Normal stem cells: The normal growth and function of mammary epithelial cells depend on interactions with the supportive stroma. Alterations in this communication can lead to the progression or expansion of malignant growth. The human mammary gland contains two distinctive types of fibroblasts within the stroma. The epithelial cells are surrounded by loosely connected intralobular fibroblasts, which are subsequently surrounded by the more compacted interlobular fibroblasts. The different proximity of these fibroblasts to the epithelial cells suggests distinctive functions for these two subtypes. We compared the gene expression profiles between the two stromal subtypes. Fresh normal breast tissue was collected from reduction mammoplasty patients and immediately placed into embedding medium and frozen on dry ice. Tissue sections were subjected to laser capture microscopy to isolate the interlobular from the intralobular fibroblasts. RNA was prepared and subjected to microarray analysis using the Affymetrix Human Genome U133 GeneChip. Data was analyzed using the Affy and Limma packages available from Bioconductor. Findings from the microarray analysis were validated by RT-PCR and immunohistochemistry. No statistically significant difference was detected between the gene expression profiles of the interlobular and intralobular fibroblasts by microarray analysis and RT-PCR. However, for some of the genes tested, the protein expression patterns between the two subtypes of fibroblasts were significantly different. This study is the first to report the gene expression profiles of the two distinct fibroblast populations within the human mammary gland. While there was no significant difference in the gene expression profiles between the groups, there was an obvious difference in the expression pattern of several proteins tested. These data also highlight the importance of studying gene regulation at both the transcriptional and post-translational level. We are continuing to advance the recapitulation of human breast epithelial morphogenesis by humanizing the mammary fat pad of NOD/ SCID mice as described by Kuperwasser et al. 2004. The stromal cells fill over 50% of the mouse mammary glands but normal epithelial cell outgrowths are very rare. The humanized gland are also used to influence the tumorigenicity of breast cancer cells. We are investigating alternative approaches to enhance epithelial cell growth such as the addition of primary human macrophages. Preliminary results show a significantly higher number of epithelial outgrowths in the presence of macrophages. Additionally, we have successfully isolated human mammary extracellular matrix. This will be useful: 1. In vivo to replace Matrigel with a more physiological matrix; 2. to study the epithelial/tumor cells in response to ECM proteins derived from specific subsets of the population (for example: age, race, parity); 3. to study matrix composition/interactions from a specific tumor subset (for example: Her2 or BRCA). Cancer stem cells: Recent data suggest that solid tumors are initiated and propagated by a rare population of cells that are unique from the tumor bulk in their ability to self-renew indefinitely, give rise to heterogeneous lineages and form tumors. In the breast, this population of cancer initiating cells has been prospectively identified as CD44+ and CD24-/dim while CD24+ cells are considered to be of a terminally differentiated luminal subtype. We have definitively illustrated, contrary to data in the literature, that (1) the presence of CD44+CD24- cells in a pleural effusion (PE) does not ensure that the PE can give rise to tumors in NOD/SCID mice, (2) in both PE and xenograft tumors, CD44+CD24- and CD44+CD24+ are equally tumorigenic and can give rise to each other and (3) CD44+CD24- cells do not faithfully recapitulate the CD44/CD24 profile of the PE in the resulting xenograft. Of 8 clinical samples, only 1 PE behaved as predicted based on the data of Al-Hajj et al. As few as 200 CD44+CD24- cells gave rise to tumors whereas 5,000 CD44+CD24+ cells failed to give rise to tumors. Contrary to these observations, CD44+CD24+ cells from several PEs were capable of initiating tumors. Furthermore, CD44+CD24- cells from 3 additional PEs failed to give rise to tumors even after 11 months. In xenografts derived from PEs and a primary tumor fragment, CD44+CD24- and CD44+CD24+ cells were consistently equally tumorigenic. CD44- cells are non-tumorigenic regardless of their source (i.e. PE vs. xenografts). With one exception that was ER+PR+HER2-, all tumors examined were of a basal phenotype (ER-, PR-, HER2+/-). In all cases, the receptor status of the primary tumor/PE was identical to that of its resulting xenograft. However, PE cells and their xenografts had markedly disparate CD24 profiles. Pleural effusions that had substantial C24- populations gave rise to xenografts that were largely CD24+. In all xenografts examined, the xenograft immunophenotype remained static after numerous passages. We have recently begun efforts to employ lentiviral mediated, doxycycline inducible shRNA production to knockdown expression of CD44. We have achieved a 4-fold reduction in CD44 protein abundance and a concomitant 6.8 fold increase in the percentage of CD44- cells in the MCF10 Ca1a.c11 cell line. While statistically significant, the biological relevance (i.e. in vivo tumorigenicity) of this knockdown is questionable given that even after shRNA induction, the fraction of CD44+ cells remains approximately 3.5%. Established Breast Cancer Cell Lines: Similarly, in breast cancer cell lines of three subtypes (ER+PR+HER2-, ER+PR+HER2+, and ER-PR-HER2-) CD44+CD24- and CD44+CD24+ cells are equally tumorigeniic at the orthotopic site with very low numbers. In vitro, however, CD24- cells possess markers characteristic of epithelial-mesenchymal transition. CD24- cells have elevated levels of Vimentin and Slug, reduced levels of E-cadherin, and increased invasiveness relative to isogenic CD24+ cells. Strikingly, CD24- cells give rise to less invasive CD24+ progeny as effectively as CD24+ cells give rise to more invasive CD24- progeny. These observations demonstrate that acquisition of CD24 expression is not permanent and not all progeny of CD24+ cells express the marker. While functionally different in vitro, in vivo tumorigenicity is similar between CD24- and CD24+ populations. Additionally, the ability to recapitulate functional heterogeneity, considered a key hallmark of the cancer stem cell, is shared by both CD44+CD24+ and CD44+CD24- cells. In summary, our observations suggest that in the majority of clinical samples and established breast cancer cell lines, cells lacking CD44 are largely non-tumorigenic, but CD24+ cells and CD24- cells are equally tumorigenic. We have also observed that cells enriched for tumorsphere forming ability are not enriched for CIC. Efforts to identify novel extracellular markers capable of enriching CIC are currently underway.
