Tumour cell heterogeneity maintained by cooperating subclones in Wnt-driven mammary cancers

Abstract

Cancer genome sequencing studies indicate that a single breast cancer typically harbours multiple genetically distinct subclones1,2,3,4. As carcinogenesis involves a breakdown in the cell–cell cooperation that normally maintains epithelial tissue architecture, individual subclones within a malignant microenvironment are commonly depicted as self-interested competitors5,6. Alternatively, breast cancer subclones might interact cooperatively to gain a selective growth advantage in some cases. Although interclonal cooperation has been shown to drive tumorigenesis in fruitfly models7,8, definitive evidence for functional cooperation between epithelial tumour cell subclones in mammals is lacking. Here we use mouse models of breast cancer to show that interclonal cooperation can be essential for tumour maintenance. Aberrant expression of the secreted signalling molecule Wnt1 generates mixed-lineage mammary tumours composed of basal and luminal tumour cell subtypes, which purportedly derive from a bipotent malignant progenitor cell residing atop a tumour cell hierarchy9. Using somatic Hras mutations as clonal markers, we show that some Wnt tumours indeed conform to a hierarchical configuration, but that others unexpectedly harbour genetically distinct basal Hras mutant and luminal Hras wild-type subclones. Both subclones are required for efficient tumour propagation, which strictly depends on luminally produced Wnt1. When biclonal tumours were challenged with Wnt withdrawal to simulate targeted therapy, analysis of tumour regression and relapse revealed that basal subclones recruit heterologous Wnt-producing cells to restore tumour growth. Alternatively, in the absence of a substitute Wnt source, the original subclones often evolve to rescue Wnt pathway activation and drive relapse, either by restoring cooperation or by switching to a defector strategy. Uncovering similar modes of interclonal cooperation in human cancers may inform efforts aimed at eradicating tumour cell communities.

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Figure 1: Evidence for distinct basal HrasmutWnt1low and luminal HraswtWnt1high subclones within some MMTV-Wnt1 tumours.
Figure 2: Rescue of basal Hrasmut iWnt tumour cells from Wnt withdrawal by heterologous luminal cWnt cells.
Figure 3: Lineage-restricted subclones recapitulate mosaiform heterogeneity in chimaeric cWnt/iWnt tumours.
Figure 4: Relapse of biclonal tumours through the evolution of either subclone.

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Acknowledgements

We thank the benefactors of the Jake Gittlen Laboratories for Cancer Research and the following Penn State College of Medicine staff: L. Budgeon of the Gittlen Histology Core, N. Sheaffer and J. Bednarczyk of the Flow Cytometry Core, D. Stanford of the Molecular Genetics Core, and J. Mohl of the Barrier Rodent Facility. This project was funded with grant support from the National Institutes of Health/National Cancer Institute, the Mary Kay Foundation, the Donald B. and Dorothy L. Stabler Foundation, and the Pennsylvania Department of Health. A.S.C. is supported by Department of Defense Predoctoral Traineeship Award W81XWH-11-1-0038.

Author information

A.S.C. designed and carried out experiments, interpreted data and co-wrote the manuscript. T.L.L. and S.A.G. performed experiments, interpreted data, and provided commentary on the manuscript. E.J.G. designed experiments, interpreted data and co-wrote the manuscript.

Correspondence to Edward J. Gunther.

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Competing interests

The authors declare no competing financial interests.

Extended data figures and tables

Extended Data Figure 1 FACS gating strategy for resolving basal and luminal subsets from mammary tumours.

Mammary tumours were mechanically and enzymatically dissociated into single-cell suspensions. a, Negative selection against Lin+ cells using Stem Cell Technologies EasySep Mouse Epithelial Cell Enrichment Kit. Resulting Lin (CD45 CD31 TER119 BP-1) cells were then immunostained with antibodies for CD49f (α6 integrin) and EpCAM and analysed by FACS. b, Exclusion of cell debris and dead/dying cells. Dead/dying cells collect as a band along the bottom of a forward scatter (FSC-A) versus side scatter (SSC-A) two-parameter plot, and these were gated out when defining population (P)1. c, Cell doublets were discarded when defining P2. d, Basal and luminal mammary epithelial cell populations were separated by immunophenotype. Basal epithelial cells are CD49fhigh EpCAMlow (P3) and luminal epithelial cells are CD49flow EpCAMhigh (P4). e, Gating tree showing gating strategy for FACS analysis as well as parent and total cell percentages within each of the gates for a representative MMTV-Wnt1 tumour.

Extended Data Figure 2 Hierarchical and biclonal MMTV-Wnt1 tumours are histologically indistinguishable.

a, Haematoxylin and eosin-stained sections from a series of MMTV-Wnt1 mammary tumours whose Hrasmut allele distribution pattern suggests hierarchical or biclonal configuration, as indicated. Scale bar, 50 μm. b, Both hierarchical and biclonal MMTV-Wnt1 tumours display mixed-lineage character. Serial sections from a hierarchical and biclonal MMTV-Wnt1 mammary tumours immunostained for smooth muscle actin (SMA) or keratin 8 (K8), which recognize basal and luminal epithelial cells, respectively. For both, brown pigment is positive staining. Sections were counterstained with haematoxylin. Scale bars, 50 μm.

Extended Data Figure 3 Tumour regression following Dox withdrawal.

a, Tumours reconstituted on wild-type or cWnt hosts following injection of iWnt/mRFP+ tumour cells were subjected to Dox withdrawal and monitored for regression. *Shown as number of tumour regressions per number of tumours subjected to Dox withdrawal. b, Northern hybridization analysis of tumour RNA with Wnt1 probe. Tumours were reconstituted on Dox-treated cWnt hosts following injection of iWnt/mRFP+ tumour cells. Depicted below are the corresponding FACS plots showing the range of contributions by donor-derived mRFP+ and host-derived mRFP cells to reconstituted tumours before Dox withdrawal. Colours indicate events within the basal (blue; CD49fhigh EpCAMlow) and luminal (green; CD49flow EpCAMhigh) gates. On rescue hosts, primary tumours that arose during Dox treatment incorporated a variable number of cWnt luminal cells, indicating that the crosstalk between heterologous cells required to seed relapse sometimes occurs early in tumour reconstitution. For one of three primary tumours analysed, the conversion to lineage-restricted chimaerism and cWnt transgene expression was essentially complete, meaning that cWnt-producing cells had replaced iWnt-producing cells despite ongoing Dox treatment. Analysis of this tumour required necropsy of the host, precluding determination of its clinical response to Dox withdrawal, which we propose would have been negligible. Concordantly, in rare cases the growth of sibling primary tumours propagated on rescue hosts continued unimpeded by Dox withdrawal, and these tumours always showed pronounced, lineage-restricted chimaerism at necropsy. Elucidating mechanisms whereby host cWnt cells compete with luminal iWnt tumour cells to become the predominant Wnt-producing subclone may offer new insights into evolutionary forces shaping tumour microenvironments.

Extended Data Figure 4 Biclonal configuration of reconstituted iWnt/mRFP+ tumours.

a, DNA sequencing chromatograms depicting a basally enriched HrasGGA12GAA mutation detected in the parental tumour. b, Evidence for distinct basal HrasmutWnt1low and luminal HraswtWnt1high tumour subclones. Sorted tumour cell subsets were analysed by DNA sequencing and by qRT–PCR for Wnt1 expression relative to Gapdh. Histograms on the left show Hras MAFs determined from chromatogram peak heights. Histograms on the right show relative Wnt1 expression with values from unsorted tumour cells set at 1. B, basal; L, luminal; Un, unsorted. Data represent mean ± s.e.m. for (from left to right) n = 2, 4, 3, 4, 1, 2, 6 or 12 explants. c, For each condition, sorted tumour cell subsets were analysed by qRT–PCR for expression of several epithelial lineage-specific genes relative to Gapdh, with values for unsorted tumour cells set at 1. Grey bars, unsorted; blue bars, basal; green bars, luminal. Data represent mean ± s.e.m. for (from left to right) n = 4, 4, 3 or 12 explants.

Extended Data Figure 5 Basal subclones from two additional iWnt/mRFP+ tumours rescued from Dox withdrawal by heterologous cWnt host cells.

a, Growth curves of tumour outgrowths derived from an iWnt/mRFP+ tumour harbouring a basally restricted HrasGGA12AGA mutation. Curves depict regression and relapse of tumours reconstituted on cWnt rescue hosts following Dox withdrawal. b, c, Top, representative FACS plots showing contributions from donor-derived mRFP+ cells and host-derived mRFP cells during tumour reconstitution. Colours indicate events within the basal (blue; CD49fhigh EpCAMlow) and luminal (green; CD49flow EpCAMhigh) gates. Bottom, DNA sequencing chromatograms showing matching, basally restricted Hras mutations present in both primary Dox-dependent tumours and chimaeric Dox-independent relapses. df, Data panels presented as in ac, showing similar results for an independent iWnt/mRFP+ tumour harbouring a distinct, basally restricted HrasCAA61CGA mutation. For both tumours shown here, northern hybridization analysis confirmed expression of donor-derived iWnt transgene before Dox withdrawal, followed by a switch to expression of host-derived cWnt transgene at relapse (data not shown).

Extended Data Figure 6 Biclonal configuration of tumours reconstituted from sorted iWnt/mRFP+ tumour cell subsets.

a, Sorted tumour cell subsets inefficiently reconstitute tumours. Three independent iWnt/mRFP+ biclonal tumours were resolved into component basal and luminal tumour cell subsets by FACS. Each tumour harboured a different basally restricted Hras mutation, as indicated. 105 sorted tumour cells were injected orthotopically into intact, post-pubertal mammary glands of wild-type host mice maintained on chronic Dox treatment. *Shown as number of reconstituted tumour outgrowths per injected gland. b, Tumour cells from a parental iWnt/mRFP+ tumour harbouring a basally restricted HrasGGA12GAA mutation were resolved into basal and luminal cell subsets by FACS. When these isolated tumour cell subsets were injected orthotopically into the mammary glands of Dox-treated wild-type hosts, few tumours were reconstituted. However, tumours that did arise always were comprised of basal HrasmutWnt1low and luminal HraswtWnt1high subsets, implicating interclonal cooperation in tumour reconstitution (n = 3 tumours reconstituted from the basal cell-enriched subset; n = 4 tumours reconstituted from the luminal cell-enriched subset).

Extended Data Figure 7 Both sorted basal and sorted luminal cell populations are required to reconstitute biclonal tumours.

Chimeric tumour relapses generated by injecting iWnt/mRFP+ tumour cells onto cWnt rescue hosts were resolved into their component basal (mRFP+/Hrasmut Wnt1low) and luminal (mRFP/Hraswt Wnt1high) cell subsets by FACS. Each sorted population was injected separately (105 basal cells per injection or 105 luminal cells per injection) or as a 1:1 admixture (5 × 104 basal cells + 5 × 104 luminal cells per injection) onto wild-type, Dox-naive hosts. All reconstituted tumours faithfully recapitulated the biclonal configuration of the source tumour. Depicted are FACS plots from parental and reconstituted tumours showing both mRFP+ and mRFP subclonal populations. Colours indicate events within the basal (blue; CD49fhigh EpCAMlow) and luminal (green; CD49flow EpCAMhigh) gates. Percentages depict mean ± s.e.m. for n = 5 clonally related parental tumour outgrowths and n = 11 tumour outgrowths reconstituted from injection of admixed cells.

Extended Data Figure 8 Increased Hras MAFs in βcatmut DITs is not due to gross copy changes at the Hras locus.

Histogram depicts Hras allele copy number relative to β-actin (Actb) determined for a cohort of clonally related Wnt tumour outgrowths. Independent relapse samples are presented in the same order depicted in Fig. 4b. Copy number values were obtained by performing qPCR on genomic DNA from tumour samples and from normal tail, with tail values set at 1. As a positive control, we included a p19Arf-deficient Wnt tumour sample (10× amplification) previously found to have approximately tenfold Hras copy number gain as determined by Southern hybridization. As Hras MAFs reproducibly exceeded βcat MAFs by approximately twofold across the βcatmut relapse set (Fig. 4b), increased Hras MAFs may reflect duplication of the Hrasmut allele (for example, via a gene conversion event) sometime in the life history of βcatmut subclones.

Extended Data Figure 9 Mixed-lineage character of DITs.

Serial sections of representative Wnt1-transgene-re-expressing and βcatmut-relapsed tumours immunostained for smooth muscle actin or keratin 8, which recognize basal and luminal epithelial cells, respectively. For both, brown pigment indicates positive staining. Sections were counterstained with haematoxylin. Scale bar, 50 μm.

Extended Data Table 1 Unsorted tumour cells efficiently reconstitute tumours

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Cleary, A., Leonard, T., Gestl, S. et al. Tumour cell heterogeneity maintained by cooperating subclones in Wnt-driven mammary cancers. Nature 508, 113–117 (2014). https://doi.org/10.1038/nature13187

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