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.
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.
©2025 The Authors; Published by the American Association for Cancer Research.

Single-molecule immunoassay is a reliable technique for the detection and quantification of low-abundance blood biomarkers, which are essential for early disease diagnosis and biomedical research. However, current single-molecule methods predominantly rely on endpoint detection and necessitate signal amplification via labeling, which brings a variety of unwanted effects, like matrix effect and autofluorescence interference. This study introduces a real-time mass imaging-based label-free single-molecule immunoassay (LFSMiA). Featuring plasmonic scattering microscopy-based mass imaging, a 2-step sandwich assay format enables background reduction, minimization of matrix effect by dynamic tracking of single binding events, and fully leveraging real-time data for improved measurement precision through a Bayesian Gaussian process model, the LFSMiA enables ultra-sensitive and direct protein detection at the single-molecule level in neat blood sample matrices. LFSMiA measurement is demonstrated for interleukin-6 and prostate-specific antigen in buffer, undiluted serum, and whole blood with sub-femtomolar detection limits and eight logs of dynamic ranges. Moreover, comparable performance is achieved with an inexpensive miniaturized setup. To show its translational potential to clinical settings and point-of-care diagnostics, N-terminal pro-B-type natriuretic peptide is examined in patient whole blood samples using the LFSMiA and results in a strong linear correlation (r > 0.99) with standard clinical lab results.
© 2025 The Author(s). Advanced Science published by Wiley‐VCH GmbH.

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.

The treatment of cancer faces a serious challenge as cancer cells within patients are heterogeneous and frequently resistant to therapeutic drugs. Here, we introduce a technology enabling the assessment of single cancer cells exposed to different drugs. PCa cells were individually sorted in self-seeding microwells, cultured for 24 h, and then exposed to several drugs to induce (R1881) or inhibit (Enzalutamide/Abiraterone) the secretion of a protein (PSA). Cell viability and PSA secretion of each individual prostate cell were monitored over a 3-day period. The PSA protein secreted by each cell was captured on a PVDF membrane through a pore in the bottom of each well. The basal PSA secretion was found to be 6.1 ± 4.5 and 3.7 ± 1.9 pg/cell/day for LNCaP and VCaP, respectively. After exposure to R1881, the PSA secretion increased by ~90% on average and was not altered for ~10% of the cells. PSA production decreased in the majority of cells after exposure to enzalutamide and abiraterone.

We report an electro-mechanical biosensor for electrical detection of proteins with disease markers using self-sensing piezoresistive micro-cantilevers. Electrical detection, via surface stress changes, of antigen-antibody (Ag-Ab) specific binding was accomplished through a direct nano-mechanical response of micro-fabricated self-sensing micro-cantilevers. A piezoresistive sensor measures the film resistance variation with respect to surface stress caused by biomolecules specific binding. When specific binding occurred on a functionalized Au surface, surface stress was induced throughout the cantilever, resulting in cantilever bending and resistance change of the piezoresistive layer. The cantilever biosensors were used for the detection of prostate specific antigen (PSA) and C-reactive proteins (CRP), which are a specific marker of prostate cancer and cardiac disease. From the above experiment, it was revealed that the sensor output voltage was proportional to the injected antigen concentration (without antigen, 10 ng/ml, 100 ng/ml, 1 microg/ml). PSA and CRP antibodies were found to be very specific for their antigens, respectively. This indicated that the self-sensing micro-cantilever approach is beneficial for detecting disease markers, and our piezoresistive micro-cantilever sensor system is applicable to miniaturized biosensor systems.