Combined treatment of pancreatic cancer xenograft with 90Y-ITGA6B4-mediated radioimmunotherapy and PI3K/mTOR inhibitor

Figure 1 Experimental treatment scheme. Group A: ⁹⁰Y-ITGA6B4 + BEZ235; Group B: ⁹⁰Y-ITGA6B4; Group C: BEZ235; Group D: Vehicle.

Figure 2 Effect of BEZ235 and ⁹⁰Y-ITGA6B4 on PI3K/Akt signaling. Expression levels of selective proteins associated with the PI3K/Akt pathway were determined at 18 h after ⁹⁰Y-ITGA6B4 exposure (185 or 370 kBq/mL). Representative western blot analyses are shown. Akt1 expression and Akt phosphorylation (p-Akt) likely induced by ⁹⁰Y-ITGA6B4 were inhibited upon BEZ235 treatment. Concordantly, phosphorylation of 4EBP1 (p-4EBP1) and S6 (p-S6), both Akt and mTOR downstream effectors, was markedly inhibited by BEZ235 pretreatment.

Figure 3 Cytotoxic effect of BEZ235 that augments the cytotoxic activity of RIT in BxPC-3 cells. Treatment with ⁹⁰Y-ITGA6B4 alone or BEZ235 alone resulted in decreased colony formation ability in cells. Moreover, pretreatment with BEZ235 before ⁹⁰Y-ITGA6B4 exposure induced stronger inhibition of the colony formation ability than that by ⁹⁰Y-ITGA6B4 or BEZ235 treatment alone; moreover, plating efficiencies (PE) of cells were significantly different between the BEZ235 (+) and (-) conditions (^bP < 0.01). Data represent mean ± SD, n = 3.

Figure 4 Enhancement of mediated radioimmunotherapeutic effects on BxPC-3 xenografts in combination with BEZ235 treatment. A: Tumor volume changes expressed as the ratio of the volume on the indicated day and the volume on the day treatment began. Compared with the control vehicle administration (light blue squares), single administration of ⁹⁰Y-ITGA6B4 (2.8 MBq) alone (red diamonds) or oral administrations of BEZ235 alone (35 mg/kg, 5 d/wk for 6 wk) (blue triangles) resulted in impaired tumor growth. When ⁹⁰Y-ITGA6B4 treatment was administered in conjunction with BEZ235 (orange squares), further reduction in tumor volume ratio was observed indicating enhanced therapeutic effect upon combined treatment. Values represent mean ± SD, ^aP < 0.05 (⁹⁰Y-ITGA6B4 + BEZ235 vs⁹⁰Y-ITGA6B4), ^bP < 0.05 (90Y-ITGA6B4 + BEZ235 vs BEZ235), ^cP < 0.05 (⁹⁰Y-ITGA6B4 + BEZ235 vs Vehicle, ⁹⁰Y-ITGA6B4 vs Vehicle); B: Average mouse body weight did not differ significantly among all 4 groups. Vertical orange arrowhead indicates the day of 90Y-ITGA6B4 injection. Values represent mean ± SD, n = 6.

Figure 5 Ki-67, p-H2AX, p-4EBP1 immunostaining of tumor sections. A: On Day 1 and; B: On Day 3 after administration of 90Y-ITGA6B4 alone or combined with BEZ235, intratumoral proliferation was determined by immunostaining for Ki-67 nuclear antigen. A marked reduction in Ki-67-positive cell numbers was observed in samples from mice treated with 90Y-ITGA6B4 + BEZ235 as well as with 90Y-ITGA6B4 alone and BEZ235 alone, compared with those in the untreated control sample. Meanwhile, increased p-H2AX-positive cell numbers were observed in samples from mice that received 90Y-ITGA6B4 treatment alone or the combined treatment than those in the control. Immunohistochemical analysis for p-4EBP1 showed that phosphorylation of 4EBP1 was decreased in the treatment groups compared with the control group. Tumor section images were acquired at 200× magnification and representative images are shown (scale bar, 50 μm). The quantitative and statistical analysis were summarized in Table 1.