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. 2016 Dec;21(3-4):99-109.
doi: 10.1007/s10911-016-9361-8. Epub 2016 Sep 28.

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

Rachel Eyre  1 , Denis G Alférez  1 , Kath Spence  1 , Mohamed Kamal  1   2 , Frances L Shaw  1 , Bruno M Simões  1 , Angélica Santiago-Gómez  1 , Aida Sarmiento-Castro  1 , Maria Bramley  3 , Mohammed Absar  3 , Zahida Saad  4 , Sumohan Chatterjee  4 , Cliona Kirwan  5 , Ashu Gandhi  5 , Anne C Armstrong  6 , Andrew M Wardley  6 , Ciara S O'Brien  6 , Gillian Farnie  7 , Sacha J Howell  6   8 , Robert B Clarke  9
Affiliations

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

Rachel Eyre et al. J Mammary Gland Biol Neoplasia. 2016 Dec.

Erratum in

Abstract

Breast cancer specific mortality results from tumour cell dissemination and metastatic colonisation. Identification of the cells and processes responsible for metastasis will enable better prevention and control of metastatic disease, thus reducing relapse and mortality. To better understand these processes, we prospectively collected 307 patient-derived breast cancer samples (n = 195 early breast cancers (EBC) and n = 112 metastatic samples (MBC)). We assessed colony-forming activity in vitro by growing isolated cells in both primary (formation) and secondary (self-renewal) mammosphere culture, and tumour initiating activity in vivo through subcutaneous transplantation of fragments or cells into mice. Metastatic samples formed primary mammosphere colonies significantly more frequently than early breast cancers and had significantly higher primary mammosphere colony formation efficiency (0.9 % vs. 0.6 %; p < 0.0001). Tumour initiation in vivo was significantly higher in metastatic than early breast cancer samples (63 % vs. 38 %, p = 0.04). Of 144 breast cancer samples implanted in vivo, we established 20 stable patient-derived xenograft (PDX) models at passage 2 or greater. Lung metastases were detected in mice from 14 PDX models. Mammosphere colony formation in vitro significantly correlated with the ability of a tumour to metastasise to the lungs in vivo (p = 0.05), but not with subcutaneous tumour initiation. In summary, the breast cancer stem cell activities of colony formation and tumour initiation are increased in metastatic compared to early samples, and predict metastasis in vivo. These results suggest that breast stem cell activity will predict for poor outcome tumours, and therapy targeting this activity will improve outcomes for patients with metastatic disease.

Keywords: Breast cancer; Mammosphere; Metastasis; Patient-derived xenografts; Stem cell activity.

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Conflict of interest statement

Compliance with Ethical StandardsFundingThis study was funded by Cancer Research UK and Breast Cancer Now. MK was funded by S cience and Technology Development Fund fellowship No. 6123.Research involving Human ParticipantsAll patients underwent fully informed consent in accordance with local research ethics committee guidelines. Ethical approval for metastatic samples was granted by the Central Office for Research Ethics Committee study number 05/Q1402/25. Early breast cancer samples were collected via the Manchester Cancer Research Centre Biobank which is licensed by the Human Tissue Authority (licence number: 30,004) and has been ethically approved as a research tissue bank by the South Manchester Research Ethics Committee (Ref: 07/H1003/161 + 5).Conflict of InterestThe authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1
Breast cancer mammosphere colony formation and in vivo tumour-initiating activity is increased in metastatic compared to early breast cancers. Cells were isolated from breast cancer samples and grown in suspension culture as mammospheres (representative mammospheres from early (EBC) and metastatic (MBC) samples shown in a). Metastatic breast cancer samples were more likely to form mammospheres (MS formation >0) than early breast cancers cultured under the same conditions b and had higher primary mammospheres forming efficiencies c (Data presented both as % mammosphere formation, and mammospheres formed/1000 cells plated). No difference in secondary mammosphere formation (self-renewal), defined as a ratio of secondary mammospheres: primary mammospheres, was observed between early and metastatic samples d. Metastatic samples had significantly higher in vivo xenotransplantation potential than early breast cancer samples, over an average period of 200 days, irrespective of tumour phenotype (p = 0.04), where in vivo growth is defined as tumour formed to size limit (1.3cm3) e. Data are presented as mean ± SEM. *p < 0.05 ****p < 0.0005. Statistical analyses: Chi Squared tests (b and e) and two tailed t-test (c and d)
Fig. 2
Fig. 2
Characterisation of Patient-Derived Xenograft tumours. Of the 144 tumours implanted in this study, 61 grew in the first passage in mice and 20 stable PDX models were created. Comparisons of representative patient tumour samples and their corresponding patient-derived xenograft in passage 1 a. Patient-derived xenografts retained ER, PR and Her2 status of the patient tumour. Representative images in b. Scale bar =100uM
Fig. 3
Fig. 3
Mammosphere colony formation predicts metastasis in PDX models. Lung metastases were detected in 14/34 models where a tumour formed in passage 1 by H&E a and confirmed by staining with a human specific mitochondrial antibody b. Lung metastases retained the ER c, PR d and Her2 e status of the primary tumour and contained ki67 positive dividing cells f. Primary mammosphere formation predicted PDX metastases, with samples which metastasised to the lungs in vivo having a significantly higher % mammosphere forming efficiency in vitro g. Metastases are more likely to form in PDX models from high grade EBC tumours (grade 3) than low grade (grade 1 and 2) h. Data are presented as mean ± SEM. *p < 0.05 Statistical analysis: Mann -Whitney U Test
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