Metastatic prostate cancer is one of the leading causes of cancer-related morbidity and mortality worldwide. It is characterized by a high mortality rate and a poor prognosis. In this work, we explore how a clinical oncologist can apply a Stackelberg game-theoretic framework to prolong metastatic prostate cancer survival, or even make it chronic in duration. We utilize a Bayesian optimization approach to identify the optimal adaptive chemotherapeutic treatment policy for a single drug (Abiraterone) to maximize the time before the patient begins to show symptoms. We show that, with precise adaptive optimization of drug delivery, it is possible to significantly prolong the cancer suppression period, potentially converting metastatic prostate cancer from a terminal disease to a chronic disease for most patients, as supported by clinical and analytical evidence. We suggest that clinicians might explore the possibility of implementing a high-level tight control (HLTC) treatment, in which the trigger signals (i.e. biomarker levels) for drug administration and cessation are both high and close together, typically yield the best outcomes, as demonstrated through both computation and theoretical analysis. This simple insight could serve as a valuable guide for improving current adaptive chemotherapy treatments in other hormone-sensitive cancers.

Our research aims to understand the adaptive-ergo potentially metastatic-responses of prostate cancer to changing microenvironments. Emerging evidence implicates a role of the polyaneuploid cancer cell (PACC) state in metastasis, positing the PACC state as capable of conferring metastatic competency. Mounting in vitro evidence supports increased metastatic potential of cells in the PACC state. Additionally, our recent retrospective study revealed that PACC presence in patient prostate tumors at the time of radical prostatectomy was predictive of future metastasis. To test for a causative relationship between PACC state biology and metastasis in prostate cancer, we leveraged a novel method designed for flow cytometric detection of circulating tumor cells (CTC) and disseminated tumor cells (DTC) from animal models. This approach provides both quantitative and qualitative information about the number and PACC status of recovered CTCs and DTCs. Specifically, we applied this approach to the analysis of subcutaneous, caudal artery, and intracardiac murine models. Collating data from all models, we found that 74% of recovered CTCs and DTCs were in the PACC state. Furthermore, in vivo colonization assays proved that PACC populations can regain proliferative capacity at metastatic sites. Additional in vitro analyses revealed a PACC-specific partial epithelial-to-mesenchymal transition phenotype and a prometastatic secretory profile, together providing preliminary evidence of prometastatic mechanisms specific to the PACC state.Implications: Considering that many anticancer agents induce the PACC state, our data position the increased metastatic competency of PACC state cells as an important unforeseen ramification of neoadjuvant regimens, which may help explain clinical correlations between chemotherapy and metastatic progression.

Chemotherapy resistance remains a major challenge in clinical oncology. The mechanisms that regulate dynamic movement through adaptive cell states to survive through chemotherapeutic stress need to be understood to advance treatment options for patients and move towards curative therapies. In response to various stressors, cancer cells can reprogram their cell cycle, uncoupling DNA replication from mitotic division to acquire a large polyploid phenotype. This alternative cell cycle of repeated G and S phases is known as an endocycle, observed across the tree of life during both development and stress response. Cells exhibiting this phenotype exist in the transient endocycling cancer cell state. Endocycling cancer cells (ECCs) may serve as an actuator of chemotherapy resistance and disease recurrence. Understanding the mechanisms regulating the mitotic-to-endocycle switch and back are crucial to enhance treatments for patients with chemotherapy resistant cancer. A major driver of endocycling described in the literature is the transcription factor, c-Myc, due to its role in governing the G1/S transition. C-Myc is dysregulated in >70% of cancers, functioning as a critical cell cycle driver through transcriptional regulation of cyclins and CDKs, as well as essential metabolic programs necessary for cycle progression. Despite the published roles of Myc in both cancer and endocycling, we have shown through single cell RNA sequencing and western blotting that ECCs exhibit a low-Myc phenotype across multiple contexts. Myc protein levels can be rescued using the proteasome inhibitor, MG132, demonstrating that the low Myc status is driven by proteasomal degradation. To test if Myc degradation serves a mechanistic function, the small molecule inhibitor, MYCi975, was used to inhibit Myc activity at various points in the mitotic cell cycle. Our data shows that loss of Myc activity in cells beyond the restriction point induces mitotic bypass, generating a 4N G1 population. Notably, cells actively replicating their DNA in S phase show high sensitivity to Myc inhibition after skipping to G1, displaying cytotoxic effects not seen in other phases of the cell cycle. Similarly, when ECCs are challenged with the Myc inhibitor, the treatment promotes mitotic bypass and enriches for G0 and G1 populations, primed to enter the next S phase upon release from Myc inhibition. Together, these data suggest a temporal aspect of Myc regulation that has not been described in the literature. Myc activity beyond the restriction point appears to commit cells to mitotic entry, playing a functional role in coupling DNA replication to mitosis. This data supports our hypothesis that the low-Myc status of endocycling cells plays a mechanistic role in permitting mitotic bypass through premature loss of activity in G2. Preliminary data suggests that rescuing Myc activity will force mitotic entry producing mitotic progeny cells. Michael Loycano, Kenneth J. Pienta, Sarah R. Amend. Temporal regulation of c-Myc in endocycling cancer cells facilitates mitotic bypass in response to chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5413.

Cancer cells in solid tumors experience hypoxia, a condition of low O 2 concentration, since their O 2 demand exceeds the supply from the surrounding vasculature. However, how these cells adapt to hypoxia requires further elucidation. Here, we use a transparent phosphorescent thin film to visualize the self-generated hypoxia field of prostate cancer cells and quantify local O 2 consumption rates, measured locally as the Laplacian of the O 2 field. Single-cell tracking on steep O 2 gradients revealed that larger cells exhibit higher motility and moderate migration bias toward O 2 -rich regions. Termination of hypoxia before cessation of O 2 consumption shifted cell distributions to larger sizes, whereas prolonged hypoxia induced apoptosis, producing cell populations of smaller areas post-hypoxia. Such resilience to hypoxia was absent for noncancerous fibroblasts. Our findings suggest that larger PC3 cells have enhanced metabolic fitness under hypoxia, identifying these cells as potential targets of cancer therapy.

Understanding and overcoming therapy resistance in cancer is crucial, as it remains a major driver of lethality. We have shown that following chemotherapy, surviving cancer cells repeatedly skip mitosis, leading to increased genomic content concomitant with an increase in cell size. This survival mechanism has been observed in multiple tumor types and in response to multiple therapy classes, suggesting a convergent phenotype. We hypothesize that this surviving cell phenotype is the actuator of therapy resistance observed in patients with cancer recurrence. It is widely known that chemotherapies elevate levels of reactive oxygen species (ROS) by indirectly disrupting the electron transport chain, leading to the generation of superoxides. When ROS levels exceed cellular antioxidant capacities, cells experience oxidative stress whereby ROS molecules directly damage important macromolecules, potentially leading to cell death. Using live cell imaging with DCFDA and CellROX dyes, we found that the cells surviving even long after therapy release contain elevated levels of ROS as compared to untreated controls across two cell lines of different tumor types (PC3 prostate cancer and MDA-MB-231 breast cancer) and following treatment with two classes of chemotherapy (cisplatin and docetaxel). In single-cell RNA sequencing datasets, we further find an enrichment for downstream targets of NRF2, a major transcription factor that regulates the response to oxidative stress in cells that survive therapy. This suggests that the cells that survive chemotherapy continue to experience oxidative stress even after therapy is removed and respond by initiating defensive mechanisms to promote survival. Stress granule formation is a common cellular response to oxidative stress that form immediately following a translational arrest. Stress granules are cytoplasmic aggregates of RNA bound by proteins, such as G3BP1, which form a stable core and recruit binding partners to form a mature granule. Stress granules are transient and offer cells the capacity to resume translation rapidly following recovery from stress. Alternatively, autophagy of stress granules during recovery offers cells a rich pool of biomolecules upon relief from stress. In both of our cell line models, we find that while stress granules are virtually absent in untreated cells, up to 18% of surviving cells induce stress granule formation, depending on cell type and chemotherapy. We further demonstrated that surviving cells that containing stress granule have enriched nuclear NRF2 levels, suggesting a positive relationship between stress granule formation and cellular response to ROS. Based on these data, we hypothesize that surviving cells experiencing oxidative stress induce stress granule formation. Future work aims to show the pro-survival role of stress granule formation through time-lapse imaging of cells expressing a G3BP1-GFP construct. Louis T. Rolle, Luke V. Loftus, Kenneth J. Pienta, Sarah R. Amend. Therapy resistant cancer cells containing stress granules display signatures of oxidative stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 1508.

PURPOSE:Biochemical recurrence (BCR) of prostate cancer (PCa) after definitive surgery and/or radiation (including salvage strategies) is a burgeoning area of clinical research inspired by ultrasensitive next-generation imaging. Most phase III trials in PCa have focused on metastatic disease, defined by conventional imaging. Despite the emergence of new imaging, clinical trial principles from metastatic studies will not optimize future BCR trials. METHODS:A Working Group convened at the National Cancer Institute on November 13, 2024 (NCI BCR WG). Key areas of discussion included nomenclature, baseline criteria for data capture, imaging considerations, delineation of high-risk populations to be targeted for trial development, requirements of metastasis-directed therapy (MDT) or hormonal therapy, quality-of-life considerations, and potential study end points. RESULTS:The NCI BCR WG defined the novel term "prostate-specific membrane antigen (PSMA)+BCR" to identify the emerging concept of recurrent PCa identifiable only on PSMA positron emission tomography (PET), overlapping with BCR and distinct from metastatic hormone-sensitive PCa as traditionally defined by conventional imaging. The WG suggested defining high-risk BCR with a prostate-specific antigen doubling time of ≤6 months, regardless of PET findings. The WG provided recommendations for baseline data capture and imaging requirements. Neither systemic therapy nor MDT were considered mandatory for control arms. The WG also discussed novel end points and quality-of-life metrics in this disease space. CONCLUSION:These discussions should inform future clinical BCR trials in this distinct disease space relative to metastatic disease defined by conventional imaging. The NCI BCR WG strongly advocates that future trials explore deintensification of treatment to minimize toxicity in this relatively indolent disease state.

Liquid chromatography (LC) and mass spectrometry (MS) are two critical components in proteomics. Advances in methods for both LC and MS have significantly enhanced protein identification and quantifications of limited amounts of proteins, particularly at the picogram-to-nanogram level of proteins. In this study, we explored various LC conditions and MS platforms to optimize protein identification and quantification using data-independent acquisition (DIA). Our investigation focused on evaluating the sensitivity for protein identification, reproducibility of quantification, and robustness across multiple models, specifically focused on analyzing proteins at pico- to nanogram levels, with an emphasis on single-cell proteomics. We further applied our approach for the proteomic analysis of HeLa single cells. Overall, we identified and quantified over 6300 proteins at the single-cell level amount of peptides with a coefficient of variation (CV) of less than 20%, and detected up to 5000 proteins from isolated single HeLa cell samples. Finally, we analyzed docetaxel-treated and nontreated PC3 cells to reveal proteome changes at the single-cell level. This study provides a comprehensive technical evaluation for LC-MS methods in protein identification and quantification for analytical applications involving single-cell proteomics from the picogram to nanogram level of proteins.

Our objectives were to assess the prognostic value of posttherapy [177Lu]Lu-PSMA (LuPSMA) SPECT/CT by visual evaluation using RECIP 1.0 during LuPSMA therapy and develop an evidence-based clinical protocol to monitor the efficacy of LuPSMA. Methods: Patients with metastatic castration-resistant prostate cancer who received at least 2 LuPSMA cycles between April 2019 and November 2023 were retrospectively included in this study. Pairs of baseline and interim LuPSMA SPECT/CT (SPECT) and PSMA PET/CT (PET) images after 2 therapy cycles were analyzed per visual RECIP 1.0. Changes in prostate-specific antigen (PSA) levels at 12 wk were categorized by Prostate Cancer Working Group Criteria 3 guidelines and combined with RECIP 1.0 reads to determine disease progression using a composite classification method (PSA + RECIP). The primary outcome was the prognostic value of posttherapeutic SPECT by RECIP 1.0 for overall survival (OS). The clinical protocol was developed on the basis of the prognostic accuracy (Harrell concordance index, or C-index) of SPECT versus PET and the combination of SPECT plus PSA (SPECT + PSA) versus the combination of PET plus PSA (PET + PSA). Results: Data from 105 patients were evaluated. Progressive disease determined by SPECT was associated with shorter OS compared with stable disease (hazard ratio, 2.5; 95% CI, 1.2-5.3; P = 0.015) and with partial response (hazard ratio, 6.5; 95% CI, 2.7-15.7; P < 0.001). Of the 73 patients who underwent PET after 2 cycles, 7 (10%), 30 (41%), 22 (30%), and 30 (41%) had tumor progression shown by SPECT, PET, SPECT + PSA, and PET + PSA, respectively. The C-index for SPECT was inferior compared with that for PET (0.54 vs. 0.66; P < 0.001), whereas the C-indices for SPECT + PSA and PET + PSA did not differ significantly (0.62 vs. 0.66, respectively; P = 0.07). Conclusion: Posttherapeutic LuPSMA SPECT/CT per RECIP 1.0 after 2 therapy cycles was prognostic for OS. LuPSMA SPECT/CT identified significantly fewer patients with RECIP-classified progressive disease; however, SPECT + PSA achieved similar prognostic accuracy to PET + PSA for LuPSMA response evaluation.

PURPOSE:Randomized clinical trials (RCTs) have shown progression-free survival (PFS) benefits of metastasis-directed therapy (MDT) without androgen deprivation therapy for oligometastatic castration-sensitive prostate cancer (omCSPC). Most patients with bone metastatic (BM) omCSPC recur with additional bone disease after MDT. We hypothesized the BM-targeting alpha-emitter radium-223 dichloride (Ra223) could target subclinical bone disease and delay progression. METHODS:This is an investigator-initiated, multicenter, open-label phase II RCT. Eligible men with recurrent omCSPC with ≥one bone metastasis (≤three on conventional imaging and/or ≤five on molecular imaging) were randomly assigned (1:1) to stereotactic ablative radiation (SABR) MDT alone or SABR MDT with Ra223 (six cycles). Primary end point was composite PFS. RESULTS:From August 9, 2019, to March 2, 2023, 64 patients were randomly assigned, 33 to SABR MDT and 31 to SABR MDT/Ra223 balancing for key covariates. Most SABR MDT/Ra223 patients (87%) received six cycles of Ra223. The median PFS was 11.8 months with SABR MDT and 10.5 months with SABR MDT/Ra223 (adjusted hazard ratio [aHR], 1.42 [95% CI, 0.79 to 2.56]; P = .24). Seven patients (11%) experienced grade 3 treatment-related adverse events (no grade 4 or 5), 2 of 33 (6%) with SABR and 5 of 30 (17%) with SABR MDT/Ra223. Patients with high-risk (HiRi) pathogenic mutations in ATM, BRCA1/2, RB1, or TP53 had worse PFS (HR, 5.95 [95% CI, 1.83 to 19.3]; P = .003). Greater T-cell receptor (TCR) unique productive rearrangements were prognostic for improved PFS independent of the treatment arm (aHR, 0.45 [95% CI, 0.21 to 0.96]; P = .04). CONCLUSION:Adding Ra223 to SABR MDT in BM omCSPC does not delay progression of disease. We provide evidence for an HiRi mutational signature and TCR repertoire as prognostic biomarkers in omCSPC treated with SABR MDT, highlighting the importance of collecting biological correlates in RCTs for omCSPC.

Cancer cells survive treatment through mechanisms that remain unclear. This study investigates the chemical changes that occur in cancer cells after treatment, focusing on lipid metabolism as a potential marker for survival and resistance. Using Time-of-Flight Secondary Ion Mass Spectrometry (ToF-SIMS) and advanced multivariate statistical analysis, we compared the chemical profiles of untreated and surviving cancer cells. Region-of-Interest (ROI) analysis revealed distinct differences in the lipid compartments, with surviving cancer cells showing significant accumulation of lipid droplets. While Principal Component Analysis (PCA) was able to differentiate the chemistry of untreated and surviving cancer cells as well as their cellular components, Multivariate Curve Resolution (MCR) provided a clearer and more detailed distinction, enabling the identification of specific cellular features such as the cytoplasm, nucleus, and lipid droplets within the surviving cells. The separation of the chemistry in nucleus and lipid droplets emphasizes the effectiveness in complex spectral analysis. Furthermore, the ability to map the distribution of lipid droplets in surviving cells can advance our understanding of how these structures contribute to cancer cell survival during treatment. The study highlights the importance of lipid droplets as potential biomarkers for cancer cell adaptation and survival post-treatment, with implications for developing new therapeutic strategies.

Prostate cancer is one of the most frequently diagnosed cancers in men. Prostate tumor staging and disease aggressiveness are evaluated based on the Gleason scoring system, which is further used to direct clinical intervention. The Gleason scoring system provides an estimate of tumor aggressiveness through quantitation of the serum level of prostate specific antigen (PSA) and histologic assessment of Grade Group, determined by the Gleason Grade of the tumor specimen. To improve our understanding of the proteomic characteristics differentiating low- versus high-grade prostate cancer tumors, we performed a deep proteomic characterization of laser microdissected epithelial and stromal subpopulations from surgically resected tissue specimens from patients with Gleason 6 (n = 23 specimens from n = 15 patients) and Gleason 9 (n = 15 specimens from n = 15 patients) prostate cancer via quantitative high-resolution liquid chromatography-tandem mass spectrometry analysis. In total, 789 and 295 grade-specific significantly altered proteins were quantified in the tumor epithelium and tumor-involved stroma, respectively. Benign epithelial and stromal populations were not inherently different between Gleason 6 versus Gleason 9 specimens. Notably, 598 proteins were exclusively significantly altered between Gleason 9 (but not Gleason 6) tumor-involved stroma and benign stroma, including several proteins involved in cholesterol biosynthesis and nucleotide metabolism. Proteomic alterations between Gleason 6 versus Gleason 9 were exclusive to the disease microenvironment, observed in both the tumor epithelium and tumor-involved stroma. Further, the molecular alterations measured in the tumor-involved stroma from Gleason 9 cases relative to the benign stroma have unique significance in disease aggressiveness, development, and/or progression. Our data provide supportive evidence of a need for further investigations into targeting stromal reservoirs of cholesterol and/or deoxynucleoside triphosphates in PCa tumors and further highlight the necessity for independent examination of the TME epithelial and stromal compartments.

BACKGROUND:This study investigates the impact of hormone therapy (HT) on the diagnostic performance of 18F-piflufolastat PET/CT in OSPREY (NCT02981368) cohort B patients with recurrent or metastatic prostate cancer. METHODS:18F-piflufolastat PET/CT was evaluated in OSPREY cohort B patients (n = 117 men) with elevated prostate-specific antigen (PSA) levels and suspected local recurrence or metastatic disease on baseline conventional imaging. Patients were stratified based on HT status, and sensitivity and positive predictive value (PPV) were determined for the subset of 93 patients with evaluable pathology. Baseline serum PSA and testosterone levels were determined within 30 days before dosing using standardized laboratory methods. RESULTS:In OSPREY cohort B, 34.4% of patients (32/93) were on at least one concomitant HT with a median exposure duration of 15.5 months. The median baseline PSA and testosterone levels for patients on concurrent HT (n = 32) were 31.6 ng/mL and 9 ng/dL, respectively. For patients not on concurrent therapy (n = 61), median PSA and testosterone levels were 6.1 ng/mL and 317.35 ng/dL, respectively. The median sensitivity of 18F-piflufolastat PET/CT across three readers was 96.4% (95%CI: 80.8%-100%) in patients receiving concurrent HT and 95.4% (95%CI: 83.7%-99.6%) in patients not receiving concurrent HT. A modest increase in median PPV was observed in patients receiving concomitant HT (median of three readers: 90.0% [95%CI: 73.6, 97.3]) compared to patients not receiving concomitant therapy (median of three readers: 77.4% [95%CI: 66.1, 88.6]). CONCLUSIONS:The diagnostic performance of 18F-piflufolastat PET/CT was unaffected by concomitant HT in OSPREY cohort B patients with recurrent and/or metastatic prostate cancer.

Cells regulate the expression of cell cycle-related genes, including cyclins essential for mitosis, through the transcriptional activity of the positive transcription elongation factor b (P-TEFb), a complex comprising CDK9, cyclin T, and transcription factors. P-TEFb cooperates with CDK7 to activate RNA polymerase. In response to DNA stress, the cell cycle shifts from mitosis to repair, triggering cell cycle arrest and the activation of DNA repair genes. This tight coordination between transcription, cell cycle progression, and DNA stress response is crucial for maintaining cellular integrity. Cyclin-dependent kinases CDK7 and CDK9 are central to both transcription and cell cycle regulation. CDK7 functions as the CDK-activating kinase (CAK), essential for activating other CDKs, while CDK9 acts as a critical integrator of signals from both the cell cycle and transcriptional machinery. This review elucidates the mechanisms by which CDK7 and CDK9 regulate the mitotic process and cell cycle checkpoints, emphasizing their roles in balancing cell growth, homeostasis, and DNA repair through transcriptional control.

PSMA-PET/CT has emerged as a superior diagnostic tool for prostate cancer, demonstrating enhanced accuracy over conventional imaging methods. Although sensitive for detecting local and metastatic prostate tumors, it can also identify other non-prostate PSMA positive lesions. Here, we report a rare case of a 67-year-old patient with metastatic prostate adenocarcinoma who was found to have an incidental Gastrointestinal Stromal Tumor (GIST), during restaging with 68Ga-PSMA-11 PET/CT. Given the broad application of PSMA PET/CT in prostate cancer, its role in diagnosing other non-prostate PSMA tumors remains uncertain, highlighting the need for further research into its application in cancer management.

Therapy resistance is one of the most common underlying causes of poor prognosis in cancer patients, ultimately driving fatal outcome. We have previously demonstrated that following chemotherapeutic stress, a subset of cancer cells undergo a mitotic skip and enter an endocycle, causing whole-genome duplication without division. This endocycling cell state is resistant to cytotoxic therapies and thus is an actuator of therapy resistance. Cells in the endocycling state eventually undergo depolyploidization and repopulate the tumor, observed clinically as a recurrence. A common feature of cancers is centrosome amplification (CA) that includes aberrations in centrosome number (>2 centrosomes/cell), size, shape, and/or positioning. Our preliminary data has shown that endocycling cancer cells exhibit CA following treatment across multiple types of chemotherapies. Based upon this data, we hypothesize that studying centrosome dynamics and abnormalities presents a window into better understanding endocycle biology and this novel mechanism of therapy resistance.We have found that the population of prostate cancer and breast cancer cells possessing CA following treatment with LD50 dose of cisplatin or docetaxel increased as a function of recovery time to over 95% by day 10, with nearly all cells clustering extra centrosomes together. Of note, the number of mitotic skips an endocycling cell undergoes has not been found to be associated with the number of centrosomes the cell has. PLK4 is a kinase essential for centriole duplication and its overexpression causes CA. Immunofluorescence analysis has revealed that endocycling cancer cells exhibit an increase in PLK4 protein expression. Endocycling cancer cells also exhibit increased protein expression of KIFC1, a mediator of centrosome clustering. Both PLK4 and KIFC1 protein expression have been found to positively correlate with DNA content. We hypothesize that endocycling cells may use clustered, supernumerary centrosomes as platforms to locally concentrate signaling molecules, lowering the activation threshold for downstream effectors. Published data from bioID proteomics of centrosomal proteins has revealed an enormous unexplored protein landscape around these organelles. Because of this, CA may be a critical mechanism for endocycling cancer cells to survive and maintain this resistant cell state. Madison M. Purkerson, Sarah R. Amend, Kenneth J. Pienta. Cancer cells may utilize centrosome amplification to promote survival as a response to chemotherapeutic stress [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 375.

As PSMA-PET/CT is routinely used for prostate cancer diagnosis due to its improved accuracy over traditional imaging techniques, an increasing number of non-prostate lesions are also being identified. Here, we report a patient with prostate adenocarcinoma, who was diagnosed with a synchronous atypical meningioma during PSMA PET/CT staging. Although PSMA PET/CT is a valuable tool in prostate cancer management, its limitations must be considered when assessing non-prostate lesions to minimize the risk of diagnostic errors.

This study delves into the proteomic intricacies of drug-resistant cells (DRCs) within prostate cancer, which are known for their pivotal roles in therapeutic resistance, relapse, and metastasis. Utilizing single-cell proteomics (SCP) with an optimized high-throughput data-independent acquisition (DIA) approach with the throughput of 60 sample per day, we characterized the proteomic landscape of DRCs in comparison to parental PC3 cells. This DIA method allowed for robust and reproducible protein quantification at the single-cell level, enabling the identification and quantification of over 1300 proteins per cell on average. Distinct proteomic sub-clusters within the DRC population were identified, closely linked to variations in cell size. The study uncovered novel protein signatures, including the regulation of proteins critical for cell adhesion and metabolic processes, as well as the upregulation of surface proteins and transcription factors pivotal for cancer progression. Furthermore, by conducting single-cell RNA-seq (scRNA-seq) analysis, we identified six upregulated and 10 downregulated genes consistently altered in drug-treated cells across both SCP and scRNA-seq platforms. These findings underscore the heterogeneity of DRCs and their unique molecular signatures, providing valuable insights into their biological behavior and potential therapeutic targets.