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. 2018 May 29;11(5):dmm032250.
doi: 10.1242/dmm.032250.

Functional and genomic characterisation of a xenograft model system for the study of metastasis in triple-negative breast cancer

Cameron N Johnstone  1   2   3   4   5   6   7 , Andrew D Pattison  6   8 , Kylie L Gorringe  9   2 , Paul F Harrison  10 , David R Powell  8   10 , Peter Lock  11 , David Baloyan  4 , Matthias Ernst  4   5 , Alastair G Stewart  7 , Traude H Beilharz  12   8 , Robin L Anderson  1   2   3   4   5
Affiliations

Functional and genomic characterisation of a xenograft model system for the study of metastasis in triple-negative breast cancer

Cameron N Johnstone et al. Dis Model Mech. .

Abstract

Triple-negative breast cancer (TNBC) represents 10-20% of all human ductal adenocarcinomas and has a poor prognosis relative to other subtypes. Hence, new molecular targets for therapeutic intervention are necessary. Analyses of panels of human or mouse cancer lines derived from the same individual that differ in their cellular phenotypes but not in genetic background have been instrumental in defining the molecular players that drive the various hallmarks of cancer. To determine the molecular regulators of metastasis in TNBC, we completed a rigorous in vitro and in vivo characterisation of four populations of the MDA-MB-231 human breast cancer line ranging in aggressiveness from non-metastatic to spontaneously metastatic to lung, liver, spleen and lymph node. Single nucleotide polymorphism (SNP) array analyses and genome-wide mRNA expression profiles of tumour cells isolated from orthotopic mammary xenografts were compared between the four lines to define both cell autonomous pathways and genes associated with metastatic proclivity. Gene set enrichment analysis (GSEA) demonstrated an unexpected association between both ribosome biogenesis and mRNA metabolism and metastatic capacity. Differentially expressed genes or families of related genes were allocated to one of four categories, associated with either metastatic initiation (e.g. CTSC, ENG, BMP2), metastatic virulence (e.g. ADAMTS1, TIE1), metastatic suppression (e.g. CST1, CST2, CST4, CST6, SCNNA1, BMP4) or metastatic avirulence (e.g. CD74). Collectively, this model system based on MDA-MB-231 cells should be useful for the assessment of gene function in the metastatic cascade and also for the testing of novel experimental therapeutics for the treatment of TNBC.This article has an associated First Person interview with the first author of the paper.

Keywords: Breast cancer; Metastasis; Mouse model; Triple-negative; Xenograft.

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Conflict of interest statement

Competing interestsThe authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
Comparison of four MDA-MB-231-derived tumour variants in vivo. (A) Primary-tumour growth rates were measured using electronic callipers. Mean tumour volume±s.e.m. is shown. 231_ATCC (n=3), 231_LNA (n=4), 231_LM2 (n=4), 231_HM.LNm5 (n=4). Differences in growth rates were determined using mixed-effects linear regression modelling (Johnstone et al., 2015): 231_ATCC vs 231_LNA (P=0.002), 231_ATCC vs 231_LM2 (P=0.002), 231_ATCC vs 231_HM (P=0.001), 231_LNA vs 231_LM2 (P=0.006), 231_LNA vs 231_HM (P=0.002), 231_LM2 vs 231_HM (P=0.168). 231_LM2 and 231_HM.LNm5 primary tumours were surgically resected at day 18, 231_LNA at day 21 and 231_ATCC at day 72 after inoculation (arrows). (B-E) In vivo bioluminescence imaging of breast cancer xenograft models. Luciferase images of live mice were captured 22 days following surgical resection of the primary mammary tumour for all models. Both local and distant tumour recurrence was present in each of the three metastatic models [(C) 231_LNA, (D) 231_LM2, (E) 231_HM.LNm5] but not in mice inoculated with 231_ATCC cells (B). Three mice are shown per model [n=4 for each model except for 231_ATCC (n=3)]. 231_LM2 cells express Firefly luciferase-2 (luc2), radiance scale 1×107 (min) to 1×109 (max). The other three models express the dimmer Firefly luciferase-1 (luc1), radiance scales 1×105 (min) to 1×107 (max). The site of original primary tumour formation in the right-side inguinal mammary gland is indicated with a red arrow in one mouse as an example (B).
Fig. 2.
Fig. 2.
Spontaneous metastasis of MDA-MB-231 variants to distant organs. Sections (3 µm) were cut from representative FFPE-fixed secondary organs obtained 22 days after primary tumour resection. H&E-stained slides were scanned using an Olympus VS120 instrument and images generated using OlyVIA software (Olympus). (A) 231_ATCC. (B) 231_LNA. (C) 231_LM2. (D,E) 231_HM.LNm5. (i) Liver, (ii) lung, (iii) spleen, (iv) metastatic deposit on the spine between the kidneys, (v) merged brightfield and fluorescent (tdTomato) images of a metastatic deposit on the spine between the kidneys (7× magnification). Metastatic lesions are indicated with a dashed blue line. T, region of tumour; K, kidney. Scale bars: (i-iii) 200 µm, (iv) 2 mm.
Fig. 3.
Fig. 3.
In vitro phenotypes of different MDA-MB-231 variants. Proliferation rate, rather than an invasive growth pattern, is correlated with metastatic ability. (A) Images of cells cultured in 3D. The indicated cell lines were seeded on top of a 50% Cultrex matrix and images captured at the indicated magnifications after 5 days. Brightfield (greyscale) and matched tdTomato fluorescent images are shown. 231_ATCC and 231_HM.LNm5 lines formed non-invasive spheroids, whereas 231_LNA and 231_LM2 formed loosely connected invasive clusters. (B) In vitro motility of MDA-MB-231 variants. Transwell migration assays using 10% FBS as a chemoattractant (n=3 per line) were completed for the indicated cell lines. Parental MDA-MB-231HM (231_HM) cells were also included for comparison. Two representative fields (×100 magnification) from two different Transwells per cell line are displayed. (C) 3D in vitro proliferation (on 50% Cultrex) was measured over 5 days using the Cell Titer Glo assay (Promega) for the indicated cell lines. Mean±s.e.m. (n=4) is presented. *P<0.05. ***P<0.001 by Student's t-test.
Fig. 4.
Fig. 4.
Cell cycle and SNP array analysis of four MDA-MB-231 cell line variants. (A-F) The indicated MDA-MB-231-derived cell lines were analysed for cell cycle distribution by flow cytometry. 231_HMcloneB2 (E) and 231_HMcloneC7 (F) are two different clonal daughter lines derived from parental 231_HM cells (D). (G) Genome-wide patterns of DNA copy number gain (blue), loss (red), loss of heterozygosity (yellow) and allelic imbalance (purple) derived from SNP array analysis are depicted linearly for the four indicated cell lines. The chromosome number is indicated at the top. The location of the homozygous deletion at 9p21.3 that is shared by the four cell lines is indicated by an arrow.
Fig. 5.
Fig. 5.
Heat map of tumour cell gene expression patterns among MDA-MB-231 mammary xenografts. Each row represents a single gene. Rows were clustered by similarity (Euclidian distance). Only genes with both a ≥twofold change (log2 fold change ≤−1 or ≥1) in expression level compared to non-metastatic 231_ATCC tumour cells (n=2) and a mean sequencing transcript count of ≥30 are shown. 231_LNA (n=6), 231_LM2 (n=6), 231_HM.LNm5 (n=6). The full dataset of differentially expressed genes is provided in Table S3.
Fig. 6.
Fig. 6.
Analysis of BMP4 and CST6 mRNA levels following treatment with demethylating agent and histone deacetylase inhibitor. (A) A CpG island (506 bp) spans the transcription start site of the human BMP4 gene. (B) Relative BMP4 mRNA levels in vehicle (DMSO)-treated 231_ATCC and 231_HM.LNm5 cells by TaqMan qRT-PCR. Expression in 231_ATCC was set to 1. Mean±s.d. (n=3). (C) TaqMan qRT-PCR analysis of BMP4 mRNA expression levels in 5-Aza-2′-deoxycytidine (5azadC)- and Trichostatin A (TSA)-treated 231_ATCC and 231_HM.LNm5 cells. Expression in vehicle-treated cells was set to 1. Mean±s.d. (n=3). (D) A CpG island (370 bp) spans the transcription start site of the human CST6 gene. (E) Relative CST6 mRNA levels in vehicle (DMSO)-treated 231_ATCC and 231_HM.LNm5 cells by TaqMan qRT-PCR. Expression in 231_ATCC was set to 1. Mean±s.d. (n=3). (F) TaqMan qRT-PCR analysis of CST6 mRNA expression levels in 5azadC- and TSA-treated 231_ATCC and 231_HM.LNm5 cells. Expression in vehicle-treated cells was set to 1. Mean±s.d. (n=3).
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