CD180 is a toll-like receptor that is highly expressed in complex with the MD-1 satellite molecule on the surface of B cells. In chronic lymphocytic leukaemia (CLL) however, the expression of CD180 is highly variable and overall, significantly reduced when compared to normal B cells. We have recently shown that reduced CD180 expression in CLL lymph nodes is associated with inferior overall survival. It was therefore important to better understand the causes of this downregulation through investigation of CD180 at the transcriptional and protein expression levels. Unexpectedly, we found CD180 RNA levels in CLL cells (n = 26) were comparable to those of normal B cells (n = 13), despite heterogeneously low expression of CD180 on the cell surface. We confirmed that CD180 RNA is translated into CD180 protein since cell surface CD180-negative cases presented with high levels of intracellular CD180 expression. Levels of MD-1 RNA were, however, significantly downregulated in CLL compared to normal controls. Together, these data suggest that changes in CD180 cell surface expression in CLL are not due to transcriptional downregulation, but defective post-translational stabilisation of the receptor due to MD-1 downregulation.

In this study, we investigated the cytotoxicity of doxorubicin (DOX)-loaded magnetic nanofluids on 4T1 mouse tumor epithelial cells and MDA-MB-468 human triple-negative breast cancer (TNBC) cells. Superparamagnetic iron oxide nanoparticles were synthesized using sonochemical coprecipitation by applying electrohydraulic discharge treatment (EHD) in an automated chemical reactor, modified with citric acid and loaded with DOX. The resulting magnetic nanofluids exhibited strong magnetic properties and maintained sedimentation stability in physiological pH conditions. The obtained samples were characterized using X-ray diffraction (XRD), transmission electron microscopy (TEM), Fourier-transform infrared spectroscopy, UV-spectrophotometry, dynamic light scattering (DLS), electrophoretic light scattering (ELS), vibrating sample magnetometry (VSM), and transmission electron microscopy (TEM). In vitro studies using the MTT method revealed a synergistic effect of the DOX-loaded citric-acid-modified magnetic nanoparticles on the inhibition of cancer cell growth and proliferation compared to treatment with pure DOX. The combination of the drug and magnetic nanosystem showed promising potential for targeted drug delivery, with the possibility of optimizing the dosage to reduce side-effects and enhance the cytotoxic effect on cancer cells. The nanoparticles’ cytotoxic effects were attributed to the generation of reactive oxygen species and the enhancement of DOX-induced apoptosis. The findings suggest a novel approach for enhancing the therapeutic efficacy of anticancer drugs and reducing their associated side-effects. Overall, the results demonstrate the potential of DOX-loaded citric-acid-modified magnetic nanoparticles as a promising strategy in tumor therapy, and provide insights into their synergistic effects.

Chronic lymphocytic Leukaemia (CLL) is a B-cell malignancy marked by biological heterogeneity, variable progression and survival outcomes. Orphan toll-like receptor CD180 (RP105) complexed with the accessory protein MD-1 and CD150, which belongs to the signalling lymphocyte activation molecule family (SLAMF1), have emerged as an important modulators of CLL cell signaling and fate. However, their functional interplay, remains insufficiently understood. This study investigated the surface and intracellular expression of CD180, CD150, and MD-1 in primary CLL cells and the MEC-1 cell line, alongside their downstream signaling responses. Flow cytometric analyses revealed that MD-1 expression is tightly associated with CD180 and CD150 positivity, which may further indicate a role for MD-1 in stabilizing CD180 surface localization. Stimulation of MD-1⁺CD180⁺ CLL cells revealed four distinct signaling phenotypes: AKT signalers, p38MAPK signalers, dual signalers, and non-responders. CD150^low cells preferentially activated AKT, whereas CD150^high cells favored p38MAPK signaling, indicating context-dependent modulation of downstream pathways. Notably, stimulation via CD150 alone induced phosphorylation of both AKT and p38MAPK in specific subsets, confirming its independent signaling capacity. In synchronized MEC-1 cells, CD180 and MD-1 expression peaked at 48 h, coinciding with enhanced AKT phosphorylation upon combined CD180/CD150 stimulation. These data suggest that the temporal dynamics of CD180 expression modulate the outcome of CD150-driven signaling, indicating a tightly regulated and interdependent interaction. Collectively, these findings define a CD180/MD-1/CD150 signaling axis that underlies the intracellular signaling heterogeneity in CLL. The relative expression balance between CD180 and CD150 may steer cells toward pro-survival or stress-associated signaling, providing novel mechanistic insight into CLL biology and identifying candidate biomarkers or therapeutic targets.

Chronic lymphocytic leukaemia (CLL) progression is critically dependent on tumour microenvironment (TME) signals, including those regulated via toll-like receptors (TLRs) that drive CLL cell survival and proliferation. We have previously shown that CD180, an orphan TLR is expressed on the surface in approximately 60 % of CLL samples, and that heterogeneity of CD180 cell surface expression and signalling impacts CLL cell survival, proliferation, and apoptosis. Interleukin-4 (IL-4), a cytokine abundantly present in the CLL microenvironment, is a well-documented pro-survival factor for CLL cells. IL-4 has also been shown to enhance sIgM expression and function by subsequent downstream signalling in a JAK3/STAT6 dependent manner within CLL samples. In this study, we investigated the impact of IL-4 on CD180 expression, CD180-mediated signalling and apoptosis of CLL cells and cell lines MEC-1 and RAMOS. Our results demonstrate that IL-4 enhances CD180 surface expression in CLL cells and RAMOS cell line, affecting downstream signalling pathways that alter CLL cell survival through the activation of AKT and p38MAPK. MEC-1 cells exhibited a less pronounced and variable response. These findings highlight the IL-4/CD180 axis as an important modulator of CLL, altering cell signalling dynamics.