Search

SR4987GFP and SR4987GFP-PTX arrest in the lung of mice upon i.v. administration. Mice were sacrificed at 6 h, 24 h and 48 h after treatments with cells. Panel (A) shows photos taken under fluorescent microscopy immediately upon enzymatic lung tissue disruption, showing the presence of several fluorescent cells until 48 h after their injection. Not significant difference were noted between mice treated with SR4987GFP and SR4987GFP-PTX (10x magnification). Panel (B) shows photos of SR4987GFP and SR4987GFP-PTX cultured for 2 and 7 days upon isolation from mice, indicating that cells maintain their viability (20x magnification). Figure S2. Sca-1 is highly expressed on SR4987 and can be used for their detection in the mice lung. In (A) FC analysis showing the high positivity of the SR4987 for Sca-1. In (B), Immunostaining for anti-Sca-1 of lungs derived from mice treated i.v. with saline , 106 SR4987 and 106 SR4987PTX cells and sacrificed 24 h after treatments. Pictures show the high presence of Sca-1+ cells (black arrows) in the parenchyma and in the lumen of lung vessels in mice treated with both SR4987 and SR4987PTX. Only, very few Sca-1+ cells were detected in control mice (CTRL) (photo 20x, in the box 40x magnifications). Figure S3. Sensibility of SR4987 to PTX and efficacy of SR4987PTX-CM to inhibit B16 proliferation. In (A) the proliferation of SR4987 in the presence of increasing concentration of PTX. The IC50 of PTX was around 34 ng/ml. In (B) is shown that viability of SR4987 is not affected by PTX even at concentration up 5000 ng/ml. In (C) is shown the growth inhibition of Molt-4 tumor cells in the presence of decreasing dilution of SR4987PTX-CM confirming that SR4987PTX release significant amount of PTX in the CM. In (D) the inhibition of proliferation of B16 cells in the presence of increasing concentration of PTX. The IC50 is 7.18 ± 4.99 ng/ml. In (E) is shown the dose dependent efficacy of SR4987PTX-CM to inhibit B16 cells proliferation with an IC50 DIL of around 1/10. Figure S4. Co-culture of SR4987 and SR4987PTX with B16 cells using transwell insert. B16 cells seeded on the top of membrane (0.4 um pore size) insert were co-cultured with different dose of SR4987 and SR4987PTX seeded on the bottom well and cultured for 5 days. The Figure shows that SR4987PTX at 1:5 ratio was able to strongly inhibit B16 proliferation as visualized by crystal violet staining of B16 in the transwell line (A) and on the membrane line (B). In line (C), photos of cultured B16 cells not stained upon co-culture or not with SR4987 and SR4987PTX (20x magnifications). Figure S5. Rosette formed by B16 + SR4987 and B16 + SR4987PTX and their ultrastructural analysis by TEM. In (A) and (B) rosette formed by SR4987GFP + B16 photographed under light and fluorescence microscopy respectively. (C-F) four different panels at TEM showing the interactions between untreated SR4987GFP and B16 cells. Note that B16 cells bound SR4987GFP appear healthy (C); the presence of area of contact between B16 and SR4987 is evident; junctional-like structure and a gap-like junction (D,E) and even nanotubule structure (F) are seen (white arrows). In (G) and (H) rosette formed by SR4987GFP-PTX + B16. Note that most of B16 cells appeared damaged and necrotic under light (G) and fluorescence (H) microscopy observation. In (I-L) four different panels at TEM showing the interaction between SR4987GFP-PTX and B16. The B16 cells bound to SR4987GFP-PTX show varying degrees of degeneration, some cells seemed even emptied of their cytoplasm (I) and, similarly to untreated SR4987, area of contact (white arrows) can be observed (J,K). In (L) a microvescicole that arise from SR4987PTX is photographed. Figure S6. Metastasis in the lung of control mice and after treatment with PTX, SR4987 and SR4987PTX. On day 21 after B16 cells injection, mice were euthanized, lungs removed and fixed in Bouin’s solution. In the figure photos of the lungs, taken under a dissecting stereomicroscope. Note the presence of several melanoma lung nodules (black arrows) in control and SR4987 treated mice. In contrast, at both dosage of SR4987PTX injected, the lung nodules are very few and are significantly less than those detected in PTX treated mice. Figure S7. Chemiotaxis of SR4987 and SR4987PTX in response to B16-CM and expression of the SDF-1 receptors CXCR4 and CXCR7. In (A) migration of SR4987PTX and SR4987 placed on the top of transwell membrane insert and stimulated by different dilution of B16-CM. No significant chemotaxis activity of both SR4987 and SR4987PTX is stimulated by B16-CM at every dilutions tested. Note that the basal migration of SR4987PTX is around three fold lower than untreated SR4987 (* p 90 %), CXCR7 is less expressed (18 %). (DOC 26 kb)

Supplementary materials and methods. (DOC 45 kb)

Glioblastomas are highly aggressive adult brain tumors with poor clinical outcome. In the central nervous system (CNS) the blood-brain barrier (BBB) is the most important limiting factor for both development of new drugs and drug delivery. Here, we propose a new strategy to treat glioblastoma based on transferrin (Tf)-targeted self-assembled nanoparticles (NPs) incorporating zoledronic acid (ZOL) (NPs-ZOL-Tf). NPs-ZOL-Tf have been assessed on the glioblastoma cell line U373MG-LUC that showed a refractoriness in vitro to temozolomide (TMZ) and fotemustine (FTM). NPs-ZOL-Tf treatment resulted in higher in vitro cytotoxic activity than free ZOL. However, the potentiation of anti-proliferative activity of NPs-ZOL-Tf was superimposable to that one induced by NPs-ZOL (not armed with Tf). On the other hand, NPs-ZOL-Tf showed a higher antitumor efficacy if compared with that one caused by NPs-ZOL in immunosuppressed mice intramuscularly bearing U373MG-LUC xenografts, inducing a significant tumor weight inhibition (TWI). The experiments performed on mice with intracranial U373MG-LUC xenografts confirmed the efficacy of NPs-ZOL-Tf. These effects were paralleled by a higher intratumour localization of fluorescently-labeled-NPs-Tf both in intramuscular and intracranial xenografts. In conclusion, our results demonstrate that the encapsulation of ZOL increases the antitumor efficacy of this drug in glioblastoma through the acquisition of ability to cross the BBB.

Systemic mastocytosis (SM) is a rare myeloproliferative neoplasm characterized by proliferation and hyperactivation of clonal mast cells. Clinical manifestations are heterogeneous and encompass cutaneous lesions, gastrointestinal alterations, osteoporosis, anaphylaxis and involvement of bone marrow and other organs due to neoplastic mast cells (MC) infiltration. As consequence, diagnosis may be difficult and patients (pts) are often evaluated by different specialists before the disease is recognized. To date, only few studies (Lim 2009, Escribano 2009, Cohen 2014) described relatively large series of pts with SM. We performed a multicentre retrospective study to evaluate clinical and biological features and therapeutic management in a large series of pts from 10 Italian centres experienced in management of SM and organized in multidisciplinary groups of specialists. We collected 455 pts diagnosed with SM according to WHO criteria. Additionally 26 pts with mastocytosis in the skin (MIS) evaluated with BM examination did not fulfil criteria for SM, leading to diagnosis of Cutaneous Mastocytosis (CM); however 2/26 pts with CM had both cKITD816V mutation and CD2/CD25 expression on MC in BM, additional 3 showed either cKITD816V or CD2/CD25. Moreover, we found 22 pts without MIS but with features of monoclonal mast cell activation syndrome. Of the 455 pts with WHO-SM (male 56%), 252 (55%) had MIS: median age at MIS diagnosis (dg) was 37 years (y) (range 0-79), while at SM dg it was 46.5 (range 18-82). Time from onset of MIS to dg of SM was 9 y (range 0-43). In 18/252 pts (7%) MIS occurred before age of 18 y (median 9, range 0-17) and persisted over childhood. Median age at dg of SM without MIS (203/455 pts, 45%) was older: 54 y, range 19-79 (p Disclosures Martinelli: Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; ARIAD: Consultancy.

Cancer stem cells (CSCs) have been identified in several solid malignancies and are now emerging as a plausible target for drug discovery. Beside the questionable existence of CSCs specific markers, the expression of CD133 was reported to be responsible for conferring CSC aggressiveness. Here, we identified two G-rich sequences localized within the introns 3 and 7 of the CD133 gene able to form G-quadruplex (G4) structures, bound and stabilized by small molecules. We further showed that treatment of patient-derived colon CSCs with G4-interacting agents triggers alternative splicing that dramatically impairs the expression of CD133. Interestingly, this is strongly associated with a loss of CSC properties, including self-renewing, motility, tumor initiation and metastases dissemination. Notably, the effects of G4 stabilization on some of these CSC properties are uncoupled from DNA damage response and are fully recapitulated by the selective interference of the CD133 expression. In conclusion, we provided the first proof of the existence of G4 structures within the CD133 gene that can be pharmacologically targeted to impair CSC aggressiveness. This discloses a class of potential antitumoral agents capable of targeting the CSC subpopulation within the tumoral bulk.