Tracking the fate of adoptively transferred myeloid-derived suppressor cells in the primary breast tumor microenvironment

<div><p>Myeloid-derived suppressor cells (MDSCs) are a heterogeneous population of immature myeloid progenitor cells that are expanded in cancer and act as potent suppressors of the anti-tumor immune response. MDSCs consist of two major subsets, namely monocytic (M-) MDSCs and granulocytic (G-) MDSCs that differ with respect to their phenotype, morphology and mechanisms of suppression. Here, we cultured bone marrow cells with IL-6 and GM-CSF <i>in vitro</i> to generate a population of bone marrow MDSCs (BM-MDSCs) similar to G-MDSCs from tumor-bearing mice in regards to phenotype, morphology and suppressive-function. Through fluorescent labeling of these BM-MDSCs and optical imaging, we could visualize the recruitment and localization of BM-MDSCs in breast tumor-bearing mice <i>in vivo</i>. Furthermore, we were able to demonstrate that BM-MDSCs home to primary and metastatic breast tumors, but have no significant effect on tumor growth or progression. <i>Ex vivo</i> flow cytometry characterization of BM-MDSCs after adoptive transfer demonstrated both organ-and tumor-specific effects on their phenotype and differentiation, demonstrating the importance of the local microenvironment on MDSC fate and function. In this study, we have developed a method to generate, visualize and detect BM-MDSCs <i>in vivo</i> and <i>ex vivo</i> through optical imaging and flow cytometry, in order to understand the organ-specific changes rendered to MDSCs in breast cancer.</p></div

Breast tumor microenvironment dynamics after adoptive transfer of DiD-BM-MDSCs.

<p><b>A</b>) Schematic of treatment regimen for TME analysis after adoptive transfer of DiD-BM-MDSCs into tumor-bearing mice. Mice were injected with PyMT-WT cells into the MFP on day 0, and i.v. injected with 4x10⁶ DiD-BM-MDSCs on day 11 and 22. Tumors were analyzed by flow cytometry 48 hours (day 24) after the second dose of DiD-BM-MDSCs. PyMT-WT tumor-bearing mice were used as controls (control n = 5; DiD-BM-MDSC n = 6 for all analyses). <b>B</b>) Tumor weight at time of flow cytometry analysis at day 24. <b>C</b>) Flow cytometry analysis of CD3 lymphocytes, CD3/CD4 and CD3/CD8 T cells, as well as CD3^-/NK1.1⁺ NK cells within the tumor at day 24. <b>D</b>) Flow cytometry analysis of the percentage of Foxp3⁺ cells within the CD3/CD4 T cell population at day 24. <b>E</b>) Flow cytometry analysis of CD11b/Ly6C/Ly6G myeloid populations as a percentage of CD45.2⁺ cells within the tumor at day 24. <b>F</b>) Flow cytometry analysis of DCs (CD11c/MHC Class II) and macrophages (CD11b/F480/MHC Class II) as a percentage of CD45.2⁺ cells within the tumor at day 24. <b>G</b>) Representative flow cytometry plots of CD11b/Ly6C populations (hi = high, med = medium and lo = low) within the CD45.2⁺ population in the tumor at day 24 and quantified in <b>H.</b> Data represented as mean ± SEM. *p<0.05; n.s. not significant.</p

<i>In vitro</i> function and localization of BM-MDSCs <i>in vivo</i>.

<p><b>A</b>) Representative histogram of CFSE-labeled CD8 OT-I T cells co-cultured with splenocytes and BM-MDSCs at various ratios as indicated (n = 3 in triplicate). <b>B</b>) RT-PCR with fold change gene expression in G-MDSCs isolated from the tumor, spleen and lungs of PyMT-WT tumor-bearing mice normalized to gene expression in BM-MDSCs (indicated by broken line; n = 3 in triplicate). <b>C</b>) Percent viability of BM-MDSCs at 3, 24 and 48 hours after initial DiD-labeling for 2, 5 or 10 min relative to unlabeled cells (broken line). Data represented as mean ± SEM. *p<0.05; **p<0.01; ***p<0.001. <b>D, E</b>) OI <i>in vivo</i> (<b>D</b>) and of <i>ex vivo</i> organs (<b>E</b>) 7 days after i.v., i.c. or i.p. injection of 1x10⁶ DiD-labeled BM-MDSCs into naïve mice. BM = bone marrow, Li = liver, LN = lymph nodes, OM = omentum majus, S = spleen, T = thymus). RE = Radiant Efficiency; R = Radiance.</p

Homing of adoptively transferred DiD-BM-MDSCs to established metastases.

<p><b>A</b>) Representative <i>in vivo</i> images of Luc-PyMT metastases (BL signal) 3 weeks after i.c. injection into C57Bl/6 mice. <b>B</b>) Representative e<i>x vivo</i> images of DiD-BM-MDSC (FL signal; top panel) localization to adrenal gland metastases (BL signal; bottom panel). DiD-BM-MDSCs were injected (i.v.) into mice from A, and images acquired 2 weeks later. Quantification of radiant efficiency (RE) of FL-signal for the adrenal gland shown on the right. <b>C</b>) Representative images of spleens (left) and RE quantification (right) from treatment groups described in <b>A and B</b>. Naïve C57Bl/6 mice were used as controls. Data represented as mean ± SEM; *p<0.05; ***p<0.001; n = 3–5 mice for all groups.</p

Fate of adoptively transferred DiD-BM-MDSC in the tumor and spleen of breast tumor-bearing mice.

<p><b>A</b>) Representative flow cytometry plots of the CD45.2/DiD-Cy5⁺ gated population in PyMT-WT tumors from control mice (primary tumor alone; left) and after adoptive transfer of DiD-BM-MDSCs (right) on day 24 as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196040#pone.0196040.g005" target="_blank">Fig 5</a><b>B and</b> <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0196040#pone.0196040.g005" target="_blank">5</a><b>C</b>) Flow cytometry analysis of CD11b/Ly6C/Ly6G (B) and CD11b/Ly6C subpopulations (C) as a percentage of CD45/Cy5⁺ cells within the tumor. <b>D</b>) Flow cytometry analysis of mature myeloid populations including granulocytes (CD11b/Ly6G), macrophages and DCs as a percentage of CD45/Cy5⁺ cells within the tumor. <b>E</b>) Representative flow cytometry plots of the CD45.2/DiD-Cy5⁺ gated population within the spleen of PyMT-WT tumor-bearing mice (control; left) and after adoptive transfer of DiD-BM-MDSCs (right). <b>F,G</b>) Flow cytometry analysis of CD11b/Ly6C/Ly6G (F) and CD11b/Ly6C subpopulations (G) as a percentage of CD45/Cy5⁺ cells within the spleen. <b>H</b>) Flow cytometry analysis of mature myeloid populations including granulocytes, macrophages and DCs as a percentage of CD45/Cy5⁺ cells within the spleen. Data represented as mean ± SEM; n = 6 for all groups.</p

DiD-BM-MDSCs home to the primary breast tumor after adoptive transfer.

<p><b>A</b>) Schematic of treatment regimen for localization of DiD-BM-MDSCs in tumor-bearing mice. Mice were injected with 2.5x10⁵ Luc-PyMT cells into the MFP on day 0, and i.v. injected with 1x10⁶ DiD-BM-MDSCs on day 14. <i>In vivo</i> optical images were obtained at 48 hours (day 16) and 7 days (day 21) post-injection of DiD-BM-MDSCs. <i>Ex vivo</i> images were taken at 7 days post-injection (day 21). <b>B</b>) Representative <i>in vivo</i> FL and BL optical images on day 16; 48 hours after injection of DiD-BM-MDSCs. Tumors outlined by broken white line. <b>C)</b> Representative <i>in vivo</i> FL and BL optical images of mice from (A) on day 21; 7 days after injection of DiD-BM-MDSCs. Treatment groups include mice with Luc-PyMT tumors and adoptively transferred DiD-BM-MDSCs (mice 1–2; left), Luc-PyMT tumors alone (mice 3–4; middle) or DiD-BM-MDSCs alone (mouse 5; right). Tumors outlined by broken white line. <b>D</b>) Representative <i>ex vivo</i> FL and BL optical images of Luc-PyMT tumor-bearing mice (left) and individual organs (right) on day 21; 7 days after DiD-BM-MDSCs injection. Tumor indicated by red arrow. BM = bone marrow, H = heart, I = intestine, Ki = kidney, Li = liver, LN = lymph nodes, Lu = lungs, S = spleen, T = tumor. RE = Radiant Efficiency; R = Radiance.</p

Characterization of <i>in vitro</i>-cultured BM-MDSCs.

<p><b>A</b>) Schematic of <i>in vitro</i> BM-MDSC generation. BMDCs flushed from the femur and tibia of naïve C57Bl/6 mice were cultured with IL-6 and GM-CSF for 5 days and isolated by FACS based on CD11b⁺/Ly6C⁺/Ly6G⁺ expression. <b>B</b>) Representative Diff-Quick stained cytospin images of CD11b⁺/Ly6C⁺/Ly6G⁺ cells. Images show <i>in vitro</i>-cultured BMDCs before FACS (left; unsorted), BM-MDSCs after FACS (middle; sorted) and DiD-labeling (right). Images were taken at 60x magnification. <b>C</b>) Gating strategy and characterization of <i>in vitro</i> cultured CD11b⁺/Ly6C⁺/Ly6G⁺ BM-MDSCs (top row) and CD11b⁺/Ly6C^med/Ly6G⁺ G-MDSCs isolated by FACS from the lung (second row), spleen (third row) and tumor (bottom row) of PyMT-MMTV transgenic mice, for the expression of various surface markers by flow cytometry.</p