Figure 1. Inhibition of AT1R with Losartan decreases mammary tumor onset and progression associated with MPA and DMBA treatment.

Nulliparous six-week-old female mice were implanted subcutaneously with MPA pellets and treated orally with DMBA. Losartan (600 mg/L) was administered in drinking water. a. Onset of mammary tumors in age-matched wildtype MPA/DMBA-treated mice control-treated (3% sucrose) or with AT₁R blocker, Losartan. Data for panels are expressed as percentage of mice free of tumors (palpable and histological assessment) after last DMBA treatment (P = 0.004, Mantel Cox Log-Rank Test). b. Tumor latency, development of palpable tumors days post-DMBA administration, data point represents individual animal. Immunohistochemistry quantification of c. Ki67 and d. Cleaved Caspase 3 in control and Losartan-treated animals. Each data point represents individual tumor analysed; and immunostain is presented as a percentage of total tumour area including tumour-associated stroma e. Representative Haematoxylin/Eosin (H&E) stained histological sections of tumors from age-matched control and Losartan treated animals. f. Representative histological sections of mammary tumor stages identified including Ductal Carcinoma in situ (DCIS), mixed DCIS and invasive ductal carcinoma (DCIS/IDC) and IDC; H&E stained (top panel) and immunostained with a differentiation marker for cancer-associated fibroblasts, α Smooth Muscle Actin antibody (αSMA, bottom panel). Intact myoepithelial layer identified by αSMA is indicated (arrowheads) in well-differentiated DCIS, while loss of myoepithelial αSMA and gain of αSMA expression in the stroma (arrowheads) is indicated in DCIS/IDC and the poorly differentiated squamous IDC. g. Tumor histopathology quantified as % of tumors assessed. Difference in incidence of invasive tumors between the 2 groups is indicated (P = 0.03, Fisher's Exact Test). Tumor-free animals are only observed in the Losartan group (green). h. Autoradiograph showing binding of I¹²⁵-[Sar¹, Ile⁸] Angiotensin II (I¹²⁵AngII) to a section (unmagnified) of the inguinal mammary containing a DCIS lesion demonstrating strong AT₁R expression. Insert: control tissue co-incubated with unlabeled AngII and I¹²⁵AngII. H&E stained serial section of DCIS lesion at 20X magnification. Data are presented as mean +SEM, n = 10-15 animals per group. *P < 0.05, Mann Whitney Test unless otherwise stated.