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Positive margin status due to incomplete removal of tumor tissue during radical prostatectomy for high-risk localized prostate cancer requires reoperation or adjuvant therapy, which increases morbidity and mortality. Adverse effects of prostate cancer treatments commonly include erectile dysfunction, urinary incontinence, and bowel dysfunction, making successful initial curative prostatectomy imperative. Current intraoperative tumor margin assessment is largely limited to frozen section analysis, which is a lengthy, labor-intensive process that is obtrusive to the clinical workflow within the operating room (OR). Therefore, a rapid method for prostate cancer margin assessment in the OR could improve outcomes for patients. Dual probe difference specimen imaging (DDSI), which uses paired antibody-based probes that are labeled with spectrally distinct fluorophores, was shown herein for prostate cancer margin assessment. The paired antibody-based probes consisted of a targeted probe to prostate-specific membrane antigen (PSMA) and an untargeted probe, which were used as a cocktail to stain resected murine tissue specimens including prostate tumor, adipose, muscle, and normal prostate. Ratiometric images (i.e., DDSI) of the difference between targeted and untargeted probe uptake were calculated and evaluated for accuracy using receiver operator characteristic curve analysis with area under the curve values used to evaluate the utility of the DDSI method to detect PSMA positive prostate cancer. Targeted and untargeted probe uptake was similar between the high and low PSMA expressing tumor due to nonspecific probe uptake after topical administration. The ratiometric DDSI approach showed substantial contrast difference between the PSMA positive tumors and their respective normal tissues (prostate, adipose, muscle). Furthermore, DDSI showed substantial contrast difference between the high PSMA expressing tumors and the minimally PSMA expressing tumors due to the ratiometric correction for the nonspecific uptake patterns in resected tissues. Previous work has shown that ratiometic imaging has strong predictive value for breast cancer margin status using topical administration. Translation of the ratiometric DDSI methodology herein from breast to prostate cancers demonstrates it as a robust, ratiometric technique that provides a molecularly specific imaging modality for intraoperative margin detection. Using the validated DDSI protocol on resected prostate cancers permitted rapid and accurate assessment of PSMA status as a surrogate for prostate cancer margin status. Future studies will further evaluate the utility of this technology to quantitatively characterize prostate margin status using PSMA as a biomarker. [ABSTRACT FROM AUTHOR]

Significance: Advanced genetic characterization has informed cancer heterogeneity and the challenge it poses to effective therapy; however, current methods lack spatial context, which is vital to successful cancer therapy. Conventional immunolabeling, commonplace in the clinic, can provide spatial context to protein expression. However, these techniques are spectrally limited, resulting in inadequate capacity to resolve the heterogenous cell subpopulations within a tumor. Aim: We developed and optimized oligonucleotide conjugated antibodies (Ab-oligo) to facilitate cyclic immunofluorescence (cyCIF), resulting in high-dimensional immunostaining. Approach: We employed a site-specific conjugation strategy to label antibodies with unique oligonucleotide sequences, which were hybridized in situ with their complementary oligonucleotide sequence tagged with a conventional fluorophore. Antibody concentration, imaging strand concentration, and configuration as well as signal removal strategies were optimized to generate maximal staining intensity using our Ab-oligo cyCIF strategy. Results: We successfully generated 14 Ab-oligo conjugates and validated their antigen specificity, which was maintained in single color staining studies. With the validated antibodies, we generated up to 14-color imaging data sets of human breast cancer tissues. Conclusions: Herein, we demonstrated the utility of Ab-oligo cyCIF as a platform for highly multiplexed imaging, its utility to measure tumor heterogeneity, and its potential for future use in clinical histopathology. [ABSTRACT FROM AUTHOR]