The treatment of choice for prostate cancer is androgen deprivation (ADT) and novel hormonal agents such as Abiraterone, Enzalutamide, or Apalutamide. Initially, this therapy is highly effective, but a significant challenge arises as most patients eventually develop resistance, resulting in castration-resistant prostate cancer (CRPC). Furthermore, the sequential use of these drugs can lead to cross-resistance, diminishing their efficacy. Tumor heterogeneity plays a pivotal role in the development of resistance to different treatments. This study utilized cellular models of CRPC to assess the response to Apalutamide when it was administered as a second- or third-line treatment. Functional and genetic analyses were conducted in various CRPC cell models exposed to Apalutamide. These analyses included real-time cell monitoring assays, flow cytometry, clonogenicity assays, and RT-qPCR. CRPC cell models were capable of continued proliferation, maintained cell cycle profiles similar to those of untreated cells, and retained their clonogenic potential. Cross-resistance to Apalutamide in models of ADT, ADT plus Enzalutamide, or Abiraterone resistance did not correlate with the expression levels of AR-V7 and AR-V9 variants. Gene expression analysis of resistant prostate cancer cell lines revealed that treatment with Apalutamide induced the emergence of more aggressive phenotypes, including cancer stem cells or neuroendocrine differentiation profiles. Most CRPC cell models developed cross-resistance to Apalutamide and were able to proliferate and retain their clonogenic capability. Apalutamide resistance was not linked to the expression of AR-V7 or AR-V9 variants but was instead associated to bypass of AR signaling pathway and the emergence of more aggressive expression profiles.

Platelets and their extracellular vesicles (EVs) have emerged as promising liquid biopsy biosources for cancer detection and monitoring. The megakaryoblastic MEG-01 cell line offers a controlled system for generating platelet-like particles (PLPs) and EVs through valproic-acid-induced differentiation. Here, we performed comprehensive characterization and proteomic validation of MEG-01-derived populations, native human platelets, and their EVs using nanoparticle tracking analysis, transmission electron microscopy, imaging flow cytometry and quantitative proteomics. MEG-01 megakaryocytic differentiation is characterized by polylobulated nuclei, proplatelet formation, and elevated CD41/CD42a expression. PLPs predominantly exhibit an activated-like phenotype (CD62P+, degranulated morphology), while microvesicles (100–500 nm) and exosomes (50–250 nm) displayed size distributions and phenotypic markers consistent with native platelet-derived EVs. Proteomics identified substantial core proteomes shared across fractions and fraction-specific patterns consistent with selective cargo partitioning during EV biogenesis. Functional enrichment indicated that MEG-01-derived vesicles preserve key hemostatic, cytoskeletal, and immune pathways commonly associated with platelet EV biology. Ingenuity Pathway Analysis showed that PLPs exhibit proliferative transcriptional programs (elevated MYC/RB1/TEAD1, reduced GATA1), while plasma exosomes display minimal differential pathway activation compared to MEG-01 exosomes. Overall, these findings suggest that MEG-01-derived EVs approximate certain aspects of megakaryocyte-lineage exosomes and activated platelet-like states, although they do not fully replicate native platelet biology. Notably, plasma exosomes show strong proteomic convergence with MEG-01 exosomes, whereas platelet exosomes retain distinct activation-related features.

In the tumor context, platelets play a significant role in primary tumor progression, dissemination and metastasis. Analysis of this interaction in various cancers, such as non-small cell lung cancer (NSCLC), demonstrate that platelets can both transfer and receive biomolecules (e.g. RNA and proteins) to and from the tumor at different stages, becoming tumor-educated platelets. To investigate how platelets are able to transfer oncogenic material, we developed in vitro platelet-like particles (PLPs), from a differentiated MEG-01 cell line, that stably carry RNA and protein of the KRAS^G12D oncogene in fusion with GFP. We co-cultured these PLPs with NSCLC H1975 tumor cells to assess their ability to transfer this material. We observed that the generated platelets were capable of stably expressing the oncogene and transferring both its RNA and protein forms to tumor cells using qPCR and imaging techniques. Additionally, we found that coculturing PLPs loaded with GFP-KRAS^G12D with tumor cells increased their proliferative capacity at specific PLP concentrations. In conclusion, our study successfully engineered an MEG-01 cell line to produce PLPs carrying oncogenic GFP-KRAS^G12D simulating the tumor microenvironment, demonstrating the efficient transfer of this oncogene to tumor cells and its significant impact on enhancing proliferation.