Establishment of a mammary carcinoma cell line from Syrian hamsters treated with N-methyl-N-nitrosourea

Abstract

Clearly new breast cancer models are necessary in developing novel therapies. To address this challenge, we examined mammary tumor formation in the Syrian hamster using the chemical carcinogen N-methyl-N-nitrosourea (MNU). A single 50mg/kg intraperitoneal dose of MNU resulted in a 60% incidence of premalignant mammary lesions, and a 20% incidence of mammary adenocarcinomas. Two cell lines, HMAM4A and HMAM4B, were derived from one of the primary mammary tumors induced by MNU. The morphology of the primary tumor was similar to a high-grade poorly differentiated adenocarcinoma in human breast cancer. The primary tumor stained positively for both HER-2/neu and pancytokeratin, and negatively for both cytokeratin 5/6 and p63. When the HMAM4B cell line was implanted subcutaneously into syngeneic female hamsters, tumors grew at a take rate of 50%. A tumor derived from HMAM4B cells implanted into a syngeneic hamster was further propagated in vitro as a stable cell line HMAM5. The HMAM5 cells grew in female syngeneic hamsters with a 70% take rate of tumor formation. These cells proliferate in vitro, form colonies in soft agar, and are aneuploid with a modal chromosomal number of 74 (the normal chromosome number for Syrian hamster is 44). To determine responsiveness to the estrogen receptor (ER), a cell proliferation assay was examined using increasing concentrations of tamoxifen. Both HMAM5 and human MCF-7 (ER positive) cells showed a similar decrease at 24h. However, MDA-MB-231 (ER negative) cells were relatively insensitive to any decrease in proliferation from tamoxifen treatment. These results suggest that the HMAM5 cell line was likely derived from a luminal B subtype of mammary tumor. These results also represent characterization of the first mammary tumor cell line available from the Syrian hamster. The HMAM5 cell line is likely to be useful as an immunocompetent model for human breast cancer in developing novel therapies.

Figure 1. Histopathologic examination of a primary mammary tumor in MNU treated hamster

Shown is a representative H&E stained section of a mammary adenocarcinoma from which the HMAM4 cell line was isolated.

Figure 2. Immunohistochemical analysis of normal mammary tissues and mammary tumors from Syrian hamsters

In the following histology images, (A) normal mammary tissue or uterus was compared to (B) mammary tissue displaying ADH and (C) a mammary tumor from a hamster treated with a 50 mg/kg dose of MNU. Shown are representative specimens stained by immunohistochemistry for pancytokeratin and HER-2/neu expression.

Figure 3. Growth characteristics of HMAM4B cells in vivo in syngeneic recipient Syrian hamsters

After s.c. injection of 1 × 10⁷ HMAM4B cells into the left and right flanks, tumor growth at each injection site was monitored by caliper measurements. The take rate observed was 50% (i.e., 3/6 sites injected with HMAM4 cells developed a tumor).

Figure 4. Analysis of HMAM5 tumor growth in vivo in syngeneic recipient Syrian hamsters

Shown are: (A) the mean body weight (n = 10) and (B) the mean tumor growth (n = 7) at each injection site after s.c. injection of 1 × 10⁷ HMAM5 cells into the right flanks of recipient Syrian hamsters. The take rate observed was 70% (i.e., 7/10 animals injected with HMAM5 cells developed a tumor). Of the 7 animals followed, one hamster was euthanized at day 65 (E1) and another at day 79 (E2) due of excessive tumor burden. Each data point represents the mean ± standard error of the body weights or tumor volume measurements at each time point.

Figure 5. Growth characteristics of HMAM5 cells in vitro

Shown are: (A) representative growth curve of HMAM5 cells in culture and (B) soft agar assay of anchorage-independent cell growth assessed by colony formation of HMAM5 cells. Cell growth and colony formation characteristics of HMAM5 cells were compared with HMAM4B and MDA-MB-231 cells. Each data point represents the mean ± standard error of three independent experiments.

Figure 6. Cytogenic analysis of the HMAM5 cell line

(A) Summary of 100 metaphases analyzed showing the distribution of chromosome numbers. In Syrian hamsters, the normal chromosome number (2n) = 44. (B) Summary of 5 karyotypes examined showing the mean chromosome number. No chromosomal aberrations were observed. Each bar point represents the mean ± standard error of 5 karyotypes.

Figure 7. Immunohistochemical analysis of normal skin tissue and mammary tumors from Syrian hamsters

In the following histology images, sections of tumors from hamsters injected s.c. with HMAM4B or HMAM5 were stained with (A) cytokeratin 5/6 or (B) p63. Staining was compared to positive controls using sections from normal hamster skin. Shown are representative specimens.

Figure 8. The effect of tamoxifen treatment on cell proliferation

The effect of tamoxifen, an antagonist of the estrogen receptor, was used as a surrogate marker to determine the ER status of HMAM5 cells. MDA-MB-231 (an ER negative cell line), MCF-7 (an ER positive cell line), and HMAM5 were untreated or treated with 3.3, 10, 33, and 100 μM tamoxifen for 40 h. Cell proliferation was measured with a BrdU labeled ELISA kit. Each bar represents the mean ± standard error of values normalized to untreated cells. Indicated (*) by the Student’s t-test, there was a statistically significant difference between the cell lines with P < 0.05.