Stromal regulation of neoplastic development: age-dependent normalization of neoplastic mammary cells by mammary stroma

Abstract

There is mounting evidence that the stroma plays a crucial role in mammary gland carcinogenesis. Here, we report that mammary gland stroma from mature and multiparous rats prevents neoplastic development and encourages normal ductal growth of grafted epithelial cancer cells. Fifty thousand epithelial cancer cells were injected into the cleared fat pads of virgin hosts at 24, 52, 80, and 150 days of age and of hosts that had undergone two cycles of pregnancy, lactation, and involution. Six months after inoculation, tumor incidence was 75%, 100%, 50%, and 18.2% in 24-, 52-, 80-, and 150-day-old virgin rats, respectively, and 0% in the twice-parous animals. Most remarkably, these neoplastic cells appeared to form normal ducts in all hosts-Ha-ras-1 mutation served as a marker to identify the tumor origin of the outgrowths. The tumor development pattern suggests a parallel to the phenomenon of age- and reproductive state-dependent susceptibility and resistance to chemical carcinogens. As susceptibility to carcinogenesis decreases, the ability of the stroma to reprogram neoplastic epithelial cells increases. Thus, the neoplastic phenotype is context-dependent, and it therefore offers the intriguing possibility that the process of carcinogenesis is amenable to normalization or cure once the mechanisms of stroma-mediated normalization are elucidated and manipulated.

Figure 1

Schematic representation of the experimental design.

Figure 2

The incidence of secondary tumors decreases with the age of the stroma. The parous host only developed normal ductal outgrowths. *Statistically different from twice-parous and 150- and 80-day-old host groups. **Statistically different from twice-parous and 150-day-old host groups.

Figure 3

Diverse results were obtained from the same tumor donor. A: Papillary carcinoma used as a donor tumor. B: Secondary tumor developed in a 24-day-old host. C: Secondary tumor developed in a 52-day-old host. D: Normal ductal outgrowth developed in an 80-day-old host. In both secondary tumors there is a noticeable increase in the deposition of extracellular matrix and the number of glands is reduced, showing a less differentiated phenotype. E to H are a representation of the phenotypically normal ducts observed in the aged and parous hosts. Areas of the whole mounts containing ducts were removed, embedded in paraffin, sectioned, and stained with H&E. Scale bars: 50 μm (G, H); 100 μm (A, B, C, E, F); 2 mm (D).

Figure 4

Examples of Ha-ras-1 expression in donor tumors and their outcomes. Each number represents one sample and its Ha-ras-1 expression: the left lane is the endogenous Ha-ras-1 and the right lane is the mutated gene. Samples 1, 3, and 5 are examples of donor tumors. Sample 2: DNA was extracted from a normal ductal outgrowth developed in an 80-day-old host injected with neoplastic cells from sample 1. Sample 4: DNA was extracted from a secondary tumor developed after the transplant of cells from sample 3. Samples 6 and 7: DNA was extracted from a normal ductal outgrowth and a secondary tumor developed in 80- and 24-day-old hosts, respectively. Both hosts were inoculated with sample 5. All donor tumors carry the codon 12 GGA to GAA mutation and the same mutation can be seen in both types of secondary outcomes, ie, tumors or normal ductal development.