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-cells" 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 that such an expression is regulated by Notch3 through -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

Notch3 sostiene CXCR4 in membrana come meccanismo nella progressione della leucemia T-ALL

Sebbene l'iperattivazione del signaling di Notch è associata allo sviluppo della leucemia linfoblastica acuta a cellule T, i meccanismi che sono alla base della diffusione delle "cellule pre-leucemiche" non sono ancora ben chiari. Obbiettivo del nostro lavoro è stato quello di identificare le potenziali "cellule pre-leucemiche" e quali possono essere i meccanismi che guidano la loro diffusione. I nostri studi sono stati effettuati utilizzando topi transgenici che sovraesprimono il dominio intracellulare attivo di Notch3. In questo modello murino abbiamo osservato un aumento progressivo ed anomalo delle cellule T CD4+CD8+ nel sangue e poi nel midollo osseo. Queste cellule erano inoltre caratterizzate da un’elevata espressione in membrana dei recettori Notch3 e CXCR4. Il trapianto di tali cellule rivela la loro competenza nell'infiltrare la milza ed il midollo osseo di topi riceventi immuno-compromessi. Inoltre, i nostri dati in vitro, ottenuti con linee cellulari di leucemia umana T, dimostrano che l'espressione in superficie di CXCR4 è fondamentale per la capacità migratoria delle cellule CD4+CD8+, e che tale espressione è regolata da Notch3 attraverso la b-arrestina. Vista l’anomala fuoriuscita di tali cellule dal timo nelle prime fasi della leucemia, abbiamo effettuato esperimenti con un antagonista del recettore CXCR4, l’AMD3100. L'antagonismo in vivo del recettore impedisce la colonizzazione del midollo osseo da parte di tali cellule CD4+CD8+Notch3+CXCR4+ nei topi transgenici per Notch3, evidenziando ulteriormente l’importanza del recettore CXCR4 nella propagazione ed infiltrazione delle cellule CD4+CD8+. Quindi, l’iperespressione dei due recettori, Notch3 e CXCR4, su queste cellule T CD4+CD8+ "pre-leucemiche" probabilmente gioca un ruolo importante nelle fasi iniziali della progressione della malattia, contribuendo alla loro propagazione ed infiltrazione in organi linfoidi. Infine, i nostri dati suggeriscono che terapie combinate che contrastino precocemente il crosstalk intratimico Notch3/CXCR4, possano impedire la diffusione di cellule "pre-leucemiche" CD4+CD8+ e la progressione della malattia

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 haematological 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 haematogical 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

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

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 <i>in vitro</i> 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