Figure 5.

Establishment of a subcutaneous xenograft model that recapitulates clinical conditions, i.e. fractionated doses of both chemotherapy and irradiation. (a) Two million SW837 cells (100 μl) were injected subcutaneously into the right flank of NMRI-Foxn1 nude mice. At a volume of ~450 mm³, mice were separated into two groups, DMSO (n = 10) and STATTIC (n = 12) and treated as indicated (black arrows pointing upward) at irradiation doses of 1.8 Gy (14×), intraperitoneal injections of 50 mg/kg 5-FU (5×) and intratumoral injections of 23% DMSO or 10 mg/kg STATTIC (9×). (b) To document tumor development, pictures of each mouse were taken once weekly. (c) To confirm inhibition of STAT3 phosphorylation in vivo, selected tumor-bearing mice were randomly sacrificed 2 hr after STATTIC or DMSO injection and subjected to Western blot analysis. Compared to DMSO-treated mice, we detected remarkably reduced pSTAT3^Tyr705 levels in STATTIC-treated mice. Displayed is a representative blot from three DMSO- and four STATTIC-treated mice with the respective densitometry data (pSTAT3/STAT3 ratios). (d) Immunohistochemical analysis also confirmed remarkably reduced pSTAT3^Tyr705 levels in STATTIC-treated mice compared to DMSO-treated mice (exemplified is one representative DMSO- and STATTIC-treated tumor, randomly selected). STATTIC- and DMSO-treated tumors showed similar rates of total STAT3 expression, proliferation (Ki-67 staining) and cellular morphologies (H&E staining).