Resident macrophages influence stem cell activity in the mammary gland

Abstract

Introduction: Macrophages in the mammary gland are essential for morphogenesis of the ductal epithelial tree and have been implicated in promoting breast tumor metastasis. Although it is well established that macrophages influence normal mammopoiesis, the mammary cell types that these accessory cells influence have not been determined. Here we have explored a role for macrophages in regulating mammary stem cell (MaSC) activity, by assessing the ability of MaSCs to reconstitute a mammary gland in a macrophage-depleted fat pad.

Methods: Two different in vivo models were used to deplete macrophages from the mouse mammary fat pad, allowing us to examine the effect of macrophage deficiency on the mammary repopulating activity of MaSCs. Both the Csf1op/op mice and clodronate liposome-mediated ablation models entailed transplantation studies using the MaSC-enriched population.

Results: We show that mammary repopulating ability is severely compromised when the wild-type MaSC-enriched subpopulation is transplanted into Csf1op/op fat pads. In reciprocal experiments, the MaSC-enriched subpopulation from Csf1op/op glands had reduced regenerative capacity in a wild-type environment. Utilizing an alternative strategy for selective depletion of macrophages from the mammary gland, we demonstrate that co-implantation of the MaSC-enriched subpopulation with clodronate-liposomes leads to a marked decrease in repopulating frequency and outgrowth potential.

Conclusions: Our data reveal a key role for mammary gland macrophages in supporting stem/progenitor cell function and suggest that MaSCs require macrophage-derived factors to be fully functional. Macrophages may therefore constitute part of the mammary stem cell niche.

Figure 1

The MaSC-enriched population from Csf1^op/op mice yields smaller outgrowths in vivo and has lower clonogenicity in vitro. (a) Wholemounts of fourth mammary glands from eight-week-old wild-type and Csf1^op/op mice showing delayed filling of the fat pad and persistence of terminal end buds in mutant animals. (b) Wholemounts of outgrowths following transplantation of CD29^hiCD24⁺ cells from wild-type or Csf1^op/op mammary glands into the cleared fat pads of syngeneic wild-type recipients. (c) CD29^hiCD24⁺ cells from Csf1^op/op mammary glands exhibit lower clonogenicity and colony size than those from control glands. Histogram (upper panel) showing colony forming capacity of 100 CD29^hiCD24⁺ cells for each genotype. Error bars show standard error of the mean for three independent experiments. Lower panel shows cultures of CD29^hiCD24⁺ cells grown on fibroblast feeder layers. Scale bars = 1 mm. Csf1 = colony stimulating factor 1; MaSC = mammary stem cell.

Figure 2

Wild-type mammary stem cells are unable to efficiently repopulate macrophage-deficient fat pads. (a) Wild-type CD29^hiCD24⁺ cells transplanted into Csf1^op/op fat pads resulted in only a single small outgrowth whereas those transplanted into wild-type recipients resulted in extensive filling of the fat pad, all harvested at eight weeks post-transplantation. (b) Histogram showing percent outgrowth and the extent of fat pad filling following transplantation of normal CD29^hiCD24⁺ cells into wild-type or Csf1^op/op fat pads. Error bars represent standard error of the mean for three independent experiments. (c) Clodronate liposomes inhibit mammary stem cell function. CD29^hiCD24⁺ cells (200) from Rosa26 mice were co-transplanted with liposomes containing either saline or clodronate into the cleared mammary fat pads of syngeneic wild-type female mice. The cells co-transplanted with clodronate liposomes resulted in no outgrowths or rudimentary structures, an example of which is shown. Scale bars = 1 mm. Csf1 = colony stimulating factor 1.