Pinnatoxin G is a cyclic imine neurotoxin produced by dinoflagellates that has been reported in shellfish. Like other members of the pinnatoxin family, it has been shown to have its effects via antagonism of the nicotinic acetylcholine receptors, with preferential binding to the α7 subunit often upregulated in cancer. Because increased activity of α7 nicotinic acetylcholine receptors contributes to increased growth and resistance to apoptosis, the effect of pinnatoxin G on cancer cell viability was tested. In a panel of six cancer cell lines, all cell types lost viability, but HT29 colon cancer and LN18 and U373 glioma cell lines were more sensitive than MDA-MB-231 breast cancer cells, PC3 prostate cancer cells, and U87 glioma cells, correlating with expression levels of α7, α4, and α9 nicotinic acetylcholine receptors. Some loss of cell viability could be attributed to cell cycle arrest, but significant levels of classical apoptosis were found, characterized by caspase activity, phosphatidylserine exposure, mitochondrial membrane permeability, and fragmented DNA. Intracellular Ca2+ levels also dropped immediately upon pinnatoxin G treatment, which may relate to antagonism of nicotinic acetylcholine receptor-mediated Ca2+ inflow. In conclusion, pinnatoxin G can decrease cancer cell viability, with both cytostatic and cytotoxic effects.
While programmed death 1 (PD-1) and programmed death-ligand 1 (PD-L1) checkpoint inhibitors have activity in a proportion of patients with advanced bladder cancer, strongly predictive and prognostic biomarkers are still lacking. In this study, we evaluated PD-L1 protein expression on circulating tumor cells (CTCs) isolated from patients with muscle invasive (MIBC) and metastatic (mBCa) bladder cancer and explore the prognostic value of CTC PD-L1 expression on clinical outcomes. Blood samples from 25 patients with MIBC or mBCa were collected at UCSF and shipped to Epic Sciences. All nucleated cells were subjected to immunofluorescent (IF) staining and CTC identification by fluorescent scanners using algorithmic analysis. Cytokeratin expressing (CK)+ and (CK)−CTCs (CD45−, intact nuclei, morphologically distinct from WBCs) were enumerated. A subset of patient samples underwent genetic characterization by fluorescence in situ hybridization (FISH) and copy number variation (CNV) analysis. CTCs were detected in 20/25 (80 %) patients, inclusive of CK+ CTCs (13/25, 52 %), CK−CTCs (14/25, 56 %), CK+ CTC Clusters (6/25, 24 %), and apoptotic CTCs (13/25, 52 %). Seven of 25 (28 %) patients had PD-L1+ CTCs; 4 of these patients had exclusively CK−/CD45−/PD-L1+ CTCs. A subset of CTCs were secondarily confirmed as bladder cancer via FISH and CNV analysis, which revealed marked genomic instability. Although this study was not powered to evaluate survival, exploratory analyses demonstrated that patients with high PD-L1+/CD45−CTC burden and low burden of apoptotic CTCs had worse overall survival. CTCs are detectable in both MIBC and mBCa patients. PD-L1 expression is demonstrated in both CK+ and CK−CTCs in patients with mBCa, and genomic analysis of these cells supports their tumor origin. Here we demonstrate the ability to identify CTCs in patients with advanced bladder cancer through a minimally invasive process. This may have the potential to guide checkpoint inhibitor immune therapies that have been established to have activity, often with durable responses, in a proportion of these patients.
Supplementary Figures 1-3 from Quantifying the Activity of Adenoviral E1A CR2 Deletion Mutants Using <i>Renilla</i> Luciferase Bioluminescence and 3′-Deoxy-3′-[<sup>18</sup>F]Fluorothymidine Positron Emission Tomography Imaging
e22034 Background: Detection and characterization of CTCs offers a minimally invasive mechanism for understanding patient response to therapy and disease evolution. Here we describe a proprietary technology platform to examine CTC incidence, N-terminal AR expression, and PTEN loss in metastatic or advanced CRPC patient samples from a phase II clinical trial (GO27983) testing the combination of the AKT inhibitor ipatasertib and abiraterone. Methods: Blood samples from CRPC patients (n = 283) were collected at screening and shipped to Epic Sciences. Upon receipt, the nucleated cells were plated onto microscope slides and stored at -80C. Two slides were thawed per patient and analyzed with an N-terminal AR CTC assay. CTCs were detected using a combination of CD45 exclusion, CK and N-terminal AR expression, and morphologic criteria. Traditional (CD45-, CK+, DAPI+) and Non-Traditional (CD45-/CK- with distinct morphology or CD45-/CK+ with fragmented nuclei) CTCs were enumerated and N-terminal AR expression was quantified for each CTC. Samples with > 2 Traditional CTCs enumerated per slide were further evaluated for PTEN loss by FISH (n = 170). Results: Traditional CTCs were detected ( ≥ 1 cell/ml) in 86% (242 of 283) of samples. Non-traditional CTCs were detected in 93% (263 of 283) of patients. N-terminal AR positivity was detected ( ≥ 1 AR+ cell/ml) in 53% (128 of 242) of Traditional CTC-positive samples, with the % positivity ranging from 0.4-100%. PTEN status was determined for 66% (160 of 243) of patients with Traditional CTCs detected. Patients with homozygous (HO) PTEN loss had a significantly higher mean CTC/mL (p = 0.0013) and AR+ CTC/mL value (p = 0.0014) than patients with PTEN non-deleted status. Conclusions: The ability of the Epic Sciences platform to detect AR+ CTCs and PTEN loss at screening provides a non-invasive approach to characterizing and monitoring CTCs identified in advanced CRPC patients. The phase II clinical trial is ongoing and we will continue to monitor longitudinal changes in CTC incidence and AR expression in relation to PTEN status.
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