Notch3-regulated microRNAs impair CXCR4-dependent maturation of thymocytes allowing maintenance and progression of T-ALL.

Malignant transformation of T-cell progenitors causes T-cell acute lymphoblastic leukemia (T-ALL), an aggressive childhood lymphoproliferative disorder. Activating mutations of Notch, Notch1 and Notch3, have been detected in T-ALL patients. In this study, we aimed to deeply characterize hyperactive Notch3-related pathways involved in T-cell dynamics within the thymus and bone marrow to propose these processes as an important step in facilitating the progression of T-ALL. We previously generated a transgenic T-ALL mouse model (N3-ICtg) demonstrating that aberrant Notch3 signaling affects early thymocyte maturation programs and leads to bone marrow infiltration by CD4+CD8+ (DP) T cells that are notably, Notch3highCXCR4high. Newly, our in vivo results suggest that an anomalous immature thymocyte subpopulation, such as CD4-CD8- (DN) over-expressing CD3ɛ, but with low CXCR4 expression, dominates N3-ICtg thymus-resident DN subset in T-ALL progression. MicroRNAs might be of significance in T-ALL pathobiology, however, whether required for leukemia maintenance is not fully understood. The selection of specific DN subsets demonstrates the inverse correlation between CXCR4 expression and a panel of Notch3-deregulated miRNAs. Interestingly, we found that within DN thymocyte subset hyperactive Notch3 inhibits CXCR4 expression through the cooperative effects of miR-139-5p and miR-150-5p, thus impinging on thymocyte differentiation with accumulation of DNCD3ɛ+CXCR4- cells. These data point out that deregulation of Notch3 in T-ALL, besides its role in sustaining dissemination of abnormal DP T cells, as we previously demonstrated, could play a role in selecting specific DN immature T cells within the thymus, thus impeding T cell development, to facilitate T-ALL progression inside the bone marrow.

Notch/CXCR4 Partnership in Acute Lymphoblastic Leukemia Progression.

Acute lymphoblastic leukemia (ALL) is the most common cancer among children. Recent advances in chemotherapy have made ALL a curable hematological malignancy. In children, there is 25% chance of disease relapse, typically in the central nervous system. While in adults, there is a higher chance of relapse. ALL may affect B-cell or T-cell lineages. Different genetic alterations characterize the two ALL forms. Deregulated Notch, either Notch1 or Notch3, and CXCR4 receptor signaling are involved in ALL disease development and progression. By analyzing their relevant roles in the pathogenesis of the two ALL forms, new molecular mechanisms able to modulate cancer cell invasion may be visualized. Notably, the partnership between Notch and CXCR4 may have considerable implications in understanding the complexity of T- and B-ALL. These two receptor pathways intersect other critical signals in the proliferative, differentiation, and metabolic programs of lymphocyte transformation. Also, the identification of the crosstalks in leukemia-stroma interaction within the tumor microenvironment may unveil new targetable mechanisms in disease relapse. Further studies are required to identify new challenges and opportunities to develop more selective and safer therapeutic strategies in ALL progression, possibly contributing to improve conventional hematological cancer therapy.

PLGA based particles as "drug reservoir" for antitumor drug delivery: characterization and cytotoxicity studies.

Doxorubicin (DOX) is commonly used to treat several tumor types, but its severe side effects, primarily cardiotoxicity, represent a major limitation for its use in clinical settings. In this study we developed and characterized biodegradable and stable poly(D,L-lactic-co-glycolic) acid (PLGA) submicrocarriers employing an osmosis-based patented methodology, which allowed to optimize the drug loading efficiency up to 99%. Proceeding from this, we evaluated on MCF-7, a human breast cancer cell line, the ability of PLGA to promote the internalization of DOX and to improve its cytotoxicity in vitro. We found that the in vitro uptake efficiency is dramatically increased when DOX is loaded within PLGA colloidal carriers, which adhere to the cell membrane behaving as an efficient drug reservoir. In fact, the particles provide a diffusion-driven, sustained release of DOX across the cell membrane, resulting in high drug concentration. Accordingly, the cytotoxic analysis clearly showed that DOX-loaded PLGA exhibit a lower 50% inhibitory concentration than free DOX. The decay time of cell viability was successfully compared with DOX diffusion time constant from PLGA. The overall in vitro results highlight the potential of DOX-loaded PLGA particles to be employed as vectors with improved antitumor efficacy.

Intrathymic Notch3 and CXCR4 combinatorial interplay facilitates T-cell leukemia propagation.

Notch hyperactivation dominates T-cell acute lymphoblastic leukemia development, but the mechanisms underlying "pre-leukemic" cell dissemination are still unclear. Here we describe how deregulated Notch3 signaling enhances CXCR4 cell-surface expression and migratory ability of CD4+CD8+ thymocytes, possibly contributing to "pre-leukemic" cell propagation, early in disease progression. In transgenic mice overexpressing the constitutively active Notch3 intracellular domain, we detect the progressive increase in circulating blood and bone marrow of CD4+CD8+ cells, characterized by high and combined surface expression of Notch3 and CXCR4. We report for the first time that transplantation of such CD4+CD8+ cells reveals their competence in infiltrating spleen and bone marrow of immunocompromised recipient mice. We also show that CXCR4 surface expression is central to the migratory ability of CD4+CD8+ cells and such an expression is regulated by Notch3 through ß-arrestin in human leukemia cells. De novo, we propose that hyperactive Notch3 signaling by boosting CXCR4-dependent migration promotes anomalous egression of CD4+CD8+ cells from the thymus in early leukemia stages. In fact, in vivo CXCR4 antagonism prevents bone marrow colonization by such CD4+CD8+ cells in young Notch3 transgenic mice. Therefore, our data suggest that combined therapies precociously counteracting intrathymic Notch3/CXCR4 crosstalk may prevent dissemination of "pre-leukemic" CD4+CD8+ cells, by a "thymus-autonomous" mechanism.

Brain Targeting by Liposome-Biomolecular Corona Boosts Anticancer Efficacy of Temozolomide in Glioblastoma Cells.

Temozolomide (TMZ) is the current first-line chemotherapy for treatment of glioblastoma multiforme (GBM). However, similar to other brain therapeutic compounds, access of TMZ to brain tumors is impaired by the blood-brain barrier (BBB) leading to poor response for GBM patients. To overcome this major hurdle, we have synthesized a set of TMZ-encapsulating nanomedicines made of four cationic liposome (CL) formulations with systematic changes in lipid composition and physical-chemical properties. The targeting nature of this nanomedicine is provided by the recruitment of proteins, with natural targeting capacity, in the biomolecular corona (BC) layer that forms around CLs after exposure to human plasma (HP). TMZ-loaded CL-BC complexes were thoroughly characterized by dynamic light scattering (DLS), electrophoretic light scattering (ELS), and nanoliquid chromatography tandem mass spectrometry (nano-LC MS/MS). BCs were found to be enriched of typical BC fingerprints (BCFs) (e.g., Apolipoproteins, Vitronectin, and vitamin K-dependent protein), which have a substantial capacity in binding to receptors that are overexpressed at the BBB (e.g., scavenger receptor class B, type I and low-density lipoprotein receptor). We found that the CL formulation exhibiting the highest levels of targeting BCFs had larger uptake in human umbilical vein endothelial cells (HUVECs) that are commonly used as an in vitro model of the BBB. This formulation could also deliver TMZ to the human glioblastoma U-87 MG cell line and thus substantially enhance their antitumor efficacy compared to corona free CLs. Thus, we propose that the BC-based nanomedicines may pave a more effective way for efficient treatment of GBM.