Multipotent luminal mammary cancer stem cells model tumor heterogeneity

Abstract

Introduction: The diversity of human breast cancer subtypes has led to the hypothesis that breast cancer is actually a number of different diseases arising from cells at various stages of differentiation. The elusive nature of the cell(s) of origin thus hampers approaches to eradicate the disease.

Methods: Clonal cell lines were isolated from primary transgenic polyomavirus middle T (PyVmT) luminal tumors. Mammary cancer stem cell (MaCSC) properties were examined by immunofluorescence, flow cytometry, differentiation assays and in vivo tumorigenesis.

Results: Clonal cell lines isolated from primary PyVmT mouse mammary luminal tumors can differentiate into luminal, myoepithelial, alveolar and adipocyte lineages. Upon orthotopic injection, progeny of a single cell follow a pattern of progression from ductal carcinoma in situ, to adenoma, adenocarcinoma and epithelial metastasis that recapitulates the transgenic model. Tumors can evolve in vivo from hormone receptor-positive to hormone receptor-negative Her2-positive, or triple negative CD44hi basal-like and claudin-low tumors. Contrary to the current paradigm, we have defined a model in which multiple tumor subtypes can originate from a single multipotent cancer stem cell that undergoes genetic and/or epigenetic evolution during tumor progression. As in human tumors, the more aggressive tumor subtypes express nuclear p53. Tumor cell lines can also be derived from these more advanced tumor subtypes.

Conclusions: Since the majority of human tumors are of the luminal subtype, understanding the cell of origin of these tumors and how they relate to other tumor subtypes will impact cancer therapy. Analysis of clonal cell lines derived from different tumor subtypes suggests a developmental hierarchy of MaCSCs, which may provide insights into the progression of human breast cancer.

Fig. 1

MaCSCs have the capacity to differentiate into multiple cell types. (a) A single Py230 cell stained with myoepithelial marker keratin 14 (K14) and luminal marker keratin 8 (K8). (b) Clonal Py230 cells grown on a glass coverslip stained with K14 and K8. Scale bar 20 μm. (c) Clonogenic efficiency of C57Bl/6 MaCSC line Py230 and FVB/N MaCSC line Py9813. (d) Hollow mammospheres of Py230 cells in suspension culture. Scale bar 50 μm. (e,f) Py230 mammosphere stained with K14 and K8. Scale bar 20 μm. (g) Py230 mammosphere grown on collagen exhibits branching structures. Scale bar 100 μm. (h) Confluent Py230 cells spontaneously form domes. (i) Domes become enlarged upon treatment with lactogenic hormones dexamethasone and prolactin. Scale bar 50 μm. (j) Expression of beta-casein by MaCSC lines Py230 and Py9813 following treatment with lactogenic hormones. Data are means ± SEM of triplicate samples. (k) Py230 cells treated with retinoic acid and rosiglitazone express genes associated with adipocyte differentiation. Data are means ± SEM of triplicate samples. Inset: Py230 cells stained with oil red O. Scale bar 10 μm

Fig. 2

Py230 mammary cancer stem cells recapitulate spontaneous luminal tumorigenesis and metastasis. a-f, H&E stained sections of Py230 MIN (a), tumor (b) and metastasis (c) compared with spontaneous PyVmT MIN, (d), tumor (e) and metastasis (f). Scale bar = 50 μm. g-j Whole mounts of normal mammary gland (NMG), pre-lactating mammary gland (PLMG), PyVmT MIN and a 10-day outgrowth of Py230 tumor cells in the cleared mammary fat pad. Scale bar = 100 μm. k-r, Representative dot plots of NMG (k,o), PLMG (l,p), PyVmT MIN (m,q) and Py230 tumor (n,r) labeled with CD24 and CD49f (k-n) or CD24 and CD29 (o-r)

Fig. 3

Py230 cells differentiate to form multiple tumor subtypes in vivo. Spontaneous PyVmT tumors (upper panels) or Py230-derived tumors representing different subtypes: luminal A, Her2, basal and claudin-low (lower 4 panels) stained for estrogen receptor (ER), progesterone receptor (PR) or Her2 expression

Fig. 4

During tumor progression Py230 cells undergo changes that upregulate nuclear p53 expression. (a) Tumor latency of Py230 tumors from first generation tumor cell injections (T1, blue) compared with Py230 tumors from third generation injections (T3, red). (b) Karyotyping of the Py230 cell line (Py230) and Py230 first generation tumors from independent animals (T1). Cells isolated from tumors of subsequent generations (T2, T3) undergo changes that result in aneuploidy. (c) Expression profiling of tumors from various generations of Py230 injections compared with spontaneous PyVmT tumors. (d) IHC staining for p53 expression in the different Py230 tumor subtypes

Fig. 5

Py230 cells differentiate to form p53-positive mesenchymal cells. (a) Py230 MaCSCs treated with 0.1 % DMSO undergo epithelial to mesenchymal transition. (b) Flow cytometry profiles of a DMSO-treated Py230 clone showing the transition over time from a luminal to a claudin-low phenotype. (c) A third generation Py230 tumor showing clonal evolution to form an invasive p53-positive tumor extension. (d) Western blot of tumor lysates. Lane 1: tumor from early passage Py230 cells, lane 2: basal-like tumor from Py117, a cell line cloned from a third generation in vivo passaged Py230 tumor, lane 3: claudin-low tumor from Py15-4, a cell line clone from a third generation in vivo passaged Py230 tumor. (e) CD24, CD44 FACS profile of Py230 and Py15-4 clonal cell lines. (f) Karyotype of clonal luminal cell line Py230 and clonal claudin-low cell lines Py15-4 and Py16-1. (g) Growth curves of Py230 luminal cell line and Py15-4 and Py16-1 claudin-low cell lines. Data are means ± SEM of triplicate cultures. Both claudin-low cell lines were derived from Py230 cells

Fig. 6

Py230 MaCSC model. Py230 MaCSCs undergo lineage differentiation in vitro in response to appropriate stimuli. We postulate that the alveolar-like cells develop from luminal cells and that adipocyte-like cells differentiate from the multipotent MaCSC, hence the dashed lines. In vivo, Py230 MaCSCs consistently form luminal tumors. With serial passaging in vivo, epigenetic and/or genetic changes taking place during tumor progression that can lead to clonal evolution and tumor heterogeneity. We have derived clonal cell lines that give only to claudin-low tumors or basal-like tumors. To date we have not isolated clonal cell lines that give rise to Her2 tumors