Verschoor, A. | bioRxiv Science

bioRxiv2025

Proteomic Remodeling During Tumor Cell-Induced Platelet Aggregation Unveils Metastatic Drivers in Colorectal Cancer

BackgroundColorectal cancer (CRC) is frequently associated with metastasis, resulting in high mortality rates. Platelets are known to play a crucial role in the metastatic cascade influencing tumor microenvironment remodeling, promoting cell transformation, facilitating metastatic niche formation, and shielding circulating tumor cells from immune surveillance. However, platelet proteomic alterations during tumor cell-induced platelet aggregation (TCIPA) remain largely unexplored. This study aims to characterize the proteomic profile of TCIPA in CRC using an in vitro model that recapitulates key aspects of CRC metastasis. MethodsTCIPA was assessed via light transmission aggregometry using an in vitro model incorporating paired primary and metastatic cell cultures. Stable Isotope Labeling with Amino Acids in Cell culture (SILAC) allowed for the discrimination of healthy platelet and tumor cell proteomes prior to and following TCIPA. Data-independent acquisition mass spectrometry was employed to analyze intra- and extracellular tumor and platelet proteomes. Comparative proteomic profiling was performed using a range of bioinformatic analyses, including clustering, differential expression, and Gene Set Enrichment Analyses (GSEA). ResultsComparison of the baseline proteome profiles of the CRC cell lines SW480 and SW620 identified 263 significant differentially expressed proteins (FDR [≤] 0.05, log2FC [≥] 1). The GSEA demonstrated enrichment of the epithelial-mesenchymal transition (FDR: 5.617 x 10-5) gene set in SW480 cells. While SW480 exhibited rapid TCIPA, SW620 did not consistently interact with healthy platelets. Following TCIPA, 34 tumor proteins showed differential expression compared to their naive status (without platelet-exposure). Notably, 17 of these proteins were significantly associated with CRC progression, particularly in the promotion of EMT, metastasis, tumor cell survival, proliferation, and metabolic reprogramming. ConclusionsThis study successfully characterized the proteomic profiles of platelets, platelet secretomes, and colorectal tumor cells following TCIPA-induced activation. The findings highlight the significant role of several tumor proteins and their metabolic effects in colorectal cancer progression, particularly with regard to metastasis.

cancer biology↗

bioRxiv2018

A novel cell-free method to culture Schistosoma mansoni from cercariae to juvenile worm stages for in vitro drug testing

BackgroundThe arsenal in anthelminthic treatment against schistosomiasis is limited and relies almost exclusively on a single drug, praziquantel (PZQ). Thus, resistance to PZQ could constitute a major threat. Even though PZQ is potent in killing adult worms, it has been shown to be limited in its activity against earlier developmental stages. Current in vitro screening strategies for new drugs depend on newly transformed schistosomulae (NTS) for initial hit identification, thereby limiting sensitivity to new compounds predominantly active in later developmental stages. Therefore, the aim of this study was to establish a highly standardized, straightforward and reliable culture method to generate and maintain advanced larval stages in vitro. We present here how this method can be a valuable tool to test drug efficacy at each discrete intermediate larval stage, reducing the reliance on animal use (3Rs).\n\nMethodology/principal findingsCercariae were mechanically transformed into skin-stage (SkS) schistosomulae and successfully cultured under serum-free and cell-independent conditions for up to four weeks with no loss in viability. Under these conditions, larval development halted at the lung-stage (LuS). Addition of human serum (HSe) propelled further development into juvenile worms within eight weeks. Skin and lung stages, as well as juvenile worms, were submitted to 96-well format drug screening assays using known anti-schistosomal compounds such as PZQ, oxamniquine (OXM), mefloquine (MFQ) and artemether (ART). Our findings showed stage-dependent differences in larval susceptibility.\n\nConclusionWith this robust and highly standardized in vitro assay, important developmental stages of S. mansoni up to juvenile worms can be generated and maintained over prolonged periods of time. The phenotype of juvenile worms when exposed to reference drugs was comparable to previously published works for ex vivo harvested adult worms. Therefore, this in vitro assay can help reduce reliance on animal experiments in the search for new anti-schistosomal drugs.\n\nAuthor SummarySchistosomiasis remains a major health threat, predominantly in developing countries. Even though there has been some progress in search of new drugs, praziquantel remains the only available drug. Probably the most important advance in the search for new drugs was in vitro transformation of cercariae and their subsequent culture. However, hit identification in compound screenings is exclusively tested in skin stage parasites and is only confirmed for more mature worms in a subsequent step. This is in part due to the lack of an easy culture system for advanced-stage parasites. We present here a reliable and highly standardized way to generate juvenile worms in vitro in a cell-free culture system. The inclusion of in vitro drug tests on advanced-stage parasites in initial hit identification will help to identify compounds that might otherwise be overlooked. Furthermore, the ability to continuously observe the parasites development in vitro will provide an important platform for a better understanding of its maturation in the human host. Taken together, this opens up new avenues to investigate the influence of specific cell types or host proteins on the development of Schistosoma mansoni and provides an additional tool to reduce animal use in future drug discovery efforts (3Rs).

microbiology↗