Liver histopathology of metastases derived from CEA-positive C15A.3 murine tumor cells (A–C) or CEA-negative parental MC38 colon tumor cells (D–F).

<p>(A, D) H&E staining of liver metastases surrounded by normal tissue. (B, E) Higher magnification, emphasizing the difference in the tumor cell growth patterns with respect to the normal-to-tumor tissue border. (C, F) Immunohistochemistry of serial sections stained for CEA with CEA-specific M5A antibody confirming the CEA-expression status of the metastases. Note many central necrotic cells as indicated by arrows in B, C and E in both metastases. Bar size (A, D) 125 µm, all other 50 µm.</p

MRI and PET scan results for liver metastases originating from C15A.3 and MC38 cells 24 h post-injection of ⁶⁴Cu-DOTA-M5A antibody.

<p>MRI images clearly show the location of the liver metastases (arrows) and were used to draw regions of interest (ROIs) for evaluating the PET data. Immuno-PET images indicate strong signals in the areas of the CEA-positive C15A.3-derived liver metastases. No enhanced tracer uptake was observed in the areas of CEA-negative MC38 derived metastases.</p

The development of liver metastases in mice monitored by micro-CT.

<p>To establish a suitable pre-screening routine, a separate group of animals was subjected to a time-series of micro-CT scans. Images were acquired 3 h post injection of a single dose of hepatocyte-specific contrast agent (ExiTron nano 6000) without the use of additional injections for follow-up. Images were collected at days 9 to 19 and days 20 to 49 for CEA-negative MC38 or CEA-positive C15A.3 metastases, respectively. Arrows indicate the same metastases at different time points for both cell lines. Note the differences in the size and number of metastases originating from MC38 (upper row) compared to C15A.3 (lower row).</p

<i>Ex vivo</i> autoradiography (A), H&E staining (B, D) and M5A-immunohistochemistry (C, E) of the CEA-positive C15A.3-derived metastases.

<p>Evaluation of the cryosections was performed 24 h post-injection of ⁶⁴Cu-DOTA-M5A antibody. (A) Autoradiography shows the heterogeneous distribution of M5A within metastases. (B, C) The location of metastases in H&E and immunostaining correlates with the location of the metastases shown in (A). Note the intense, dark staining of the C15A.3 tumor cells near the tumor-to-normal border (D, E arrows). Bar size A-C 3 mm and C-D 200 µm respectively.</p

Uptake of the ⁶⁴Cu-DOTA-labeled M5A antibody in CEA-positive C15A.3- and CEA-negative MC38-derived liver metastases.

<p>(A) Depicted are the %ID/cc of the labeled M5A antibody in either C15A.3 or MC38 derived metastases and the uptake of the control antibody hu14.18 at three different time points post injection of antibodies. For blocking experiments, 500 µg of unlabeled M5A was injected 3 h prior to labeled M5A. (B) The ratio of the antibody uptake between metastases and healthy liver. For A+B n = 8 animals for C15A.3+M5A, n = 7 animals for MC38+M5A, n = 2 animals for blocking and n = 4 animals for control antibody (hu14.18). All animals were scanned at every time-point. (C) %ID/cm³ of ⁶⁴Cu-DOTA-labeled M5A antibody in several organs at different time points. (D) Focus on the %ID/cm³ of ⁶⁴Cu-DOTA-labeled M5A in the right ventricle. For C+D n = 7 at all time points. Statistically significant results are designated with * when p≤0.05.</p

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

PET imaging of the proliferation in <i>Ye</i>-infected mice and the histological findings.

<p>(A) Coronal [¹⁸F]FLT-PET and fused PET and MR images from PBS-treated (n = 3), low- (n = 7) and high dose- (n = 8) infected mice 3 days <i>p</i>.<i>i</i>. The arrows indicate the positions of the spleens, which showed no increased uptake of [¹⁸F]FLT in <i>Ye</i> infected animals compared to PBS-treated controls. (B) Immunohistochemical stainings of spleen tissue sections from the PBS-treated, low dose-, and high dose-infected mice 3 days <i>p</i>.<i>i</i>. Sections were stained for H&E, Ki 67 and Tk-1.The encircled areas indicate germinal centers. A: abscess, RP: red pulp, WP: white pulp. Data are representative of 6 analyzed mice.</p

<i>In vivo</i> biodistributions of [¹⁸F]FLT in the PBS-treated control, low-dose and high-dose Ye-infected mice.

<p>The averages and standard deviations are given for indicated organs based on the ROIs of the PET images. Static 10 min PET scans of PBS-treated, low-dose- and high-dose infected mice were acquired 1, 2 and 3 days <i>p</i>.<i>i</i>. The errors indicate one standard deviation. 11 to 13 MBq of [¹⁸F]FLT were administered to each mouse.</p

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