Figure 1.
Breast cancer rate of metastasis to bone is not correlated to in vitro or in vivo growth rate.
A. COX2 expression, measured by quantitative RT-PCR, by TM40D, TM40D-MB, TM40D-COX2, and TM40D-MB-shCOX2 cells. B. Metastasis to bone from primary TM40D, TM40D-COX2, highly aggressive TM40D-MB, or TM40D-MB-shCOX2 cells which also over-express COX2 was assessed at maximum tumor size by homogenizing both hind leg bones followed by in vitro growth under selection media and complemented with genomic DNA PCR for GFP. C. MTT cell proliferation assay performed on TM40D and TM40D-COX2 cells to measure in vitro growth. D. Mice were implanted with either low tumorigenic/COX2 expressing TM40D cells or COX2 over-expressing TM40D-COX2 cells into fourth mammary fat pad and monitored for in vivo tumor growth rates. N = 1/10, 4/8, 5/9, and 0/5 for TM40D, TM40D-COX2, TM40D-MB, and TM40D-MB-shCOX2, respectively. *p<0.05 compared to TM40D and TM40D-MB-shCOX2.
Figure 2.
Tumor immune profile reveals elevated Tregs levels.
TM40D (grey bar) or COX2 over-expressing TM40D-COX2 (black bar) cells were implanted in the fourth mammary pad of Balb/c mice. At maximum tumor volume tumors were assessed for (A) CD4+ and CD8+ T-cells, (B) immature monocytes (CD11b+ F4/80+ Ly6g+), tumor associate macrophages (CD11b+ F4/80+ Ly6g−), G MDSCs (CD11b+ Ly6clow Ly6g+) and M MDSCs (CD11b+ Ly6Chi Ly6g−). Tregs (FoxP3+CD4+ CD25+) were assessed via flow cytometry of spleens from TM40D and TM40D-COX2 challenged mice (C). Quantitative analysis of Tregs observed in the tumor of tumor challenged mice (D). Representative Treg levels in the primary tumor of mice challenged with TM40D or TM40D-COX2 mammary tumor cells (E). Quantitative analysis of Tregs observed in the spleen of TM40D versus TM40D-COX2 challenged mice (F). p<0.05 compared to TM40D group, n = 4–5 animals per group. *p<0.05 compared to TM40D.
Figure 3.
TM40D-COX2 tumors preferentially recruit Tregs to the tumor.
Histological sections of TM40D and TM40D-COX2 tumors were stained with (A and B) H&E or (C and D) AlexaFluor 594 for CD4 (RED) AlexaFluor 488 for FoxP3 (GREEN), or Dapi for nucleus (BLUE). Arrows in panels C and D represent Tregs. E. Quantitative analysis of panels C and D measuring the percent of CD4+ cells that have FoxP3 co-localized to the cell. N = 3 samples per group, 8–10 sections per sample. *p<0.01 compared to TM40D. Bar = 20 µm.
Figure 4.
PGE2 directly involved in inducing Treg migration.
CD4+ CD25+ Tregs were enriched by magnetic column (MACS) from naïve thymus and lymph nodes (A). Treg migration was measured using media alone, cell-free supernatant collected from TM40D, TM40D-COX2, or TM40D-MB cells (B). Additionally, the role of PGE2 was assessed by anti-PGE2 blocking antibody added to TM40D-COX2 media. Purified GFP-FoxP3+ cells were isolated from spleen following CD4+ T cell enrichment (C) and analyzed for expression of the PGE2 receptors EP1, EP2, EP3, or EP4 via RT-PCR with GAPDH as a control (D). #p<0.05, *p<0.01 compared to Media alone, N = 3 samples per group.
Figure 5.
TM40D-COX2 tumors have increased apoptotic CD8+ T cells.
Histological sections of TM40D and TM40D-COX2 tumors were stained with AlexaFluor 594 for CD8 (RED) AlexaFluor 488 for Cleaved Caspase 3 (ClCasp3+) (GREEN), and Dapi for nucleus (BLUE) (A and B, respectively). C. Quantitative analysis of panels A and B measuring the percent of CD8+ cells that have cleaved Caspase 3 co-localized to the cell. N = 3 samples per group, 8–10 sections per sample. *p<0.01 compared to TM40D.