TL-118 and Gemcitabine Drug Combination Display Therapeutic Efficacy in a MYCN Amplified Orthotopic Neuroblastoma Murine Model – Evaluation by MRI

<div><p>Neuroblastoma (NB) is the most common extra-cranial pediatric solid tumor with up to 50% of NB patients classified as having high-risk disease with poor long-term survival rates. The poor clinical outcome and aggressiveness of high-risk NB strongly correlates with enhanced angiogenesis, suggesting anti-angiogenic agents as attractive additions to the currently insufficient therapeutics. TL-118, a novel drug combination has been recently developed to inhibit tumor angiogenesis. In the current study, we used the SK-N-BE (2) cell line to generate orthotopic NB tumors in order to study the combinational therapeutic potential of TL-118 with either Gemcitabine (40 mg/kg; IP) or Retinoic acid (40 mg/kg; IP). We show that TL-118 treatment (n = 9) significantly inhibited tumor growth, increased cell apoptosis, reduced proliferation and extended mouse survival. Moreover, the reciprocal effect of TL-118 and Gemcitabine treatment (n = 10) demonstrated improved anti-tumor activity. The synergistic effect of these drugs in combination was more effective than either TL or Gemcitabine alone (n = 9), via significantly reduced cell proliferation (p<0.005), increased apoptosis (p<0.05) and significantly prolonged survival (2-fold; p<0.00001). To conclude, we demonstrate that the novel drug combination TL-118 has the ability to suppress the growth of an aggressive NB tumor. The promising results with TL-118 in this aggressive animal model may imply that this drug combination has therapeutic potential in the clinical setting.</p></div

Treatment effect on tumor growth and mouse survival.

<p>A. Tumor volume (mm³) for each individual mouse, as measured from T₂W MRI images as a function of days post cell inoculation in control (n = 19), Gemcitabine (Gem; n = 6), TL-118^1/4 (n = 9) and TL^1/4+ Gem combination (n = 10) treated mice. The dashed line indicates the maximal survival day of the control-treated mice. The b-values represent the average exponential coefficients of each treatment group. The b-values of all the treated groups (Gem, TL^1/4 and TL^1/4+ Gem) were significantly lower compared to control (p<0.0001). B. Representative T₂W anatomical axial images of Control, Gem, TL-118^1/4 and TL^1/4+ Gem treated tumors that were acquired on the indicated days (Bar = 1 cm) C. Kaplan-Meier survival analysis for each of the treated groups (*p<0.05; **P<0.0001; ***p<0.00001 compared to control). D. Box and Whisker plots of mean calculated b-values for each treated group (black square – median; * p<0.0001).</p

Effects of the different therapies on NB tumor proliferation and apoptosis.

<p>Representative histological sections of control (1^st column), TL-118^1/4 (2^nd column), Gem (3^rd column) and TL^1/4 + Gem (4^th column) treated mice. Slides were immuno-stained for proliferation (BrdU) (A) and apoptosis (TUNEL) (B); Quantification of BrdU positive (C) and TUNEL positive (D) cells/HPF analyzed from 10 randomly selected HPF/tumor ; n = 3–6 mice/group. Original magnification is indicated on the right image of each row. * p = 0.01, ** p<0.005. The histological sections were taken at the end of the experiments when the tumor load/mouse reached ethical limits.</p

TL-118^¼ drug composition.

<p>TL-118^¼ drug composition.</p

Effects of TL therapy combinations on NB tumor vascularization and perfusion.

<p>A. Representative T₂W fast SE images (top) and the corresponding ΔSco₂ (middle row) and ΔSo₂ (bottom) maps of control (left), TL-118^1/4 (middle column) and TL^1/4 + Gem (right) treated tumors (Bar = 1 cm). B. Mean ΔSo₂ values of the tumor and liver-ROI's calculated for control (blue), TL-118^1/4 (green) and TL^1/4 + Gem (purple) treated mice. C. Representative histological slides immuno-stained with the smooth muscle marker α-SMA (upper-rows) and with the endothelial cell marker CD31 (lower-rows), of control (top), TL-118^1/4 (middle) and TL^1/4 + Gem (bottom) treated tumors. Photographs were taken from the peripheral (left column) and central (right column) regions (original magnification ×20) demonstrating the higher vascularity in tumor periphery. D. Quantification of α-SMA positive vessels/HPF was determined from the tumor center (dark) and peripheral (light) areas for each of the treatment groups. (Mean ± SE; n = 5–7 mice/group).</p