AICAR suppressed tumor angiogenesis and inflammatory cells infiltration.

<p>(A, B) Microvessel density in tumor tissues was determined by immunofluorescent staining by an endothelial-specific antibody CD31. (A) Control group and (B) AICAR-treated group. (C) Quantitative analysis of fluorescent-positive area (per 4000 µm²) in tumors. Vessel density was significantly suppressed in AICAR-treated mice group (**p<0.01). (D, E) Macrophage- and neutrophil- infiltration in Y79 xenografts. Typical photomicrographs of immunofluorescent staining for CD11b (red) in Y79 xenografts. Nuclei were stained with propidium iodide (blue). Y79 cells isolated from control mice (D) and AICAR-treated mice (E). (F) Quantitative analysis of the CD11b (+) cells/DAPI (+) cells ratio in tumors. The number of CD11b (+) cells was significantly lower in the AICAR-treated mice group than in the control mice group (**p<0.01). Each column represents the mean ± SEM. Scale bars (A, B, D, E), 200 µm.</p

AICAR does not alter the levels of cyclins A, D and E in retinoblastoma in vivo.

<p>Quantitative RT-PCR analysis of tumors treated with AICAR in comparison with control shows no significant difference. Each column represents the mean ± SEM.</p

Proposed mechanism of action for AICAR in human retinoblastoma in an in-vivo xenograft model.

<p>AICAR administration leads to activation of AMPK decreased tumor vessel density and decreased CD11b (macrophage) infiltration. Activated AMPK inhibits mTOR pathway, protein synthesis and cell growth. In addition, AICAR administration results in decreased levels of p21, which was recently found to inhibit apoptosis and promote cell proliferation. Overall signaling changes leads to loss of viability due to apoptosis, proliferation block and inhibition of tumor progression.</p

AICAR treatment of retinoblastoma is associated with activation of AMPK, inhibition of mTORC1 and decrease of p21.

<p><b>A.</b> AICAR treatment of retinoblastoma is associated with activation of AMPK. Western blot analysis of phosphorylated ACC (Ser-79) (a downstream effector of AMPK) showed significant increase of pACC in tumours from AICAR treated mice comparing to control (**p<0.01, n = 19). <b>B and C.</b> Treatment with AICAR resulted in the inhibition of the mTORC1 pathway. Western blot analysis of tumor xenografts harvested from mice treated with AICAR showed significant decrease of mTOR pathway downstreams, pS6RP (Ser235/236) and the p4E-BP1 (Ser65) when compared to PBS-treated mice (***p<0.001 for both, n = 17 for pS6RP and n = 23 for p4EBP1). <b>D.</b> AICAR down-regulates p21WAF1/Cip1 in AICAR treated tumors as shown via Western blot analysis (*p<0.05, n = 23). Density values bands are graphically expressed relative to control. GAPDH was used as a loading control in all panels. Multiple bands represent separate biological samples. Each column represents the mean ± SEM.</p

AICAR suppressed proliferation and induced apoptosis of retinoblastoma in vivo.

<p>(A, B) Immunofluorescent analysis for Ki67 of tumors of Y79 cells isolated from control mice (A) and AICAR-treated mice (B). Nuclei were stained with propidium iodide (red). (C) Quantitative analysis of Ki67 (+) cells/PI (+) cells ratio in tumors. Values are significantly lower in the AICAR-treated mice group than in the control mice group (**p<0.01). (D,E) Apoptotic cells in retinoblastoma xenografts. Typical photomicrographs of apoptotic cells using TUNEL assay (green) in Y79 xenografts. Nuclei were stained with propidium iodide (red). Y79 cells isolated from control mice (D) and AICAR-treated mice (E). (F) Quantitative analysis of the apoptotic cell percentage in tumors. Note that the number of TUNEL (+) cells was significantly higher in the AICAR-treated mice group than in the control mice group (**p<0.01). Each column represents the mean ± SEM. Scale bars (A, B, D, E), 200 µm.</p

Bioimaging with NP-angiography showing GFP expression using the Topcon camera with fluorescein angiography filter settings.

<p>Late phase FAs (A and D) show the CNV lesions prior to injection of NP. Autofluorescent images taken prior to injection of NP reveal minimal background fluorescence of the CNV lesions (B and E). Injection of targeted NP carrying a GFP plasmid (NP-GFPg) causes increased fluorescence of the CNV lesions from GFP expression (C) whereas non-targeted NP carrying a GFP plasmid (ntNP-GFPg) does not cause any increase in the intensity of fluorescence of the CNV over background autofluorescence (F).</p

Late phase fluorescein angiography (FA) and choroidal flatmounts (<i>x10</i>) two weeks after laser photocoagulation.

<p>Representative lesions are from the control group (A–D) and the NP-ATPμ-Raf treated group (E and F). Group (A) received no treatment; (B) received intravenous injection of non-targeted NP containing ATPμ-Raf on days 1, 3, and 5 after laser CNV creation; (C) received intravenous injection of α_νβ₃ targeted-NP without ATPμ-Raf gene on days 1,3, and 5; (D) received injection of ATPμ-Raf gene without NP on days 1, 3, and 5; (E) received injection of α_νβ₃ targeted-NP containing ATPμ-Raf (NP-ATPμ-Raf) on days 1, 3, and 5; and (F) received injection of NP-ATPμ-Raf on days 3, 5, and 7. NP-ATPμ-Raf treated groups (E and F) had significantly lower grade CNV lesions on FA grading and smaller CNV size compared to the control group (A–D). No statistically significant difference in size was noted between the control groups A–D. Quantification of the CNV size on choroidal flat mounts is shown in (G). *P<0.01. Data are expressed as the mean ± SE.</p

Choroidal flatmounts showing accumulation of rhodamine labeled NP and expression of GFP plasmid in the CNV.

<p>The CNV lesions are delineated by arrowheads in bright field images with false blue color (A and E). FITC-filtered images highlight the GFP expression one day after systemic injection of Rd-NP-GFPg (B) whereas non-targeted NP (Rd-ntNP-GFPg) does not induce GFP expression in CNV (F). Cy3-filtered images highlight that rhodamine-labeled NP (Rd-NP-GFPg) accumulates in the CNV (C), while rhodamine-labeled non-targeted NP (Rd-ntNP-GFPg) does not (G). Some particles can be visualized circulating in the choroidal vessels. Overlay of images A–C is presented in panel D and overlay of E–G is shown in H.</p

Increased macrophage infiltration at the site of treated CNV.

<p>Macrophage infiltration was highest on day 3 with gradual decrease on days 5 and 7. Significantly higher number of macrophages were observed with the NP-ATPμ-Raf treated group compared to the control group on days 5 and 7 (A and B). There was a statistically significant reduction of CNV size noted on day 7(C). Immunofluorescent staining of representative frozen sections (<i>x20</i>) obtained at 3, 5, and 7 days after laser photocoagulation for ED 1, a marker for macrophage (D). *P<0.01. Data are expressed as the mean ± SE.</p

Evaluation of endothelial cell apoptosis with TUNEL staining in frozen sections.

<p>Quantification of TUNEL positive cells showed significantly more TUNEL(+) cells/lesion (A) and TUNEL (+) cells/mm² (B) with treatment of NP-ATPμ-Raf compared to the control group on day 3 and 5 after laser injury. There was a statistically significant reduction of CNV size noted on day 7(C). Double-immunofluorescent staining of frozen sections (<i>x20</i>) obtained at 3, 5 and 7 days after laser photocoagulation for the endothelial cell marker CD31 and TUNEL stain (D). *P<0.01. Data are expressed as the mean ± SE.</p