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. 2023 Oct 24;26(12):108308. doi: 10.1016/j.isci.2023.108308

Figure 3.

Figure 3

EnnA exhibits a powerful antitumor activity in vivo

(A–C) E0771 cells (1x105) were surgically implanted into the mammary fat pad of seven-week-old female B6 mice. Post-operative day 10 (POD10), mice were injected with either EnnA (10 mg/kg) or vehicle every 48 h. Tumor volumes (A) and body weight (B) were measured on the days indicated. C. Tumor lysates from six control (1–6) and six EnnA-treated mice (1–6) were used for Western blot analysis using specific antibodies to the indicated proteins.

(D) E0771 cells (5x104) were surgically implanted into the mammary fat pad of B6 mice. POD7, mice were injected with EnnA (10 mg/kg), 17-AAG (50 mg/kg), or vehicle every 48 h; tumor volumes were measured on days indicated.

(E) 4T1 cells (5x104) were surgically implanted into the mammary fat pad of BALBc mice. POD7, mice were injected with EnnA (10 mg/kg), 17-AAG (25 mg/kg), or vehicle every 48 h; tumor volumes were measured on days indicated.

(F) H&E staining of E0771 tumors from control and EnnA-treated mice from A. The scale bar represents 300 μm for 4x and 50 μm for 20x. G. Average of the number of immune-like cells found in two cm square necrotic areas from three controls and three EnnA-treated tumors from A.

(H) H&E staining of 4T1 tumors from control and EnnA-treated mice from E. (I) Average of the number of immune-like cells found in two cm square necrotic areas from three controls and three EnnA treated tumors from E. The scale bar represents 100 μm for 4x and 20 μm for 20x.

(J) Immunohistochemistry analysis of immune cell infiltration using anti-CD45 antibody staining of tumors from control and EnnA-treated mice from A. The scale bar represents 300 μm for 4x and 50 μm for 20x. (K) The corrected total cell fluorescence (CTCF) intensity was calculated, as indicated in Method, from 100 CD45+ cells from three different microscopic fields of control and EnnA-treated tumors.

(L) E0771 cells (5x104) were surgically implanted into the mammary fat pad of seven-week-old female nude mice. POD7, mice were injected with either EnnA (10 mg/kg) or vehicle every 48 h, and tumor volumes were measured on days indicated. Data are represented as the mean of at least two independent studies ±S.E.M. ∗∗p < 0.01 and ∗∗∗∗p < 0.0001, as determined by one-way unpaired tailed Student’s t test. n represents the number of mice per group.