The Rodent Liver Undergoes Weaning-Induced Involution and Supports Breast Cancer Metastasis

Abstract

Patients with postpartum breast cancer are at increased risk for metastasis compared with age-matched nulliparous or pregnant patients. Here, we address whether circulating tumor cells have a metastatic advantage in the postpartum host and find the postlactation rodent liver preferentially supports metastasis. Upon weaning, we observed liver weight loss, hepatocyte apoptosis, extracellular matrix remodeling including deposition of collagen and tenascin-C, and myeloid cell influx, data consistent with weaning-induced liver involution and establishment of a prometastatic microenvironment. Using intracardiac and intraportal metastasis models, we observed increased liver metastasis in post-weaning BALB/c mice compared with nulliparous controls. Human relevance is suggested by a ∼3-fold increase in liver metastasis in patients with postpartum breast cancer (n = 564) and by liver-specific tropism (n = 117). In sum, our data reveal a previously unknown biology of the rodent liver, weaning-induced liver involution, which may provide insight into the increased liver metastasis and poor prognosis of women diagnosed with postpartum breast cancer.

Significance: We find that patients with postpartum breast cancer are at elevated risk for liver metastasis. We identify a previously unrecognized biology, namely weaning-induced liver involution, that establishes a prometastatic microenvironment, and which may account in part for the poor prognosis of patients with postpartum breast cancer. Cancer Discov; 7(2); 177-87. ©2016 AACR.This article is highlighted in the In This Issue feature, p. 115.

Conflict of interest statement

Conflict of Interest Statement: The authors have declared that no conflict of interest exists

©2016 American Association for Cancer Research.

Figures

Figure 1. Evidence for weaning induced liver involution

(A) Rat livers were harvested for biochemical and IHC analyses (red arrows) from nulliparous (N), early (P2–4), mid (P11–13), and late (P18–20) pregnancy, lactation day 10 (L), and post-weaning days 2–10 and 28 (Inv2-Inv10, R). (B) Liver weights from age-matched rats across the reproductive cycle; rats/grp: Nullip (N), n=25; P2–4, n=5; P11–13, n=4; P18–20 & L, n=10; Inv2, n=9; Inv4, n=8; Inv6 & Inv10, n=6; Inv8, n=7; R, n=14. (C) Representative Ki67 IHC (top left) and dual Ki67/Heppar-1 IHC (top right) images from P18–20 liver; Ki67+ hepatocytes (arrows); Ki67 hepatocytes (asterisk); Ki67 non-parenchymal cells (arrow-heads); scale bar=20 µm. Quantification of Ki67+ hepatocyte IHC by reproductive stage (bottom panel); n=4 rats/grp. (D) Heatmap of UHPLC-MS metabolomics by reproductive stage (top) and Z-scores of anabolic/reducing (bottom left) and catabolic/stress (bottom right) metabolites; n=4–6 rats/grp. (E) Partial least squares discriminate analysis (PLS-DA) of rat liver metabolomics data (see Table S1A). (F) Cleaved caspase-3 immunoblot (top; n=4 rats/grp) and densitometry (bottom). (G) Representative apoptotic hepatocyte detected by TUNEL (inset; scale bar=20 µm), and TUNEL quantification across the reproductive cycle; N, n=7; L & Inv6, n=5; Inv4, Inv10, & R, n=4. Graphs show mean with SEM. One-way ANOVA with Tukey multiple comparisons test. *=p-value<0.05, **=p-value<0.01, ***=p-value<0.001.

Figure 2. Extracellular matrix remodeling accompanies weaning-induced liver involution

(A) Absolute quantification of rat liver ECM proteins by QconCAT based MS-MS proteomics; n=4–6 rats/grp. (B) Partial least squares discriminate analysis (PLS-DA) of rat liver ECM proteomics data from N, L, Inv6, and R stages (See Fig. S2, Table S1B). (C) Box-and-whisker plots of TNC, collagen 1-α1, and collagen 4-α1 obtained from QconCAT proteomics in (A). (D) TNC expression across involution by immunoblot (top, and Fig. S3E), with densitometry normalized to GAPDH (bottom, and Fig. S3E); quantification is of 4 technical replicates, n=4–6 rats/grp. (E) Representative liver TNC IHC (brown stain) at L (upper left) and Inv8 (lower left) and IHC quantification across reproductive stage (upper right); scale bar=25 µm, n=4–6 rats/grp. (F) TNC fragment length measured in N, L, and Inv8 livers; n=5 rats/grp. (G) Representative H&E stained rat liver sections from N (left), Inv6 (middle), and R (right) stages; scale bar=150 µm. Graphs show mean with SEM. One-way ANOVA with Tukey multiple comparisons test. *=p-value<0.05, **=p-value<0.01.

Figure 3. Immune populations increase in the liver during weaning-induced involution

(A) IHC quantification of CD68 positivity (left), and representative CD68 IHC images (right); scale bar=40 µm, n=4–6 rats/grp. (B) Flow cytometric quantification of Balb/c mouse liver immune cell populations; CD45+ leukocytes (top left), CD11bloF4/80+Ly6CLy6G mature macrophages (top right), CD11bhiF4/80Ly6C+Ly6G monocytes (bottom left), and CD11bhiF4/80Ly6C+Ly6G+ neutrophils (bottom right); Nullip (N), n=19; L, Inv4, & Inv6, n=14; R, n=9; Inv2, n=7 mice/grp. (C) F4/80 IHC quantification (left), and representative F4/80 IHC images (right); n=5 mice/grp. (D) Ly6C IHC quantification (left), and representative Ly6C IHC images (right); n=5 mice/grp. (E) Ly6G IHC quantification (left), and representative Ly6G IHC images (right); scale bars for c-e=40 µm, n=5 mice/grp. Graphs show mean with SEM. One-way ANOVA with Tukey multiple comparisons test. *=p-value<0.05, **=p-value<0.01, ***=p-value<0.001, ****=p-value<0.0001.

Figure 4. Evidence for a pro-metastatic microenvironment in the postpartum liver

(A) Intracardiac metastasis model, Nullip (N), n=24; Inv2, n=25. (B) Percent mice with tumor cells in liver (p=0.03; Chi-squared, RR 1.6 [95% CI:0.9–9.6]); (C) Tumor Cell Latency. (D) Percent mice with tumor cells in lung (p=0.81), bone marrow (p=0.51), and brain (p=0.36), Chi-squared. (E) Representative H&E image of liver micro-metastasis and staining for Ki67, CD45, and Heppar-1/CK18; scale bars=25 µm. (F) Portal vein metastasis model, Nullip, n=18; Inv2, n=17; R, n=8. (G) Percent of mice with overt metastasis at study end (**p=0.001, RR 3.9 [95% CI: 1.3–11.6]; *p=0.03, RR 2.0 [1.08–3.66]; two-tailed Fisher’s exact). (H) Representative Ki67, CD45, and Heppar-1/CK18 staining on overt liver metastases (T) from Inv2 mice, dashed lines denote tumor border; scale bars=25 µm. (I) Frequency of liver metastasis in young breast cancer patients (≤45 years of age); N, n=185; PPBC<5, n=205; PPBC 5-<10, n=174 (p=0.038; multivariate logistic regression, OR=4.05 [95% CI:1.08–15.12]). (J) Subset analysis of site-specific metastases in women with metastatic disease (N, n=34; PPBC<10, n=83). Frequency of liver metastasis (p=0.04, one-sided Fisher’s Exact; p=0.058, two-sided Fisher’s Exact, OR: 4.12 [95% CI:0.90–18.94]), and lung (p=1.00), brain (p=1.00), & bone (p=0.11) metastasis by Fisher’s exact (see Table S6). (K) Model of the postpartum involuting liver pro-metastatic microenvironment.