/ Acepromazine (100mg/kg + 2.5mg/kg + 2.5mg/kg body weight) using a 1 ml syringe and sterile 22 G needle. At these dosages the mice remain deeply anesthetized for approximately 30 minutes, which allows sufficient time to perform the intracranial injections. At 5 minutes post-anesthesia, lower limb reflexivity was checked: deeply anesthetized animals will not response to a toe pinch. Corneal reflex was also be used to assess depth of anesthesia during surgery. After surgery, mouse were kept warm with gauze pads and a heat lamp and monitored until recovering fully from the procedure. No animals were exposed to unreasonable pain or distress. Analgesia in the form of subcutaneous injections of Buprenorphine SR was administered as necessary based on routine veterinary care guidelines. Mice were monitored daily for the development of brain tumor-related deficits, including lack of coordination, lethargy and cachexia. Upon the development of tumor-related deficits in the first animal, all mice in the cohort were immediately euthanized by intraperitoneal injection of a lethal dose of Avertin (1 mg/10g body weight). The chest cavity was exposed by an incision above the diaphragm. A 22G needle was inserted into the left ventricle followed by continued perfusion with 4% PFA/PBS. Brains were post-fixed overnight followed by experimental analyses.Healthy male NCR-nu/nu mice between 6–12 weeks of age were purchased from Jackson Laboratories. NCR-nu/nu mice were injected intracranially with 2 x 10eviously . Alterna5 U87 GBM cells were implanted into the flank of NCR-nu/nu athymic mice (n = 5) in a 100 μl suspension of growth factor-reduced matrigel (BD Biosciences) and serum free culture media. To develop U87-bev and U87-IgG xenografts, five subcutaneous U87 tumors were treated with bevacizumab (10 mg/kg) or IgG from human serum (Sigma) for four weeks. The largest tumor was extracted, dissociated and then reinjected subcutaneously (n = 5 NCR-nu/nu mice), at which point the process was repeated two more times. The final xenografts were named U87-bev and U87-IgG based on the agent they had been serially treated with. Mice with tumors were monitored daily. Euthanasia occurred when mice reached institutional euthanasia criteria . These animal procedures were approved by the IACUC at MD Anderson Cancer Center. The final tumor lysates collected from bevacizumab-treated and IgG-treated mice represent the bevacizumab-resistant U87-bevR and -sensitive U87-bevS xenografts, respectively.For subcutaneous tumor experiments, 5 x 10Brains were dissected, embedded in paraffin, and sectioned coronally. To compare the size of tumors formed by LN229 cells expressing Nrp1-specific shRNA versus NT shRNA, FFPE brain sections were stained with hematoxylin and eosin. The cross-sectional area of each tumor was measured at its largest point. Measurements from at least five tumors were averaged from either condition. To quantify Nrp1-dependent vascular densities in LN229 GBM-bearing mice (n = 3 mice per cell type injected), numbers of CD31-expressing blood vessels were counted in five randomly selected 20x fields. Alternatively, anti-GFAP immunohistochemical staining intensity was quantified in randomly selected fields from mice harboring tumors formed from LN229 cells (n = 3 control tumors and n = 3 Nrp1 shRNA tumors). We quantified the relative Nrp1-dependent sizes of GSC6-27-generated brain tumors (n = 3 mice per cell type injected) by measuring GFP fluorescent signal with ImageJ in brain sections sliced coronally in 1 mm intervals through the injection point (n = 3 slices per tumor).Immunoblotting was performed on detergent-soluble lysates using standard protocols. The following primary antibodies were used: rabbit anti-pSMAD3 , rabbit anti-total SMAD2/3 , human specific goat anti-Nrp1 , goat anti-mCherry , rabbit anti-Nrp2 , rabbit anti-phospho-Histone H3 , and mouse anti-αν integrin . Immunoblots were overlayed with donkey anti-rabbit 800, donkey anti-mouse 680, and donkey anti-goat 800 secondary antibodies purchased from Licor . Antibodies were added in blocking buffer comprising 3% bovine serum albumin (BSA) in Tris buffer saline containing 0.1% Tween-20 (TBST). Immunofluorescence and immunohistochemistry was performed on formalin-fixed paraffin-embedded (FFPE) tissue according to standard protocols using the following primary antibodies: rat anti-CD34 , goat anti-IBA1 , rabbit anti-GFAP , human-specific goat anti-vimentin , and rabbit anti-TGFβR2 . Alexa-conjugated goat anti-rabbit 488, goat anti-rabbit 594, and goat anti-mouse 594 secondary antibodies (1:200) were used. Antibodies were incubated in blocking buffer comprising 1% bovine serum albumin (BSA) in phosphate buffer saline containing 0.1% TritonX-100 (PBST).Nrp1 expression was analyzed in FFPE human tissue samples by incubating slides with in PBS containing 20 μg/ml Proteinase K for 20 minutes at room temperature. Primary antibody overlay was performed with rabbit anti-Nrp1 , which reacts with Nrp1 from multiple species, in blocking buffer comprising 10% swine serum in PBST. Slides were incubated in PBS containing 3% hydrogen peroxide, and then incubated with swine anti-rabbit biotinylated  secondary antibody in blocking buffer. Tissue staining was visualized using the Vector Labs Vectastain ABC (cat#PK-4000) and Vector Labs DAB (cat#SK-4100) reagent kits, and counterstained with hematoxylin. Images of stained tissue sections and cells were captured using an Olympus confocal microscope.5' TGT GCC AAA GAT GTC AGA GA 3' and 5' ACC TGG TGT TTT CTG TCC AC 3'.pGIPZ lentiviral vectors encoding human Nrp1-specific or non-targeting (NT) shRNAs (Dharmacon) have been described elsewhere . PlasmidLN229 cells were seeded onto 60mm plates and serum starved overnight. Cells were treated with 0.1 ng/ml rhTGFβ1 (R&D cat#240-B-002/CF) for 0, 15, and 30 minutes. For VEGF-A competition experiments, 100 ng/ml rhVEGFA (Biolegend cat#583704) was used. Cells were lysed in 50 mM Tris, pH 7.4, 150 mM NaCl, 1% NP40, 1 mM EDTA containing a cocktail of protease and phosphatase inhibitors (Roche), and analyzed by western blotting. Band densities were quantified using ImageJ software.3 cells per 8-well chamber slide). Cells were serum starved overnight, treated with 5 ng/ml TGFβ1 for 0, 5, 15, and 30 minutes. After stimulation cells were immediately fixed with 4% PFA/PBS in the absence of detergent to prevent membrane permeabilization. Anti-TGFβR2 primary antibodies and goat anti-rabbit Alexa594-conjugated secondary antibodies were use to label TGFβR2 on the cell surface. Three independent experiments were performed and 3 wells were stimulated with TGFβ1 for each time point. Anti-TGFβR2 fluorescence signal was quantified at each time point for at least 10 randomly selected cells (based on DAPI staining) per well. 80x magnification images were taken for analysis. ImageJ software was used to calculate the average TGFβR2 staining intensity per cell.LN229 GBM cells expressing GFP as well as control shRNAs or Nrp1 shRNAs were plated on laminin-coated slides .To determine functions for Nrp1 in GBM we first analyzed levels of Nrp1 gene expression in human brain tumor samples by querying The Cancer Genome Atlas (TCGA) GBM database. As shown in Next, we used an anti-Nrp1 antibody to immunoblot detergent-soluble lysates from a non-cancerous brain tissue sample or from six different primary GBM samples. Nrp1 protein levels were elevated in five of the six tumor lysates analyzed . We alsoWe also compared Nrp1 and Nrp2 protein levels in six different human GBM cell lines. As shown in In addition to analyzing Nrp1 regulation of tumor growth in the LN229 GBM cell line, we also analyzed primary stem-like GBM cells (GSCs) derived from patient tumors. These cells growth as neurosphere-like spheroids in serum-free media and generate highly malignant tumors in the mouse brain . We detePrior reports have shown that Nrp1 can serve as a TGFβ co-receptor and modulate TGFβ receptor signaling in various cell types . TransfePrior reports have shown that Nrp1 can modulate canonical TGFβ receptor signaling in various cell types . To deteElevated VEGF-A levels and receptor signaling correlate with uncontrolled angiogenesis and hemorrhage in GBM . Activatin vivo for acquired bevacizumab resistance [S) versus bevacizumab-resistant (U87-bevR) GBM cells. In comparison to U87-bevS tumors, human Nrp1 protein was significantly down regulated in all four U87-bevR tumor xenografts, as revealed by immunoblotting lysates with a human-specific anti-Nrp1 antibody . As shown in Collectively, these data reveal that tumor growth, progression and recurrence following anti-vascular therapies such as bevacizumab are dependent, in part, on the Nrp1-TGFβ signaling pathway. We propose a model in which Nrp1 promotes interactions between GBM cells and blood vessels in the brain tumor microenvironment via the TGFβ pathway as well as VEGF-A. This leads to differential Nrp1-dependent TGFβ receptor signaling in tumor cells versus endothelial cells and possibly other stromal cells. Diminished expression of Nrp1 in GBM cells leads to deregulation of this signaling balance, leading to VEGF-A independent angiogenesis and tumor recurrence following bevacizumab treatment .Here, we have analyzed functions for Nrp1 in GBM growth, progression and recurrence after anti-angiogenic therapy. Our data reveal the following novel findings: (i) NRP1 RNA and protein expression increases as brain tumors progress from low grade to high grade GBM ; (ii) RNNrp1 as a TGFβ receptor signaling component has been reported previously in fibroblasts, endothelial cells and immune cells, and has been implicated as a driver of tumor progression , 40. DesNrp1-dependent angiogenesis pathologies in GBM are more likely related to latent-TGFβ activation and signaling. In GBM cells Nrp1 promotes TGFβ signaling, whereas in endothelial cells Nrp1 suppresses TGFβ signaling. Hence, the exact cellular context of the Nrp1 interaction with TGFβ receptors plays a critical role in determining TGFβ signaling outputs. A prior report has shown that cis interactions between Nrp1 and VEGF receptors can enhance signaling, whereas trans interactions with Nrp1 can inhibit VEGF receptor signaling by promoting internalization . InteresNrp1-mediated TGFβ signaling in GBM cells is competitively blocked by treatment with VEGF-A. These results match other reports showing that VEGF-A and TGFβ ligands compete for binding to Nrp1 . AutocriPatients treated with the anti-VEGF-A inhibitory antibody bevacizumab or other anti-angiogenic drugs that target the VEGF-A pathway show improvements in progression-free survival. However, overall patient survival rates have not improved due to acquired resistance and tumor recurrence. A significant percentage of patients with recurrent GBM develop unusually robust patterns of invasive cell growth , 43. MolLastly, these data showing important roles for Nrp1 signaling in GBM cells and effects on blood vessels in the tumor microenvironment have direct links to what has been reported in brain vascular development. Our group and others have demonstrated that αvβ8 integrin and its latent TGFβ ECM protein ligands are essential for angiogenesis in the developing brain –51. TGFβS1 Fig(A); Analysis of GBM TCGA database reveal that NRP2 RNA expression levels are not statistically different in GBM versus normal brain or lower grade brain tumors. (B); NRP2 is enriched in mesenchymal GBM samples based on analysis of the GBM TCGA database. (C); Comparative expression of NRP1 and NRP2 in various tumor regions based on IVY GBM database queries. (D); Overall survival plot taken from TCGA dataset for patients with primary GBM. Patients were segregated based on high levels of NRP1 expression (n = 195) versus low levels of NRP1 expression (n = 153). Note that high versus low levels of NRP1 mRNA do not lead to statistical differences in overall patient survival.(JPG)Click here for additional data file.S2 Fig(A-D); Immunohistochemistry stains of two different GBM samples using control IgG  an anti-Nrp1 antibody  reveals specificity of the anti-Nrp1 antibody.(JPG)Click here for additional data file.S3 Fig(A-C); Formalin fixed paraffin embedded sections through cerebral cortices of the human fetal brain were immunohistochemically labeled with anti-Nrp1 antibodies  or control IgG (C). Note that Nrp1 protein is expressed mainly in intracerebral blood vessels (arrows) in the developing human brain. Scales bars, 50 μm.(JPG)Click here for additional data file.S4 Fig(A); Nrp1-dependent proliferation was quantified in cells expressing control (NT) shRNAs or Nrp1 shRNAs by counting cell number every 24 hours over 4 days. Note that silencing Nrp1 expression does not impact LN229 cell proliferation in vitro. (B-E); Intracranial implantation on LN229 cells reveals a striking Nrp1-dependent difference in GBM cell growth. Shown are representative images, revealing that Nrp1 silencing leads to more robust tumor cell growth as revealed by H&E staining coronal brain sections. Note the hemorrhage within the tumors derived from Nrp1 shRNA cells (arrows). Panels D, E are higher magnification images of boxed areas in B, C. (F); Quantitation of Nrp1-dependent GBM growth in vivo, revealing that LN229 cells expressing Nrp1 shRNAs generate intracranial tumors that are nearly twice as large as control tumors. Error bars represent standard deviation, ***p<0.001 for Nrp1 shRNA versus control shRNA. ; Analysis of Nrp1-dependent proliferation as determined by double immunofluorescence with anti-vimentin to label GBM cells (green) and anti-pS10 Histone H3 to identify mitotic cells (red) in control and Nrp1 shRNA orthotopic brain tumors. (I); Quantitation of Nrp1-dependent GBM cell proliferation as determined by counting vimentin-expressing tumor cells that are also immunoreactive for pS10 Histone H3. For these experiments we analyzed 5 randomly selected fields in tumors expressing control shRNAs or Nrp1 shRNAs. There are no statistically significant Nrp1-dependent differences in tumor cell proliferation.(JPG)Click here for additional data file.S5 Fig(A-D); Margins of intracranial tumors formed from LN229 cells expressing control shRNAs or shRNAs targeting Nrp1 were labeled with antibodies recognizing human vimentin to visualize tumor cells and GFAP to visualize astrocytes . Alternatively tumor sections were labeled with anti-vimentin to image tumor cells in combination with anti-Iba1 to visualize astrocytes and microglial cells .(JPG)Click here for additional data file.S6 Fig(A); Anti-Nrp1 immunoblot of six different primary GSC cultures reveals varying levels of Nrp1 protein expression. (B); Lentivirus expressing non-targeting control shRNAs or Nrp1 shRNAs were used to silence Nrp1 expression in GSC7-2 cells, as revealed by anti-Nrp1 immunoblots. (C); Images of GSCs expressing GFP in combination with control shRNAs or Nrp1 shRNAs. (D); GSC proliferation assay results using the Alamar Blue reagent reveals no Nrp1-dependent growth differences in GSCs. ; Images of mouse brains harboring tumors generated from GSC7-2 cells expressing control shRNAs (D) or shRNAs targeting Nrp1 (E), imaged by bright field microscopy (top) or with GFP fluorescence (bottom). (G); Nrp1-dependent brain tumor volumes were quantified by measuring GFP fluorescence intensity in coronal slices from tumors derived from GSC7-2 expressing control shRNAs (n = 3) or Nrp1 shRNAs (n = 3), *p<0.05 for Nrp1 shRNA versus control shRNA.(JPG)Click here for additional data file.S7 Fig(A); LN229 cells expressing control shRNAs or shRNAs targeting Nrp1 were stimulated with TGFβ1 for varying times, and Smad3 phosphorylation was analyzed by immunoblotting. (B); Quantitation of Nrp1-dependent canonical TGFβ signaling based on one representative immunoblot. Note that RNAi-mediated silencing of Nrp1 leads to reduced Smad3 phosphorylation in response to TGFβ1. (C); Detergent-soluble lysates from non-transfected HEK-293T cells were treated with 5 ng/ml TGFβ1 for varying times. Detergent-soluble lysates were immunoblotted with anti-Nrp1, anti-pSmad2 and anti-pSmad3 antibodies. (D); HEK-293T cells transiently transfected with a pcDNA3.1 plasmid to overexpress Nrp1 and then stimulated with TGFβ1 for varying times. Detergent-soluble lysates were immunoblotted with anti-Nrp1, anti-pSmad2 and anti-pSmad3 antibodies. Note the time-dependent increased levels of Smad2 and Smad3 phosphorylation after Nrp1 overexpression.(JPG)Click here for additional data file.S8 Fig(A); LN229 GBM cells forcibly expressing Cherry (top) or a Nrp1-Cherry fusion protein (bottom) were treated with 5 ng/ml TGFβ1 for varying times. Cell surface levels of TGFβR2 were analyzed by labeling fixed, non-permeabilized cells by immunofluorescence. (B); Quantitation of Nrp1-dependent TGFβR2 cell surface protein levels following TGFβ1 treatment, **p<0.01 for time 0 versus 15 and 30 minutes for Cherry control and **p<0.001 for time 0 versus 5, 15 and 30 minutes for Nrp1-Cherry. (C); Detergent-soluble lysates from LN229 cells expressing control Cherry or Nrp1-Cherry fusion protein were analyzed by immunoblotting. Cells were stimulated TGFβ1 for varying times, and Smad3 phosphorylation was analyzed by immunoblotting. (D); Quantitation of Nrp1-dependent TGFβ signaling via Smad3. Note that forcibly expressing Nrp1 leads to increased enhanced signaling via Smad3.(JPG)Click here for additional data file.S9 Fig(A); Detergent-soluble lysates from HUVECs expressing control shRNAs or Nrp1 shRNAs were immunoblotted with Nrp1 antibodies, revealing diminished Nrp1 expression following RNAi-mediated silencing. (B); HUVECs were stimulated with 5 ng/ml TGFβ1 for varying times and detergent-soluble lysates were immunoblotted with anti-pSmad3 antibodies. Note the increased levels of pSmad3 in the absence of Nrp1. (C); HUVECs were treated with TGFβ1 for varying times and TGFβR2 internalization was quantified by immunofluorescence. Note that TGFβ1 does not induce time-dependent TGFβR2 internalization. (D); HUVECs were labeled with Phalloidin-Alexa594, revealing Nrp1-dependent F-actin cytoskeletal defects.(JPG)Click here for additional data file.


This study was intended to determine if there is variation among physiatrists in referral preferences for postacute rehabilitation for stroke patients based on physician demographic characteristics or geography.A cross-sectional survey study was developed with 5 fictional case vignettes that included information about medical, social, and functional domains. Eighty-six physiatrist residents, fellows, and attendings were asked to select the most appropriate postacute rehabilitation setting and also to rank, by importance, 15 factors influencing the referral decision. Chi-square bivariate analysis was used to analyze the data.Eighty-six surveys were collected over a 3-day period. Bivariate analysis (using chi-square) showed no statistically significant relationship between any of the demographic variables and poststroke rehabilitation preference for any of the cases. The prognosis for functional outcome and quality of postacute facility had the highest mean influence ratings , whereas location of postacute facility and insurance had the lowest mean influence ratings .Physiatrists’ referral preferences did not vary with any identified practitioner variables or geographic region; referral preferences only varied significantly by case. Stroke is the leading cause of serious long-term disability in this country. Given the impact on individuals with stroke and the substantial resources devoted to their care, it is important to gain a greater understanding of which poststroke interventions lead to the best outcomes. One area of controversy is the type of rehabilitation facility where persons with stroke should receive their rehabilitative care.Nearly 800,000 individuals experience a stroke each year in the United States, at a cost of 33.6 billion dollars.Poststroke rehabilitation options include inpatient rehabilitation facilities (IRFs), skilled nursing facilities (SNFs), long-term acute care hospitals (LTACHs), home therapy, and outpatient therapy. The process of assessing rehabilitation needs and selecting the most appropriate rehabilitation option for a person with acute stroke is complex and not well-studied. Depending on the institution, this determination may be made by nurses, case managers, social workers, physical therapists, occupational therapists, speech and language pathologists, and/or physicians . Physiatrists’ role in this process varies among hospitals, with some hospitals involving physiatry routinely, and others rarely or never. Physiatrists’ role includes the medical and functional assessment as it encompasses all of the rehabilitation needs, through a strong relationship within the interdisciplinary rehabilitation team. Physiatrists are arguably the physicians with the most specific training in stroke rehabilitation, and it is therefore important to better understand their referral preferences for these patients.,3 When referral to an IRF is being considered, the question of whether or not a patient will be able to participate in and benefit from the 3 hours of therapy that are mandated in an IRF is of particular concern.Many factors may be considered when determining the most appropriate poststroke rehabilitation option for a given patient. These factors may include the severity and nature of neurological and functional deficits, medical comorbidities, provider and facility relationships, insurance coverage, cost, geographical proximity and location of available facilities, and patient and family preference.Assessment protocols are not standardized, and there is little reassurance that patients are reliably receiving the most appropriate rehabilitation. Furthermore, there exist no standardized criteria or guidelines to assist referral teams in predicting which poststroke discharge option is optimal for each patient. To optimize patient outcomes after stroke, more information is needed about which patients benefit most from rehabilitation in each setting. Knowing who is making these referral decisions and how they are making them is an important first step towards reaching this goal. Measures of activities of daily living (ADLs) ability after stroke are predictive of discharge home versus a rehabilitation institution, but do not distinguish between patients discharged to SNF and patients discharged to IRF.Given the large number of individuals involved in making decisions regarding rehabilitation level of care, and the many factors that contribute to this decision, it is unsurprising that research has found variation in referral patterns. After stroke, patients are more likely to be evaluated for rehabilitation needs if they are hospitalized in a stroke unit. Ilet et al further found that the likelihood of discharge to a rehabilitation unit is influenced by variation in practice among hospitals. Geographic proximity to an IRF has been shown to be a substantial predictor of the likelihood of discharge to IRF. Variation in the utilization and intensity of poststroke rehabilitation services has also been demonstrated by Medicare beneficiaries’ payment analysis.,10When rehabilitation consultation teams assist in making the referral decision, patient outcomes improve.,12 There is also some indication in the literature that patients admitted to IRF experience better functional recovery than those admitted to SNF.–17 To date, studies comparing IRF to SNF outcomes in the United States have all been observational in nature, and no randomized studies have been performed. As a result, comparing IRF to SNF stroke rehabilitation outcomes is complicated by the differences between the patient populations referred for these 2 different types of care. Multiple factors known to influence outcomes after stroke  have also been found to be different in those receiving postacute stroke rehabilitation in IRFs and those receiving this rehabilitation in SNF.Patients who suffer a stroke benefit from early rehabilitation.We sought to examine postacute stroke rehabilitation referral preferences among physiatrists. We hypothesized that there is variation among physiatrists in referral preferences based on demographic variables and/or geographic location, leading to patients with similar backgrounds and functional limitations being referred to different types of rehabilitation. Given that different rehabilitation options have different outcomes, this variation in referral preferences may lead to suboptimal rehabilitation outcomes for some stroke patients.22.1This study was approved by the Institutional Review Board of Columbia University Medical Center. Three of the authors administered a survey to physiatrists attending the American Academy of Physical Medicine and Rehabilitation (AAPM&R) Annual Assembly . Participants were approached by investigators in common areas of the conference  and asked to complete the survey. This sampling method was selected to avoid the low response rates typically seen with surveys distributed electronically or by mail. Participants were given the option of completing the survey on paper or on a computer tablet (via Survey Monkey).The survey collected basic demographic information about respondents and presented 5 fictional poststroke cases. Demographic data included trainee status, board certification, academic affiliation, extent of active involvement in the care of stroke patients, and practice affiliation(s). It also included state of practice, age, and number of years in practice. The 5 case vignettes included information about medical, social, and functional domains as seen in Table 2.2Participants (n = 86) included residents, fellows, and attending physicians out of the possible reported 2638 conference attendees.2.3Demographic variables were collapsed for analysis, including location of practice (grouped into regions using the US Census Bureau categories), age , and years in practice . Statistical analysis was completed using SPSS  Bivariate analysis using the chi-square test was used to assess the relationship between referral preferences and all of the demographic variables. Subsequently, the Cramér V was used to further determine the strength of association between referral pattern and practice affiliation .3Eighty-six surveys were collected over a 3-day period. Demographic data for the 86 respondents are described in Table As shown in Table Figure 4To the best of our knowledge, this is the first published study examining postacute stroke rehabilitation referral preferences by physiatrists in the United States. Although physiatrists are not always part of the referral decision in every medical institution, we believe that their participation is an important component in optimal patient outcomes. Our results indicate that there is substantial practice variation for certain case vignettes, perhaps representing patients whose characteristics overlap “ideal” patient characteristics for 2 or more levels of care. We did not, however, find any regional practice differences in this study, nor did we find other demographic variables that were associated with referral preferences. Whereas we do not have any “gold standard” to determine the appropriate discharge destination for the patients represented in the vignettes, the pattern of referral by respondents seems to favor referral to an IRF to a notable degree.,18 In this study, referral to IRF was generally favored over other options, showing that the majority of physiatrists refer poststroke patients to IRF. This may reflect greater familiarity with IRF care, given the key role that IRFs play in physiatry training and practice. It may also reflect an explicit preference for IRF among physiatrists because of belief in superior care or superior functional outcomes for persons with stroke. It would be interesting to conduct a similar survey of other rehabilitation professionals, such as physical and occupational therapists, whose training and practice may be less IRF-centric.Medicare data have shown that the largest number of stroke patients in the United States receive rehabilitation care in a SNF after discharge (32%), followed by IRF (22%) and home health care (15%).Future study in this field has the potential to lead to standardized assessment tools and protocols for healthcare practitioners who make poststroke referral decisions. Such tools may reduce undesirable variation in care, and help provide the most appropriate level of rehabilitation to stroke survivors.4.1This study has several important limitations. As would be expected in a small survey study, the respondent sample does not represent physiatrists in general. The survey was administered only to a convenience sample of physiatrists with the time, availability, and financial resources to attend the AAPM&R Annual Assembly. Both academic physiatrists and young, male physiatrists who were relatively new to the field were over-represented in the sample. Also, as with any survey study, there is inherent recall bias, and responses may reflect more of an idealized version of practice preferences rather than actual experience. Since some individuals chose not to participate in the survey, and some responders did not complete every question, the data are limited by nonresponder bias. Additionally, whereas the case vignettes were designed to highlight key patient variables that may influence referral decisions, and whereas the respondents rated the importance of different variables in general as seen in Fig. Future research should further clarify which patient factors are most influential for physiatrists (and other providers) as they make referral decisions. Additionally, more information is needed about which patient variables are most predictive of success in different rehabilitation settings. Once these pieces are in place, guidelines can be created to guide practitioners in these decisions.5Physiatrists’ referral preferences did not vary with any identified practitioner variables; referral preferences only varied significantly by case. Further study is needed to determine the patient factors that most influence referral decisions, and to determine which factors should be used to guide referral decisions for optimal patient outcomes.The authors gratefully thank Lauri Bishop DPT, for her advice regarding data analysis.


Androgen Receptor (AR) is a key driver in prostate cancer. Direct targeting of AR has valuable therapeutic potential. However, the lack of disease relevant cellular methodologies capable of discriminating between inhibitors that directly bind AR and those that instead act on AR co-regulators has made identification of novel antagonists challenging. The Cellular Thermal Shift Assay (CETSA) is a technology enabling confirmation of direct target engagement with label-free, endogenous protein in living cells. We report the development of the first high-throughput CETSA assay (CETSA HT) to identify direct AR binders in a prostate cancer cell line endogenously expressing AR. Using this approach, we screened a pharmacology library containing both compounds reported to directly engage AR, and compounds expected to target AR co-regulators. Our results show that CETSA HT exclusively identifies direct AR binders, differentiating them from co-regulator inhibitors where other cellular assays measuring functional responses cannot. Using this CETSA HT approach we can derive apparent binding affinities for a range of AR antagonists, which represent an intracellular measure of antagonist-receptor Ki performed for the first time in a label-free, disease-relevant context. These results highlight the potential of CETSA HT to improve the success rates for novel therapeutic interventions directly targeting AR. As such, the suitability of a molecule for progression as a drug is frequently assessed from indirect functional cellular responses, which can be influenced by interactions with co-regulators and components of associated signalling pathways. In recent years, the absence of direct target engagement technologies has manifested as clinical failure of drugs which have not demonstrated conclusive evidence of binding to the intended target2.A key requirement for a small molecule drug to exert a pharmacological effect is to bind with sufficient affinity and duration to its target protein. Despite this, few options are available to directly measure a compound binding to a protein within the more complex cellular and 6. Hence there is an urgent need for cellular assays which measure direct target engagement in a disease-relevant setting, enabling more predictive translation into clinical efficacy. The Cellular Thermal Shift Assay (CETSA®) is a technology capable of fulfilling these requirements. It relies on the inherent thermal stability of the target protein within the cell, and a change in thermal stability induced upon compound binding7.The low success rate of translating an early drug discovery program into clinical efficacy has led to increased focus on the disease-relevance of screening assays. For cell-based assays, recent reports highlight the drive towards primary cell types, endogenous target expression and label-free systems8. AR is a nuclear hormone receptor which responds to androgens by undergoing a conformational change and translocating to the nucleus where it acts as a transcription factor to modulate gene expression10. AR is modular in structure and is composed of a N-terminal domain, a DNA-binding domain and a ligand-binding domain11, against which several small molecule inhibitors have been developed12. AR’s role in driving prostate cancer was defined following the observation that androgen starvation by castration can halt disease progression. In the majority of cases however, relapse to CRPC is observed, a process reliant on AR-driven transcription13. A range of AR antagonists are in development or approved to treat CRPC, but are hampered by resistance through amplification, truncation or single nucleotide polymorphisms within the AR gene12. Novel AR antagonists able to overcome resistance may offer new, much-needed therapies to treat CRPC.Androgen Receptor (AR) is a well-validated target for the treatment of prostate cancer and a key driver of castration resistant prostate cancer (CRPC)15. Upon activation AR recruits a variety of proteins including numerous epigenetic regulators which act as part of an AR signalling complex to facilitate modulation of gene transcription. Bromodomain-containing proteins such as BRD416 and ATAD217, and epigenetic regulators such as Enhancer of zeste homologue 2 (EZH2)18 and lysine specific demethylase 1 (LSD1)19 influence transcription of androgen-responsive genes, while components of the mixed-lineage leukemia (MLL) complex facilitate AR transcriptional activation via an interaction with menin20. AR-driven transcription is further modulated by post-translational modification mediated through receptor tyrosine kinase signalling10. The variety of factors affecting AR-driven transcription can make interpretation of functional responses difficult21, a common problem for many intracellular drug targets.Current cellular assay technologies typically measure changes in the functional consequence of AR agonism, namely transcription of androgen-responsive genes. However, AR-driven transcription is influenced by co-regulators from a complex network of pathways and interactions. Like other steroid receptors, in the absence of ligand AR is complexed with chaperones including Heat shock protein 90 (Hsp90) and co-chaperones such as p23To facilitate identification of direct AR binders, we sought to apply CETSA to measure direct cellular target engagement. We report the first high-throughput CETSA (CETSA HT) assay for the determination of intracellular target engagement with AR, measured in disease-relevant cell lines derived from primary human prostate cancers. Using this screening-compatible CETSA HT assay we profiled a training set of compounds in parallel with cellular assays measuring AR-driven transcription. We demonstrate the ability of CETSA HT to correlate functional responses with AR target engagement, and more importantly to differentiate cellular responses driven through co-regulators. In addition, we observed distinct AR thermal profiles depending on the receptor ligand occupancy. This enabled ligand competition experiments to determine parameters for apparent intracellular binding affinity. We therefore demonstrate for the first time that CETSA HT can determine intracellular receptor-ligand binding affinity, using label-free compounds, with receptor expressed in its endogenous context within a prostate cancer cell line.The ability to confidently identify direct AR binders, and to derive physiologically relevant measures of intracellular binding affinity, has the potential to expedite the identification of novel and improved AR antagonists for the treatment of CRPC. This method might have broad application in drug discovery.7. To develop a CETSA assay for AR, we first investigated AR thermostability using quantitative western blot as a readout to detect the amount of soluble thermostable protein following heat shock, a methodology henceforth referred to as CETSA Classics. For these experiments we utilised a monoclonal cell line, CWR22Pc-R1-AD124, derived from a primary human prostate cancer, CWR2225. This prostate-cancer derived cell line expresses a single copy of AR, resolved as a single species by western blot . This allows analysis of changes in the thermal stability of cellular AR following compound addition as evidence of target engagement.CETSA is based on measurements of remaining soluble target protein against a background of thermally denatured and precipitated proteins following a heat challenge27 and mass spectrometry analysis confirming intracellular compound accumulation , plate-based CETSA assay compatible with high-throughput applications  using AlphaScreenCETSA)7, whereby ligand concentration was varied in the presence of a heat shock at 46 °C. This temperature provided a suitable window between unbound and agonist-bound AR where the majority of cellular AR was thermally destabilised in the absence of agonist target engagement  for in-cell AR target engagement of 1.7 and 1.2 nM respectively. The partial AR agonist Andrenosterone showed reduced potency of target engagement, with an EC50 of 50.6 nM. The inactive metabolic products of testosterone, dehydroisoandrosterone 3-sulphate and trans-dehydroandrosterone, showed only partial or no target engagement respectively  determined as 666.8 nM and IC50 for ARN-509 determined as 20.6 nM  prior to heatshock at 46 °C. In this format, we observed a concentration-dependent destabilisation of agonist-stabilised AR, which then allowed us to rank relative antagonist potency, with the Enzalutamide 50% inhibitory concentration  Table . To capt33 supporting the concept that our CETSA HT agonist competition assay is able to identify direct AR binders. Our comparative analysis was also able to discriminate compounds that are not reported to directly bind AR but instead are inhibitors of AR co-regulators. While several of these compound classes showed activity in functional AR transcriptional assays, all were inactive in CETSA HT, reinforcing the idea that CETSA HT can exclusively detect direct AR binders.As expected, our screen identified known AR antagonists Enzalutamide, Hydroxyflutamide and MK-2866 as active in both CETSA HT and functional AR transcriptional assays Table . Much ev20. Consistent with this, the menin inhibitors MI-136 and MI-503 reduced ARE luciferase response in the low-to-mid µM range after a 24 hour treatment (Table The menin subunit of the mixed-lineage leukemia (MLL) complex has been reported to act as a co-regulator of ARnt Table . This efnt Table . As expe34. Danusertib and Entrectinib, two kinase inhibitors with reported TrkA activity, and a TrkA inhibitor reported by Bayer36, were active in the ARE-luciferase assay, decreasing DHT-stimulated AR transcriptional responses, but were inactive in CETSA HT  was recently identified as an AR-binding partner and is implicated as a co-regulatorHT Table . The tra37, as has Niclosamide38. The use of a no heat-shock control in the absence of exogenous DHT confirmed reduction in total cellular AR rather than reduction of AR thermal stability caused by competition of DHT . Given that our functional reporter assay was limited to 24 hours to avoid indirect effects on AR-dependent transcription, the lack of AR-dependent transcriptional effects for these inhibitors is not surprising.Compounds within the pharmacology set showing no engagement with AR and no effect on AR-driven transcription included inhibitors of components of the WNT signalling pathway implicated in CRPCIn conclusion, the application of a CETSA HT assay, coupled with a control to measure changes in total AR, allowed differentiation of AR transcriptional responses from AR target engagement. Prominent examples from the pharmacology test set included menin, TrkA, p23 and BRD4 inhibitors Table . These c®. As expected, DHT caused a concentration dependent increase in thermostable AR, demonstrating target engagement with an EC50 of 0.95 nM. The addition of increasing concentrations of Enzalutamide effectively antagonised the DHT-mediated thermal stabilisation, with the highest Enzalutamide concentration, 100 µM, reducing the EC50 of DHT target engagement to 110 nM . Four temperatures were selected from the melt curve Fig.  where a 47. Additionally, quantification of compound binding to AR has been limited by an inability to generate sufficient purified recombinant full-length protein48. CETSA therefore provides a feasable alternative to measure binding to a target where traditional biophysical methods are not possible, for example when recombinant protein cannot be produced.As a measure of cellular target engagement, CETSA offers unique benefits through application to physiologically relevant samples without the requirement for any modifications to either the target protein or the test compound. CETSA assesses engagement with the target in a setting where it is endogenously expressed and is not modified by any tags or reporter genes. The present comparative study demonstrates the advantages of CETSA HT assays for target-driven early drug discovery, where it provides a more precise measure of direct binding than functional cellular assays, in a more physiologically relevant manner than alternative cellular target engagement technologies49. Our application of CETSA Classics to AR revealed that target engagement of antagonist does not modulate AR thermal stability, while target engagement of agonist causes a thermal stabilisation. The thermal stability of AR is only altered when the ligand binding domain is occupied by an agonist. The reasons for this are not clear, although structural data suggests agonists and antagonists can occupy different regions of the ligand binding domain50. However, target engagement of antagonist was able to reverse agonist-mediated thermal stabilisation, presumably through direct competition of agonist from the ligand binding domain as competition experiments show responses typical of competitive binding . Similarly, Hydroxyflutamide and other antagonists did not show any evidence of binding as an agonist in LNCaP cells harbouring the T877A mutation which has also been linked to paradoxical agonism54  supplemented with 10% foetal bovine serum (FBS) in a humidified incubator at 37 °C, 5% CO26 to establish melt curves and ligand induced shifts. Intact, viable cells (CWR22Pc-R1-AD1) were harvested and resuspended to a final concentration of 20 × 106 cells/mL in Hank’s balanced salt solution (Thermo Scientific). Cells were divided into separate aliquots and treated with DMSO control, 100 nM DHT, 100 µM Enzalutamide or by a combination of Enzalutamide (30 or 100 µM) and 100 nM DHT. Following 1 hour incubation at 37 °C, the treated samples were aliquoted into a PCR plate  and heated at temperatures ranging from 37 to 63 °C for 3 minutes , before they were snap frozen in liquid nitrogen. All samples were supplemented with Complete protease inhibitor cocktail (Roche) before lysis by 3x freeze-thaw using liquid nitrogen and a water-bath. Samples were clarified by centrifugation at 20,000xg for 20 minutes, and thereafter 40 µl of each supernatant was collected and mixed with 20 μl gel loading buffer . Protein separation was done using NuPAGE 4–12% BisTris gels (Thermo Scientific), where 10 μl of each sample mixture was loaded per lane, and run in MOPS buffer (Thermo Scientific). Western blots were performed using an iBlot2 device (Thermo Scientific) on nitrocellulose membranes. Transfer was optimised at 25 V for 8 minutes. Blocking and dilution of antibodies were performed in 5% non-fat milk in Tris-Buffered Saline and TWEENTM20. The mouse derived anti-AR antibody  was diluted to 1:1000 and incubated overnight at 4 °C. Specific protein bands of AR were detected using horseradish peroxidase (HRP) conjugated secondary antibody  together with Clarity Western ECL substrate (Bio-Rad Laboratories).CETSA Classics experiments were performed according to the general CETSA protocol® is described in Supplementary Methods and Supplementary Fig. ® reagents to yield optimal signal, as described in Supplementary Fig. The process for identification and optimisation of antibody pairs for quantification of AR by AlphaScreen6 cells/mL in complete media. Cells were split into separate pools and treated with DMSO control, 100 nM 5α-Dihydrotestosterone  or 10 µM Enzalutamide before seeding 20 µL/tube into 24x MicroAmp® 8-tube strips, 0.2 mL PCR tubes . Samples were prepared such that each PCR strip contained 4x compound treated samples and 4x vector control treated samples, with each PCR strip subsequently being heat shocked at a single temperature. Following 2 h incubation under tissue culture conditions, heat shocks were performed at indicated temperatures for 3 minutes followed by a 3 minute cool at 25 °C using a Veriti SimpliAmp (ThermoFisher). Samples were stored at 4 °C until all heat shocks were complete before addition of 20 µL/tube 2x SureFire Lysis Buffer. Following 10 minute incubation, samples were briefly clarified. Samples were mixed by a 10 µL aspirate and dispense  and 3 µL of lysate was transferred to a 384w ProxiPlate. A solution of 1x ImmunoAssay Buffer was prepared containing 0.08 µg/mL Dako Mouse anti-AR antibody, 0.15 µg/mL MerckMillipore Rabbit anti-AR antibody, 120 µg/mL Anti-mouse IgG Alpha Donor beads and 30 µg/mL Anti-rabbit IgG AlphaLISA Acceptor beads. 6 µL of this solution was added under subdued light to the lysate in the ProxiPlate, sealed and incubated 16 h in the dark before analysis using an EnVision platereader.CWR22Pc-R1-AD1 cells were harvested and resuspended to 6.25 × 10CETSA experiments were performed in a plate-based, automation-compatible assay setup, as described in Supplementary Fig. ® 555 acoustic dispensor or a hp Tecan D300e digital dispensor. Internal standard controls were included on each plate . Cells were lysed by addition of 10 µL/well 2x SureFire Lysis Buffer using a Multidrop Combi. Following 10 minute incubation at room temperature, the solution was mixed (10 × 7 µL aspirate and dispense) and 3 µL lysate was transferred to a 384w ProxiPlate using a Bravo liquid handling system (Agilent). Antibody and AlphaScreen reagent, prepared as above, were added by Multidrop Combi (6 µL/well). Plates were incubated in the dark 16 h and analysed using an EnVision platereader.ITDRF4 cells/well were seeded in 15 µL into a 384w white luminescence plate pre-dosed with compound dose responses using an Echo® 555 acoustic dispensor, and incubated 24 h. 15 µL 2x Steady-Glo® reagent (Promega) was added per well and incubated 15 min in the dark before reading luminescence using an EnVision platereader. Experiments were performed in phenol red free RPMI media supplemented with 10% charcoal stripped FBS.CWR22Pc-R1-AD1 cells stably expressing ARE-firefly Luciferase vector pGL4.23 Luc2/miniP (vector sequences available on request) were maintained in T175 cell culture flasks. Cells were harvested and DHT added to 1 nM final concentration. 2 × 107 cells/mL and 5 µL/well seeded using a Multidrop Combi dispensor. Plates were incubated, heat shocked, and transferred to endpoint as described above. Data was plotted in GraphPad Prism 6 to fit a nonlinear regression and determine 50% effective concentration (EC50). The EC50 determined at each antagonist dose (A’) was normalised to the EC50 determined in the absence of antagonist (A) as [Log10 ((A′/A) − 1)] to derive the fold-shift in agonist EC50 at each antagonist dose. Fold shifts greater than 2-fold, yielding a positive value for [Log10 ((A′/A) − 1)], were plotted against the logarithm of the antagonist dose, and a linear trendline fitted using Prism 6. The X intercept when Y = 0 was determined as pA2, and anti-logged to derive Ki.Antagonist was dosed into a 384w PCR plate using a hp Tecan D300e digital dispensor. A 2X DHT dose response was prepared in media and 5 µL added per well on top of the antagonist in the 384w PCR plate. CWR22Pc-R1-AD1 cells or LNCaP cells were harvested to 3.5 × 10The authors declare that all data supporting the findings of this study are available within the article and Supplementary Information, or are available from corresponding authors upon request.Supplementary Information


Sesamum indicum L.) is an ancient oilseed crop known for its nutty seeds and high-quality edible oil. It is an unexplored crop with a great economic potential. The present study deals with assessment of genetic diversity in the crop. Twenty two RAPD and 18 SSR primers were used for analysis of the 47 different sesame accessions grown in different agroclimatic zones of India. A total of 256 bands were obtained with RAPD primers, of which 191 were polymorphic. SSR primers gave 64 DNA bands, of which all of were polymorphic. The Jaccardʼs similarity coefficient of RAPD, SSR, and pooled RAPD and SSR data ranged from 0.510 to 0.885, 0.167 to 0.867, and 0.505 to 0.853, respectively. Maximum polymorphic information content was reported with SSRs (0.194) compared to RAPDs (0.186). Higher marker index was observed with RAPDs (1.426) than with SSRs (0.621). Similarly, maximum resolving power was found with RAPD (4.012) primers than with SSRs (0.884). The RAPD primer RPI-B11 and SSR primer S16 were the most informative in terms of describing genetic variability among the varieties under study. At a molecular level, the seed coat colour was distinguishable by the presence and absence of a group of marker amplicon/s. White and brown seeded varieties clustered close to each other, while black seeded varieties remained distanced from the cluster. In the present study, we found higher variability in Sesamum indicum L. using RAPD and SSR markers and these could assist in DNA finger printing, conservation of germplasm, and crop improvement.Sesame (The online version of this article (doi:10.1007/s13205-016-0578-4) contains supplementary material, which is available to authorized users. Sesamum indicum L. , commonly known as sesame or Gingelly, is an ancient oilseed crop cultivated in almost all continents. It is a source of high-quality edible oil that contains, in addition, medicinally important antioxidant lignans, namely, sesamin, sesamolin, and sesamol , 0.2 mM dNTP mix, 1 µM of each of forward and reverse primer, 1U Taq Polymerase, and 200 ng of template DNA. Reaction mixture was diluted to final volume by nuclease-free water. The amplification was carried out using the PCR programme: 94 °C for 3 min, then 30 cycles each of 94 °C for 30 s, 55 °C for 45 s, and 72 °C for 1 min followed by 10 cycles of 94 °C for 30 s, 53 °C for 45 s, and 72 °C for 1 min and a final extension of 72 °C for 10 min.Eighteen pairs of SSR primers selected from previous reports  or 3% (for SSR), agarose gel prepared in 1× TAE buffer. GeNei™ Low Range DNA Ruler plus  was used for RAPD, and 100 bp marker from either Promega (Cat No. G2101) or Merck Bioscience (Cat No. 61265267050173) was used for SSR, as molecular weight marker. Electrophoresis was run using 80–100 V. The gel was stained with ethidium bromide and viewed under a Gel Doc . The images were photographed and stored in computers. Reproducibility of the amplification products was ascertained by running at least two independent PCRs for each primer (RAPD) or primer pairs (SSR). Only consistent bands were taken for analysis.nu is the number of alleles and N is total number of individuals sampled. Gene diversity at the lth locus was estimated as f is breeding incoefficient, and pi is the frequency of the ith allele. EMR was obtained by multiplying the proportion of polymorphic markers (β) and the total number of polymorphic fragments (n) obtained , where p is the proportion of the total genotypes containing a particular band. It is useful in calculating the resolving power (RP), which in turn enabled us to know that the ability of a primer to distinguish various genotypes is presented as ∑Ib , gene diversity (GD), polymorphic information content (PIC), effective multiplex ratio (EMR), marker index (MI), and resolving power (RP), were estimated using the 3.25 version of the Power Marker statistics software  and Jaccardʼs distance (D). Subsequently, ‘S’ was used for construction of the dendrogram following UPGMA (Unweighted Pair Group Method with Arithmetic Mean) method. Principal coordinate analysis (PCoA) was done using the software PAST, version 2.15  was found negative correlated with FA . PIC, however, showed a positive correlation of 0.998 (p = 0.01) with GD. MI, which was maximum for the primer RPI-B11 and minimum for RPI-B8 and showed a positive correlation with PIC .Summary of binary data and estimates of the marker attributes for different RAPD primers is presented in Table p = 0.01) with FA. PIC showed a positive correlation with GD . The values for MI and RP were maximal for primer S16 and minimal for S14 and S15. The RP and MI one again showed a positive correlation of 0.856 with MI (p = 0.01).DNA profile of the accessions using some SSR primers is shown in Fig. S1B. A total number of 64 amplicons were obtained with SSR with all of them being polymorphic Table . The maxMatrix analysis of RAPD data showed a Jaccardʼs similarity coefficient in the range of 0.510–0.885. This reflected the presence of a high genetic variability among the accessions under study. Similarity was the highest between the accessions T13 and TKG55 and least between N32 and GT10. Clustering and PCoA confirmed this observation. UPGMA grouping reveals that the sesame germplasm under study can be divided into two distinct clusters . Cluster A was smaller and comprised of 11 accessions. Cluster B was larger which diverged further into two subclusters, namely, B1 and B2. Cluster B1 further subdivided into B1a comprising of 24 accessions and B1b comprising of 11 varieties. Cluster B2 was represented by solitary TC289.Jaccards similarity coefficient obtained from SSR data ranged from 0.167 to 0.867, indicating a high genetic variability among the accessions. In contrast to RAPD, SSR reveals that RT127 and JT7 were the closest and PRACHI and YLM17 were highly dissimilar. The UPGMA of SSR data grouped the accession into two clusters . There was complete realignment of the varieties observed. Cluster A was represented by the solitary PRAGHTI. Remaining 46 accessions together formed cluster B, which further got split into B1 and B2 subclusters. Cluster B1 was the largest with 40 accessions, and cluster B2 consisted of six cultivars. The cophenetic correlation was 0.8 for both RAPD and SSR clusters which was considered as a good fit for the data matrix and the resultant clusters . Analysis revealed that the first two principal coordinates accounted for about 30% of the total variance. PCoA divided the cultivars into three groups. Group A included 24 varieties . Group B consisted of 11 varieties . Group C consisted of 12 varieties .For SSR data, the PCoA showed that the first two principal coordinates accounted for about 25% of the total variance. However, there observed a complete realignment of the accessions leading to clustering of accessions into three groups . Group A consisted of 34 varieties . Group B consisted of seven varieties . In addition, Group C included six varieties .PCoA of pooled data (RAPD and SSR) gave a grouping pattern that resembled the one obtained with RAPD data as seen earlier with dendrogram plots. There were two distinct major groups observed. Group A had 11 varieties, and Group B consisted of 36 accessions, including TC289 that remained solitary in dendrogram  and codominant markers (SSR and RFLP) for classification, fingerprinting, genetic diversity analysis, gene mapping, molecular breeding, and germplasm evaluation  was comparable with earlier reports Below is the link to the electronic supplementary material.