Abstract A030: Systematic optimization of multiplex immunofluorescence of bone marrow eliminates false positives in the detection of disseminated tumor cells
Authors: Axelrod Haley , Pienta Kenneth J. , Valkenburg Kenneth C. 
Published: February 2019

Abstract
Approximately 30% of prostate cancer (PCa) patients develop biochemical recurrence after radical prostatectomy or radiation therapy; 40% of these patients develop metastasis. Importantly, 100% of men who die of PCa have cancer in the bone. Our goal is to determine whether we may identify disseminated tumors cells (DTCs) in the bone marrow (BM) of PCa patients. This will allow us to study and characterize the biology of BM-DTCs, allowing us to develop innovative strategies to target these cells before they develop overt lethal metastatic lesions. However, the identification of BM-DTCs has been hampered by strong background staining of non-tumor BM cells in immunofluorescence (IF) assays. This prevalent false positive staining calls into question the true identity of what had previously been classified as DTCs. The purpose of this study was to rigorously and systematically optimize our multiplex IF staining protocol so as to eliminate the background signal, thereby increasing confidence that positively stained cells are truly BM-DTCs. The IF protocol we employ involves applying BM (spiked with PCa cells as a positive control) onto positively charged agar-coated slides following a red blood cell lysis step. We then apply heat to promote proper seeding of the cells onto the slide, followed by fixation and permeabilization. We then use indirect IF with primary and fluorescently labeled secondary antibodies before staining the nucleus and mounting a coverslip. We primarily stained pan-cytokeratin (CK) to label cancer cells, CD45 to label white blood cells (WBCs), and DAPI to label the nuclei. This base protocol resulted in false positive staining of many WBCs in the fluorescent channel reserved for CK-positivity. In addition, we observed ubiquitous autofluorescent signal throughout the BM, making it difficult to detect true positives. We then went through the protocol step by step to determine the source of the background signal. We assessed fixation time, permeabilization, antigen retrieval, blocking reagents, primary antibody concentration, secondary antibody concentration, nuclear stain, wash reagents, and reagents to eliminate autofluorescence. Each step provided us with information about background signal. After these optimizations, our current novel protocol has eliminated autofluorescence and false positive signal. We now consistently observe high signal-to-noise ratio for CK-positivity in the tumor cells and no CK-positivity in the WBC population. We have also performed this protocol on a small number of PCa patient BM and blood samples and saw no background signal. These data lead us to conclude that we have, for the first time, a multiplex IF protocol that can be used to stain patient BM tissue to identify BM-DTCs. Future studies will determine the true number of BM-DTCs present at different time points during PCa progression, as well as study the biology of these rare cells in relation to development of metastasis and patient prognosis.
Citation Format: Haley Axelrod, Kenneth J. Pienta, Kenneth C. Valkenburg. Systematic optimization of multiplex immunofluorescence of bone marrow eliminates false positives in the detection of disseminated tumor cells [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A030.