Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The . gov means it’s official. Federal government websites often end in . gov or . mil. Before sharing sensitive information, make sure you’re on a federal government site VSports app下载. .

Https

The site is secure. The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely V体育官网. .

. 2014 Dec 23:5:5871.
doi: 10.1038/ncomms6871.

DNA barcoding reveals diverse growth kinetics of human breast tumour subclones in serially passaged xenografts

Long V Nguyen  1 , Claire L Cox  1 , Peter Eirew  2 , David J H F Knapp  1 , Davide Pellacani  1 , Nagarajan Kannan  1 , Annaick Carles  3 , Michelle Moksa  3 , Sneha Balani  1 , Sohrab Shah  2 , Martin Hirst  3 , Samuel Aparicio  2 , Connie J Eaves  1
Affiliations

DNA barcoding reveals diverse growth kinetics of human breast tumour subclones in serially passaged xenografts (VSports在线直播)

Long V Nguyen et al. Nat Commun. .

Abstract

Genomic and phenotypic analyses indicate extensive intra- as well as intertumoral heterogeneity in primary human malignant cell populations despite their clonal origin. Cellular DNA barcoding offers a powerful and unbiased alternative to track the number and size of multiple subclones within a single human tumour xenograft and their response to continued in vivo passaging. Using this approach we find clone-initiating cell frequencies that vary from ~1/10 to ~1/10,000 cells transplanted for two human breast cancer cell lines and breast cancer xenografts derived from three different patients. For the cell lines, these frequencies are negatively affected in transplants of more than 20,000 cells. Serial transplants reveal five clonal growth patterns (unchanging, expanding, diminishing, fluctuating or of delayed onset), whose predominance is highly variable both between and within original samples. This study thus demonstrates the high growth potential and diverse growth properties of xenografted human breast cancer cells VSports手机版. .

PubMed Disclaimer

VSports手机版 - Figures

Figure 1
Figure 1. Experimental design.
Serial passages of individual tumours derived from separate samples of barcoded MDA-MB-231 and SUM-149 cells (nine and four, respectively) are shown in purple, and for cells from patient-derived xenografts in green. White boxes indicate early passages of patient-derived xenografts, before barcoding cells for subsequent clonal analyses. The total number of cells (or fraction of the previous tumour) used to initiate each subsequent tumour, the time before removing the tumour for analysis, and the number and frequency of uniquely barcoded clones (expressed as a proportion of the estimated input number of barcoded cells) are shown on the right. CIC frequencies were calculated as the no. of clones divided by the total no. of barcoded cells transplanted based on the 30% transduction efficiency measured by FACS analysis of input cells.
Figure 2
Figure 2. Variation in clone size in primary and metastatic tumour xenografts.
(a) An inverse linear relationship is seen between the numbers of cells transplanted and the number of clones detected, for both cell lines tested (Δ SUM-149 cells; ○ MDA-MB-231 cells). Values shown are the geometric mean±s.e.m. of the frequency of CICs calculated for each of the tumours identified in Fig. 1. (b) Cumulative distributions of clone sizes in primary xenografts generated from different numbers of S1–S4 (upper panel) and M1–M9 cells (lower panel). (c) Overlap between clones present in the tumour (M8) that arose at the site of injection (black) and in simultaneously assessed liver metastases (brown). (d) Comparison of the distributions of the 52 overlapping clones identified in panel c (upper panel) and for the 816 and 140 clones detected simultaneously at the injection site (black) and liver (brown), respectively (lower panel). The x-axis represents the size of clones binned in log2-increments.
Figure 3
Figure 3. Diverse in vivo clonal growth patterns of human breast cancer cell lines.
(a) Growth patterns of individual clones in primary and secondary derivative tumours generated from M3 cells. In each plot, a separate line portrays the growth activity of an individual clone in successive passages. Clones that remained relatively constant in size between passages are shown in shades of red, and those whose size increased or decreased are shown in shades of yellow and blue, respectively. The area in each plot shaded in grey represents the relative clone size below the threshold used for detecting barcoded clones. In cases where replicate tumours had different limits of detection, and are represented on the same plot, the higher limit is shown. (b) Relative proportions of the different clonal growth patterns at each passage from M3, S3 and S4 cells. Colours in each sector correspond to the colour-coded clonal patterns described in a.
Figure 4
Figure 4. Delayed growth of M4 clones in serially transplanted mice.
(a) Growth patterns of individual clones in primary, secondary and tertiary tumours derived from M4 cells. In each plot, a separate line portrays the growth activity of an individual clone in successive passages. Clones that remained relatively constant in size between passages are shown in shades of red, and those whose size increased or decreased are shown in shades of yellow and blue, respectively. Clones that first became detectable in secondary and tertiary tumours are shown in grey and black, respectively. The area in each plot shaded in grey represents the relative clone size below the threshold used for detecting barcoded clones. In cases where replicate tumours had different limits of detection, and are represented on the same plot, the higher limit is shown. (b) Relative proportions of the different M4 clonal growth patterns at each passage. Colours in each sector correspond to the same colour-coded clonal patterns described in a.
Figure 5
Figure 5. Diverse in vivo clonal growth patterns of patient-derived tumour xenografts.
(a) Growth patterns of individual clones in primary, secondary and tertiary tumours generated from T1-11 cells. In each plot, a separate line portrays the growth activity of an individual clone in successive passages. Clones that remained relatively constant between passages are shown in shades of red, and those whose size decreased are shown in blue. Clones that first became detectable in secondary tumours or that fluctuated in size between passages are shown as grey. The area in each plot shaded in grey represents the relative clone size below the threshold used for detecting barcoded clones. In cases where replicate tumours had different limits of detection, and are represented on the same plot, the higher limit is shown. (b) Relative proportions of the different clonal growth patterns exhibited by T1-11 and T1-12 cells. Colours in each sector correspond to the colour-coded clonal patterns described in a. (c) Relative proportions of the different clonal growth patterns exhibited by T2-111 and T2-1121 cells. Colours in each sector correspond to the colour-coded clonal patterns described in a, except for clones that increased in size between passages that are shown in yellow.
Figure 6
Figure 6. Replicate xenografts include both symmetric and asymmetric clonal growth patterns.
(a) Venn diagrams showing the proportion of clones that demonstrated symmetrical (non-overlapping parts of the circle), and asymmetrical growth patterns (overlap between two circles) in replicate tumours derived from parental M3, M4 and S3 tumours. Different colours are used to identify each of the five clonal growth patterns detected as follows: constant (red), increasing (yellow), diminishing (blue), fluctuating (first appearing in secondary tumours, grey) and delayed (first appearing in tertiary tumours, black), and the size of each circle reflects the relative abundance of clones displaying the growth pattern it represents. The numbers shown are the absolute numbers of clones whose replicate derivatives displayed symmetrical or asymmetrical growth patterns. (b) Venn diagrams showing the proportion of clones that demonstrated symmetrical (non-overlapping parts of the circle) and asymmetrical growth patterns (overlap between 2 circles) in replicate tumours derived from parental T1 and T2 tumours, using the same colour coding and illustrative principles as in panel a.
See this image and copyright information in PMC

References (V体育官网入口)

    1. Visvader J. E. & Stingl J. Mammary stem cells and the differentiation hierarchy: current status and perspectives. Genes Dev. 28, 1143–1158 (2014). - PMC (VSports在线直播) - PubMed
    1. Meacham C. E. & Morrison S. J. Tumour heterogeneity and cancer cell plasticity. Nature 501, 328–337 (2013). - VSports注册入口 - PMC - PubMed
    1. Eirew P. et al.. A method for quantifying normal human mammary epithelial stem cells with in vivo regenerative ability. Nat. Med. 14, 1384–1389 (2008). - "V体育官网入口" PubMed
    1. Lim E. et al.. Aberrant luminal progenitors as the candidate target population for basal tumor development in BRCA1 mutation carriers. Nat. Med. 15, 907–913 (2009). - PubMed
    1. Al-Hajj M., Wicha M. S., Benito-Hernandez A., Morrison S. J. & Clarke M. F. Prospective identification of tumorigenic breast cancer cells. Proc. Natl Acad. Sci. USA 100, 3983–3988 (2003). - PMC - PubMed

Publication types