Fig 1.
Gene transcription of PSMA across cell lines.
(A) Validation of canine-specific TaqMan PSMA primer efficiency across 5-log orders of RNA concentration using CPA, a canine prostatic carcinoma cell line. (B) Comparative PSMA gene expression across canine cell lines. Relative PSMA expressions for six cHSA cell lines (red) compared to the non-malignant canine endothelial cell line, CAoEC (green). Positive and negative control cell lines include CPA (blue) and ACE-1 (purple), respectively. Representative data presented from 3 independent biologic replicates with 5 technical replicates for each cell line.
Fig 2.
Antibody validation for canine PSMA protein expression.
Validation of antibodies for detecting PSMA in human and canine cell lines by (A) confocal fluorescent microscopy, and (B) western blot analysis. Positive and negative controls for canine and human include (CPA and ACE-1) and (LNCaP and PC3), respectively.
Fig 3.
Comparative PSMA protein expressions across cell lines.
Comparison of PSMA protein expressions and associated histograms for six cHSA cell lines (red) relative to the non-malignant CAoEC line (green) by (A and B) western blot analysis, and (C and D) by confocal fluorescent microscopy. Canine positive and negative controls are CPA (blue) and ACE-1 (purple), respectively. Significance p < 0.05 and p < 0.01 denoted by “*” and “**”, respectively.
Fig 4.
Characterization of PSMA in cHSA tumor samples.
Spontaneously-arising cHSA tumor samples involving the spleen, liver, and lung tissues, with representative (A) histology by H&E and PSMA immunohistochemistry (Score 3; magnification 400x), and comparison (B) of staining properties for cHSA by CD31 and PSMA (magnification 100x).
Fig 5.
Optimization of qualitative PCR methodology and dynamic range of detection for PSMA.
(A) Differential PSMA amplicon generation by qualitative PCR with the lowly expressing PSMA cHSA cell line (FITZ; red) compared with CAoEC (green) across different thermocycles ranging from 24 to 30. Determination of qualitative PCR sensitivity for detecting PSMA amplicons at 27 cycles with a logarithmic titration of (B) FITZ cells only or (C) when FITZ cells are spiked into whole blood. Lowest cell density capable that produces faintly visible amplicons is 104 FITZ cells, alone or in addition to whole blood nucleated cells. Amplicon production expressed as a ratio of PSMA/β actin pixels/area. Abbreviations FITZ (FZ; red) and CAoEC (CE; green).
Fig 6.
Tandem cytologic evaluation and PSMA amplicon generation from hemorrhagic effusions.
(A) Cytologic characterization of hemorrhagic effusions collected from five dogs, three with hemoabdomen (Dogs 1, 2, and 4) and two with hemorrhagic pericardial effusion (Dogs 3 and 5). Note the identification of atypical cells in Dogs 1–3 and 5 (right, top panel). (B) Generation of visible PSMA amplicons (27 cycles) from hemorrhagic effusions overtly positive for Dogs 1–3 and 5, and weakly positive for Dog 4. (C) Immunohistochemical evaluation of primary tumors from Dogs 1–3 and 5, confirming that primary tumors presumed to be the source of exfoliative cHSA cells stain positively for PSMA.
Fig 7.
Generation of PSMA amplicons from non-cHSA tumor histology.
Immunohistochemical evaluation of tumor from Dog 4, showing immunopositivity for vimentin, neuron-specific enolase, and PSMA only. Dotted black line separates normal liver tissue (right) from tumor tissue (left). CD18 and CD31 immunohistochemistry fail to demonstrate immunoreactivity within tumor cells, but do stain resident macrophages and normal blood vessels, respectively. Magnification 400x; abbreviations CK, cytokeratin; VM, vimentin; NSE, neuron-specific enolase.