In prostate cancer (PCa), cancer‐associated fibroblasts (CAFs) promote tumor progression, drug resistance, and metastasis. Although circulating tumor cells are studied as prognostic and diagnostic markers, little is known about other circulating cells and their association with PCa metastasis. Here, we explored the presence of circulating CAFs (cCAFs) in metastatic castration‐naïve prostate cancer (mCNPC) patients. cCAFs were stained with fibroblast activation protein (FAP), epithelial cell adhesion molecule (EpCAM), and receptor‐type tyrosine‐protein phosphatase C (CD45), then FAP⁺EpCAM⁻ cCAFs were enumerated and sorted using fluorescence‐activated cell sorting. FAP⁺EpCAM⁻ cCAFs ranged from 60 to 776 (389 mean ± 229 SD) per 2 × 10⁸ mononuclear cells, whereas, in healthy donors, FAP⁺ EpCAM⁻ cCAFs ranged from 0 to 71 (28 mean ± 22 SD). The mCNPC‐derived cCAFs showed positivity for vimentin and intracellular collagen‐I. They were viable and functional after sorting, as confirmed by single‐cell collagen‐I secretion after 48 h of culturing. Two cCAF subpopulations, FAP⁺CD45⁻ and FAP⁺CD45⁺, were identified, both expressing collagen‐I and vimentin, but with distinctly different morphologies. Collectively, this study demonstrates the presence of functional and viable circulating CAFs in mCNPC patients, suggesting the role of these cells in prostate cancer.

Circulating tumor cell (CTC)- and/or tumor-derived extracellular vesicle (tdEV) loads in the blood of metastatic castration-resistant prostate cancer (CRPC) patients are associated with worse overall survival and can be used as predictive markers of treatment response. In this study, we investigated the quantity/quality of CTCs and tdEVs in metastatic castration-naive prostate cancer (CNPC) and CRPC patients, and whether androgen deprivation therapy (ADT) affects CTCs and tdEVs. We included 104 CNPC patients before ADT initiation and 66 CRPC patients. Blood samples from 31/104 CNPC patients were obtained 6 months after ADT. CTCs and tdEVs were identified using ACCEPT software. Based on the morphology, CTCs of metastatic CNPC and CRPC patients were subdivided by manual reviewing into six subclasses. The numbers of CTCs and tdEVs were correlated in both CNPC and CRPC patients, and both CTCs (p = 0.013) and tdEVs (p = 0.005) were significantly lower in CNPC compared to CRPC patients. Qualitative differences in CTCs were observed: CTC clusters (p = 0.006) and heterogeneously CK expressing CTCs (p = 0.041) were significantly lower in CNPC patients. CTC/tdEV numbers declined 6 months after ADT. Our study showed that next to CTC-load, qualitative CTC analysis and tdEV-load may be useful in CNPC patients.

The clinical utility of circulating tumor cells (CTCs) is hampered by the low number of cells detected. Diagnostic leukapheresis (DLA) offers a solution but, due to the observed non‐specific binding and clumping, processing of DLA samples using the CellSearch system only allows for the processing of aliquots consisting of ~ 2% of the total DLA sample per test. Here, we introduce a flow enrichment target capture Halbach‐array (FETCH)‐based separation method in combination with a DNase preprocessing step to capture CTCs from larger fractions of DLA products without clumping. To evaluate the FETCH method, we processed peripheral blood samples from 19 metastatic castration‐naïve prostate cancer (mCNPC) patients with CellSearch, and processed 2% aliquots of leukapheresis samples from the same patients with CellSearch as well as FETCH with or without DNase preprocessing. Using 2% aliquots from six patients, the use of FETCH with fewer immunomagnetic epithelial cellular adhesion molecule (EpCAM) conjugated ferrofluids was tested, whereas 20% aliquots from four patients were used to evaluate the processing of 10‐fold larger DLA samples using FETCH. Results show that the cell clumping normally seen after immunomagnetic enrichment of DLA material was greatly reduced with the use of DNase pretreatment, while the number of CTCs detected was not affected. The number of CTCs detected in 2% aliquots of DLA using FETCH was unchanged compared to CellSearch and did not decrease when using down to 10% of the volume of immunomagnetic anti‐EpCAM ferrofluids normally used in a CellSearch test, whereas the number of co‐enriched white blood cells reduced a median 3.2‐fold. Processing of a 20% aliquot of DLA with FETCH resulted in a 14‐fold increase in CTCs compared to the processing of 2% aliquots of DLA using CellSearch and a total 42‐fold median increase in CTCs compared to peripheral‐blood CellSearch.

Background: Metastatic prostate cancer (mPCa) is marked by heterogeneity and therapy resistance, which arise from prolonged therapy regimens. This heterogeneity is reflected in various morphologic and genetic characteristics, biomarker expression, and other molecular mechanisms, thereby contributing to the complexity of the disease. Methods: To investigate tumor heterogeneity, the effects of androgen targeting therapy (ADT) on single-cell PSA secretion was assessed by analyzing the prostate cancer cell lines using a modified ELISpot platform. The FACS and cytospin techniques were employed to understand the influence of the cell cycle on PSA secretion patterns. Additionally, a proteome array was used to identify potential biomarkers from different PCa cell lines with varying metastatic potential. Results: Among the various PCa cell lines examined, PSA expression and secretion could be visualized only from the LNCaPs. PSA secretion from circulating tumor cells (CTCs) further confirmed the validity of this assay. These LNCaPs exhibited heterogeneity in single-cell intracellular and extracellular PSA expression and in their ADT responses. LNCaPs in the G1 phase showed higher PSA secretion than in the S or G2/M phase. Apart from PSA, Cathepsin D, Progranulin, IL-8, Serpin E1, and Enolase 2 were identified as secretome markers from the metastatic PCa cell lines. Conclusions: We observed variability in PSA secretion in LNCaP in response to anti-androgen treatment and a cell cycle-dependent secretion pattern. The notable presence of Progranulin and Cathepsin D in metastatic cell lines makes them promising candidates for use in multiplexing and single-cell platforms, potentially advancing our understanding and treatment of this disease.

PSA is the most common biomarker used in the screening and monitoring of prostate cancer. However, changes in PSA do not always reflect disease dynamics in every patient, and antihormonal agents may modulate its levels without significant antitumor effects. Changes in circulating tumor cells (CTC) have been described as a more objective measure of treatment response. Differences between PSA and CTC may be explained by heterogeneity in tumor cells producing PSA. To explore this, we measured the PSA secretion from a single CTC to gain insights into the PSA secretion heterogeneity between tumor cells. CTCs were enriched using EpCAM-based immunomagnetic enrichment in diagnostic leukapheresis of 18 patients with metastatic castration-naïve prostate cancer (mCNPC) not pretreated with any therapy, including androgen deprivation therapy. Calcein+ CD45− cells were sorted by flow cytometry and deposited as single cells on a nanowell array to measure the PSA secretion after 24 hours. In nine of 18 patients, PSA secretion was detectable and observed from both prostate-specific membrane antigen–positive and prostate-specific membrane antigen–negative CTCs. In these patients, 29% to 100% (mean, 52; median, 47) of CTCs secreted PSA, with average PSA secretion levels ranging from 4 to 11.68 pg/cell (mean, 6.38 ± 2.29; median, 6.05). Notably, a strong heterogeneity in PSA secretion was observed within each patient. Our study demonstrates that CTC in mCNPC, even before therapy, produces varying amounts of PSA and often no PSA. These findings may explain the shortcomings of PSA as a biomarker for therapy response. Significance: This study reveals heterogeneity in PSA secretion among individual CTCs from patients with mCNPC prior to any therapeutic intervention, thereby highlighting the limitations of PSA as a biomarker.

Table 1 from PSA Secretion from Single Circulating Tumor Cells of Metastatic Castration-Naïve Prostate Cancer Patients