Patient-derived luminal breast cancer xenografts retain hormone receptor heterogeneity and help define unique estrogen-dependent gene signatures

Abstract

Bypassing estrogen receptor (ER) signaling during development of endocrine resistance remains the most common cause of disease progression and mortality in breast cancer patients. To date, the majority of molecular research on ER action in breast cancer has occurred in cell line models derived from late stage disease. Here we describe patient-derived ER+ luminal breast tumor models for the study of intratumoral hormone and receptor action. Human breast tumor samples obtained from patients post surgery were immediately transplanted into NOD/SCID or NOD/SCID/ILIIrg(-/-) mice under estrogen supplementation. Five transplantable patient-derived ER+ breast cancer xenografts were established, derived from both primary and metastatic cases. These were assessed for estrogen dependency, steroid receptor expression, cancer stem cell content, and endocrine therapy response. Gene expression patterns were determined in select tumors ±estrogen and ±endocrine therapy. Xenografts morphologically resembled the patient tumors of origin, and expressed similar levels of ER (5-99 %), and progesterone and androgen receptors, over multiple passages. Four of the tumor xenografts were estrogen dependent, and tamoxifen or estrogen withdrawal (EWD) treatment abrogated estrogen-dependent growth and/or tumor morphology. Analysis of the ER transcriptome in select tumors revealed notable differences in ER mechanism of action, and downstream activated signaling networks, in addition to identifying a small set of common estrogen-regulated genes. Treatment of a naïve tumor with tamoxifen or EWD showed similar phenotypic responses, but relatively few similarities in estrogen-dependent transcription, and affected signaling pathways. Several core estrogen centric genes were shared with traditional cell line models. However, novel tumor-specific estrogen-regulated potential target genes, such as cancer/testis antigen 45, were uncovered. These results evoke the importance of mapping both conserved and tumor-unique ER programs in breast cancers. Furthermore, they underscore the importance of primary xenografts for improved understanding of ER+ breast cancer heterogeneity and development of personalized therapies.

Fig 1

Retention of ER and PR expression in patient-derived luminal breast cancer xenografts over multiple passages. Patient-derived xenografts were established in NOD/SCID or NOD/SCID/ILIIrg^−/− mice, and subsequently propagated via direct transplantation of solid tumor pieces into new recipient mice. Tumors were grown under continuous estrogen supplementation. Sections of five xenograft tumor lines were stained by IHC for ER (left panels) and PR (right panels) at passage 1, and at subsequent passages 2–7 as indicated. The intensity (1–3) and percent of ER+ and PR+ cells are indicated on each panel. Scale bars, 100 µM

Fig 2

Growth kinetics of ER+ breast cancer xenografts in the absence or presence of estrogens. Tumors were transplanted into recipient mice either in the absence of exogenously added hormones (placebo), or in the presence of estrogen (17β-estradiol, E). a Tumor volumes were measured weekly (once attainable) and plotted versus the number of days of incubation ±SEM. Final tumor mass at necropsy ±SEM was plotted for each hormone treatment. Sample numbers (tumors): PE4 placebo (6), E (8); AS9 placebo (2), E (5); PT12 placebo (6), E (8); PT15, placebo (4), E (10); PT16 placebo (2), E (4). Tumor volumes at each time point, and final tumor masses, were compared using Student’ s t test. For tumor volume, *p < 0.05 E compared to placebo. For final tumor mass, *p < 0.05. b Sections from tumors grown in mice containing placebo versus estrogen (E) pellets were stained by IHC for ER. The staining intensity and percent of ER positive cells are indicated on each panel. Scale bars, 100 µM

Fig 3

Endocrine therapy response of previously untreated primary tumor xenografts. a Tumors PT12 and PT15 were grown under estrogen supplementation (E) until they reached 300 mm³ (40–50 days), then treated for 3 weeks with tamoxifen, 0.6 mg/kg IP 3×/ week (E + Tam), or removal of the estrogen pellet (PT12, estrogen withdrawal, EWD). Arrows indicate the start of tamoxifen injections and EWD. Tumor volumes are plotted versus number of days of incubation. Final tumor masses are shown on the right panel. *p < 0.05 PT15 tumor volume and tumor mass (E + Tam compared with E). b Sections of PT12 and PT15 tumors from untreated (E), tamoxifen treated (E + Tam), or EWD animals (PT12 only) were stained by H&E or IHC for ER. For PT12 H&E, the percent of tumor cellularity is indicated; the intensity and percent of ER staining is indicated for PT12 and PT15. Scale bars, 100 µM

Fig 4

Relative abundance of cancer stem cell markers in ER+ tumor xenografts. a Fresh pieces of tumors (all from estrogen-treated animals except for PT18, no treatment) were processed into single cell suspensions, and stained with a cocktail of antibodies to exclude murine host cells, and antibodies to CD44 (AlexaFLuor⁶⁴⁷) and CD24 (AlexaFLuor⁴⁸⁸). Dead cells were excluded by PI. Unstained cells from each tumor were used to set gates. The percent of cells with the cancer stem cell signature CD449+CD24(−/low) for each tumor is indicated in the upper left quadrant. b Sections of tumors were stained by IHC for CK5. The percent of cells stained is indicated on each panel. Scale bars, 100 µM

Fig 5

Estrogen-regulated genes and signaling networks in luminal tumor xenografts PE4 and PT12. The gene expression profiles of tumors PE4 and PT12 grown without (placebo) or with continuous estrogen (E) were determined using Affymetrix human gene 1.1 ST arrays. An n of 3 tumors were profiled for all groups except for PT12 E, n of 4. A list of estrogen-regulated genes in MCF7 cells was assembled by performing a union of significantly changed genes in data sets GSE848 or GSE3834 (supplementary methods). a Venn diagrams depicting E up- and down-regulated genes in xenografts PE4, PT12, and MCF7 cells (p < 0.05, > 1.5-fold). b The top 20 E up-and down-regulated genes in PE4 or PT12 are depicted by hierarchical cluster. Genes that also changed in MCF7 cells in the same direction are indicated by underlines/asterisks. c Common E up- (36) and down- (35) regulated genes in PE4 and PT12 graphed by hierarchical cluster. Underlines/asterisks indicate genes that were also regulated in the same direction in MCF7 cells. d Schematic map of ER-regulated pathways, based on cellular location, which are activated (red) or deactivated (green) in PE4 or PT12 tumors. Pathways also significantly affected in MCF7 cells are circled

Fig 6

Gene expression networks in endocrine therapy treated PT12. The gene expression profiles of tumor PT12 grown with continuous estrogen for 8 weeks (E), plus tamoxifen for the last 3 weeks (E + Tam), or EWD for the last 3 weeks, were determined using Affymetrix human gene 1.1 ST arrays. An n of 3 tumors were profiled for all groups except for PT12 E, n of 4. a Venn diagrams comparing estrogen (E) up-regulated genes (compared with placebo) and tamoxifen (E + Tam) and EWD down-regulated genes (compared with E), and E down-regulated genes (compared with placebo) and E + Tam and EWD up-regulated genes (compared with E). b Hierarchical cluster of E up- and down-regulated genes reversed by both endocrine therapies (Pl placebo, T E+ tamoxifen, W EWD). Genes changed in the same direction in MCF7 cells with both tamoxifen and EWD are indicated by boxes and either tamoxifen or EWD are indicated by asterisks. c Normalized expression (log2) plots of four genes that significantly changed in both tamoxifen and EWD PT12 tumors, and tamoxifen-treated MCF7 cells (24 h E vs. 24 h E + 4-hydroxy-tamoxifen [GSE848]), and EWD MCF7 tumors (E vs. 48 h EWD [GSE3834]). d Schematic map based on cellular location of the top significant gene networks altered in tamoxifen (E + Tam) or EWD PT12 tumors

Fig 7

Estrogen-regulated target genes in PE4. a Relative expression levels of the genes for PR (PGR) and cancer/testis antigen family 45 (CT45) members A1-6 in placebo versus estrogen (E) treated PE4 tumors. Expression derived from Affymetrix microarray data analysis, n of 3 each, *p < 0.05 E compared with placebo. b Tumor volume (left) and tumor mass (right) ±SEM of PE4 grown under placebo, estrogen (E), or estrogen plus MPA (E + MPA) conditions. *p < 0.05, E + MPA tumor volume compared with E, tumor mass E relative to placebo or E + MPA. c Sections of PE4 tumors treated with placebo, E, or E + MPA were stained by IHC for PR (left). Intensity and percent of cells stained are indicated. Scale bars, 100 µM. qPCR for PGR was performed on cDNA prepared from duplicate samples of PE4 tumors treated with placebo, E, or E + MPA. *p < 0.05. d qPCR for CT45 (probes cover all members) on cDNA prepared from duplicate placebo, E, or E + MPA-treated PE4 tumors (left) or four luminal breast cancer cell lines (right). Values were normalized to β-actin and plotted as fold change relative to placebo. *p < 0.05 E compared with placebo or E + MPA