The effect of colchicine on cholesterol crystal formation, expansion and morphology: a potential mechanism in atherosclerosis.

Background: Inflammation is pivotal to the progression of atherosclerosis. Cholesterol crystals (CCs) that grow and enlarge within the plaque core can cause plaque rupture and trigger inflammation as they deposit into the atherosclerotic bed. Thus, agents that affect CC formation, expansion, and morphology may reduce cardiovascular (CV) risk independent of lipid-lowering and anti-inflammatory therapy. Objective: Because colchicine is highly concentrated in leukocytes that can enter the atherosclerotic plaque core, we tested its effect on the formation and growth of CCs in bench experiments to determine whether it may have direct effects on CCs, independent of its known anti-inflammatory actions. Method: Different dosages of colchicine mixed with cholesterol (0.05-5 mg/ml/g of cholesterol) were used to influence the formation CCs and volume expansion in vitro. These were compared to control samples with cholesterol in ddH2O without colchicine. In an ex vivo study, fresh atherosclerotic human plaques were incubated with and without colchicine in a water bath at 37°C for 48 h to assess the impact of colchicine on CC morphology. Scanning electron microscopy (SEM) was utilized to analyze CC morphology in samples from the various treatment groups. Results: The addition of colchicine to cholesterol caused a substantial dose-dependent reduction in volume (p < 0.05). Pairwise comparisons of volume reduction, showed a significant reduction in volume at 5 mg/ml/g when compared to control (p < 0.02) but the calculated Cohen's d effect size was large for five of the six pairwise comparisons. By SEM, CCs from both in vitro and ex vivo samples treated with colchicine had evidence of dissolution and changes in their morphology as evidenced by the loss of their sharp edges. In contrast, CCs in untreated specimens retained their typical geometric structure. Conclusions: Colchicine can reduce CC formation and expansion and alter CC morphology. These previously unappreciated effects of colchicine may contribute to its clinical benefit in patients with CV disease independent of its anti-inflammatory effects.

Machine learning-assisted elucidation of CD81-CD44 interactions in promoting cancer stemness and extracellular vesicle integrity.

Tumor-initiating cells with reprogramming plasticity or stem-progenitor cell properties (stemness) are thought to be essential for cancer development and metastatic regeneration in many cancers; however, elucidation of the underlying molecular network and pathways remains demanding. Combining machine learning and experimental investigation, here we report CD81, a tetraspanin transmembrane protein known to be enriched in extracellular vesicles (EVs), as a newly identified driver of breast cancer stemness and metastasis. Using protein structure modeling and interface prediction-guided mutagenesis, we demonstrate that membrane CD81 interacts with CD44 through their extracellular regions in promoting tumor cell cluster formation and lung metastasis of triple negative breast cancer (TNBC) in human and mouse models. In-depth global and phosphoproteomic analyses of tumor cells deficient with CD81 or CD44 unveils endocytosis-related pathway alterations, leading to further identification of a quality-keeping role of CD44 and CD81 in EV secretion as well as in EV-associated stemness-promoting function. CD81 is coexpressed along with CD44 in human circulating tumor cells (CTCs) and enriched in clustered CTCs that promote cancer stemness and metastasis, supporting the clinical significance of CD81 in association with patient outcomes. Our study highlights machine learning as a powerful tool in facilitating the molecular understanding of new molecular targets in regulating stemness and metastasis of TNBC.