Immune system cells, including neutrophils, are recruited by the tumor microenvironment as a site of chronic inflammation and begin to favor tumor growth. Neutrophils present in the tumor site are called tumor-associated neutrophils (TAN) and can present two phenotypes: N1 (antitumor) or N2 (pro-tumor). Evidence shows the high capacity of immune system cells to interact with extracellular vesicles (Evs) released by tumor cells. Evs can modulate the phenotype of cells within the immune system, contributing to tumor development. Here, we investigated the role of MDA-MB-231-derived Evs upon the polarization of neutrophils towards an N2 phenotype and the underlying mechanisms. We observed that neutrophils treated with Evs released by MDA cells (MDA-Evs) had their half-life increased, increased their chemotactic capacity, and released higher levels of NETs and ROS than neutrophils treated with non-tumoral Evs. We also observed that neutrophils treated with MDA-Evs released increased IL-8, VEGF, MMP9, and increased expression of CD184, an N2-neutrophil marker. Finally, neutrophils treated with MDA-Evs increased tumor cell viability. Our results show that MDA-Evs induce an N2-like phenotype, and the blockage of phosphatidylserine by annexin-V may be an essential agent counter-regulating this effect.

Altered extracellular matrix (ECM), a hallmark of solid tumors, affects cellular survival, migration and differentiation. Typically viewed as tumor-suppressive, evidence suggests that apoptosis can also generate pro-tumoral signals. We previously showed that ECM from high-grade astrocytomas induces extensive endothelial anoikis, while a surviving subpopulation fails to form tubular structures (tubulogenesis-defective endothelial cells, or TDECs). We combined functional assays with whole-cell proteomics to investigate this response. Using real-time video microscopy, we found that apoptotic endothelial cells induced by tumor ECM attracted migrating endothelial cells and guided sprouting. Conditioned media from apoptotic endothelial cells contained a 2.8-fold increase in extracellular vesicles (EVs) relative to autologous ECM-primed endothelial cells. Although both EV populations improved TDEC tubulogenesis, only EVs produced upon tumor-ECM stimulation induced TDEC migration—a property lost when using EVs secreted by endothelial cells growing on TN-C-depleted matrices. Proteomic profiling revealed that TDECs shift from an adhesion-anchored to a microtubule-rich and glycolytically rewired phenotype, with upregulation of vesicle-trafficking regulators (ARF1/3/4, ANXA2/5), migration drivers (RAC1/3, RHOA/C, WDR1, FSCN1) and glycolytic enzymes (ENO1, ALDOA, PKM, LDHA), alongside the suppression of integrin- and cytoskeletal-anchoring proteins. Collectively, these findings indicate that tumor-ECM-driven endothelial apoptosis generates reversible reprogramming and an EV-mediated autocrine mechanism that may favor angiogenic balance.

Abdominal adiposity is associated with increased risk of abdominal aortic aneurysm (AAA) development. Calpains are non‐lysosomal calcium‐dependent cysteine proteases that are highly expressed in human and experimental AAAs. Using a pharmacological inhibitor and genetically deficient mice, we previously demonstrated that calpain‐2 (a major ubiquitous isoform) deficiency mitigated angiotensin II (AngII)‐induced AAA formation in hypercholesterolemic mice. In addition, we also demonstrated that calpain inhibition strongly suppressed adipose tissue inflammation in obese mice. Here, we evaluated the contribution of adipocyte‐specific calpain‐2 on obesity‐accelerated AAA in mice. Calpain‐2 protein is highly expressed in the periaortic adipose tissue (PAAT) of AngII‐induced AAAs in obese mice. To determine the relative contribution of calpain‐2 in obesity‐accelerated AAA development, calpain‐2 floxed mice were bred to mice with a tamoxifen‐inducible form of Cre under control of either the ubiquitous promoter, chicken β‐actin, or adipocyte‐specific promoter, Adipoq. Ubiquitous or adipocyte‐specific depletion of calpain‐2 in mice significantly suppressed Ang II–induced AAA formation in obese mice. In addition, calpain‐2 depletion reduced the incidence of AngII‐induced AAAs in mice. Furthermore, calpain‐2 deficiency prevented AngII‐induced aortic medial elastin fragmentation, adventitial collagen disruption, and periaortic leukocytic accumulation. These results suggest that adipocyte‐derived calpain‐2 plays a critical role in AngII‐induced AAA development in diet‐induced obese mice.