Immortalized, premalignant epithelial cell populations contain long-lived, label-retaining cells that asymmetrically divide and retain their template DNA

Clonal populations of immortalized, premalignant cells were derived by using flow cytometry for side-population (SP) cells from COMMA-D1 murine mammary epithelial cells (Kittrell et al., unpublished data). COMMA-D1 murine mammary epithelial cells were originally isolated from the mammary glands of midpregnant Balb/C mice [17]. Derived clonal populations included "Non-Side Population 2" ("NSP2"), "Non-Side Population 3" ("NSP3"), and "Side Population 3" ("SP3"). The clonal population NSP2, interpreted to be a ductal progenitor cell, yields a ductal outgrowth that fills the mammary fat pad but does not differentiate in response to hormones (Kittrell et al., unpublished data). NSP3 cells have poor growth in vivo and, when successfully implanted, typically fill less than 10% of the mammary fat pad with a nest of surviving cells (Kittrell et al., unpublished data). Finally, the population SP3 cells typically fills 20% to 59% of the mammary fat pad on injection (Kittrell et al., unpublished data). Cells were grown in Dulbecco's Minimal Essential Growth Medium (Gibco, Carlsbad, CA) mixed with F12 Nutrient Medium (Gibco), supplemented with 2% fetal bovine serum (Gibco), 1% 1 M HEPES Buffer (Sigma, St. Louis, MO), 1% 100× antibiotic antimycotic (Gibco), 5 ng/ml EGF (per 500 ml; Sigma), and 10 μg/ml insulin (per 500 ml; Sigma) at 37°C with 5% CO₂.

Smith et al. [18‐20] previously described in detail the procedure used to clear the mammary epithelium from the inguinal fat pad of 3-week-old host mice, as well as the subsequent transplantation of cells. In brief, three mice per condition (six control (thoracic) glands; six inguinal fat pads) were anesthetized; both inguinal mammary fat pads, cleared; and a cell suspension, injected. Twenty thousand NSP2 cells, 50,000 NSP3 cells, or 50,000 SP3 cells suspended in phosphate-buffered saline were inoculated in 10-μl volumes by using a Hamilton (Reno, NV) syringe equipped with a 30-gauge needle. Control mammary glands (thoracic) remained intact.

Two weeks after the implant surgery (mouse age of 5 weeks), 1 mg (100-μl volume), 5-bromo-2-deoxyuridine (5-BrdU) was administered via intraperitoneal injection for 2 consecutive days per week for 5 weeks (Figure 1). The 5-BrdU injections were then stopped for 3 weeks (mouse age, 13 weeks), when some nulliparous mice were inoculated with [³H]-thymidine (³H-TdR) (50 μCi, 100 μl) and euthanized within 90 minutes (Figure 1). Remaining mice (mouse age, 12 to 13 weeks) were mated with male Balb/C mice. Female mice were subsequently inoculated with ³H-TdR (50 μCi, 100 μl) and euthanized within 90 minutes at either 4 to 6, 8 to 10, or 12 to 15 days after coitus (Figure 1). Mammary fat pads that were either intact (that is, control thoracic) or containing the implanted cells were collected, fixed in neutral buffered formalin, and prepared for autoradiography and immunohistochemistry. Small intestine from the same mice served as a non-mammary control for autoradiography.

After immunohistochemical processing, slides were transferred to distilled water and then coated with Kodak NTB-2 liquid emulsion (Carestream Health, Inc., Rochester, NY) diluted 1:1 with distilled water. After drying for 1 hour, slides were stored in lightproof slide boxes at 4°C for 7 days. After exposure, the slides were developed in Kodak D-19 (Carestream Health, Inc., Rochester, NY), washed in distilled water, and fixed in Kodak rapid fixer diluted 1:1 with distilled water. Slides were washed once more in distilled water and dehydrated through a series of graded ethanols and xylenes. Sections were mounted with Permount (Fisher Scientific, Pittsburgh, PA), and slides subsequently observed and evaluated for autoradiographic grains and for immunostaining.

Cells were plated in 25-mm² tissue flasks, cultured as described earlier, and allowed to incubate overnight. Cells were then treated with culture medium plus 0.5 μM 5-ethynyl-2-deoxyuridine (EdU) diluted in phosphate-buffered saline (PBS) for imaging by using the Click-iT EdU Imaging Kit (Invitrogen). Twenty-four hours later, the EdU solution was removed, cells were washed with PBS, and either the cells were immediately fixed at room temperature for 15 minutes with 4% paraformaldehyde, washed twice with Tris-buffered saline (TBS), and processed for EdU imaging, or the culture medium was replaced. Unfixed cells were then grown to confluence and serially passaged 5 times (NSP2 and NSP3 cells serially passaged at 1:3; SP3 cells serially passaged at 1:6). At the fifth passage, cells were washed with PBS and fixed with 4% paraformaldehyde at room temperature. Fifteen minutes later, the fixative was removed, and the cells were washed twice with TBS. For EdU imaging, cells were permeabilized for 20 minutes at room temperature with 0.2% Triton X-100 and subsequently washed twice with TBS. Freshly prepared Click-iT^® reaction cocktail (2 ml; Invitrogen, prepared as described by the manufacturer) per 25-mm² surface area was added to the cells, which were then incubated in the dark for 30 minutes. The reaction cocktail was subsequently removed, cells were washed twice with TBS, and nuclei were stained for 4 minutes at room temperature with diamno-2-phenylindole (1:1,000; DAPI; Invitrogen). DAPI was removed, cells washed twice with TBS, and flask bottoms were mounted by using Fluoromount-G (Southern Biotech, Birmingham, AL). EdU-positive cells were visualized by using fluorescence microscopy.

The mouse mammary gland is a dynamic organ that has been shown to regenerate itself from as little as one cell [14]. Consequently, it is likely that the mammary gland contains stem or progenitor cells allowing self-renewal to occur. Previously, it was shown that a population of cells exists within the epithelium of the mouse mammary gland that retains a ³H-TdR label, deemed label-retaining cells [9]. With this knowledge, we sought to identify and locate long-lived label-retaining mouse mammary epithelial cells in vivo as well as to determine the presence of long label retention in immortalized, premalignant cell populations maintained in epithelium-divested mammary fat pads. Three-week-old Balb/C female mice were anesthetized, and their inguinal mammary glands were cleared of host epithelium by using protocols described by Smith et al. [18‐20] (three mice per condition; six cleared mammary fat pads). Either 20,000 or 50,000 immortalized, premalignant cells (NSP2, 20,000 cells; NSP3 and SP3, 50,000 cells) were inoculated in 10-μl volumes. Control (thoracic) glands remained intact (three mice per condition; six intact glands). Subsequently, to identify label-retaining cells present in the mouse mammary gland, 5-BrdU was administered to the 5-week-old female mice during allometric mammary ductal growth for 2 consecutive days per week for 5 weeks. The 5-BrdU injections were then stopped for 3 weeks (mouse age, 13 weeks). Some of the nulliparous female mice were then inoculated with ³H-TdR to distinguish 5-BrdU-label-retaining cells that remain in the cell cycle, and subsequently euthanized. The remaining 13-week-old mice were mated, inoculated with ³H-TdR, and euthanized at either 4 to 6, 8 to 10, or 12 to 15 days after coitus (Figure 1). Glands were collected and prepared for autoradiography and immunohistochemistry. Sections were further stained for ER-α or PR. Mammary cells within the ducts and alveoli that retain both a 5-BrdU and ³H-TdR double-label are indicative of long-lived label-retaining cycling epithelial cells (LRECs).

Sections of mouse mammary fat pads implanted with immortalized, premalignant cells were examined for evidence of outgrowths containing label-retaining cells. Of the implanted fat pads, 12 of 16 NSP2, 0 of 18 NSP3, and 10 of 18 SP3 yielded outgrowths that filled between 10% and 50% of the fat pad. In both pregnant and nulliparous mice, outgrowths containing NSP2-implanted cells typically filled 10% to 40% of the fat pad, yielding ductal structures with blunted side branches. Outgrowths containing SP3 cells filled 20% to 50% of the fat pad with both ductal and lobular structures. Finally, of specific note, NSP3 cells implanted into the mammary fat pads of athymic nude mice yielded only focal areas of epithelial cells. These results suggest that the NSP3 cells had indeed survived, but did not produce outgrowths consisting of ducts or alveoli or both, consistent with those found with NSP2 or SP3 cell implants. These spherical areas of epithelial cell growth typically filled only 3% to 5% of the fat pad (thus, it was not recorded as an outgrowth). In the NSP2 and SP3 outgrowths, occasional areas of atypical epithelial hyperplasia (~0.1 to 1.5 mm in diameter), with nuclei that appeared to be normal, were apparent (Table 1).

Among the outgrowths and intact glands, cells labeled with 5-BrdU (long-lived label-retaining; template DNA strand) or ³H-TdR (cell cycling; newly synthesized DNA strand) were detected (Figure 2a-f: BrdU, green arrows; ³H-TdR, orange arrows). These single-labeled cells, which included 0.1% to 23.8% labeled with 5-BrdU and 0.4% to 14.3% labeled with ³H-TdR, were typically detected along the branching epithelium of mature mouse mammary glands in both pregnant and nulliparous mice (Table 2). Specifically, in the intact glands of mice during early pregnancy (4 to 6 days after coitus), we found that approximately 4.5% to 5.4% intralobular epithelial cells were labeled with ³H-TdR (cell cycling; newly synthesized DNA strand). At 8 to 10 days after coitus, the number of intralobular epithelial cells labeled with ³H-TdR decreased to 1.0% to 3.4% of the cells. By 12 to 15 days after coitus, alveolar differentiation was substantial, with 5.0% to 7.5% intralobular epithelial cells labeled with ³H-TdR. These results are consistent with those described by Traurig [21], who found that the highest rates of mammary epithelial cell proliferation occurred on day 4 after coitus and day 12 (after the onset of placental progestin secretion).

The frequency of epithelial cells labeled with ³H-TdR in fat pads implanted with NSP2 cells (ductal progenitor) was much less in pregnant mice than in nonpregnant (1.9% versus 14.3%, respectively; Table 2). These values were in contrast to fat pads implanted with the lobular progenitor cells NSP3 and SP3, in which the numbers of ³H-labeled cells increased in response to pregnancy. Cells containing double-labeled nuclei (both stained with 5-BrdU and containing autoradiographic grains (³H-TdR; LREC)) were found within all intact glands (Table 3) and were frequently located in mammary outgrowths containing implanted premalignant cells (Table 4). Representative images of double-labeled cells are shown in the insets of Figure 2a-f, which were similar for both nulliparous and pregnant mice whose glands were either intact or fat pads were implanted with immortalized premalignant cells.

Asymmetric division (Figure 3) would be particularly useful for stem and other long-lived cells, whereby a cell's archetype is retained for future generations. Being specifically interested in whether this phenomenon occurred within cells present in immortalized populations, we examined outgrowths from fat pads implanted with premalignant cells and labeled with both 5-BrdU and ³H-TdR. Asymmetric division with selective DNA segregation was rare, but occurred in the NSP2, NSP3, and SP3 cells that were implanted into cleared fat pads in both nulliparous (Figure 4a) and pregnant (Figure 4b) hosts. Furthermore, it was found that the number of immortalized, premalignant cells that contain a single label (either 5-BrdU or ³H-TdR) increased up to 108% after chase, providing further support that label-retaining cells within immortalized, premalignant populations undergo asymmetric division and pass the newly synthesized DNA strand to their daughters. A similar increase was observed in the intact control glands after the chase period.

It was next determined whether a percentage of epithelial cells present in immortalized, premalignant cell populations express estrogen receptor-α (ER-α) or progesterone receptor (PR). In the fat pads of nulliparous mice implanted with immortalized, premalignant cells, ER-α and PR expression was rare, being detected in 0 to 6% of immortalized cells counted per field of view (Figure 5a, c; Tables 5 and 6). In the intact glands of nulliparous mice, however, ER-α and PR expression was found in considerably larger cell numbers than in implanted fat pads: approximately 17% to 21% of cells counted per field of view (Supplemental figure S1 in Additional file 1; ER-α (a-c) and PR (d-f), red arrows) (Tables 5 and 6). These data demonstrate that although occasional cells express ER-α and PR, the number of immortalized, premalignant cells that express the steroid receptors is considerably smaller than the number found in normal mammary epithelium.

During pregnancy, ER-α was expressed in approximately 6% to 7.5% of the cells counted per field of view in fat pads with outgrowths of immortalized, premalignant cells (Figure 5b; Table 5). Conversely, PR expression in the immortalized, premalignant cells in pregnant mice was substantially less than ER-α expression: 0% to 1% of cells counted per field of view (Figure 5d; Table 6). In contrast, 5% to 7% of epithelial cells present within the intact glands of pregnant mice expressed ER-α and PR (Supplemental Figure 1) (Tables 5 and 6). Therefore, these data indicate that immortalized, premalignant cells implanted into the fat pads of pregnant mice that express PR are present in small numbers.

Cells positive for ER-α (Figure 5a and 5c; green arrows, inset) or PR (Figure 5b and 5d; green arrows, inset) and a ³H-TdR label were rare (<1%) both in outgrowths of immortalized, premalignant cells and in the intact gland (Tables 7 and 8). Nevertheless, these results suggest that some of the cells expressing the hormone receptors were also progressing through the cell cycle.

Although it is evident that label-retaining epithelial cells are present in the regenerating mammary gland, it may be possible that these cell populations are also present in other tissues. Booth et al. [10] described that label-retaining cells were found in several nonepithelial mammary tissues, including nerve, fatty stroma, and endothelial tissue. Consistent with that observation, label-retaining cells, in this study, were also located among cartilage (Supplemental figure S2a in Additional file 2), adipose tissue (Supplemental figure S2b in Additional file 2), skeletal muscle (Supplemental figure S2c in Additional file 2), and periductal cells (Supplemental figure S2d in Additional file 1). These results indicate that label-retaining cells are present in tissues in addition to the mammary epithelium.

To verify whether label-retaining cells were present in immortalized, premalignant cell populations in vitro, clones were cultured and treated with 0.5 μM EdU. EdU-labeled cell nuclei were found in all immortalized, premalignant cell populations in vitro at both passage 0 and passage 5. In passage 5 cell cultures, a twofold to 5.5-fold decrease was found in the number of EdU-label retaining cells present compared with passage 0 (Table 9; Figure 6). One cause for the decrease in number of label-retaining cells might be the result of dilution of noncycling cells during serial passage. At passage 0, approximately 20% of immortalized, premalignant cells are EdU positive. If dilution of noncycling cells were in play, then, after five passages, cells seeded at a 1:3 (NSP2, NSP3) or 1:6 dilution (SP3) would have one cell in 243 (3⁵) or one cell in 7,776 (6⁵), respectively, as a label-retaining cell. This was not the case, as five NSP2 cells of 34 cells (14.7%), seven NSP3 cells of 157 cells (4%), and 21 SP3 cells of 561 cells (3.8%) were positive for EdU after five serial passages (Table 9). These results show that the label-retaining, premalignant populations are selectively preserved throughout serial passage in vitro. These data imply that label-retaining, premalignant cells were not out of the cell cycle during passage, but nevertheless retained their original DNA label. In addition, label-retaining cells in the fifth passage were occasionally found juxtaposed to unlabeled cells suggesting selective label retention during mitosis (Figure 6).