Analysis of the Secretome of Primary CLL Cells in Different in Vitro Co-Culture Systems
Authors: Osswald Lena , Giansanti Piero , Bassermann Florian , Schmidt-Supprian Marc 
Published: December 2024

Introduction
Chronic lymphocytic leukemia (CLL) is the most frequent hematologic malignancy with a heterogeneous clinical course. CLL cells are highly dependent on their microenvironment. When cultured in vitro, patient-derived CLL cells rapidly undergo apoptosis unless they are in co-culture with supporting feeder cells. In patients, CLL cells traffic between peripheral blood, bone marrow and lymph nodes where they receive pro-survival and proliferative stimuli. Secreted proteins play a major role in the bidirectional interplay between CLL cells and the surrounding microenvironment mediating cell-cell communication, activation, migration and proliferation. Therefore, the characterization of the secretome of CLL cells will help to better understand CLL-microenvironment interactions and to identify new potential therapeutic targets.
Here we present, for the first time, a comprehensive analysis of the secretome of primary CLL cells in different in vitro co-culture systems mimicking the bone marrow and lymph node microenvironment.
Methods
Patient-derived CLL cells (n= 20) were co-cultured in serum-free medium with two different feeder cells: EL08 stroma cells that support survival of CLL cells without inducing proliferation and follicular dendritic cells overexpressing CD40L and interleukin 21 (“FDC CD40L IL21”) that successfully induce proliferation in primary CLL cells mimicking the proliferative centers in lymph nodes. Supernatants were centrifuged, filtered and processed by acetone precipitation before being analyzed by high-sensitivity mass-spectrometry-based proteomics. A data-independent acquisition method with a label-free quantification strategy was used to determine relative abundance of secreted proteins.
Results
We identified up to 5000 proteins in the secretome of primary CLL cells. Among the most abundant proteins in both co-culture settings, we found beta-2 microglobulin which is known to correlate with CLL stage and burden and the proteoglycan serglycin which plays a role in inflammatory processes as well as in tumorigenesis but has not been described in CLL yet. The secretome of proliferating CLL cells (in co-culture with FDC CD40L IL21) was characterized by an upregulation of multiple inflammatory chemokines (CXCL1, CXCL3, CXCL6, IL-8, CCL2) compared to resting CLL cells. We further identified several secretory proteins that have been shown to play a role in CLL pathogenesis previously, including members of the PI3K-AKT pathway (CSF1, LAMA4, IL6), the pattern recognition molecule PTX3 and tissue factor pathway inhibitor (TFPI). Moreover, several proteins that are known to promote tumorigenesis and metastasis in solid tumors by inducing epithelial-mesenchymal transition (EMT) such as the protease inhibitors serpinE2, testican-1 and TIMP-1 as well as ADAM12 were more abundant in the secretome of CLL cells in co-culture with FDC CD40L IL21 which implies an activation of EMT-like pathways in proliferating CLL cells.
As the IGHV mutational status is a crucial prognostic factor in CLL patients, we also investigated its impact on the composition of secretory proteins. We found an increased secretion of the anti-inflammatory cytokine interleukin-10 in IGHV mutant samples compared to IGHV unmutant samples suggesting that a stronger immunosuppression might contribute to the more aggressive course of disease in IGHV mutant patients. Moreover, we found increased levels of the cysteine protease cathepsin H in the secretome of IGHV mutant samples which is a key regulator of TLR3 signaling and is considered to promote tumor progression in different cancers, however, its role in CLL has not been explored yet.
Conclusion
We present for the first time an extensive characterization of the secretome of primary CLL cells which gives new important insights into the cross-talk of CLL cells with their microenvironment and identifies new factors that contribute to disease progression and therefore could serve as potential therapeutic targets (e.g., inhibition of EMT-like programs). Further experiments are performed to investigate the specific role of differentially secreted proteins and to elucidate the impact of drug treatment on the composition of the CLL secretome.