Journal: Translational Oncology

Article Title: HER2 as a potential therapeutic target on quiescent prostate cancer cells

doi: 10.1016/j.tranon.2023.101642

Figure Lengend Snippet: Proteomics of membrane proteins in G 0 vs. G 1 PC3 cells. (A) Experimental schema. (B) Volcano plot of 354 differentially expressed (DE) genes plotted as measured expression log 10 fold change against statistical significance of the change. All genes had non-zero expression detected in G 0 and G 1 , fold change ≥4 or ≤ 0.25 and p ≤ 0.1 Fisher's exact test. Data is from three pooled biologic replicates. (C) Gene ontology analyses of DE genes using KEGG database, shown are the ten biological pathways most significantly impacted by DE genes. (D) Theoretical network analyses for nucleocytoplasmic transport—the most significantly enriched pathway from panel (C). (E) Gene ontology analyses of DE genes using the gene ontology cellular components database. (F) Theoretical network analyses for nuclear body—the most significantly associated cellular component from panel (E). For the network analyses in (D) and (F), modes of theoretical interaction and numbers of interaction are shown in the legend. On the network analysis web, nodes with increased expression in G 0 are blue and increased expression in G 1 are red. (G) Gene ontology analyses of DE genes using gene ontology biological processes database. (H) Gene ontology analyses of DE genes using the molecular functions database.

Article Snippet: Cell culture : The human prostate cancer cell lines, PC3 (Cat #: CRL-1435), DU145 (Cat #: HTB-81), LNCaP (Cat #: CRL-1740), and an osteoblast precursor cell line, MC3T3-E1 (Cat #: CRL-2593) were obtained from the American Type Culture Collection.

Techniques: Membrane, Expressing

Journal: Translational Oncology

Article Title: HER2 as a potential therapeutic target on quiescent prostate cancer cells

doi: 10.1016/j.tranon.2023.101642

Figure Lengend Snippet: Cell surface HER2 expression on quiescent prostate cancer cells. (A) Cell surface HER2 expression on PCa cell lines (PC3, DU145, C4-2B, LNCaP) and benign prostate epithelial cells (PNT2) cultured in 10% FBS measured by flow cytometry and mean fluorescence intensity (MFI). Open histograms represent an isotype control antibody. Shaded histograms are a HER2 antibody. The vertical line and arrow represent positive expression as defined by the isotype control antibody. (B) Flow cytometry for surface HER2 and BrdU incorporated into DNA of PC3 cells (C) Histograms showing HER2 cell surface expression on the same cell lines cultured in the presence or absence of FBS. (D) Quantification of HER2 surface expression from panel (B) as defined by mean fluorescence intensity (MFI). (E) Immunofluorescence imaging of PC3 (left) or C4-2B (right) cells cultured in either 10% FBS or serum starved conditions showing endogenous p27 and HER2 expression. HER2 (red), p27 (green) and nuclei (DAPI, blue). Scale bar, 50 μm. (F) Cell surface HER2 as measured by flow cytometry of PCa cells cultured with either 1 µM abemaciclib or 0.01% ethanol vehicle. (G) Western blot for HER2 from indicated cell lines cultured for 2 days with or without FBS. β-actin was a loading control. Data represent mean ± S.D. of triplicate wells. A representative biological replicate experiment is shown.

Article Snippet: Cell culture : The human prostate cancer cell lines, PC3 (Cat #: CRL-1435), DU145 (Cat #: HTB-81), LNCaP (Cat #: CRL-1740), and an osteoblast precursor cell line, MC3T3-E1 (Cat #: CRL-2593) were obtained from the American Type Culture Collection.

Techniques: Expressing, Cell Culture, Flow Cytometry, Fluorescence, Control, Immunofluorescence, Imaging, Western Blot

Journal: Translational Oncology

Article Title: HER2 as a potential therapeutic target on quiescent prostate cancer cells

doi: 10.1016/j.tranon.2023.101642

Figure Lengend Snippet: Cell surface HER2 expression is associated with p27 expression. Left: PC3, Right: C4-2B. Top: Flow cytometry histograms demonstrating cell surface (labeling before fixation) HER2 expression on PC3 and C4-2B cells. Cells were gated by lowest 10% or highest 10% of HER2 levels (HER2 low and HER2 high) populations. Middle: Example flow cytometry histograms of p27 labeling from either the lowest (blue) or highest (red) 10% of HER2 surface expression. Bottom: Quantified p27 expression in the low vs. higher HER2 populationsp27 levels were measured as mean fluorescence intensity (MFI). Data represent mean ± S.D. of triplicate wells. A representative biological replicate experiment is shown.

Article Snippet: Cell culture : The human prostate cancer cell lines, PC3 (Cat #: CRL-1435), DU145 (Cat #: HTB-81), LNCaP (Cat #: CRL-1740), and an osteoblast precursor cell line, MC3T3-E1 (Cat #: CRL-2593) were obtained from the American Type Culture Collection.

Techniques: Expressing, Flow Cytometry, Labeling, Fluorescence

Journal: Translational Oncology

Article Title: HER2 as a potential therapeutic target on quiescent prostate cancer cells

doi: 10.1016/j.tranon.2023.101642

Figure Lengend Snippet: Microenvironmental induction of cell surface HER2. (A) Human PC3 PCa cells were cultured with murine OBs, MC3T3-E1 cells for 3 days in the presence of 10% FBS, and PCa cells were identified as murine MHC negative populations. (B) Relative HER2 expression (MFI) on PC3, DU145, or C4-2B PCa cells co-cultured with OBs, compared to PCa cells cultured alone. Data represent mean ± S.D. of triplicate wells. A representative biological replicate experiment is shown. (C) DTCs were identified in BM and liver using FACS as human HLA positive and mouse MHC negative populations. (D) Cell surface HER2 expression on DTCs isolated from BM and liver. Data are shown as mean ± S.D. (N=3). * indicates p < 0.05 compared to control cells.

Article Snippet: Cell culture : The human prostate cancer cell lines, PC3 (Cat #: CRL-1435), DU145 (Cat #: HTB-81), LNCaP (Cat #: CRL-1740), and an osteoblast precursor cell line, MC3T3-E1 (Cat #: CRL-2593) were obtained from the American Type Culture Collection.

Techniques: Cell Culture, Expressing, Isolation, Control

Journal: Translational Oncology

Article Title: HER2 as a potential therapeutic target on quiescent prostate cancer cells

doi: 10.1016/j.tranon.2023.101642

Figure Lengend Snippet: Impact of T-DM1 treatment on metastasis free survival in a prostate cancer left ventricle injection xenograft model. (A) Experimental design for the in vivo experiment. GFP/luciferase-expressing PC3 cells were injected into male SCID mice by left ventricle intracardiac (i.c.) injection. Human IgG (15 mg/kg) or T-DM1 (15 mg/kg) were injected by intraperitoneal (i.p.) injection every 3 days until 12 days after tumor injection IgG: n = 12. T-DM1: n = 14. (B) Kaplan-Meier analysis of time to formation of metastases visible by bioluminescence imaging or death. (C) Representative bioluminescence images of control mice (human IgG injected) and T-DM1 treated mice 124 days after tumor injection. (D) Antibody dependent cellular cytotoxicity (ADCC) assays using flow cytometry. PC3 cells were labeled with DiD for subsequent identification, and were treated with T-DM1 before addition of splenocytes from SCID mice. PC3 cells were identified as DiD positive and non-viable cells were identified as positive for DAPI. (E) Dead PC3 cells (%) quantified from data in panel (D) Data represent mean ± S.D. (N=3).

Article Snippet: Cell culture : The human prostate cancer cell lines, PC3 (Cat #: CRL-1435), DU145 (Cat #: HTB-81), LNCaP (Cat #: CRL-1740), and an osteoblast precursor cell line, MC3T3-E1 (Cat #: CRL-2593) were obtained from the American Type Culture Collection.

Techniques: Injection, In Vivo, Luciferase, Expressing, Imaging, Control, Flow Cytometry, Labeling