Mammary glands exhibit molecular laterality and undergo left-right asymmetric ductal epithelial growth in MMTV-cNeu mice.

Significant left-right (L-R) differences in tumor incidence and disease outcome occur for cancers of paired organs, including the breasts; however, the basis for this laterality is unknown. Here, we show that despite their morphologic symmetry, left versus right mammary glands in wild-type mice have baseline differences in gene expression that are L-R independently regulated during pubertal development, including genes that regulate luminal progenitor cell renewal, luminal cell differentiation, mammary tumorigenesis, tamoxifen sensitivity and chemotherapeutic resistance. In MMTV-cNeu(Tg/Tg) mice, which model HER2/Neu-amplified breast cancer, baseline L-R differences in mammary gene expression are amplified, sustained or inverted in a gene-specific manner and the mammary ductal epithelium undergoes L-R asymmetric growth and patterning. Comparative genomic analysis of mouse L-R mammary gene expression profiles with gene expression profiles of human breast tumors revealed significant linkage between right-sided gene expression and decreased breast cancer patient survival. Collectively, these findings are the first to demonstrate that mammary glands are lateralized organs, and, moreover, that mammary glands have L-R differential susceptibility to HER2/Neu oncogene-mediated effects on ductal epithelial growth and differentiation. We propose that intrinsic molecular laterality may have a role in L-R asymmetric breast tumor incidence and, furthermore, that interplay between the L-R molecular landscape and oncogene activity may contribute to the differential disease progression and patient outcome that are associated with tumor situs.

INTRODUCTION

Cancers that initiate in paired organs and other bilaterally symmetric tissues exhibit an unusual feature, which is that tumors occur with non-equivalent incidence on the left versus right sides (1–5). The basis for this laterality has not been addressed at the cellular or molecular level, an oversight that may be significant because patient survival is reported to differ according to primary tumor situs (1–2). The side with elevated tumor incidence is organ-dependent and not necessarily the same side that is associated with poorer disease outcome. For breast cancer, the majority of occurrences are unilateral, with higher tumor incidence on the left (3). Left-side predominance also occurs in bilateral cases, in which more tumors develop first in the left breast or are larger than those on the right (3). Yet, despite the increased incidence and larger average tumor size of left-sided breast cancer, right-sided breast cancer may be associated with worse prognosis. Right-sided breast tumors are prone to earlier onset of bone metastasis and give rise to higher numbers of sites with metastatic involvement (6). This suggests that disease progression is related to the side of tumor formation, which could result in differential patient survival. Although studies directly addressing breast cancer patient survival relative to tumor laterality are limited and have generated contradictory findings (3), there is some indication that lower survival rates occur in patients with right-sided disease (7).

The left-sided excess of breast cancer and potential relationship between tumor laterality and patient prognosis suggests that mammary tissues harbor L-R differences that are relevant to oncogenesis. To address this we have used normal and neoplastic MMTV-cNeu mice to probe for L-R differences at the beginning and end of puberty--a period when the rapidly growing ductal epithelium (8) is vulnerable to genetic, hormonal, and other environmental perturbations that heighten risk for developing breast cancer later in life (9–11). Here we provide evidence that mouse mammary glands have baseline L-R differences in gene expression that are L-R discordantly altered by HER2/Neu and that are accompanied by asymmetric ductal epithelial growth and patterning. Furthermore, we used comparative genomic analysis to show that the L-R differences in gene expression that we identified in mouse mammary glands are predictive of breast cancer patient outcome, with right-side expression profiles associated with significantly poorer long-term patient survival.

RESULTS AND DISCUSSION

Thoracic mammary glands are molecularly L-R asymmetric

Ductal epithelial networks in thoracic mammary glands (TMGs) of early pubertal (4-week) and post-pubertal (10-week) wild type (WT) mice (Fig. 1A, B) were quantified by image and fractal analysis as described previously (12). Despite increases in network area and number of branch points between weeks 4 and 10, as well as changes in TEBs, which decrease in number and initiate regression by week 10 (13), all of these morphological parameters were statistically equivalent for left and right TMGs at both timepoints, indicative of L-R symmetry (Fig. 1C). By contrast, microarray analysis yielded approximately 161 transcripts that were L-R differentially expressed (i.e., up-regulated or down-regulated) with >1.2 fold change (q-value<0.05, Fig. 1D), including genes and pathways that have established roles in oncogenesis and/or therapeutic sensitivity (Table S1). Several of the transcripts identified in the array were examined by qRT-PCR (Fig. 1E), which confirmed that relative to left-side expression, some genes were increased and others were decreased in expression levels on the right side. For example, Gata-3 and FoxM1, which regulate luminal progenitor cell differentiation and renewal (14–15), and which also have opposing protective and causative roles in tumorigenesis in the breast and other organs (16–17), were more highly expressed on the left side (Fig. 1E). By the end of puberty, both genes were down-regulated; however, the fold decrease was significantly greater for left-side glands which resulted in net symmetric expression (Fig. 1E). Asymmetric expression also was found for notch-1, another regulator of mammary luminal progenitor cell commitment (18) that is involved in breast tumorigenesis (19) (Fig. 1E). Notch-1 was right-side elevated, and by 10-weeks it showed slightly higher fold decrease in right-side glands compared to left (Fig. 1E). To determine if asymmetric expression of genes with dual roles in ductal growth and tumorigenesis is a general property of TMGs, we examined estrogen receptor alpha (ERα). ERα was L-R equivalently expressed at both the start and end of puberty, consistent with it not being identified as a candidate by microarray (Fig. 1E). We also examined CD24, a pan-epithelial marker in mouse mammary glands (20), which showed modest left-side elevation in 4-week TMGs, but not in 10-week TMGs (Fig. 1E), raising the possibility that subtle differences in epithelial cell number could be present during early puberty, despite equivalent ductal network growth and morphology.

Figure 1

Morphological and molecular analysis of TMGs

Wild type mouse TMGs such as the representative L-R matched pairs shown at 4-weeks (A) and 10-weeks (B) were processed as carmine red stained whole mounts and composite images were manually traced, isolated, and converted to 8-bit gray scale images for analysis with MetaMorph® image analysis software (scale bar = 1mm). Thresholds and the total area of ductal networks were determined by the integrated morphometry analysis subroutine, and the fractal dimension, a measure of complexity of the network’s morphology, was determined using HarFa analytical software by applying the box counting method (12). Branch points and terminal end buds (TEBs), which are shown in higher magnification insets for 10-week glands (arrowheads indicate TEBs; scale bar = 5 μm), were quantified by manual counting. Color-coding can be used to follow matched L-R pairs harvested from the same mouse in all graphs. No significant L-R differences (C) were found in ductal network area, fractal dimension, relative density, branch points or TEBs at 4 or 10 weeks as determined by one-tailed paired student’s t-test. Microarray analysis of left versus right TMGs (D) using left as the baseline reference was performed using RNA pooled from 3–4 intact 4-week TMGs [#3 and #8 glands as diagrammed in Veltmaat et al (41)] for cDNA synthesis and hybridization to Affymetrix GeneChip Mouse Genome 430 2.0 Arrays. The arrays were preprocessed and normalized using RMA (42). Each array experiment was completed in biological and technical triplicate. Differentially expressed probesets were identified based on a fold-change (increase or decrease in right side compared to left) of at least 1.2, and a q-value of less than 0.05. Pathway analysis was carried out for each set of laterality associated genes (left or right) by probing the NCI Pathway interaction database (43). SYBR Green-based qRT-PCR of select array candidates was performed with primers listed in Table S2 (E). Real-time PCR miner was used to calculate Ct values and replication efficiency(44) and fold changes relative to GAPDH mRNA were determined by delta-delta Ct. Fold changes across groups were determined using the lower level of 4-week expression as baseline as indicated by the horizontal grey line. Bars represent mean ± SEM of ≥5 mice; *p<0.05, **p<0.01; *** p<0.001 (two-tailed paired student’s t-tests).

Genes involved in therapeutic sensitivity also were represented in the microarray. Elevated right-side expression was detected for retinoic acid-inducible G-protein coupled receptor 5D (GPRC5D), a gene that enhances sensitivity to an estrogen receptor antagonist, tamoxifen, in MCF7 breast cancer cells (21) and that was decreased by the end of puberty (Fig. 1E). In addition, stathmin-1 (Stmn-1), a microtubule destabilizing protein that confers chemoresistance in breast and other tumor types (22–26), was modestly left-side elevated in 4-week TMGs, followed by inversion to modest right-side elevated expression in 10-week TMGs (Fig. 1E). Given the many L-R differences in gene expression in TMGs, it was surprising that microarray analysis did not uncover connections to any known laterality genes (Table S1), including nodal and Pitx2, regulators of embryonic L-R patterning that also are expressed in breast cancer and other tumor types (4). Thus, we assessed these genes by qRT-PCR, which confirmed symmetric expression (Fig. 1E). Together, these findings demonstrate that despite symmetric nodal and Pitx2 expression, the left and right TMGs of WT mice are molecularly lateralized with asymmetric expression of other genes that may impart differential predisposition to oncogenesis.

HER2/Neu causes L-R asymmetric ductal growth and alters L-R gene expression in TMGs

To address the possibility that mammary ductal epithelium might be primed for differential growth during neoplasia, we quantified ductal networks in MMTV-cNeu mice, which are a commonly used model of HER2+ breast cancer (27). Compared to WT, the ductal network area was smaller in 4-week MMTV-cNeu TMGs and in particular, left-sided MMTV-cNeu networks were significantly smaller than their right-sided counterparts (Fig. 2A, C). Left-sided networks also contained fewer branch points, and had higher fractal dimension, relative density, and number of TEBs (Fig. 2A, C). Morphological asymmetry persisted through the end of pubertal development, with left-sided networks maintaining decreased area and higher numbers of branch points and TEBs (Fig. 2B, C). Given the L-R differences in MMTV-cNeu ductal network growth and pattern, we evaluated whether MMTV-cNeu TMGs have asymmetric ErbB2/Neu expression or activity. Although qRT-PCR showed that Neu expression was elevated in right-side TMGs, endogenous ErbB2 expression was L-R equivalent, as was Numb, a notch inhibitor whose expression is regulated by ErbB2 (28) (Fig. 2D). Moreover, phospho-ErbB2/Neu immunoprecipitation showed equivalent levels in left and right side TMGs, suggesting similar activation of ErbB2/Neu signaling on both sides (Fig. 2D).

Figure 2

Morphological and molecular analysis of MMTV-cNeu TMGs

TMGs from MMTV-cNeu mice such as the representative L-R matched pairs shown at 4-weeks (A) and 10-weeks (B) were processed for morphometric analysis and data for matched L-R pairs in individual mice were color coded as described in Fig. 1 and tested by Grubb’s Outlier test, which indicated an absence of outliers. Ductal network area, fractal dimension, relative density, branch points, and TEBs exhibited significant L-R differences at 4 weeks (C) as determined by one-tailed paired student’s t-test (*p<0.05; **p<0.01). Ductal network area, branch points, and TEBs remained significantly L-R different at 10 weeks (C). SYBR Green-based qRT-PCR showed asymmetric expression of Neu, but symmetric mRNA expression of ErbB2 and Numb (D). Bars represent mean ± SEM. N ≥ 5, *p = 0.003. Results were confirmed with a second primer set listed in Table S1. Total ErbB2/Neu protein was immunoprecipitated from left or right TMGs (Antibody #4290, Cell Signaling), immunoblotted, and probed with anti-phospho-ErbB2/Neu (Antibody #2243, Cell Signaling). Densitometry of triplicate results indicated no significant L-R differences (D). SYBR Green-based qRT-PCR analysis of gene expression in left vs. right TMGs of MMTV-cNeu mice was performed as described in Fig. 1E. Bars represent mean ± SEM of 5 mice, *p<0.05; **p<0.01; *** p<0.001 (two-tailed paired student’s t-tests).

Further analysis of MMTV-cNeu TMGs indicated that molecular laterality was amplified, sustained, or inverted in a gene-specific manner by comparison to WT. Notch-1 expression was approximately 3-fold higher in right-sided 4-week MMTV-cNeu TMGs (Fig. 2E), which is an amplification of the modest Notch-1 asymmetry that was present in WT TMGs (Fig. 1E and Fig. S1). In 10-week MMTV-cNeu TMGs, asymmetric Notch-1 expression was inverted, with approximately 2-fold higher expression in left-side glands (Fig. 2E). Because Notch influences breast cancer cell sensitivity to several therapeutic agents, including trastuzumab, gefitinib, docetaxel, and tamoxifen (29), the L-R uncoupled regulation of Notch-1 expression in MMTV-cNeu TMGs may be important in the context of differential disease progression. In addition, FoxM1 and Gata-3, which were left-side elevated in 4-week WT TMGs (Fig. 1E), were decreased on both sides in MMTV-cNeu TMGs; however, the fold decrease for FoxM1 was greater on the left side (Fig. S1) resulting in net L-R symmetric expression (Fig. 2E). Analysis of 10-week MMTV-cNeu TMGs showed that FoxM1 expression was further decreased, albeit the fold decrease was greater for right-side glands (Fig. 2E and Fig. S1). Given the additional role of FoxM1 in modulating endocrine and chemotherapeutic resistance in breast cancer cells (30–32), the L-R uncoupled regulation of FoxM1 expression in MMTV-cNeu TMGs was notable. We also found similar L-R asymmetric regulation of Gata-3 in MMTV-cNeu TMGs, which resulted in modestly higher left-sided expression by 10-weeks (Fig. 2E and Fig. S1).

Genes with symmetric expression in 4-week MMTV-cNeu TMGs included ERα, CD24, nodal, and Pitx2 (Fig. 2E). However, by 10 weeks their expression was elevated in left-sided glands, with the exception of nodal, which was elevated on both sides (Fig. 2E). GPRC5D, which was right-side elevated in 4-week WT TMGs (Fig. 1E), also was right-side elevated in 4-week MMTV-cNeu TMGs (Fig. 2E), despite an overall marked reduction in expression on both sides (Fig. S1). Stmn-1 was asymmetric in 4-week MMTV-cNeu TMGs (Fig. 2D, Fig. S1) but by week-10 was increased only on the left-side, resulting in inverted asymmetric expression (Fig. 2E).

IMGs are refractory to HER2/Neu-induced asymmetric growth and show delayed L-R asymmetric gene expression

Although mouse TMGs share more similarity with human mammary glands than inguinal mammary glands (IMGs) (3), IMGs are more commonly used in experimentation because of their larger size and easier accessibility (33–34). Therefore, we also examined IMGs. Like TMGs, IMGs showed no significant L-R differences in morphology at either 4-weeks or 10-weeks (Fig. 3A–C). Unlike TMGs, early pubertal IMGs showed an absence of significant molecular asymmetry except for Stmn-1, which was modestly right-side increased (Fig. 3D). However, by the end of puberty, 10-week IMGs had developed molecular L-R asymmetry similar to that observed in 4-week WT TMGs, with left-side elevated expression of FoxM1, Gata-3, Notch-1, ERα, and CD24 (Fig. 3D).

Figure 3

Morphological and molecular analysis of IMGs

L-R pairs of IMGs from 4-week (A) and 10-week (B) wild type and 4-week (E) and 10-week (F) old MMTV-cNeu mice were processed and analyzed as described in Fig. 1A–C. Ductal network area, fractal dimension, relative density, branch points, and TEBs did not exhibit significant L-R differences (C, G) as determined by one-tailed paired student’s t-test. SYBR Green-based qRT-PCR analysis of gene expression of left vs. right IMGs of wild type (D) and MMTV-cNeu (H) mice was performed as described in Fig. 1E. Pitx2 was not detectable in wild type IMGs at either age nor in MMTV-cNeu IMGs at 4 weeks. Bars represent mean ± SEM of ≥3 mice, *p = 0.01 (two-tailed paired student’s t-tests).

As previously reported (35), ductal networks in MMTV-cNeu IMGs were smaller compared to WT, and we found symmetric morphology at both 4 and 10-weeks (Fig. 3A–C). Although there were overall changes in gene expression relative to WT (Fig. S1), 4-week MMTV-cNeu IMGs did not exhibit molecular L-R asymmetry, with the exception of modest right-side elevation of Notch-1 and a more robust 3.5-fold left-side elevation of Pitx2 (Fig. 3H). Although Pitx2 was not associated with asymmetric ductal growth per se in either TMGs or IMGs, given that altered Pitx2 methylation occurs in breast and other cancer types (4) the overall changes in Pitx2 expression nevertheless suggest a potential role in HER2/Neu-induced neoplasia. By the end of puberty, MMTV-cNeu IMGs showed pronounced molecular asymmetry, as exemplified by right-side elevated FoxM1 and Gata-3 expression and left-side elevated ERα and Notch-1 (Fig. 3H). Thus by comparison to TMGs, both WT and MMTV-cNeu IMGs were temporally delayed in developing molecular asymmetry, which may account for their remaining refractory to HER2/Neu-induced asymmetric epithelial growth and morphogenesis.

TMG molecular laterality is associated with differential breast cancer patient survival

To determine if L-R differences detected in mouse mammary glands are clinically relevant, the genes identified in our microarray experiment (Fig. 1D) were evaluated in a large number of breast tumor gene expression data sets for which corresponding patient outcome is also known (n=1334). Of the 161 transcripts identified in the microarray, we were able to map 96 of them by Unigene ID to their human transcript counterpart for each patient.

Because the sidedness of tumor location was not available in the clinical annotation files, patients were assigned to left (n=642) or right-side (n=692) groups based on whether their tumor gene expression profiles more closely matched with the left or right profiles identified in the mouse microarray. Notably, right-side gene expression was linked to poorer patient survival (Fig. 4A). We next analyzed subsets of patients with HER2+ and HER2− tumors. Whereas the relationship between L-R gene expression and outcome fell just short of significance in the HER2 over-expressing subset (Fig. 4B), the relationship was significant in the HER2− subset (Fig. 4C). It should be noted that because HER2 status was not available in the clinical annotation files, we assigned patients to the HER2+ and HER2− subsets based on mean ERBB2 transcript levels. For this reason, and also because of statistical power limitations due to the HER2+ subset containing far fewer patients (n=276) than the HER2− subset (n=1058), the relationship between HER2+ patient survival and L-R gene expression may be unclear and require additional investigation with a larger HER2+ test cohort.

Figure 4

Comparative genomic analysis of mouse L-R mammary gene expression profiles with human breast tumors and the relationship to breast cancer patient survival

We compiled a large cohort of breast cancer patients from multiple studies available through the Gene Expression Omnibus (http://www.ncbi.nlm.nih.gov/geo/) to test the association between laterality associated genes and patient survival. Hazard ratios (HR) are indicated for all patients (A), and HER2+ (B), HER2− (C), ER+ (D), ER− (E) subsets. A 20-gene subset of the 96 L-R TMG gene expression set (F) is a robust predictor of outcome among all breast cancer patients (G). References are provided for genes previously implicated in oncogenesis; those with none available (N/A) are indicated. Our combined cohort comprised patients from the GSE2034 (45), GSE7390 (46), GSE4922(47), GSE25055 (48), and GSE3494 (49) cohorts (n=1334). For all patients, clinical outcome data as well as the gene expression profile of their respective tumors was available. Whenever possible we used 10-yr disease free survival as the clinical endpoint in our study; however, when disease free survival was not available we alternatively used either distant metastasis free or overall survival as the clinical endpoint. The arrays for each separate cohort were preprocessed and normalized using RMA(42). ER status was assigned based on the clinical annotation files, and HER2 status was assigned based on the mean ERBB2 transcript levels (probe set ID 216836_s_at) within each study cohort independently. Affymetrix GeneChip Mouse Genome 430 2.0 Arrays probe sets were mapped to their human counterpart genes by Unigene IDs. When multiple probe sets recognized the same gene transcripts, only the probe with the highest mean intensity was used. To assign signature scores to patients, the expression values for each gene were standardized such that the mean and standard deviation were set to 0 and 1 in each individual patient cohort, respectively. Subsequently, we calculated signature scores for each patient as previously described (50–51), where positive scores were considered to indicate that a tumor had ‘right-sided’ gene expression and negative scores were considered to indicate that a tumor had ‘left-sided’ gene expression. Survival curves were graphed using Graphpad Prism® 5 and statistical tests were completed in R.

Since ER status also is tightly linked to breast cancer patient outcome (36), we evaluated L-R gene expression patterns in ER+ and ER− patient subsets. In both subsets, right-side gene expression was associated with decreased survival (Fig. 4D, E). Lastly, we performed univariate Cox-regression survival analyses with each of the L-R transcripts, which identified a 20-gene subset that likely drove the predictive capacity of the complete 96-gene set (*p<0.05, Cox-regression) (Fig. 4F). Indeed, the evaluation of these 20 genes among the 1334 patient cohort outperformed the original 96 L-R gene set (Fig. 4G). Thus, the L-R gene expression profiles identified in mouse mammary glands are significantly linked to breast cancer patient survival rates, and demonstrate that right-sided gene expression is associated with poorer survival.

In summary, our results indicate that despite their morphological symmetry, mammary glands are molecularly lateralized. Although left and right glands express the same genes, the relative levels of gene expression significantly differ and are subject to L-R uncoupled regulation during pubertal development. Our results also demonstrate that many of the genes associated with the left side are down-regulated, yet remain elevated or amplified on the right-side in TMGs of MMTV-cNeu mice, consistent with more aggressive disease progression reported for right-sided breast tumors (6). Moreover, the L-R uncoupled gene expression is accompanied by asymmetric growth and morphogenesis of the ductal epithelium. The molecular laterality of mammary glands at the start of puberty appears to be important in potentiating HER2/Neu oncogene-mediated asymmetric growth since IMGs, which exhibit L-R differences in gene expression at the end of puberty, but not at the start, fail to undergo L-R asymmetric growth in MMTV-cNeu mice. From the perspective of modeling human breast development and cancer, these results confirm there are significant differences between thoracic and inguinal glands and provide the first evidence that each mammary pair is independently L-R regulated regardless of its anterior or posterior location. By analogy to anterior-posterior differences that underlie differential development and neoplastic susceptibility of mouse TMGs versus IMGs (3), as well as the processes that establish molecular L-R differences in other bilaterally symmetric tissues (37–39), we hypothesize that mammary laterality may be rooted in embryonic patterning. Therefore, future investigation to determine the connections between positional differences in gene expression, axial patterning, and the relationship to mammary development and tumorigenesis will be revealing. Furthermore, given the roles of ErbB2/HER2 in normal and neoplastic mammary development (40), as well as the significant link we found between L-R gene expression and breast cancer patient survival, our findings highlight laterality as a parameter that warrants greater consideration in experimental design in mouse mammary models as well as clinical analysis of breast cancer patients.

Table S1. L-R mammary microarray analysis

Transcripts identified in the L-R mouse TMG microarray are listed and the side with elevated expression is indicated (A). Results from pathway analysis of microarray data also are indicated (B).

Table S2. qRT-PCR primers

Primer sets used for qRT-PCR are listed. For experiments that were confirmed by alternate primer sets, the primers used to generate the results presented in the Figures are indicated (*).

Figure S1. qRT-PCR analysis of mammary gene expression in wild type versus MMTV-cNeu mice

SYBR Green-based qRT-PCR was used to assess gene expression in L-R paired 4-week TMGs (A), 10-week TMGs (B), 4-week IMGs (C), and 10-week IMGs (D). For 4-week TMGs, data were normalized relative to WT right-sided glands for all genes except Notch-1, ERα, and GPRC5D, which were normalized relative to WT left-sided glands. Comparison of fold differences between WT and MMTV-cNeu glands indicated that FoxM1, Gata-3, Notch-1, CD24, GPRC5D, and Stmn-1 were independently up or down-regulated in left versus right-sided glands. For 10-week TMGs, data were normalized relative to WT left-sided glands for all genes except FoxM1, Gata-3, Nodal, and Pitx2, which were normalized relative to WT right-sided glands. Comparison of fold differences between WT and MMTV-cNeu glands indicated that FoxM1, Gata-3, Notch-1, CD24, GPRC5D, and Pitx2 were independently up or down-regulated in left versus right-sided glands. For 4-week IMGs, data were normalized relative to WT right-sided glands, except for Notch-1, ERα, and Stmn-1, which were normalized to WT left-sided glands. Comparison of fold differences between WT and MMTV-cNeu glands indicated that all genes examined were similarly up or down-regulated in left versus right-sided glands except Notch-1, which was slightly more elevated in MMTV-cNeu right-sided glands compared to WT. SYBR Green-based qRT-PCR showed gene expression in 10-week left vs. right IMGs (D). For 10-week IMGs, data were normalized relative to WT right-sided glands for all genes. Comparison of fold differences between WT and MMTV-cNeu glands indicated that FoxM1, Gata-3, Notch-1, ERα, and CD24 were independently up or down-regulated in left versus right-sided glands. Bars represent mean ± SEM of ≥ 3 mice, *p<0.05; **p<0.01; *** p<0.001, two-tailed paired student’s t-tests.