Myeloid-derived suppressor cells in mammary tumor progression in FVB Neu transgenic mice - PubMed (original) (raw)

Myeloid-derived suppressor cells in mammary tumor progression in FVB Neu transgenic mice

Fuminori Abe et al. Cancer Immunol Immunother. 2010 Jan.

Abstract

Female mice transgenic for the rat proto-oncogene c-erb-B2, under control of the mouse mammary tumor virus (MMTV) promoter (neuN), spontaneously develop metastatic mammary carcinomas. The development of these mammary tumors is associated with increased number of GR-1(+)CD11b(+) myeloid derived suppressor cells (MDSCs) in the peripheral blood (PB), spleen and tumor. We report a complex relationship between tumor growth, MDSCs and immune regulatory molecules in non-mutated neu transgenic mice on a FVB background (FVB-neuN). The first and second tumors in FVB-neuN mice develop at a median of 265 (147-579) and 329 (161-523) days, respectively, resulting in a median survival time (MST) of 432 (201 to >500) days. During tumor growth, significantly increased number of MDSCs is observed in the PB and spleen, as well as, in infiltrating the mammary tumors. Our results demonstrate a direct correlation between tumor size and the number of MDSCs infiltrating the tumor and an inverse relationship between the frequency of CD4(+) T-cells and MDSCs in the spleen. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assessment of enzyme and cytokine transcript levels in the spleen, tumor, tumor-infiltrating non-parenchymal cells (NPCs) and mammary glands revealed a significant increase in transcript levels from grossly normal mammary glands and tumor-infiltrating NPCs during tumor progression. Tumor NPCs, as compared to spleen cells from wild-type (w/t) mice, expressed significantly higher levels of arginase-1 (ARG-1), nitric oxide synthase (NOS-2), vascular endothelial growth factor (VEGF-A) and significantly lower levels of interferon (IFN)-gamma, interleukin (IL)-2 and fms-like tyrosine kinase-3 ligand (Flt3L) transcript levels. Transcript levels in the spleens of tumor-bearing (TB) mice also differed from normal mice, although to a lesser extent than transcript levels from tumor-infiltrating NPCs. Furthermore, both spleen cells and NPCs from TB mice, but not control mice, suppressed alloantigen responses by syngeneic control spleen cells. Correlative studies revealed that the number of MDSCs in the spleen was directly associated with granulocyte colony stimulating factor (G-CSF) transcript levels in the spleen; while the number of MDSCs in the tumors was directly correlated with splenic granulocyte macrophage stimulating factor (GM-CSF) transcript levels, tumor volume and tumor cell number. Together our results support a role for MDSCs in tumor initiation and progressive, T-cell depression and loss of function provide evidence which support multiple mechanisms of MDSC expansion in a site-dependent manner.

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Figures

Fig. 1

Fig. 1

The induction and growth, as well as, survival characteristics of mammary tumors in FVB-neuN mice are shown. a Cumulative hazard assessment for the development of the first and second palpable tumors. b A Kaplan Meier cumulative survival plot is shown. c Tumor growth rates for the mice that developed tumors using the average time to the development of a palpable tumor for time 0. The average ± SEM tumor volume is plotted for the first and second tumors

Fig. 2

Fig. 2

Hematoxylin and eosin stained, grossly normal mammary tissue from: a 2-month-old FVB-neuN mouse, b 4-month-old FVB-neuN mouse, c 6-month-old FVB-neuN mouse and d “Normal” mammary tissue in neuT mouse with gross neoplastic disease. ×200 magnification

Fig. 3

Fig. 3

a–c Photomicrographs (×200) are from non-necrotic foci of a mammary tumor in an FVB-neuN mice. a An immunohistochemical (IHC) stain of a primary tumor with an antibody to GR-1. The insert is an hematoxylin and eosin (H&E) stain shown ring and histiocytic cells marked by arrows. b Microtubules infiltrating the primary tumor (IHC using an antibody against CD31). c Tumor cell apoptosis as revealed by TUNNEL staining. d H&E stain of a pulmonary metastasis (×25)

Fig. 4

Fig. 4

Flow cytometric analysis using four color staining panels including: a Non-T-cell lymphocyte subsets, b T-cell subsets and, c myeloid subsets. Results are expressed as frequency and absolute number of the cellular subset in the spleens of FVB-neuN mice. Analysis of myeloid cells includes MDSCs (Gr-1+CD11b+), DC2s (CD11c+CD11b+), DC1s (CD11c+CD11b−) and plasmacytoid DCs (CD11c+CD11b−B220+). The results presented include spleen cells from wild-type (W/t) FVB mice, FVB-neuN mice at 2 months of age (Tg 2 m), 4 months of age (Tg 4 m), 6 months of age (Tg 6 m) and from the spleens of mice with gross tumor burdens (Tumor bear). N = 6–8 in each cohort. An asterisk represents a cohort significantly different from w/t spleens, while a pound sign represents a significant difference from NPC isolated from the tumor. The mean ± SEM is shown for the tumor NPCs, spleen cells from TB mice and from the spleens of w/t FVB mice

Fig. 5

Fig. 5

(a) Cellular analysis of the tumor-infiltrating non-parenchymal cells (NPCs) from FVB-neuN mice versus spleen cells from w/t versus TB (Tumor spleen) mice and (b, c) suppressive activity of spleen cells and NPCs from TB mice (b) Con-A mitogenic activity and (c) MLR coculture responses with irradiated allogeneic spleen cells from BALB/c. The tumors were surgically excised, reduced to 1 mm fragments using cross scalpel blades, collagenase/DNase dissociated and the NPCs separated from parenchymal cells by Ficoll hypaque density gradient centrifugation. The frequency of the various subpopulations are shown herein as an average ± SEM for the tumor NPCs (a) and incorporation of 3H-thymidine into stimulated spleen cells or NPCs from the TB animals and from the spleens of w/t FVB mice (b, c). And asterisk represents a significant difference from w/t mice; while a pound sign represents significantly different from the NPCs in TB mice

Fig. 6

Fig. 6

Expression of cytokine and enzyme transcript levels in (a) spleen cells and (b) mammary tumors and mammary tissues of FVB-neuN mice. The results presented represent the transcript levels compared to w/t spleens in w/t (FVB mice, FVB-neuN mice at 2 months of age (Tg 2 m), FVB-neuN mice at 4 months of age (Tg 4 m), FVB-neuN mice at 6 months of age (Tg 6 m) and from the spleens of mice with gross tumor burdens (Tumor bear). In the mammary tissue studies, (b) grossly normal tissue was also studied. N = 6–8 in each cohort. An asterisk represents a significant difference from w/t mice, while a pound sign represents a significant difference from TB mice. The mean ± SEM of the ΔΔCT versus w/t spleen cells is shown with the exception of mammary tissue which is compared to w/t mammary tissue

Fig. 7

Fig. 7

Analysis of enzyme and cytokine transcript levels (mean ± of the ΔΔCT) in spleen cells and tumor NPCs from TB mice versus w/t spleen cells. The results represent studies in w/t mice, spleens of mice with gross tumor burdens (tumor bear) and NPCs from tumors. N = 6–8 in each cohort. An asterisk represents a significant difference from w/t mice while a pound sign represents a significant difference from the NPCs in TB mice. The mean ±

SEM is shown in each instance

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