Molecular characterisation of ALK+ ALCL - derived exosomes
Here we analyse for the first time the molecular content of the ALK+ and ALK- ALCL-derived exosomes using an in vitro model of ALK+ (Karpas 299, SUP-M2) and ALK- (Mac1, Mac2) ALCL. Exosomes were isolated using well established ultracentrifugation protocols (Suppl. methods). Abundant exosome secretion was seen in both ALK+ and ALK- ALCL cell lines by Nanoparticle Tracking Analysis (NTA), which measures the size and the relative particle concentration, and by transmission electron microscopy (Figure 1a-b). CD30 receptor was detected in the ALCL-derived exosomes by immuno-electron microscopy and Western blot analysis (Figure 1c). Hansen and co-workers have demonstrated a CD30 vesicle-containing network in lymphoid tissue of classical Hodgkin lymphoma (cHL), which might facilitate the communication between distant cell types in cHL tissue and allow a functional CD30–CD30L interaction in trans.(5) Better understanding of the potential role of CD30 in tumor cell microenvironment may have therapeutic implications in the era of CD30-targeted therapy.
In this study, we also found for the first time, that activated ALK and critical components of the ALK signaling are present in the exosomes secreted by ALK+ ALCL cells indicating activation of Ras/ERK, JAK/STAT3, PI3K/AKT/mTOR and Sonic Hedgehog pathways. (Figure 1d-f). Of note, JunB(6), known to be upregulated by NPM-ALK, was enriched at a higher level in ALK+ compared to ALK- ALCL exosomes. Activated STAT3 and AKT, which represent common signaling components in both ALK+ and ALK- ALCL are detected at a variable level in all ALCL-derived exosomes (Figure 1d-f). Interestingly, PD-L1, a crucial regulator of the immune checkpoint, which is differentially expressed among the ALK+ and the ALK- ALCL cell lines and tumors(7) is present in the exosomes (Figure 1d-f). Whether exosomal PD-L1 functionally interacts with the PD-1 on the T cells in TME and may affect the response to immune checkpoint immunotherapy certainly merits further investigation. Mac2A cells expressed higher levels of exosomal PD-L1 than Mac1, and notably, Mac2A were derived from the same patient (as Mac1) cells at a later, more advanced stage of disease. To the best of our knowledge, detection of PD-L1 has not been demonstrated in any lymphoma-derived exosomes to date.
ALCL-derived exosomes can be uptaken by lymphoid cells
The ALCL-derived exosomes were readily uptaken by various cell types including murine pro-B lymphoid cells, Ba/F3 (Figure 2a-b). The uptake was time-dependent and reached a level of 75% at 6 hours. Proteins present in the exosomes such as the CD30 receptor were detected in Ba/F3 cells following exosome uptake and, furthermore, the recipient cells acquired anti-apoptotic properties such expression of cFLIP, which has been previously shown to confer resistance to FAS-mediated apoptosis in ALCL (Figure 2c).(8) Notably, proliferation of Ba/F3 cells was increased in the presence of ALCL-derived exosomes with the highest level being observed at 8 hours, and at this point the proliferation fraction exceeded that of IL-3 – treated cells (Figure 2d). Taken together, these findings suggest that the ALK+ ALCL-derived exosomes are enriched in oncogenic signals, and their uptake by recipient cells leads to anti-apoptotic and cell proliferation effects. Export of oncogenic signaling by exosomes with functional properties has been described in aggressive B-cell lymphomas as well.(9) Moreover, we show that exosomes secreted by the Ba/F3 cell line stably transfected with NPM-ALK are potently uptaken by paternal Ba/F3 cells in a time dependent manner (Figure 2e-j). These findings provide evidence of functional exosome uptake by non-neoplastic lymphoid cells (Ba/F3) as well.
ALCL-derived exosomes interact with the tumor microenvironment cells
To investigate the role of ALCL-derived exosomes on the tumor stroma, we examined the uptake of ALCL derived exosomes by the bone marrow stroma-derived L88 fibroblasts. We found that incubation of the L88 cells with ALCL-derived exosomes confers morphological and molecular changes (expression of aSMA) in the fibroblasts that undergo transformation to cancer-associated fibroblasts (CAF) (Figure 3a-c). It is well-established that the tumor cells induce CAF phenotype, and the activated fibroblasts in turn, produce a number of growth factors and cytokines that promote, among other cellular functions, cell proliferation and angiogenesis.
Having shown the functional interaction of ALCL-secreted exosomes with L88 fibroblasts, we sought to investigate whether exosome-mediated activation of stromal cells altered the cytokine profile of the TME. Using a cytokine array, the level of certain cytokines including MCP-1/CCL2, CCL5/RANTES, IL-6 and IL-8 was substantially increased in the medium following incubation with ALK+ ALCL-secreted exosomes (Figure 3d) with the highest relative increase being observed for IL-8 and MCP-1/CCL2. Notably, MCP-1/CCL2 and IL-6 levels were found to be elevated in the plasma of SCID/beige mice injected with two ALK+ (SUPM2, Karpas 299) and one ALK- (Mac1) ALCL cell lines (Fig 3e-g). In a previous study, Dejean and co-workers (10) showed that high-mobility-group box-1 (HMGB-1), a proinflammatory cytokine, is released by ALK+ ALCL cells, and extracellular HMGB-1 stimulated secretion of the IL-8 chemokine by other cell types as epithelial cells of the skin (keratinocytes). IL-8 was capable of inducing invasiveness and dissemination of ALK+ ALCL cells. This is of particular interest, since ALK+ ALCL express the IL-8 receptors CXCR1 and CXCR2. Therefore, it is tempting to speculate that ALK+ ALCL-secreted exosomes induce IL-8 production by the CAFs, which are secreted in the microenvironment and IL-8, in turn, interacts with the tumor cells bearing the IL-8 receptors. The role of monocyte chemoattractant protein-1 / CCL2 (MCP-1/CCL2) in ALCL pathogenesis and progression is not yet known. The MCP-1/CCL2 and its receptor CCR2 regulate migration from the blood across the vascular endothelium and tissue infiltration of monocytes and macrophages, which play a significant role in immunological surveillance and inflammation.