Previous studies have shown that the i.duc administration of 4-hydroxytamoxifen and PLD to rodents with carcinogen-induced mammary tumors, and of PLD to Her2/neu mice was associated with regression of established mammary tumors and prevention of new lesions [7
]. In this study, we investigated the safety of i.duc PLD and mechanism underlying PLD’s protective effects. We found that i.duc PLD, despite an additional pregnancy, remained protective against tumorigenesis in the Her2/neu mice by reducing spontaneous tumor incidence and prolonging latency compared with vehicle treated mice. In the wild type FVB/N mice, i.duc PLD resulted in reduction in the number of MaSC compared to untreated controls. However, development of mammary tumors in the treated mice after long-term follow up showed that PLD is carcinogenic in the normal mouse mammary gland.
Whole mount analysis in Her2/neu transgenic mice showed no effects on the structure of the mammary glands, such as the number of end buds () or ductal dilatation of primary ducts (). Structural changes observed in whole mount analysis and histopathological findings, depend on aging, duration after injection, and estrus phase [5
]. Therefore, a retrospective analysis based on the long-term follow-up data did not provide an adequate explanation for death of pups born to i.duc PLD-treated Her2/neu mice. Prospective studies on the effect of i.duc PLD on milk production are necessary to resolve this question. In fact, MNU-treated rats treated by i.duc PLD examined within 4 weeks showed a loss of density of ductal outgrowth and periductal inflammation compared to untreated controls [8
]. These results are consistent with a severe depletion of stem cells in the mammary gland and decrease of the number of MRUs in i.duc PLD-treated mice seen in this study. Taken together, the major reason for death of all pups born to PLD-treated mice appears not to be due to structural abnormalities, but possibly a compromised functioning of mammary gland epithelial cells.
I.duc PLD significantly protected mice by prolonging the latency and decreasing incidence of spontaneous mammary tumor development in Her2 mice (). A likely explanation for the protection could be a reduction in MaSC function. However, formation of tumors in wild type FVB/N mice that received i.duc PLD over the long term is a serious pitfall. PLD is a formulation of doxorubicin in poly (ethylene glycol)-coated (stealth) liposomes with a prolonged circulation time and unique toxicity profile [13
]. Numerous anti-neoplastic agents including doxorubicin are known carcinogens, and rare occurrence of second malignancies in cancer patients treated with single or multiple drugs has been documented [14
]. Rodents are particularly susceptible to anthracycline-induced mammary tumors [16
]. Howell et al. [19
] reported that four of 12 rats given an intramammary injection of daunorubicin (4 or 8 µg) developed mammary tumors in the injected area within 6.5 months of injection. The inability of rodent mammary tissue to metabolize the drug was cited as a possible cause of drug-induced tumors in rats [19
]. Similarly, it is possible that i.duc PLD-treated mouse mammary glands were unable to metabolize PLD which led to its accumulation in the mammary glands. However, unlike rodents, metabolism of PLD does occur in human breast tissue. Our recent clinical trial showed the presence of metabolites such as doxorubicinol in the breast tissue and blood of women who received PLD into one duct prior to mastectomy [8
]. Obviously, metabolism of anthracyclines in breast tissue between rodents and humans is different; this needs to be addressed with comparative studies on pharmacokinetics of PLD in the mammary gland and serum in the future. Transfer of milk contaminated with poorly metabolized PLD in the mammary gland from the mother to the pups as a cause of their death also needs to be ruled out. However, at the present time, the risk of developing cancer in the breast after a long latency due to PLD exposure remains a valid concern.
One can speculate on how PLD causes malignant mammary tumors in i.duc exposed mice. As stem cells self-renew throughout life and are long-lived, one possibility is that accumulating genetic anomalies over time could compromise their genomic integrity and potentially give rise to cancer [1
I.duc treatment has a significant potential for both breast cancer prevention and treatment. Treating patients with a family history or with other high risk factors for developing breast cancer holds the promise of reducing the cancer burden in this vulnerable population [20
]. Therefore before proceeding with clinical translation of this novel technique, the first priority is the selection of appropriate treatment agents which are proven safe and fully metabolized in mammary glands when administered intraductally.
In conclusion, we have presented data to demonstrate that i.duc PLD protects Her2/neu mice despite the tumor-promotional effects of an additional pregnancy. Some of the protective effects could be attributed to a reduction in normal MaSC population in the mouse mammary gland. Upon long-term follow up, i.duc PLD-treated mice developed malignant mammary tumors. This finding under-scores the need for further research into metabolism of PLD in the human breast, and a continuing search for new agents for intraductal use.