Effects of human mesenchymal stem cells after intratumoral injection in neuroblastoma tumors.

<p>Panel A. Athymic mice were injected with human NB ACN cells (3×10⁶ cells/mouse) subcutaneously (s.c.) into the right flank. When the tumor size reached 100 mm³, hMSCs (1×10⁶ cells/mouse, 5 mice, red bars) or saline solution (control group: 5 mice, black bars) were injected intratumorally. Tumor volume was measured at different times and p values were calculated using Student's t test with Welch's correction. A representative experiment out of two performed is shown. Inset panel A shows a representative CD90 immunohistochemical staining of hMSC-treated ACN tumors surgically removed 1 day or 35 days after hMSC inoculum. Arrows indicate MSCs positive for CD90. Original magnification 40x. Panel B. Survival curves of ACN-bearing mice untreated or treated with hMScs were constructed by using the Kaplan–Meier method. Statistical analysis of different treatment groups was performed by Peto's log-rank test. This is a representative experiment out of two performed. Panel C. Paraffin-embedded tumor sections from control and hMSC-treated mice were stained for the cell proliferation marker Ki67. Data are expressed as mean value of Ki67 positive cells counted in 10 fields/slide. Inset panel B shows a representative staining of control and hMSC-treated ACN tumors with Ki67. Original magnification 100x. P values were calculated using Unpaired t test with Welch's correction. Panel D. Paraffin-embedded tumor sections from control and hMSC-treated mice were stained for detection of apoptosis with terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling [TUNEL] assay. Data are expressed as mean value of TUNEL positive cells counted in 10 fields/slide. p values were calculated using Unpaired t test with Welch's correction. Panel E. Representative staining of control and hMSC-treated ACN tumors with TUNEL. Green cells are TUNEL positive. Panel F. Representative immunofluorescent staining of control and hMSC-treated ACN tumors with anti-caspase-3 mAb. Arrow indicate positive cells with nuclear activated caspase-3 localization; arrowheads indicate positive cells with cytosolic activated caspase-3 localization.</p

Survival curves and murine mesenchymal stem cell distribution within neuroblastoma bearing immunocompetent mice.

<p>Panel A. A/J mice were injected in the tail vein with ffLUC-NXS2 cells (2×10⁵ cells/mouse; n = 5) and imaged by bioluminescence imaging 1 and 7 days after tumor cell inoculum. Panel B. A/J mice were i.v. injected or not with NXS2 cells (2×10⁵ cells/mouse) and treated with ffLUC-mMSC (1×10⁶ cells/mouse) 7 days after tumor cell inoculum. Control group included 3 healthy mice treated with mMSCs whereas NXS2 bearing mice group includes 4 mice injetced with NXS2 cells and treated with mMSCs. The animals were imaged by bioluminescence 8, 15, 22, 29 days after tumor cell inoculum. This is a representative experiment out of three performed. Panel C. A/J mice were inoculated in the tail vein with NXS2 cells (2×10⁵ cells/mouse) and treated with syngeneic ffLUC-mMSC (1×10⁶ cells/mouse) or saline seven days after tumor cell inoculum. Control group included 6 mice only injected with tumor cells, whereas mMSC-treated groups included 6 mice. Survival curves were constructed by using the Kaplan–Meier method. Statistical analysis of different treatment groups was performed by Peto's log-rank test. This is a representative experiment out of three performed.</p

Close Interactions between Mesenchymal Stem Cells and Neuroblastoma Cell Lines Lead to Tumor Growth Inhibition

<div><p>Mesenchymal stem cells (MSCs) have attracted much interest in oncology since they exhibit marked tropism for the tumor microenvironment and support or suppress malignant cell growth depending on the tumor model tested. The aim of this study was to investigate the role of MSCs in the control of the growth of neuroblastoma (NB), which is the second most common solid tumor in children. <em>In vivo</em> experiments showed that systemically administered MSCs, under our experimental conditions, did not home to tumor sites and did not affect tumor growth or survival. However, MSCs injected intratumorally in an established subcutaneous NB model reduced tumor growth through inhibition of proliferation and induction of apoptosis of NB cells and prolonged the survival of hMSC-treated mice. The need for contact between MSCs and NB cells was further supported by <em>in vitro</em> experiments. In particular, MSCs were found to be attracted by NB cells, and to affect NB cell proliferation with different results depending on the cell line tested. Moreover, NB cells, after pre-incubation with hMSCs, acquired a more invasive behavior towards CXCL12 and the bone marrow, i.e., the primary site of NB metastases. In conclusion, this study demonstrates that functional cross-talk between MSCs and NB cell lines used in our experiments can occur only within short range interaction. Thus, this report does not support the clinical use of MSCs as vehicles for selective delivery of antitumor drugs at the NB site unless chemotherapy and/or radiotherapy create suitable local conditions for MSCs recruitment.</p> </div

Invasiveness of neuroblastoma cells after contact with human mesenchymal stem cells.

<p>SH-SY5Y, Htla-230 and GI-LI-N NB cells incubated or not with hMSCs in a transwell system for 24 hours, were placed in the upper wells on Matrigel®-coated invasion chambers and separated by an 8 μm membrane from CXCL12 or bone marrow sample. After 24 hours, NB cells migrated into the lower chamber were stained and counted after May Grunwald-Giemsa. Panels A–C show the invasiveness of SH-SY5Y (Panel A), GI-LI-N (Panel B) and Htla-230 (Panel C) to CXCL12. Panels D shows the invasiveness of SH-SY5Y to CXCL12 in presence or absence of CXCR4 antagonist AMD3100 that was added to some wells. Panel E shows the invasiveness of SH-SY5Y to the bone marrow of an healthy donor. The data are expressed as mean values obtained from the count of 10 fields/well. Bars are SD. Statistical analysis was performed by Unpaired t test with Welch's correction.</p

Survival curves and immunohistochemical analysis of human Htla-230 neuroblastoma bearing mice treated with human mesenchymal stem cells.

<p>Panel A. Athymic mice (Nude-nu) were i.v. injected with ffLUC-Htla-230 (3×10⁶ cell/mouse; n = 15). Tumor establishment occurred 14 days after tumor cell inoculum as assessed by bioluminescence imaging. Panel B. Athymic mice (Nude-nu) were injected in the tail vein with Htla-230 (3×10⁶ cell/mouse) and treated i.v. with hMSCs (1×10⁶ cells/mouse, n = 6 or 3×10⁶ cells/mouse, n = 5) or saline solution (n = 6) 14 days after tumor cell inoculum. Survival curves were constructed by using the Kaplan–Meier method. Statistical analysis of different treatment groups was performed by Peto's log-rank test. Panel C. Representative hematoxylin-eosin-staining of tumors surgically removed from control and hMSC-treated Htla-230-bearing mice is shown. Original magnification 10x. Panel D. Representative immunohistochemical CD90 staining of lungs and tumors surgically removed from hMSC-treated Htla-230 bearing mice at different times after hMSC i.v. inoculum. Arrows indicate hMSCs positive for CD90. Original magnification 40x.</p

Human neuroblastoma cells attract human mesenchymal stem cells <i>in vitro</i>.

<p>Human MSCs, placed in the upper wells of transwell system, were separated by an 8 μm membrane from NB cells or NB cell conditioned medium that were dispensed in the lower well. Negative and positive controls were represented by serum-free medium (DMEM) and PDGF-BB at 100 ng/ml, respectively. Migration was quantified by counting migrated cells under the microscope after cell staining with May Grunwald. Panel A shows representative images of migrated hMSCs to DMEM, PDGF-BB, Htla-230 NB cells, Htla-230 conditioned medium. Original magnification 100x. Panel B shows quantification of migrated hMSC to different NB cells and NB conditioned medium. The data represent mean values obtained from the count of 10 fields/well. Bars represent SD. Panel C shows the results obtained by pooling all the data obtained from hMSC migration to NB cell lines and NB cell conditioned medium. Statistical analysis was performed by Unpaired t test with Welch's correction.</p

Effects of human mesenchymal stem cells on neuroblastoma cell proliferation.

<p>SH-SY5Y (Panel A) and Htla-230 (Panel B) and ACN (Panel C) NB cells were cultured in 96-well plates for 3 days in presence or absence of hMSCs at different ratio. After 3 days of co-culture, the cells were pulsed with ³H-thymidine for 18 hours and analyzed. The data are expressed as mean value ± SD from three different experiments. Statistical analysis was performed by Student's t test with Welch's correction.</p

SIRT1 at the crossroads of AKT1 and ERβ in malignant pleural mesothelioma cells

In this report, we show that malignant pleural mesothelioma (MPM) patients whose tumors express high levels of AKT1 exhibit a significantly worse prognosis, whereas no significant correlation with AKT3 expression is observed. We provide data that establish a phosphorylation independent role of AKT1 in affecting MPM cell shape and anchorage independent cell growth in vitro and highlight the AKT1 isoform-specific nature of these effects. We describe that AKT1 activity is inhibited by the loss of SIRT1-mediated deacetylation and identify, by mass spectrometry, 11 unique proteins that interact with acetylated AKT1. Our data demonstrate a role of the AKT1/SIRT1/FOXM1 axis in the expression of the tumor suppressor ERβ. We further demonstrate an inhibitory feedback loop by ERβ, activated by the selective agonist KB9520, on this axis both in vitro and in vivo. Our data broaden the current knowledge of ERβ and AKT isoform-specific functions that could be valuable in the design of novel and effective therapeutic strategies for MPM

Supplementary Data from z-Leucinyl-Leucinyl-Norleucinal Induces Apoptosis of Human Glioblastoma Tumor–Initiating Cells by Proteasome Inhibition and Mitotic Arrest Response

Supplementary Figures S1-S6 and Supplementary Tables S1-S3.</p

Cell survival of the PT4, MM1 and FO-1 cell cultures containing TICs.

<p>Survival of PT4 cells (A, E); MM1 cells (B) and FO-1 cells (C, D) after 6 days of treatment with the indicated substances and solvent controls. The medium renewal schedule was identical to that used for the cultures containing GBM TICs (see Introduction). Cell survival is expressed in arbitrary units as evaluated by MTT analysis. Standard deviations are indicated as vertical bars (n = 3 independent assays). DMSO concentration in (D) was 0.1% vol/vol. Drug concentrations in (D) were: NAC 20 mM, PLX4032 10 µM. Gefitinib final concentration in (E) was 3.9 µM. #The unpaired t-test was significant at P<0.05. §The unpaired t-test was significant at P = 0.01 or less. *The unpaired t-test was significant at P<0.001. **The unpaired t-test was significant at P<0.0001.</p