Myofibroblasts represent the most abundant cell type in the tumor stroma of invasive breast carcinomas (for a review, see [3
]). Myofibroblasts were shown originally to derive mainly from interstitial and perivascular fibroblasts, except for a subpopulation of highly smooth-muscle-differentiated myofibroblasts from venous smooth muscle cells [11
] (). Bone-marrow-derived mesenchymal stem cells or fibrocytes might also contribute to reactive stroma. Experimental models have demonstrated that mesenchymal stem cells enhance invasion, motility and metastasis of human breast carcinoma cell lines [12
]; however, the relevance of this interaction in breast carcinomas in humans still awaits confirmation. Likewise, although recruitment of fibrocytes is a pronounced phenomenon in wound healing and fibroses [13
], their contribution to human breast cancer has not been documented convincingly.
Figure 2 Vascular smooth muscle cells recruited from venous blood vessels might contribute to reactive stroma. Using triple-staining of cryosections of human breast tumors, we demonstrated that (a) in tumors with no or few smooth-muscle-differentiated myofibroblasts, (more ...)
Upon transplantation of a breast cancer cell line to mice transplanted with bone marrow from double-mutant recombinase-activating gene-1 (RAG-1)-/-
β-gal transgenic and green fluorescent protein (GFP) transgenic mice, 20% of the myofibroblasts in the resultant tumors were bone-marrow-derived [15
]. In another study, SV40 large T antigen-, human telomerase reverse transcriptase (hTERT)- and RasV12-transfected human breast cells recruited Rag-1null
and GFP transgenic bone-marrow-derived cells to comprise ~90% of the tumor-associated cells, of which the majority expressed α-smooth muscle (α-sm) actin [16
]. Given that cancer is a tissue-specific disease and that human breast stroma differs appreciably from the stroma of mouse mammary gland [3
], we believe that additional markers of human fibrocytes are needed. This is particularly pertinent to breast tissue. Currently, the most widely used marker of fibrocytes, CD34, is expressed by all fibroblasts in normal breast but is lost in α-sm-actin-positive myofibroblasts [17
We have shown previously that resident, normal fibroblasts readily undergo conversion to myofibroblasts in response to tumor cells in culture (reviewed in [3
]). Multiple studies have documented that the converted stroma in turn supports cancer cell growth and metastasis [3
]. That the reactive stroma itself, once converted by any means, might also signal to epithelial cells and eventually convert them to cancer cells is beginning to be appreciated.
A comparison of stromal cells isolated from tumors and normal tissues, respectively, suggests that stromal cells might indeed provide cues for malignancy. In recombination studies of human prostate-derived cells, normal fibroblasts support growth arrest and normal histology of the epithelium, whereas so-called cancer-associated fibroblasts (CAFs) direct immortalized prostatic epithelial cells towards adenocarcinomas [23
]. The myoepithelial cells isolated from ductal carcinoma in situ
(DCIS) were shown to be grossly abnormal and to secrete many cytokines and matrix metalloproteinases (MMPs) that normal myoepithelial cells did not [24
]. Thus, stromal as well as myoepithelial cells might be a switch for overt malignancy in luminal epithelial cells. Conversely, an experimental model of DCIS has demonstrated that only myoepithelial cells isolated from normal glands can inhibit progression of DCIS to invasive carcinoma [24
Evidence that not only certain cell populations but also changes in the microenvironment as a whole can promote tumorigenesis also comes from studies of irradiated mouse mammary gland stroma. Ionizing radiation is a known albeit weak carcinogen in the mammary epithelium. In addition to its widely accepted DNA-damaging (i.e. mutagenic) effect, irradiation elicits persistent microenvironmental alterations in ECM composition and cytokine activities. Transplantation of a p53-null mouse mammary epithelial cell line to cleared mammary fat pads showed that tumor incidence was 81% in irradiated animals but only 19% in sham-irradiated animals. Experiments in animals with one-sided irradiation confirmed that tumors formed only on the irradiated side [25
It is intriguing to note the dramatic epigenetic changes that were clearly transmitted to progenies after combined irradiation and transforming growth factor-β (TGF-β) treatment of single human breast epithelial cells [26
]. There was a heritable disruption of epithelial cell polarity and multicellular organization in three-dimensional (3D) assays in the entire surviving population, indicating non-mutational but heritable mechanisms [26
]. Of note, the stroma itself also has been suggested as a target of the chemical carcinogen N-nitrosomethylurea, which was previously thought to exert its effect only through direct mutation induced in the epithelium [27
]. However, confirmation of these latter findings via the use of marked stromal cells would be essential for interpretation of the data.
The stromal compartment also includes mast cells, macrophages and T-cell subtypes, all of which are emerging as essential modulators of tumor growth, and more research clearly needs to be done in this area. The influence of immune cell infiltrates on tumor development might be favorable or adverse, depending on the cell types involved. Mast cells are attracted to tumors by tumor-derived chemoattractants and affect tumor development in accordance with local tumor conditions. In a mouse tumor model, recruitment of mast cells was required for angiogenesis and expansion of Myc-induced pancreatic islet tumors [28
], whereas in human breast cancer, the presence of mast cells might be an independent, favorable prognostic factor [29
]. Macrophages, as a major population of infiltrating cells in the tumor stroma, are considered to have an overall tumor-supporting role. The attraction of tumor-associated macrophages is thought to be caused by hypoxia, which stimulates macrophages to perform pro-angiogenic functions [30
]. Pollard and coworkers, who were the first to describe the importance of macrophages in breast tumors, developed a transgenic mouse model that displays a progression series from benign tumor to malignancy. The results show a marked increase in macrophage infiltration prior to the transition to malignancy, which is associated with angiogenesis, thus showing that the microenvironment is directly involved in the development of the high-density tumor vasculature that is required for tumor progression (reviewed in [31
]). These findings highlight the crucial role that the host stromal reaction, including the inflammatory cell infiltrate, has in modulating cancer progression. Immune cells, which seem to be very widely involved in tumor development, are easily accessible, making them a potential and attractive tool for tumor therapy, as discussed below.
Transgenic mouse models have also pointed to the importance of ECM and TGF-β signaling in tumor development. Disruption of the ECM in the mammary gland of normal mice ([32
], discussed in more detail below) led to the formation of reactive stroma [33
] long before tumors were formed [34
]. Importantly, knockout of TGF-β receptor II revealed prostatic hyperplasia in the stroma, as well as in adjacent epithelium, leading to prostatic intraepithelial neoplasia, a presumed forerunner of carcinoma [35
]. In spite of the fact that TGF-β signaling has so far not been found to be inactivated during the normal course of tumor pathogenesis, these latter studies provide a small but growing number of counter-examples to the assumption that mutations in epithelial cells are a required initiating event for neoplastic change.
Collectively, these studies in our view lend credence to the fact that a physiologically abnormal microenvironment could promote, as well as possibly initiate, mammary or other types of epithelial tumors.