Journal: Molecular cancer research : MCR

Article Title: Sympathetic Signaling Reactivates Quiescent Disseminated Prostate Cancer Cells in the Bone Marrow

doi: 10.1158/1541-7786.MCR-17-0132

Figure Lengend Snippet: (A–C) PCa cells (PC3 and DU145) cultured with NE, Propranolol, or vehicle control and evaluated for Ki67 expression using flow cytometry (N=3; Student’s t-test). (D–E) Immunocytochemistry for Ki67 expression by PCa cells following in vitro culture of PC3 cells and GAS6+/+ OB co-culture in the presence of NE or vehicle control (N=3; Student’s t-test). (F–G) PC3-FUCCI cells cultured in vitro with NE or vehicle control and evaluated for cell cycle phase using flow cytometry (N=3; Student’s t-test).

Article Snippet: ELISA An ELISA assay was performed following the manufacturer’s instructions (R&D Systems, Dy986) to evaluate GAS6 expression in OBs (MC3T3-E1s and primary murine OBs) in the presence of vehicle or NE.

Techniques: Cell Culture, Control, Expressing, Flow Cytometry, Immunocytochemistry, In Vitro, Co-Culture Assay

Journal: Molecular cancer research : MCR

Article Title: Sympathetic Signaling Reactivates Quiescent Disseminated Prostate Cancer Cells in the Bone Marrow

doi: 10.1158/1541-7786.MCR-17-0132

Figure Lengend Snippet: (A) Expression of mRNA dormancy-associated genes and adhesion/homing-associated genes expressed by OBs were determined in MC3T3-E1 OB cultures treated with NE or vehicle control using qPCR (N=2; Student’s t-test). Values were normalized to GAPDH levels. (C) Expression of GAS6 in PCa cells (PC3) and OBs (MG63, MC3T3-E1, and GAS6+/+ OB) was determined by an ELISA in culture conditions with NE and vehicle control (N=3; student’s t-test). (D) Ki67 expression of OBs (MG63, MC3T3-E1, and GAS6+/+ OBs) cultured with NE or vehicle control was determined by flow cytometry (N=3; Student’s t-test).

Article Snippet: ELISA An ELISA assay was performed following the manufacturer’s instructions (R&D Systems, Dy986) to evaluate GAS6 expression in OBs (MC3T3-E1s and primary murine OBs) in the presence of vehicle or NE.

Techniques: Expressing, Control, Enzyme-linked Immunosorbent Assay, Cell Culture, Flow Cytometry

Journal: Molecular cancer research : MCR

Article Title: Sympathetic Signaling Reactivates Quiescent Disseminated Prostate Cancer Cells in the Bone Marrow

doi: 10.1158/1541-7786.MCR-17-0132

Figure Lengend Snippet: (A–B) GAS6 expression in GAS6+/+ OBs cultured under conditions with NE or vehicle control was determined by immunocytochemistry and quantified (N=3; student’s t-test). GAS6−/− OBs were cultured under the same conditions, serving as a negative control. (C) PC3 cells and (D) DU145 cells were co-cultured with GAS6+/+ and GAS6−/− OBs in the presence of NE or vehicle control. Ki67 expression was determined by FACS (N=3; ANOVA). (E) PC3-FUCCI cells were co-cultured with GAS6+/+ and GAS6−/− OBs in the presence of NE or vehicle control. Cell cycle phase was determined by FUCCI vector expression using flow cytometry (N=3; ANOVA).

Article Snippet: ELISA An ELISA assay was performed following the manufacturer’s instructions (R&D Systems, Dy986) to evaluate GAS6 expression in OBs (MC3T3-E1s and primary murine OBs) in the presence of vehicle or NE.

Techniques: Expressing, Cell Culture, Control, Immunocytochemistry, Negative Control, Plasmid Preparation, Flow Cytometry

Journal: Molecular cancer research : MCR

Article Title: Sympathetic Signaling Reactivates Quiescent Disseminated Prostate Cancer Cells in the Bone Marrow

doi: 10.1158/1541-7786.MCR-17-0132

Figure Lengend Snippet: (A) Pictorial diagram demonstrating the experimental protocol for the ex vivo culture model. (B,C) Representative sample of the flow plot gates found from PC3-FUCCI cells injected into ex vivo GAS6+/+ femurs treated with vehicle (B) or NE (C) added to the culture conditions. Live cells were negatively gated for murine IgG2b b haplotype (mH-2Db) (BioLegend, Cat# 111516, PE/Cy7) and mCD45 (BioLegend, Cat# 103112, APC), which were then positively gated for HLA-A,B,C (Biolegend, Cat# 311426, APC/Cy7). After these gates were applied, cells were plotted on the FUCCI spectrum. (D) PC3-FUCCI cells were isolated following injection into ex vivo GAS6+/+ or GAS6 −/− femur explants and cell cycle was determined using flow cytometry (N=3; Student’s t-test).

Article Snippet: ELISA An ELISA assay was performed following the manufacturer’s instructions (R&D Systems, Dy986) to evaluate GAS6 expression in OBs (MC3T3-E1s and primary murine OBs) in the presence of vehicle or NE.

Techniques: Ex Vivo, Injection, Isolation, Flow Cytometry

Journal: Molecular cancer research : MCR

Article Title: Sympathetic Signaling Reactivates Quiescent Disseminated Prostate Cancer Cells in the Bone Marrow

doi: 10.1158/1541-7786.MCR-17-0132

Figure Lengend Snippet: Model of NE signaling within the bone marrow may activate dormant PCa DTCs through both direct and indirect methods. The direct method hypothesis includes NE binding to PCa DTCs via Adrenergic Receptors, inhibiting cell cycle inhibitors p21, p27 and p38 causing PCa entry into the proliferative cell cycle. The indirect method hypothesis includes NE binding to local OBs via Adrenergic Receptors, resulting in decreased secretion of the dormancy regulating molecule GAS6. ADR = Adrenergic Receptors; NE= Norepinephrine; PCa =Prostate Cancer; DTC = Disseminated Tumor Cell; OBs = Osteoblast Cell.

Article Snippet: ELISA An ELISA assay was performed following the manufacturer’s instructions (R&D Systems, Dy986) to evaluate GAS6 expression in OBs (MC3T3-E1s and primary murine OBs) in the presence of vehicle or NE.

Techniques: Binding Assay