Patient-derived Mammosphere and Xenograft Tumour Initiation Correlates with Progression to Metastasis

195 early breast cancer surgical specimens were collected by the Manchester Cancer Research Centre Biobank from patients undergoing surgery for primary tumour removal at University Hospital of South Manchester, Salford Royal and The Pennine Acute Hospitals NHS Trusts. 112 unrelated metastatic samples (pleural effusion or ascitic fluid) were collected from patients during standard therapeutic drainage procedures at The Christie NHS Foundation Trust. All patients underwent fully informed consent as either “basic consent” or “animal consent” in accordance with local research ethics committee guidelines (see Compliance with Ethical Standards section for consenting procedures). Clinical information for samples used in this study (grade, nodal involvement, oestrogen receptor (ER) status, Her2 receptor status, radiotherapy, chemotherapy and endocrine therapy prior to sample collection, Nottingham Prognostic Index (NPI) score) is detailed in Table 1. Cells from samples with basic consent were isolated (see below) and cancer stem cell activity was assessed in vitro only. Samples with animal consent were also implanted into mice (see Compliance with Ethical Standards section for animal ethics approvals) to assess tumour initiation in vivo (See Supplementary Fig. 1 for sample pathway).

120 early breast cancer samples and 24 metastatic breast cancer samples were implanted into female NSG (NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mice in accordance with the UK Home Office Animals (Scientific Procedures) Act 1986. All tumours were implanted subcutaneously bilaterally into at least two mice per patient sample, either fresh or following one freeze/thaw. Early breast cancers were implanted as 2x2mm³ fragments, and metastatic samples were injected as 1 × 10⁶ isolated cancer cells. Metastatic cells were injected in 100 μl of a 50:50 mix of matrigel and mammosphere media (components detailed below). The addition of matrigel to early breast cancer fragments was tested but this did not improve tumour take rate, therefore early breast cancers were implanted without matrigel. The implantation procedure did not change during the duration of the study. Oestrogen supplementation was provided in drinking water for mice with ER positive tumours at a concentration of 8 μg/ml. Tumour growth was measured twice weekly using callipers. When tumours reached 1.3cm³ mice were culled and tissue fragments were either implanted into a further generation of mice, frozen for later use, or fixed and examined histologically.

Early breast cancers were disaggregated by mincing with a scalpel, prior to digestion in 4.7 ml RPMI medium plus enzymes from the Miltenyi tumour dissociation kit (130–095-929) on a rotating platform at 150 rpm for 2 h. Optimisation was carried out by splitting tumours in half and comparing the following methods: rotating platform vs. gentleMACS tubes, 2 h digestion vs. overnight digestion, Miltenyi tumour digestion enzymes vs. collagenase. In all cases the number of viable cells (assessed using Trypan Blue on a haemocytometer) were counted following digestion. Two hours digestion on a rotating platform with Milteniyi tumour digestion enzymes was found to be optimal. Following digestion, cells were strained through a 70 μM filter (BD, 352340), using a plunger from a 5 ml syringe (Terumo, SS05SE1) to very gently massage the undigested tissue over the filter, and the filter was then rinsed with 3 × 1 ml RPMI medium. Cells were then further strained through a 40 μM filter (BD, 352340), the filter was rinsed with 3 × 1 ml RPMI medium and centrifuged at 1000 g for 5 min at 4 °C to pellet cells. Supernatant was removed and the cell pellet resuspend in 100 μl of ice-cold PBS. Live cells were counted using Trypan Blue (Gibco, 15,250–061) on a haemocytometer, and cells were then cultured as mammospheres (see below). Metastatic samples were first centrifuged at 1000 g for 10 min to pellet cells. Pellets were resuspended in PBS and blood cells were removed by centrifugation of the cell suspension through 0.5 volumes of Lymphoprep solution (Axis Shield, Dundee, UK) at 800 g for 20 min. Epithelial cells were removed from the interface and diluted with PBS before centrifugation at 800 g for 2 min at 4 °C to pellet cells. The supernatant was removed and a cell count performed using Trypan Blue on a haemocytometer. Cells were then cultured as mammospheres (see below).

Mammosphere culture was performed as previously described [12]. A single cell suspension was prepared by manual disaggregation (25 gauge needle) and a total of 500 cells/cm² were plated in appropriate polyHEMA (Poly (2-hydroxyethylmethacrylate)) coated tissue culture plates in mammosphere medium (phenol red-free DMEM/F12 (Gibco, 21,041)) containing B27 supplement (no vitamin A; Invitrogen, 12,587), rEGF (20 ng/ml; Sigma, E-9644) and Pen-Strep). Methylcellulose was not added to mammosphere media. Cells were cultured for seven days before mammospheres with diameter greater than 50 μm were counted by visual inspection at ×40 magnification using a microscope fitted with a graticule. Percentage primary mammosphere-forming efficiency (MFE) was calculated by dividing the number of mammospheres formed by the number of cells plated and expressed as a percentage. To assess self-renewal, mammospheres were counted, centrifuged (115×g), and dissociated into a single cell suspension by incubation for 2 min at 37 °C in trypsin EDTA 0.125 % (Sigma), followed by mechanical dissociation (25 gauge needle). Single cells were re-plated at 500 cells/cm² and the number of secondary mammospheres counted after 7 days. Mammosphere self-renewal was calculated by dividing the number of secondary mammospheres formed by the number of primary mammospheres formed.

All tumours/cell pellets were fixed in formalin and paraffin embedded. Tumour staining for ER, PR, Ki67 and Her2 was performed in The Christie Hospital Pathology Department. Antibodies used were anti-ERα (Thermo, SP1), anti-PgR (Dako, M3569), anti-Ki67 (Dako M7240) and anti-Her2 (Vector Laboratories, VP-C380). Antigen retrieval was performed either using Target Retrieval Solution pH 9 (Dako S2367, for ER, PR and Ki67) or in 10 mM Citrate buffer (Her2). Antibodies were detected using Dako EnVision Detection System Peroxidase/DAB, Rabbit/Mouse (Dako, K5007) and sections were counterstained with haematoxylin. Hormone receptor positivity was defined as follows; oestrogen receptor (ER) > 5 % positive cells, progesterone receptor (PR) > 5 % positive cells, Her2 receptor 3+ or 2+ confirmed by positive FISH analysis. Human cells were detected in PDX models using a human specific anti-mitochondrial antibody (Abcam, ab92824). Lungs, livers, femurs and lymph nodes were examined for metastases post-mortem in each mouse where subcutaneous tumour growth (P1) was seen. 5 x step sections (40 μM) of each organ were stained with human specific mitochondrial antibody. Sections were stained on a Leica Bond system using standard protocol F and primary antibody conditions of 1:1000 for 15 min. Metastases were detected by visual examination of stained sections. Organs where at ≥1 human cell was observed were classed as metastases positive.

Statistical analysis was performed using SPSS. Data are represented as mean ± SEM. Statistical significance was measured using parametric testing, assuming equal variance, in the majority of experiments with standard t-tests for two-paired samples used to assess difference between test and control samples. Chi squared analysis was used for categorical variables, The Mann-Whitney U test was used for data which was not normally distributed. Differences were considered statistically significant if the probability value (p) was ≤0.05.