Radiation Dose and Quality Induced Stem Cell Signature
We previously showed that the MaSC signature defined by Visvader and colleagues  is enriched in mammary mRNA expression profiles up to 1 month after exposure to 10 cGy γ-radiation . To assess the generality and durability of this response, we analyzed mammary mRNA expression profiles from mice exposed to different doses and radiation qualities. Radiation quality is important since densely ionizing is more carcinogenic than sparsely ionizing radiation . Tissues analyzed at 1 and 4 weeks, but not 12 weeks, following exposure to 100 cGy sparsely ionizing γ-rays or 10 or 30 cGy densely ionizing radiation were enriched for both a Notch-dependent signature  and the MaSC signature  compared to control profiles (Supporting Information Fig. 1). Ingenuity Pathway Analysis identified NOTCH1, CTNBB1, TGFB1, TGFB2, and WNT16 as upstream regulators (p < .05) for all radiation treatments at Week 1. At Week 4, only CTNNB1 and TGFB1 persisted. E-cadherin (CNTBB1) is highly regulated during EMT, while Notch and TGFβ are both implicated as signals in stem cell self-renewal. These data suggest that a durable but transient stem cell signature is present in irradiated mammary gland.
Figure 1. In silico mammary model and MASCa frequency predictions as a consequence of different radiation mechanisms. (A): Schematic of the mammary lineage components in the model. MASCa and PROGa can both self-renew. Differentiated EPITHa have two possible hormone receptor states (HR +/−). (B): An in silico gland consists of DUCTa, each of which is a lattice to contain MASCa (red), PROGa (yellow), and EPITHa (blue). Shown in the top inset is a growing DUCTa with MASCa and PROGa actively migrating to the tip and dividing to elongate the lattice. During symmetric division, MASCa create a nonmigratory daughter MASCa. After cell agents fill the DUCTa lattices, the DUCTa bifurcate to create two new elongating DUCTa. (C): At week 9, morphogenesis ceases with all migratory MASCa and PROGa differentiating to EPITHa. (D): The rate of MASCa symmetric division, freqSymDiv_MASCa was estimated to be 0.12 in order to match the 1/2,000 MASCa frequency we previously reported . The heat map shows MASCa frequency of a simulated sham-irradiated gland measured at week 18. (E–K): The effects on MASCa frequency after simulating different radiation mechanisms in a simulated “juvenile” mammary gland (during morphogenesis at week 3) were tested (n = 25). Simulations indicate that a transient increase in MASCa symmetric division or transient dedifferentiation, but not cell inactivation through death or cell senescence, could induce the radiation-induced twofold increase in MaSC frequency in irradiated “juvenile” mice previously reported. (L–R): Simulations of irradiated mature glands (after completion of morphogenesis at week 12) were performed to further discriminate between self-renewal and dedifferentiation. (Q): The model predicts MASCa frequency is not affected by transiently increasing self-renewal after morphogenesis is complete. (R): In contrast, dedifferentiation initiated at week 12 increased MASCa frequency 4.5-fold. ***, p < .001. Abbreviations: DUCTa, duct agents; ER, estrogen receptor; EPITHa, epithelial cell agent; MASCa, mammary stem cell agent; PROGa, progenitor agent.
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In Silico Mammary Epithelial Population Dynamics
Given the experimental challenges of testing whether radiation increases (a) cell inactivation (i.e., death) that then triggers proliferation; (b) signals for stem cell self-renewal, or (c) dedifferentiation, we turned to modeling to evaluate these potential mechanisms. ABMs previously developed for mammary acinar morphogenesis  were modified to simulate mammary lineage population dynamics during puberty. Mammary ABM consisted of interacting agents (computer programs) simulating mammary stem cells, progenitor cells, and differentiated cells (Fig. 1A). In this model, mammary stem cell agents (MASCa) can self-renew by symmetric division to gives rise to two MASCa or can asymmetrically divide to give rise to one MASCa and one progenitor agent, PROGa. In contrast to MASCa, PROGa can only self-renew for a limited number of times before becoming a fully differentiated epithelial cell agent (EPITHa) (Supporting Information Table S1). Fully differentiated EPITHa have limited proliferative potential. Although stem cells are thought to reside in the basal cell layer, our simplified model lacks this positional information. All cell agents reside within duct agents (DUCTa), each of which is a monolayer grid corresponding to a duct (Fig. 1B) with pre-ordained dimensions derived from measurements in mammary gland whole mounts.
During simulated puberty, MASCa and PROGa agents actively divide and migrate to fill the DUCTa lattice (Fig. 1B). MASCa agents undergoing symmetric division produce nonmigratory MASCa, resulting in the random distribution of MASCa, consistent with analysis of mammary epithelial composition using multiscale in situ sorting . Proliferation is initiated during puberty (i.e., week 3 in the mouse), and cell agents divide, filling DUCTa lattices. When a grid is full, bifurcation leads to two DUCTa, and the process is repeated, until simulated week 9, representative of an adult gland when morphogenesis is complete (Fig. 1C). We swept the range for each unknown parameter in the ABM to identify values that resulted in branch growth rates that matched analysis of in vivo mammary outgrowth. Identified and fixed parameters are shown in Supporting Information Table S1. A key parameter was the rate of MASCa symmetric division, freqSymDiv_MASCa, which was found to be 0.12 (Fig. 1D). This value was obtained by selecting simulations leading to a MASCa frequency of approximately 1/2,000 to match the frequency of mammary repopulating cells we previously reported using limiting dilution assay .
Irradiating mice during puberty (4 weeks of age) with either 10 or 100 cGy doubled the stem cell frequency measured in adult mammary gland . We simulated radiation-induced cell inactivation, self-renewal, or dedifferentiation in a “juvenile” mammary gland (i.e., has not completed morphogenesis) to determine their impact on MASCa frequency at simulated week 18. Radiation-induced cell senescence was modeled by randomly assigning agents to undergo permanent growth arrest; even extremely high values for random cell senescence (60% of cells) did not affect MASCa frequency (Fig. 1E). Radiation-induced cell death was simulated by randomly deleting agents. We also modeled selective cell death, where MASCa, PROGa, or EPITHa agents were preferentially deleted. High values (60%) of random and selective MASCa killing resulted in modest but significant decreases in MASCa frequency, 0.84-fold change and 0.76-fold change, respectively (Fig. 1F, 1G). In contrast, neither differential cell killing of PROGa or EPITHa resulted in a significant change in MASCa frequency (Fig. 1H, 1I). Thus, no mode of cell inactivation increased MASCa frequency.
As expression profiling showed that Notch signaling was present at 1 and 4 weeks after irradiation but resolved by 12 weeks, we simulated a transient increase in self-renewal. Increasing the rate of MASCa symmetric division in simulated juvenile glands from 0.12 to 0.16 for a duration equivalent to 4 weeks was sufficient to double MASCa at 18 weeks (Fig. 1J). Dedifferentiation was modeled by enabling a random fraction of EPITHa to become nonmigratory MASCa starting at the time of simulated radiation. TGFβ and E-cadherin, which are involved in EMT, were transiently invoked in expression profiles. Therefore, dedifferentiation was also transiently initiated for a duration equivalent to 4 weeks. A dedifferentiation rate of 0.0001% in simulated juvenile tissue led to an approximately two-fold increase in MASCa frequency (Fig. 1K). Thus, ABM simulations suggest that increased self-renewal or dedifferentiation, but not compensatory regeneration, are mechanisms that could increase stem cell frequency in irradiated juvenile mice.
Radiation therapy results in a much lower risk of breast cancer in adult women and the smaller risk is not associated preferentially with ER negative cancer . Therefore, we used the ABM to predict the effect of radiation on MASCa after morphogenesis was complete, comparable to an adult gland. Radiation time was set at simulated week 12, and MASCa frequencies were predicted for week 18. As in the juvenile setting, neither random cell senescence nor selective PROGa cell death had any effect on MASCa frequency (Fig. 2L, 2O). Random cell death and selective MASCa death led to greater decreases in MASCa frequency in simulated adult versus simulated juvenile gland (Fig. 2M, 2N). Differential EPITHa death in simulated irradiated adult glands led to a small (1.2-fold) but significant increase in MASCa frequency (Fig. 2P). In contrast to simulations of irradiated juveniles, transiently increasing self-renewal in adults did not affect MASCa frequency (Fig. 1Q), because significant proliferation is a necessary condition. On the other hand, dedifferentiation initiated at week 12 increased MASCa frequency 4.5-fold (Fig. 1R). Thus, modeling predicted that dedifferentiation could increase MaSC frequency in irradiated adult mice, but that self-renewal would not.
Figure 2. Lineage marker status of MCF10A treated with radiation and/or TGFβ. MCF10A outgrowths in humanized mouse fat pad consist of morphologically bilayered epithelium. (A): Whole mounts (left) and histology (middle, ×10; right, ×40). (B): CK14 (red) and CK18 (green) dual stain. (C): Human specific DNA, COT1, in situ hybridization of humanized fat pad. Note that both fibroblasts and epithelium are human. (D): Negative control for COT1 in situ hybridization in mouse mammary gland. (E): Dual immunostaining of basal CK14 (red, left panel) and luminal CK18 (green, middle panel) in MCF10A cells. Nuclei are counterstained with DAPI (blue). Rightmost panel is merged to show the presence of double CK14/18 positive cells. Scale bar = 50 µm. Cells in which both markers simultaneously colocalize appear yellow in the merged image. (F): Quantitation of basal CK14, luminal CK18, and double positive populations under four treatment conditions (sham, treatment with TGFβ, and/or 200 cGy γ-radiation). Frequencies are calculated as the total number of cells expressing each or both of the CK markers over the total number of cells in the population. Three biological replicates were randomly imaged for a total analysis of 6,935 cells. The frequency of cells expressing both CKs were significantly increased (p < .001, χ2) after treatment with TGFβ or radiation and TGFβ compared to the sham irradiated controls. ***, p < .001. (G): Quantitation (mean ± S.D.) of cells positive for both basal CK14 and luminal CK18 in MCF10A cultured with TGFβ as a function of graded doses of γ-radiation (blue) or Si particle radiation (red) compared to γ-irradiated cells cultured without TGFβ. Very low doses of either radiation type significantly increased the frequency of double positive cells. Abbreviations: CK, cytokeratin; DAPI, 4′,6-diamidino-2-phenylindole; IR, irradiation; TGFβ, transforming growth factor β.
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Based on the prediction of different outcomes depending on the mechanism of response, we irradiated 12-week-old adult mice with 10 or 100 cGy. Six weeks later, mammary epithelial cells were isolated and analyzed. Similar cell numbers were recovered from irradiated mouse mammary glands, as is expected given these low doses. The mammary stem cell enriched lin−/Cd24med/Cd49hi fraction was analyzed by flow cytometry . This population increases in cells isolated from mice irradiated as juveniles . However, the lin−/Cd24/Cd49 profiles of sham and irradiated adult mammary cells were comparable (Supporting Information Fig. 2A, 2B). Functional analysis of repopulating potential using limiting dilution into cleared mammary fat pads is the gold standard to assess mammary stem cells  and is increased in cells isolated from mice irradiated as juveniles . Consistent with the lineage analysis, mammary repopulating activity was similar between irradiated adult mice and contemporaneous sham-irradiated mice (Supporting Information Fig. 2C, 2D). Therefore, the prediction that dedifferentiation would increase mammary stem cells in adult mice is wrong, which suggests that increased self-renewal signaling is the most likely mechanism.
Mammary Epithelial Lineage Commitment
To test the prediction that self-renewal was more likely than dedifferentiation, we turned to the MCF10A nonmalignant human mammary epithelial cell line, which can be primed by radiation to undergo TGFβ-mediated EMT [26, 27]. MCF10A cell cultures consist of distinct CK18 and CK14 expressing cells and reportedly contain a small population of cells expressing stem cell markers . We found that MCF10A exhibit a remarkable capacity to generate histiotypic ductal outgrowths in a humanized mammary gland (Fig. 2A–2D). Ducts formed by MCF10A consist of luminal CK18 and basal CK14 bilayer (Fig. 2B), which were confirmed as human by in situ hybridization for COT1 DNA (Fig. 2C, 2D). The human breast epithelium consists of luminal, CK18 epithelial cells and basal, CK14 epithelial cells and a rare population in which basal and luminal CKs colocalize is thought to represent a pluripotent progenitor cell . Both single and double positive CK18 and CK14 cells are evident in MCF10A cultures (Fig. 2E). Thus, we used CK18 and CK14 CK expression in MCF10A as a model to further study radiation effects on epithelial lineage commitment.
EMT, elicited by overexpression of transcription factors or exposure to TGFβ, is strongly associated with acquisition of stem cell markers and function . Here we tested whether EMT primed by radiation affected the CK14/18 distribution of MCF10A cells. The distribution of CK14, CK18, and CK14/18 positive cells in cultures arising from irradiated cells was not significantly different from sham-irradiated populations (Fig. 2F). Significantly more CK14/18 cells and less CK18 cells were present in cultures exposed to TGFβ compared to control cultures or irradiated cultures. In contrast, nearly a third of the cells in irradiated cultures treated with TGFβ were CK14/18, which was significantly higher than in single treatment or control cultures (p < .001, Fig. 2F). The dose-response for EMT following either densely or sparsely ionizing radiation is switch-like, that is, doses as low as 3 cGy are sufficient and higher doses are quantitatively similar . Thus we examined the frequency of CK14/18 cells following graded doses of either sparsely or densely (350 MeV/amu Si) ionizing radiation. The frequency of CK14/18 cells exhibited a very similar dose-response (Fig. 2G), which was substantied by nonsignificant Pearson and linear regression coefficients. The correlation between EMT and CK14/18 cells seemed to support a dedifferentiation mechanism.
We reasoned that a dedifferentiation mechanism would mean that CK14/18 cells are the progeny of lineage restricted, single keratin positive cells, while increased self-renewal would increase symmetric division of double positive cells to produce similar double-positive daughters. To test this, we analyzed the lineage characteristics as a function of time in culture. The percentage of CK14/18 dual-labeled cells was unexpectedly greatest at 24 hours after plating in control and irradiated cultures and gradually decreased over time while CK14 cells increased (Fig. 3). TGFβ-treated cultures began with and maintained more double-positive cells than those cultured without TGFβ. As above, irradiated, TGFβ-treated cultures contained the highest percentage of CK14/18 positive cells. To test whether symmetric division of pre-existing progenitors could account for the population distribution over time, we formulated an ABM consisting of bipotent cell agents (BPa), representing CK14/18 positive cells, defined by their ability to undergo symmetric self-renewal or asymmetric lineage commitment to either a basal (BCa) or luminal (LCa) cell agent type (Fig. 4A). Monolayer culture lag and log phase were simulated by assigning each agent with one cell cycle time for the first three simulated days a second cell cycle time for the remainder of the week-long experiments. Contact inhibition was included such that a cell agent could divide only if there was neighboring space. Dedifferentiation of BCa or LCa to BPa was also included (Fig. 4A). A parameter sweep determined that turning off dedifferentiation led to the best fit of the simulated treatment conditions (Fig. 4B, 4C), suggesting that this mechanism is the least likely to explain the in vitro results. Moreover, the best-fit simulation results using dedifferentiation are noticeably worse than the fit using increased symmetric division (Supporting Information Fig. S3A–S3C). The fitted parameters indicated that the probability of symmetric self-renewal increased in TGFβ-treated cells and the greatest difference was observed between controls and irradiated, TGFβ-treated cells (Fig. 4B). As the in vivo ABM predicted that self-renewal would only be effective during morphogenesis when there is considerable proliferation, MCF10A were grown to confluence (7 days) to reduce proliferation, then irradiated and cultured for additional 5 days with and without TGFβ (Supporting Information Fig. S4A). Under these conditions of low proliferation, neither radiation nor treatment with TGFβ affected the proportion of CK14/18 cells compared to control cultures (p = .323, Supporting Information Fig. S4B).
Figure 3. The distribution of MCF10A CK subpopulations changes after radiation and/or TGFβ exposure. To evaluate lineage commitment, control or 200 cGy γ-irradiated MCF10A cells were cultured with TGFβ and fixed at the indicated time prior to immunostaining for CK18 and CK14 lineage markers. Ten random images were acquired from each biological triplicate at 24, 48, 72, 120 and 168 hours after radiation and/or TGFβ treatment. Assignment of CK status is represented as mean ± SE at each time point. Abbreviations: CK, cytokeratin; IR, irradiation; TGFβ, transforming growth factor β.
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Figure 4. Comparison of in vitro lineage distribution with in silico modeling after radiation and/or TGFβ exposure. (A): The in silico model consists of BPa, LCa, and BCa cell agents. (B): The best fit parameters for the model. The parameter α is the probability for BPa symmetric division. The parameter γ dictates the probability for asymmetric luminal division. The parameters δL and δB determine the probability for dedifferentiation of LCa or BCa to BPa, respectively. Tc1 and Tc2 are cell agent cycle times for the first 3 days and the remaining days in culture, respectively. The best fit parameters indicate that both TGFβ treatment alone and radiation with TGFβ treatment increases self-renewal, but the greatest difference in the probability of symmetric self-renewal is observed between sham and cells that were irradiated and TGFβ treated. (C): Experimental data points indicating the number of cells (mean ± SE) counted for a set surface area randomly scanned (n = 3). Red stars, CK14+ cells; green squares, CK18+ cells; yellow diamonds, double positive cells. Shaded areas indicate simulation results (red, BCa; green, LCa; yellow, BPa; mean ± SD, n = 50) using the model with the best fit parameters yielding the lowest squared residual error for all three cell type growth kinetics and for all four experimental conditions. Insets show example simulations at 24-hour, 96-hour, and 168-hour time points (red, BCa; green, LCa; yellow, BPa) along with an in vitro image at 168-hour for each treatment (CK14, red; CK18, green; nuclear counter stain, DAPI blue). Abbreviations: BPa, bipotent cell agent; BCa, basal cell agent; IR, irradiation; LCa, luminal cell agent; TGFβ, transforming growth factor β.
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To further test whether TGFβ is a necessary signal for self-renewal in vivo, Tgfb1 heterozygote and wild-type littermates were irradiated with 10 cGy at 4 weeks of age. Although null genotypes are embryonic or perinatal lethal, Tgfb1 heterozygote mammary gland has 70%–90% less TGFβ than wild-type tissue . As previously reported , mammary repopulation frequency doubled in the mammary gland of irradiated wild-type mice. In contrast, the mammary repopulating activity of irradiated Tgfb1 heterozygote mice was similar to unirradiated mice (Supporting Information Fig. S5). These data provide functional validation of the requirement for radiation-induced TGFβ in vivo. Together, ABMs of in vivo and in vitro mammary cell fate decisions and experimental data support the hypothesis that radiation stimulates self-renewal, which requires both TGFβ and active proliferation.
Radiation-Induced Notch and TGFβ Promote Self-Renewal
As a further test of this hypothesis, MCF10A were stably transduced with the retrovirus Let7c-reporter. Stem cells downregulate Let-7 miRNA . Ibarra et al. constructed a reporter in which Let-7c miRNA silences Ds-Red expression in differentiated cells. Therefore, the lack of Let-7c in stem/progenitor cells leads to Ds-Red cells  (Fig. 5A). As evident in dual phase-fluorescence micrographs, monolayer cultures contain few Ds-Red cells (Fig. 5B) and mammospheres grown in non-adherent, growth restricted conditions contained only one or two Ds-Red cells (Fig. 5C). To test whether Ds-Red positive progenitor cells underwent self-renewal, we viably sorted the population using flow cytometry into Ds-Red positive and negative populations and cultured them independently. The Ds-Red positive sorted population gave rise to cultures containing both Ds-Red positive cells and negative cells but the Ds-Red negative sorted population did not yield Ds-Red cells. The percentage of Ds-Red positive cells in cultures initiated from sorted Ds-Red-cell increased from 1.7% ± 0.08% to 3.7% ± 0.18%, indicative of self-renewal.
Figure 5. Radiation and TGFβ increase the frequency of cells expressing the Let-7 reporter in a Notch-dependent fashion. (A): Diagram of the assay in which Let-7c expression represses Ds-Red reporter in differentiated cells. Cells lacking Let-7c are red. (B): Phase/fluorescence merged images of MCF10A expressing the Let-7c reporter after radiation and/or TGFβ treatment. Scale bar = 100 µm. (C): Representative images of MCF10A mammospheres expressing the Let-7c reporter after radiation and/or TGFβ treatment. Mammospheres treated with GSI contained DS-Red cells. (D): Fluorescence-activated cell sorting analysis of Ds-Red-cell frequency of cells from sham, after radiation and/or TGFβ treatment (gray) compared to those treated with GSI (black). Ds-Red cells increased in cultures treated with TGFβ or radiation and TGFβ compared to the sham controls (mean ± SE from three biological replicates), which was blocked by Notch inhibition. Ds-Red positive sorted population continued to give rise to both Ds-Red positive cells and negative cells, whereas the Ds-Red negative cells did not yield Ds-Red progenitor cells under any condition. (E): Representative images of Notch and β-catenin immunostaining illustrating the nuclear localization of Notch induced by 200 cGy γ-radiation and TGFβ. Scale bars = 50 µm. (F): The frequency of cells that exhibit nuclear Notch staining is significantly increased by either single or double treatments. Incubation (4-hour) with GSI exhibit Notch immunoreactivity, evidence of antibody specificity. *, p < .05; **, p < .01; ***, p < .001; χ2. Abbreviations: DMSO, dimethylsulfoxide, vehicle control; DAPI, 4′,6-diamidino-2-phenylindole; GSI, gamma secretase inhibitor; IR, irradiation; TGFβ, transforming growth factor β.
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Radiation alone did not affect the frequency of Ds-Red cells as measured by flow cytometry (Fig. 5D). In contrast, TGFβ treatment significantly increased the Ds-Red population (p < .01), which was further enhanced by irradiation (p < .001). When Ds-Red cells were sorted from irradiated cultures treated with TGFβ, 39.6% ± 0.48% of the subsequent population was Ds-Red, consistent with increased self-renewal. In contrast, sorting Ds-Red negative cells from sham, TGFβ, radiation or irradiated, TGFβ treated cultures generated cultures without Ds-Red positive cells, which support the conclusion that dedifferentiation does not contribute to the increase in Ds-Red cells that we observed after exposure to radiation and TGFβ. We then modeled the self-renewal capacity of Ds-Red positive progenitor cells and found that, in accord with the results for the CK14/18 double positive population, the probability of symmetric divisions of these cells increased after radiation and TGFβ exposure (0.68 in sham cultures; 0.99 in cultures treated with radiation and TGFβ).
Stem cells, radiation, and EMT can be linked through their common association with the Notch pathway . Notch activation precedes increased mammary repopulating activity in irradiated tissue based on expression profiling, expression of Notch target genes, and protein localization . We localized nuclear Notch in MCF10A cells in sham and double treated cultures (Fig. 5E). As shown by quantitation (Fig. 5F), all treatments significantly increased cells exhibiting nuclear Notch, even though only double treatments induce EMT or CK14/18 dual labeled cells. We next tested whether this was functionally required for the increase in Let-7c reporter cells by using a GSI, which prevented the nuclear localization of Notch, indicative of activation (Fig. 5F). GSI blocked the increase in the frequency of Ds-Red cells in TGFβ treated cultures (Fig. 5D), but did not affect the presence of Ds-Red cells in mammospheres (Fig. 5C), which indicates that concurrent Notch and TGFβ induced by radiation co-operate to stimulate self-renewal reported by Let-7c.
Radiation Does Not Affect Tumor ER Status After Ductal Morphogenesis Is Complete
Our prior studies showed that the expansion of stem cells in mice irradiated during puberty correlates with development of aggressive, hormone receptor negative tumors arising in the radiation chimera model . The current modeling and experiments shows that proliferation is necessary for stem cell expansion, thus irradiation of adult mice does not increase stem cells. If so, irradiation after cessation of ductal morphogenesis should not shift Trp53 tumor type in the mammary chimera model. To test this, we compared tumors arising in mice that were transplanted with Trp53 null mammary tissue and irradiated after morphogenesis was complete to those occurring in mice that were irradiated and subsequently transplanted with Trp53 null mammary tissue (i.e., ductal morphogenesis comparable to puberty ensues after irradiation). In both cases, the mice were 10 weeks old when exposed to sparsely or densely ionizing radiation and Trp53 null tumors arose 9–15 months later. Significantly more ER negative tumors arose in mice irradiated prior to Trp53 null transplantation compared to sham-irradiated mice (Fig. 6), as shown previously . In contrast, the proportion of ER negative tumors was unaffected in mice that were irradiated after Trp53 ductal morphogenesis was complete. Thus, the increased frequency of ER-negative cancer correlates with increased mammary stem cells as a function of radiation exposure in young animals, while neither occurs when adult mice are irradiated.
Figure 6. Radiation exposure before completion of morphogenesis increases ER-negative tumors while irradiation after morphogenesis does not affect ER status. Tumors that arose from Trp53 null fragments undergoing morphogenesis before or after irradiation were scored for ER status using the Allred scoring system. (A): As previously reported, ER-negative tumors were significantly increased (χ2, p = .03, 95% CI) when morphogenesis occurred after exposure to either sparsely ionizing (36%, n = 25) or densely ionizing (42%, n = 64) radiation compared to sham (63%, n = 51). (B): In contrast, exposure to either sparsely or densely ionizing radiation after morphogenesis was complete did not affect the ER status of subsequent tumors (χ2 p [mt] .05, 95% CI). *, p < .05. Abbreviations: ER, estrogen receptor; IR, irradiation.
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