Circulating Tumor Cell Dynamics in Patients Undergoing Pluvicto Therapy for Metastatic Prostate Cancer

Introduction: Prostate cancer is the most common malignancy diagnosed in men and the second leading cause of cancer related deaths. While the 5-year survival rate of localized prostate cancer is >99%, the rate significantly drops to 31% for metastatic disease. Despite considerable advancements in diagnostic and therapeutic techniques, the management of advanced prostate cancer, particularly metastatic castration-resistant prostate cancer (mCRPC), poses a major challenge as conventional androgen deprivation therapies (ADT) become ineffective. Pluvicto (177Lu-vipivotide tetraxetan), a novel targeted radioligand therapy recently approved by the FDA, offers a promising solution by specifically targeting prostate-specific membrane antigen (PSMA) on malignant cells. PSMA is an ideal therapeutic and diagnostic target due to its high expression on prostate cancer cells. By binding to PSMA-positive cells, Pluvicto delivers localized radiation, selectively inducing apoptosis while sparing healthy tissue. Despite this medical advancement, assessing therapeutic efficacy remains complex due to the dynamic nature of tumor cell characteristics. Circulating tumor cells (CTCs) are cells that migrate from the primary tumor site and into the bloodstream. The process of isolating CTCs from peripheral blood samples, often referred to as "liquid biopsies," provides a non-invasive and real-time approach to monitoring treatment response by quantifying and characterizing CTCs. Moreover, a substantial body of research has established the prognostic importance of CTC counts and their association with treatment outcomes in mCRPC. The present work evaluates CTC levels and PSMA expression in mCRPC patients treated with Pluvicto to provide insights into the prognostic and therapeutic implications of CTC characterization.

Materials and

Methods: The therapeutic regimen of Pluvicto comprises a series of six systemic infusions administrated approximately six weeks apart. Whole blood samples were obtained from mCRPC patients undergoing Pluvicto therapy before the start of treatment and prior to each infusion. Samples were processed using Ficoll-Paque density gradient centrifugation to isolate the buffy coat, then incubated with magnetic beads coated in antibodies targeting CD45 antigen, as CD45 is a common leukocyte marker that is not expressed on the surface of CTCs. Then, CTCs were enriched via negative selection as the sample was then passed through magnetic columns, leaving CD45- cells and enhancing the purity of the CTC population. Isolated CTCs were cytospun onto glass slides, fixed, permeabilized, and immunofluorescently stained with antibodies targeting PSMA and Pan-cytokeratin. High magnification images were acquired using an LSM900 confocal microscope. A custom MATLAB image processing algorithm was employed to extract physical properties of each cell, including area, diameter, circularity, and fluorescence data from segmented cell regions of interest (ROIs). All fluorescence data are presented in terms of the Corrected Total Cell Fluorescence (CTCF), calculated by the subtracting the integrated density of the ROI by the product of the ROI area and mean background intensity.

Results, Conclusions, and Discussions: The prognostic potential of liquid biopsies in mCRPC has been extensively studied, as CTC count and prostate-specific antigen (PSA) levels are measurable entities in peripheral blood, offering real-time insights into disease progression and therapeutic response. Therefore, we sought to isolate and characterize CTCs from whole blood samples obtained from mCRPC patients throughout their Pluvicto treatment regimen (Figure 1A). To establish a robust baseline for PSMA and cytokeratin expression, two immortalized cell lines, Jurkat and LNCaP, were imaged to serve as a negative and positive control of PSMA/cytokeratin expression (Figure 1B). This approach facilitated the determination of PSMA and cytokeratin expression thresholds for reliable CTC identification. As anticipated, the subsequent quantitative analysis showed that both PSMA and cytokeratin were expressed at significantly higher levels in the LNCaP cell line when compared to the Jurkat line, exhibiting more than a tenfold increase (Figure 1C). Following CTC enrichment and immunofluorescent staining for PSMA and cytokeratin, confocal images of samples were acquired and analyzed using the MATLAB image processing algorithm (Figure 1D). A significant reduction in CTC PSMA expression (Figure 1E) and CTC diameter was observed across all patients throughout their treatment, underscoring Pluvicto’s ability to effectively target high PSMA expressing cells. Moreover, this decrease in PSMA expression was irrespective of patient therapeutic response and did not always correlate with traditional biomarkers of disease progression, such as PSA. This observation suggests that surviving CTCs may inherently express lower PSMA levels, thereby evading Pluvicto’s targeting mechanism, or they may adapt to the therapeutic pressure imposed by Pluvicto and downregulate PSMA as a survival mechanism. Additionally, trends between PSA serum levels and CTC counts generally paralleled therapeutic efficacy (Figure 1F). Overall, this study offers insights into the potential of using CTC characterization and PSMA expression as predictive and monitoring tools for assessing the therapeutic efficacy of Pluvicto treatment in patients with advanced mCRPC. Future research into the molecular changes in mCRPC patient CTCs may enable improved patient outcomes through more personalized and adaptive treatment strategies.

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