A renewable tissue resource of phenotypically stable, biologically and ethnically diverse, patient-derived human breast cancer xenograft models

Abstract

Breast cancer research is hampered by difficulties in obtaining and studying primary human breast tissue, and by the lack of in vivo preclinical models that reflect patient tumor biology accurately. To overcome these limitations, we propagated a cohort of human breast tumors grown in the epithelium-free mammary fat pad of severe combined immunodeficient (SCID)/Beige and nonobese diabetic (NOD)/SCID/IL-2γ-receptor null (NSG) mice under a series of transplant conditions. Both models yielded stably transplantable xenografts at comparably high rates (∼21% and ∼19%, respectively). Of the conditions tested, xenograft take rate was highest in the presence of a low-dose estradiol pellet. Overall, 32 stably transplantable xenograft lines were established, representing 25 unique patients. Most tumors yielding xenografts were "triple-negative" [estrogen receptor (ER)-progesterone receptor (PR)-HER2+; n = 19]. However, we established lines from 3 ER-PR-HER2+ tumors, one ER+PR-HER2-, one ER+PR+HER2-, and one "triple-positive" (ER+PR+HER2+) tumor. Serially passaged xenografts show biologic consistency with the tumor of origin, are phenotypically stable across multiple transplant generations at the histologic, transcriptomic, proteomic, and genomic levels, and show comparable treatment responses as those observed clinically. Xenografts representing 12 patients, including 2 ER+ lines, showed metastasis to the mouse lung. These models thus serve as a renewable, quality-controlled tissue resource for preclinical studies investigating treatment response and metastasis.

Figure 1. Comparisons of representative patient biopsies and their resulting xenografts

Hematoxylin-eosin (H&E) stained sections. Patient biopsies are depicted in the left column; corresponding xenograft samples are depicted in the right column. Scale bar = 50 μm.

Figure 2. Biomarker expression in representative xenografts

Five representative patient samples demonstrating retained biomarker status as xenografts. Xenograft line designations are shown above the column to which they apply. Biomarker designations are shown to the left of the row to which they apply. Inserts show the corresponding biomarker status in the tumor of origin. Scale bar = 50 μm.

Figure 3. Histological analysis of lung metastases

12 xenograft lines demonstrating lung metastases are shown. Insets show higher magnification images of the metastatic lesion. Scale bar = 50 μm.

Figure 4. Global gene expression analyses of the BCM xenografts combined with 337 breast samples of the UNC337 breast cancer dataset

(A) Semi-unsupervised hierarchical clustering of 31 BCM xenografts and 337 breast samples using the 1900 intrinsic list [25, 26]. Localization of the BCM xenografts is shown by the black rectangles below the array tree. Expression of selected genes is shown in the heatmap. Each colored square represents the relative transcript abundance (in log2 space) for each sample with highest expression being red, average expression being black and lowest expression being green. (B) Detailed clusters of the BCM xenografts (one line per patient depicted) with the biomarker expression of each sample. Estrogen receptor (ER), progesterone receptor (PR), HER2, cytokeratin 19 (CK19), cytokeratin 5/6 (CK5/6) and EGFR.

Figure 5. Transcriptome and Proteome Stability

Xenograft lines are phenotypically stable over multiple transplant generations with respect to gene expression by Affymetrix microarray and Reverse Phase Proteomic Analysis (RPPA). A. Hierarchical cluster analysis using all probesets with detectable expression. Xenograft designation ([BCM-]####), and transplant generation number (TG#), are shown for each branch of the dendrogram. Patient of origin/xenograft association are designated by color. B. Box and whisker plot of Pearson correlation distances within patient and between patients. C. Hierarchical cluster analysis using 161 antibodies in RPPA. Xenograft designations are as shown for panel A. D. Box and whisker plot of Pearson correlation distances within patient and between patients.