Fig 1.
Description of the Ex ovo CAM model.
(A) Fertilized eggs day 3 post-fertilization (3dpf) were removed from the shell and grown ex ovo. (B) Embryos were grown for an additional 7 days at 37°C in humidified chambers. (A’) Meanwhile, tumor cells were grown in culture and then (B’) incorporated along with various drugs into 3-dimensional collagen 1 based onplants incorporating a larger and smaller mesh whereby tumor-derived NV (red lines) will sprout from the CAM vessels and branch upward into the onplant. (C) On day 10 post-fertilization, six tumor onplants were applied onto each CAM at evenly spaced intervals, using at least 5 different CAMs per group (≥30 replicates total). (D) Image of a day 13 post-fertilization ex ovo embryo with six tumor onplants (indicated by arrows). (E) On day 13, CAM vessels were perfused with DIL, embryos were euthanized and tumor onplants were excised. (F) Excised onplants were fixed to a 6-well microscope slide and imaged using confocal microscopy (G). The area of NV within the upper mesh was then quantified manually (H) or by automated quantification (I). (J) The onplants containing tumor cells resulted in 5 – 10x the amount of quantified vasculature within the onplant compared to the negative control onplants.
Table 1.
List of angiostatics obtained and tested in our CAM model for screening for synergistic combinations.
Fig 2.
Comparison of manual quantification vs. automated quantification.
(A) Images from manual quantification are shown (left) with their corresponding images from the automated quantification (right) for 6 samples representative of low, medium, and high levels of NV. Note that in the images from the automated quantifications, vessels outside of the upper mesh area of the onplant are not displayed as a feature of the software. (B—C) Comparison of 100 quantified images using the manual vs. automated computational methods demonstrates strong correlation throughout the range of onplant vascularization (B), as well as a comparison of the percent of vascularization for lower levels of vascularization (below 6%) (C).
Fig 3.
Monotherapy dosing of angiostatics in the CAM model.
Monotherapies were tested at multiple doses in order to ensure activity based on the inhibition of tumor-induced neovascularization in our model system. Once this functional activity was confirmed, the maximum dose of no effect was identified for each of the various angiostatic monotherapies that demonstrated activity in the CAM model. The indicated maximum ineffective doses were subsequently used to screen for synergistic combinations ensuring that any observed tumor NV inhibiting activity was due to exponential benefits in combination. Asterisks indicate significant reduction in tumor vascularization compared to the untreated controls.
Fig 4.
Combining bevacizumab (Avastin®) and temsirolimus (Torisel®) results in significant activity blocking tumor-induced NV.
(A) Representative images of the tumor onplants from the different treatment groups are shown with the original image on the left and the image showing the results of quantification on the right, followed by quantification of tumor NV (B). As expected, Avastin® and Torisel® did not demonstrate significant reduction in tumor NV individually using the maximum doses of no effect found in the earlier dosing studies (Fig 3). However, significant reduction in tumor-induced angiogenesis was observed when combining Avastin® and Torisel® at these same doses. The resulting angiogenesis levels are comparable to the no-tumor negative control levels. X-axis groups indicate what was included in the collagen onplant. Data include results from six separate experiments with over thirty individual onplants per group per each experiment. Error bars represent standard deviation. ** indicates p values < 0.01 between the indicated groups. The calculated CI value indicates a synergistic effect when combination compared to each monotherapy.
Fig 5.
Synergistic angiostatic activity of bevacizumab and temsirolimus is observed at 10-fold lower doses.
To further demonstrate synergistic activity of combining bevacizumab (Avastin®) and temsirolimus (Torisel®) when blocking tumor-induced NV, each were combined at concentrations ten-fold below their maximum ineffective doses. Individual treatment with Avastin® or Torisel® had no effect on tumor NV, as expected, but in combination, tumor NV was completely blocked. (A) Representative images of the tumor onplants from the different treatment groups are shown with the original image on the left and the image showing the results of quantification on the right (B) Quantification of tumor NV demonstrated strong, synergistic angiostatic activity against tumor-derived NV (CI value = 2.42E-5). (C) Similar results were observed using the human glioblastoma cell line U87. Error bars represent SEM. X-axis groups indicate what was included in the collagen onplant.