CD44posCD49fhiCD133/2hi defines xenograft-initiating cells in estrogen receptor-negative breast cancer - PubMed (original) (raw)

CD44posCD49fhiCD133/2hi defines xenograft-initiating cells in estrogen receptor-negative breast cancer

Matthew J Meyer et al. Cancer Res. 2010.

Abstract

Defining the populations of tumor-initating cells that are present in tumors is a first step in developing therapeutics to target these cells. We show here that both CD44(pos)CD24(neg) and CD44(pos)CD24(pos) cell populations in estrogen receptor (ER) alpha-negative breast tumors are tumorigenic in murine xenograft models. We also describe a third population of xenograft-initiating cells (XIC) enriched in CD44(pos)CD49f(hi)CD133/2(hi) cells that display heightened tumorigenicity, self-renewal in vivo, and the capacity to give rise to functional and molecular heterogeneity. Consistent with their capacity for self-renewal, these cells express elevated levels of Sox2, Bmi-1, and/or Nanog and their CpG islands are hypermethylated relative to nontumorigenic cells. These differences in methylome regulation may be responsible for the dramatic functional differences between the two populations. The identification of CD44(pos)CD49f(hi)CD133/2(hi) XIC in ER-negative tumors may lead to expanded understanding of these tumors and ultimately the development of therapeutics designed to specifically target the cells.

Copyright 2010 AACR.

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Conflict of interest statement

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Figures

Figure 1

Figure 1

Sorting strategy used for the isolation of CD44posCD49fhiCD133/2hi cells. Cells were depleted of nontumor cells from pleural effusions or mouse cells from xenografts by negative selection as described in Materials and Methods. Live cells were identified by their ability to exclude DAPI and discriminated from debris by their forward scatter (FSC) versus DAPI profile. Following depletion of doublets, CD44posCD49fhi cells were plotted by their side scatter (SSC) versus CD133/2 profile permitting identification of CD133/2hi cells. Gates were established (insets of CD44 versus CD49f and CD133/2 versus SSC) after compensation with live, lineage-negative cells not exposed to CD44, CD49f, or CD133/2 antibodies.

Figure 2

Figure 2

CD44posCD49fhiCD133/2hi cells recapitulate the immunohistochemical heterogeneity of the parental tumor. A, expression profile of CD44, CD49f, and CD133/2 in a pleural effusion (top) and xenograft (bottom) and their xenografts resulting from sorted CD44posCD49fhiCD133/2hi cells. B, tumor tissues from passage 1 SH12 xenograft and passage 2 SH12 xenograft initiated with CD44posCD49fhiCD133/2hi cells were subjected to immunohistochemical analysis with the specific antibodies indicated.

Figure 3

Figure 3

Molecular heterogeneity within tumors. A, transcript abundance of K14 and K18. Within each tumor, mRNA abundance in CD44pos CD49fhiCD133/2hi cells is expressed as a fold change relative to CD44pos and negCD49fneg/low CD133/2neg/low cells. *, P < 0.05. B, transcript abundance of Bmi-1, Nanog, and Sox2. Within each tumor, mRNA abundance in CD44posCD49fhiCD133/2hi cells is expressed as a fold change relative to CD44pos and negCD49fneg/low CD133/2neg/low cells. *, P < 0.05. C, ratio of promoter CpG methylation to global CpG methylation in sorted and unfractionated tumors. Histogram bar height corresponds to the ratio of cutting by methylation-sensitive restriction enzymes _Bss_HII and _Hpa_II. Lower restriction enzymatic activity is associated with hypomethylation. a, b, and c, within tumor, values with dissimilar letters are different at P < 0.05.

Figure 4

Figure 4

Localization of CD44, CD49f, and CD133/2 in primary ER-negative breast tissue and normal breast tissue. Confocal microscopy images of representative patient tumors and a normal breast sample illustrating localization of DNA (DAPI; blue), CD44 (red), CD133/2 (green), and CD49f (purple). In the merged images, cells simultaneously positive for all three markers appear white.

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