Intraductally administered pegylated liposomal doxorubicin reduces mammary stem cell function in the mammary gland but in the long term, induces malignant tumors

Abstract

Previously, we have shown that the intraductal (i.duc) administration of pegylated liposomal doxorubicin (PLD) to Her2/neu transgenic mice is associated with mammary tumor regression and prevention. Exploring the mechanism underlying the protection afforded by PLD, we studied: the effects of i.duc PLD-treatment with a subsequent pregnancy on outgrowth of tumors in Her2/neu mice; whether the i.duc PLD antitumor effect was mediated partially through changes in normal mammary stem cells (MaSCs); and the long-term safety of i.duc PLD into the normal mouse mammary gland. Her2/neu mice were treated with two i.duc injections of PLD given four weeks apart; pregnancy was induced and mice were followed up for changes in physiology, and tumor formation. We found that all pups born to i.duc PLD-treated Her2/neu mice died without weight gain within 7 days after birth. Despite an additional pregnancy, compared to vehicle control PLD-treated Her2/neu mice had a significantly longer latency and lower frequency of tumor development. Mammary epithelial cells isolated from untreated and i.duc PLD-treated 6-8 months-old multiparous FVB/N mice were analyzed for their repopulating ability in mammary fat pads of naïve recipients. Mice were also monitored for abnormalities in mammary gland morphology and function, including tumor formation. PLD-treated FVB/N mice displayed histomorphologic changes and a significant reduction in the outgrowth potential of cells from the mammary glands. Thus, i.duc PLD administration altered the mammary gland structurally and functionally by reducing the MaSC population, which could compromise milk production. Followed long term, i.duc PLD-treated FVB/N mice developed malignant mammary tumors, confirming similar published findings on doxorubicin injected into the mammary gland of rats. Unless there are fundamental species differences in PLD metabolism in rodents and humans, this finding seriously limits the consideration of i.duc PLD use in the clinic for treatment or prevention of breast cancer.

Fig. 1

Protective effect of i.duc PLD with subsequent pregnancy on tumor incidence and latency in Her2/neu mice. Kaplan–Meier plot of mouse tumor-free survival (a) and gland tumor-free survival (b) with i.duc PLD compared with vehicle by log-rank test. The timing of pregnancies before and after PLD is indicated by black ovals. c Mammary gland whole mount analysis of untreated and PLD-treated mice. Bar 10 mm. d Comparison of numbers of end buds between groups. I.duc vehicle (75.8 ± 6.2, n = 6 specimens × 4 fields = 24) and i.duc PLD (65.6 ± 4.1, n = 6 specimens × 4 fields = 24), P = 0.179. e A representative image shows 4 fields (5 × 5 mm) of a whole mount specimen. Bar 10 mm. f Comparison of the diameters of primary ducts. I.duc vehicle (77.2 ± 11.8, n = 9) and i.duc PLD (60.3 ± 7.7, n = 16), P = 0.225. ns not significant. g PLD #1, 100 µm in diameter of the largest primary duct adjacent to the central lymph node. h Vehicle #5, 50 µm in diameter

Fig. 2

Mammary gland reconstitution in transplanted mammary fat pads. Mammary epithelial cells were transplanted into cleared mammary fat pads of 3 week-old recipient female FVB/N mice. Outgrowths in the recipients were analyzed in whole mounts 8 weeks after transplantation. Outgrowths from: a 10⁶ mammary gland epithelial cells isolated 31 weeks after i.duc PLD; b 10⁴ mammary gland epithelial cells isolated 18 weeks after i.duc PLD; c 10⁶ and d 10⁴ epithelial cells isolated from untreated, age and parity matched (to a and b) FVB/N mouse mammary glands

Fig. 3

Histopathology of mammary glands and tumors in FVB/N mice. a Lymphocytic infiltration and b fibrosis were determined in each specimen. Representative histology is shown of: c invasive undifferentiated mammary carcinoma arising in mfp transplanted with epithelial cells from PLD-treated mouse, d mammary intraepithelial neoplasia, and e–h mammary tumors developing in i.duc PLD treated mice, e poorly differentiated epithelial carcinoma, f carcinoma, solid pattern with necrosis (arrow), g carcinoma invading lymph nodes (arrow) and h sarcoma-like neoplasm with EMT and local invasion of skeletal muscle (arrow). Bars 20 µm