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1.
Figure 4

Figure 4. From: EGFR targeting monoclonal antibody combines with an mTOR inhibitor and potentiates tumor inhibition by acting on complementary signaling hubs.

Combination therapy shows more differentiation within the tumors as shown by H and E evaluation. Five-micrometer-thick sections after formalin fixation and wax embedding were taken and stained using standard H and E techniques. Four animals from each group were evaluated for keratinization the animals were based on the two larger and smaller tumors from each group. (I) The control and the Sirolimus groups behave similarly with less differentiation and less keratinization. The amount of differentiation increases in the Nimotuzumab-treated groups while with the combination there is most differentiation with large areas of keratinization. (II) Depicts the amount of keratinization as a percent of the total tumor section. Each bar is an average of four sections from four different animals evaluated in each group. All the groups show significant keratinization as compared with the control. However, the amount of keratinization was maximum in the combination group. Error bars are standard deviation around the mean in all the figures.

Roshan James, et al. Cancer Med. 2012 October;1(2):114-127.
2.
Figure 3

Figure 3. From: EGFR targeting monoclonal antibody combines with an mTOR inhibitor and potentiates tumor inhibition by acting on complementary signaling hubs.

Significant reduction in tumor volume using Nimotuzumab and even better with the combination in different tumor models. (A) BALB/c nude mice carrying xenografts of A-431 cells respond significantly to Nimotuzumab but not to Sirolimus. However, the combination inhibits better than Nimotuzumab alone. Stars indicate significance using the Friedman's test followed by Dunn's multiple comparison tests. (B) A-431 cells (5 × 106 cells/dose) were injected in SCID mice and the dosing were identical to that in the BALB/c nude mice except that the dosing was started earlier on day 7 without the tumors attaining 200 mm3 as in (A). The mice were then sacrificed on day 18. Graphs show standard error around the mean.

Roshan James, et al. Cancer Med. 2012 October;1(2):114-127.
3.
Figure 5

Figure 5. From: EGFR targeting monoclonal antibody combines with an mTOR inhibitor and potentiates tumor inhibition by acting on complementary signaling hubs.

Nimotuzumab and Sirolimus in combination downregulates various signaling proteins downstream to EGFR as shown by immunohistochemistry. (A) Five-micrometer-thick sections were taken and stained using standard immunohistochemistry techniques from formalin-fixed and wax-embedded tumor sections. Primary antibodies were incubated for 2 h followed by washes in buffer and developed using a secondary antibody developing kit from Vectastain. Panel I (A–D) shows the expression of PCNA in tumor sections. Panel II (E–H) shows the expression of pAKT, Panel III (I–L) shows the expression of pMAPK while panel IV (M–P) depicts pSTAT3 expression. (B) Shows the percent cells positive as measured from three independent 20× photomicrographs taken from the microscope. Error bars show standard deviation. *P < 0.05, **P < 0.01, ***P = 0.001, respectively, relative to control by ANOVA. Error bars are standard deviation around the mean in all the figures.

Roshan James, et al. Cancer Med. 2012 October;1(2):114-127.
4.
Figure 6

Figure 6. From: EGFR targeting monoclonal antibody combines with an mTOR inhibitor and potentiates tumor inhibition by acting on complementary signaling hubs.

Tumors from combination therapy cluster differently in a microarray analysis compared with Sirolimus and Nimotuzumab group. (IA) Interarray clustering after removing the outlier control sample 2.8B showed that the clusters were spontaneously formed based on the treatment received. While the controls and the Sirolimus-treated groups were clustered together, the Nimotuzumab and the combination group clustered separately. One from the combination group was eliminated as RNA quantity obtained was insufficient. (IB) Principle component analysis shows that the Sirolimus and the control group are clustered together as compared with the Nimotuzumab and the combination group. Outlier 2.8B is shown. (IIA–C) The volcano plots show the expression of genes relative to the control. These graphs are generated using the R program. This plot shows the negative log 10 of P-value versus log of fold change. The horizontal purple line distinguishes significant P-value genes (above purple line). The genes falling in between two vertical lines are those having fold change value <−1 to >1. While the upper right quadrant shows significant with fold >1 and P-value <0.05 the upper left quadrant significant with fold >1 and P-value <0.05. The figures inside the graph show the total number of genes which are significantly downregulated or upregulated relative to the control biopsies (left side down and right side is upregulated). (III) Venn diagrams show the degree of similarity in the treated groups.

Roshan James, et al. Cancer Med. 2012 October;1(2):114-127.
5.
Figure 1

Figure 1. From: EGFR targeting monoclonal antibody combines with an mTOR inhibitor and potentiates tumor inhibition by acting on complementary signaling hubs.

The combination of Nimotuzumab with Sirolimus enhances inhibition of EGFR-expressing cells as compared with the individual drugs alone. (A) Sirolimus shows a dose-dependent inhibition of proliferation in A-431 cells using SRB assay. In combination with a fixed concentration of Nimotuzumab (83 nmol/L), the level of inhibition is sustained across the Sirolimus range (25–1.56 nmol/L). (B) Nimotuzumab shows a dose-dependent inhibition across the concentration range 83–5.1 nmol/L. In combination with a fixed concentration of Sirolimus (25 nmol/L), the level of inhibition is sustained even at lower concentrations of Nimotuzumab. The gray line across each data point is significantly different from the dark line (t-test analysis). (C) The combination of Nimotuzumab and Sirolimus on A-431 cells is synergistic. A theoretical bliss curve (dotted line in the figure) demonstrating additive response was calculated for combined cytotoxicity. The experimental curves obtained with varying concentrations of Sirolimus (400–0.78 nmol/L) and fixed concentration of Nimotuzumab (83 nmol/L) and with varying concentration of Nimotuzumab (400–0.78 nmol/L) and a fixed concentration of Sirolimus (25 nmol/L) lie above the theoretical additive curve suggesting synergistic inhibition. The x-axis shows the log-transformed values of drug concentration in molar (mol/L). (D) Quantification of the EGFR receptor on A-431 and BxPC-3 cells was done using a flow cytometer Beckman Coulter Cyan ADP. Anti-EGFR antibody Nimotuzumab was used at a similar concentration range in both the cell lines. A-431 shows at least fourfold higher mean fluorescence intensity as compared with BxPC-3 cells. (E) Nimotuzumab (3.33–0.05 μmol/L) with a fixed concentration of Sirolimus (25 nmol/L) shows much higher cytotoxicity as compared with the drug alone. The threshold of Sirolimus seems to be important in these cells, as lower concentration of Sirolimus with fixed amount of Nimotuzumab has around 20% cytotoxic effect (Fig. S1). Figure represents one of the four independent experiments. Error bars are standard error around the mean in all the figures.

Roshan James, et al. Cancer Med. 2012 October;1(2):114-127.
6.
Figure 2

Figure 2. From: EGFR targeting monoclonal antibody combines with an mTOR inhibitor and potentiates tumor inhibition by acting on complementary signaling hubs.

Nimotuzumab and Sirolimus in combination independently downregulate signaling proteins downstream to EGFR and mTOR. (A) One million A-431 cells were seeded in a 6-well plate and incubated for 24 h with the drugs alone or in combination in presence of 1% serum. The cells were then spiked with 10 ng/mL of EGF for 10 min. The cell lysates were prepared and the total protein was estimated using Bradford's method. The proteins were then separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), transferred overnight to the polyvinylidene fluoride (PVDF) membrane. The expressions of various proteins were checked by standard Western blot analysis. The blots were developed by enhanced chemiluminescence as per the manufacturer's protocol. Two blots were used and the expression is normalized to their respective controls in the blots. The numbers below the blot are relative to the respective controls from arbitrary values generated from the Alpha view software. (B) mTOR expression is high in A-431 cells unstimulated or stimulated with EGF. (C) Cetuximab showed higher inhibition of proliferation of A-431 cells at equimolar concentrations when compared with Nimotuzumab using SRB assay. Error bars are SD around the mean. (D) Nimotuzumab and Cetuximab were incubated for 2 h at 333 nmol/L and in the presence of EGF at 10 ng/mL for 10 min. Cetuximab at these equimolar concentrations is a better inhibitor of EGFR downstream signaling molecules. Lane C is a control treated with EGF and a polyclonal irrelevant antibody. This was run on the same blot at a different lane from lanes 1 and 2 (Fig. S2).

Roshan James, et al. Cancer Med. 2012 October;1(2):114-127.

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