Optimizing the time of Doxil injection to increase the drug retention in transplanted murine mammary tumors

Abstract

Sex hormonal milieus during the female fertility cycle modulate the tumor vascular permeability of breast cancer. It has been proposed that the liposomal formulated doxorubicin (ie, Doxil), given at the menstrual/estrous stage with the predicted highest tumor vascular permeability, allows significantly increased drug retention in the breast tumor. In the current study, syngeneic murine 4T1 mammary tumors were established on the backs of female BALB/c mice and Doxil was administered at particular mouse estrous cycle stages. The results indicated that Doxil administration during certain times in the mouse estrous cycle was crucial for drug retention in 4T1 tumor tissues. Significantly higher drug concentrations were detected in the tumor tissues when Doxil was administered during the diestrus stage, as compared to when the drug injection was given at all other estrous stages. Our study also showed that the tumor-bearing mice exhibited nearly normal rhythmicity of the estrous cycle post drug injection, indicating the feasibility of continual injection of Doxil at the same estrous cycle stage. By using 4T1 cells cultured in vitro, we showed that progesterone (P4) significantly inhibited cell proliferation and the production of six tumor-derived cytokines, eg, sTNF-RI, CXCL-16, GM-CSF, MIP-1alpha, MIP-1gamma, and Flt3-L. Some of these factors have been shown to be vascular modulators in diverse tissues. In this report, we demonstrated that the concentration of P4 in the plasma and/or estrous cycle stage of 4T1 tumor-bearing mice can be used to select the best time for administrating the liposomal anticancer drugs.

Keywords: Doxil; breast cancer therapy; menstrual cycle; mouse mammary tumor; progesterone.

Figure 1

The time of Doxil administration during mouse estrous cycle and its effects on drug retention in 4T1 tumors. A) The Dox concentrations in 4T1 tumors when Doxil is injected during diestrus stage or other estrous stages. B) The dynamic changes of serum progesterone and estrogen levels during the estrous cycle. The concentrations of P4 and E2 in mice were previously published. The best time for Doxil injection is indicated by the black arrow and bar. The worst times for Doxil injection are indicated by small triangles and dotted bars.

Figure 2

Doxil administration and its effects on the growth of 4T1 tumors and estrous cycle of tumor-bearing mice. A) Length of estrous cycles that occur before and after Doxil injection. B) Length of estrous cycle stages in control and tumor-bearing mice prior to and after Doxil administration. C) Effect of Doxil treatment on 4T1 tumor size.

Figure 3

Immunohistochemical analysis of PR and mPRα protein expression in 4T1 tumor tissues. A) 4T1 tumor stained with H & E. B) Negative control of 4T1 tumor tissue stained without primary antibody. Magnification of all images is 400×. C) 4T1 tumor tissue stained with anti-PR. D) 4T1 tumor tissue stained with anti-mPRα. Abbreviations: H & E, hematoxylin & eosin; PR, progesterone receptor; 4TI, mouse mammary cell line.

Figure 4

P4 treatment and cytokine expression in vitro. Scanned signal intensity of the hybridized protein array spots from a cytokine microarray assay of conditioned medium from 4T1 mouse mammary cell cultures treated with progesterone for 24 hours. Each bar represents the average values from two hybridized spots of duplicate assays with subtraction of the baseline reading (baseline reading value = 100).

Figure 5

Effect of sex hormones on cell proliferation and cell death in vitro. A) Cell proliferation index measured for 4T1 cultures treated with P4 or E2, compared to controls. The data were obtained from three experiments. B) Appearance of 4T1 cultures treated with various doses of P4. A. control; B. E2; C. P4 15 ng/ML; D. P4 30 ng/ML; E, P4 60 ng/ml. Magnification of all images is 100×. Abbreviation: 4TI, mouse mammary cell lines.