PMC

Characterization of MaSC-containing and luminal progenitor containing compartments. Mammary epithelial cells isolated as in Figure 4A, were incubated with PE-conjugated anti CD24 Ab, or APC-conjugated anti CD29 Ab and analyzed as described in Methods. Gated: upper left (FVB) and upper second from left (RARα1/KO) for CD24^high/CD29^low (luminal progenitors, upper left square) and for CD24^low/CD29^high (MaSC, lower right square). The third and fourth upper squares-negative controls, cells incubated with conjugated IgGs. The results shown are of one experiment, which was repeated once.

Mammary gland transplantation. Fragments of epithelium containing mammary glands of eight to ten weeks old virgin mice (wt or RARα1/KO) were transplanted into number 4 glands pre-cleared of epithelium of three weeks old wild type or RARα1/KO animals (see Materials and methods). At week 11 (eight weeks after transplantation) the transplant-recipient glands and intact glands number 3 from the same mouse, as control, were processed for whole mounts. >90% of wt-FVB transplants repopulated the recipient gland while <50% of the FVB-RARα1/KO did (see Table 1).

Characterization of the stem/progenitor cell compartment of wt-FVB and RARα1/KO. (a) Representative histograms of flow cytometry analysis of CD24^low/ALDH^high compartment in mammary epithelium. Primary mammary epithelial cells (approximately1 × 10⁶) were incubated with ALDH substrate with inhibitor (first and third from left upper and lower panels) or without inhibitor (second and fourth from left upper and lower panels) followed by incubation with anti-CD24 Ab and detected with secondary Ab (APC-conjugated) as described in Materials and methods. Inclusion of the DEAB inhibitor reduced the ALDH activity by 82 and 87%, in FVB and RARα1/KO cells, respectively. Gated: CD24^low/ALDH^high (b) Summary of seven individual experiments. Experiments were performed as in A, shown are median, 3.9% and 6.7% respectively, (mean 4.4% and 6.6%, respectively, P = 0.04, unpaired t-test).

The effect of RARα1/KO on neonatal and pubertal gland development. (a) Effect on the size of the neonatal glands. Number 4 glands were dissected from five to six days old wt and RARα1/KO C57Bl/6 or FVB, four mice per group, and whole mounts were prepared as described in Materials and methods. The area taken up by the mammary tree was analyzed using the Image J software. Bars are mean and SD of four glands per group. (b-e) Effect on pubertal mammary tree branching morphogenesis and terminal end buds. Number 4 mammary glands from seven to eight weeks old wt or RARα1/KO C57Bl/6 (b-d) and FVB pubertal mice (e) were dissected and processed for whole mounts and paraffin sections. Branching points were counted along the entire length of the three longest ducts. Bars show mean and SD of seven pairs (14 total) of number 4 glands per group (P = 0.0001, t-test). The same glands were used to count the peripheral terminal end buds (TEBs) (P = 0.00001, t-test).

Wnt1-tumorigenesis and wt and RARα1/KO transplanted tumor growth. (a) Representative histograms of flow cytometry analysis of CD24^low/ALDH^high cells in FVB-wnt1 and FVB-wnt1-RARα1/KO- mammary glands. Primary mammary cells were isolated from glands of seven-week-old mice and processed for flow cytometry analysis as described in Figure 3. (b) Summary of flow cytometry experiments. The experiments were carried out as in A; each dot represents individually processed sample. Statistical analysis, two way ANOVA, P = 0.0025. (c) Tumor-free survival (Kaplan-Meier curve). Forty female mice transgenic for Wnt1 and wt-RAR and 42 RARα/KO were allowed to age and were examined at regular intervals for the appearance of a palpable tumor nodule. The difference in disease-free survival (percent of tumor-free mice), plotted as a function of post-natal age (Kaplan-Meier curve) was statistically significant (P = 0.0002). (d) Transplanted MMTV-RARα1/KO-wnt1 tumor fragments have slower growth rate than MMTV-wnt1 tumors. Fragments of randomly chosen pairs of MMTV-wnt1/wt and MMTV-wnt1/RARα1/KO tumors were transplanted into the opposite flanks of wt-FVB hosts. The tumors were measured every three days. The bars show mean and SE (n = 12 per group, P = 0.01. two-way ANOVA test). (e) Inoculation of MMTV-wnt1/RARα1/KO cell suspensions produces tumors after longer latency. Cell suspensions (10⁵ and 10⁴) prepared from a pair of similar size MMTV-wnt1/RARα1/KO and MMTV-wnt1 tumors were inoculated into eight to ten FVB-mice, (as in D) and appearance of palpable tumors was recorded and plotted as fraction of tumor-free mice vs. days post-inoculation (Kaplan-Meier).

Growth, composition and regulation of primary mammospheres. (a) Comparison of mammosphere growth. Primary cells obtained from wt and RARα1/KO mammary glands, were inoculated at 2.5 to 5 × 10⁴ under conditions of ultra-low adhesion and serum-free medium and eight days later, the mammospheres were disassociated and the cells counted (see Materials and methods). The bars show mean (4,750 and 8,833 cells respectively, and SD of three individual experiments and three to four samples per group; P = 0.04 by unpaired t-test. (b) Bi-potential cells in mammospheres. Primary mammary epithelial cells were incubated for eight days until most single cells died; mammospheres were dissociated, inoculated into 96 wells, at 1cell/well (see Materials and methods) and when colonies formed stained for CK18 and CK14. (Red - CK18; green - CK14; blue - DAPI). Total number of colonies stained = 21. Scale bar = 10 um. (c) Regulation of mammosphere growth by retinoids. Cells isolated from mammary glands of seven- to eight-week old FVB mice were inoculated under conditions of mammosphere formation and eight to ten days later were treated with 10 nM of Am580, 200 nM of atRA, and/or 10-fold excess (100 nm and 2 μM, respectively) of RARα antagonist, Ro41-5253 for seven to eleven days. Each symbol represents an individual well; there were two to four wells per experiment, and a total of four experiments. Kruskal-Wallis test P = 0.0002; *indicates significance at P < 0.05 by Dunn's Multiple Comparison Test. (d) RARγ1 expression. RNA was extracted from freshly isolated and partially purified mammary epithelial cells and wt and RARα1/KO, and subjected to Q-PCR analysis as described in Materials and methods. The bars show mean of three experiments, two samples in each.