<i>Csfr1</i> mRNA is reduced in monocytes from <i>Ccr2</i>^<i>-/-</i> mice.

<p>Monocytes were purified from the blood of wild type, <i>Ccl2</i>^<i>-/-</i>, and <i>Ccr2</i>^<i>-/-</i> mice (5 mice in each group). mRNA was isolated and mRNA encoding CSF-1R was quantified by PCR. * p > 0.05 by t-test; ** p < 0.05 by t-test.</p

Measurement of tumor-associated leukocytes and endothelial cells.

<p>Tissue microarrays were prepared from MMTV-<i>neu</i>-driven tumors in wild type, <i>Ccl2</i>^<i>-/-</i>, and <i>Ccr2</i>^<i>-/-</i> mice at 60, 80, 100, 120 and 140 days of age. Four or five mice were included per data point. Tissue microarray slides treated with fluorescent antibodies against (<b>A</b>) Ki-67, (<b>B</b>) vWF, (<b>C</b>) CD31, and (<b>D</b>) Mac2 and imaged by fluorescence or immunohistochemistry. Quantified stains were compared using one-way ANOVA. *, p < 0.05; **, p < 0.01; ***, p < 0.001.</p

CCL2’s effects on mammary carcinoma cells are non-cell autonomous.

<p>(<b>A</b>) <i>In vitro</i> growth of mammary carcinoma cell lines derived from MMTV-<i>neu</i>-induced tumors in wild type, <i>Ccl2</i>^<i>-/-</i>, and <i>Ccr2</i>^<i>-/-</i> mice. Two representative cell lines were tested from each genotype. (<b>B</b>) Neutralization of CCL2 in cultures of wild type murine tumor cells or addition of exogenous CCL2 to <i>Ccl2</i>^<i>-/-</i> tumor cells did not affect cell proliferation <i>in vitro</i> as measured by ³H-thymidine incorporation. Etoposide was used as positive control for growth inhibition. (<b>C</b>) Concentration of CCL2 protein in medium or cell lysates of cultured MCF7, SK-BR-3 or MDA-MB-231 human breast cancer cell lines determined by ELISA. (<b>D</b>) Immunoblot detection of full-length and cleaved poly(ADP-ribose) polymerase (PARP) in lysates from MCF7, SK-BR-3 or MDA-MB-231 cells treated with CCL2-neutralizing antibody. Etoposide was used as a positive control for PARP cleavage. Blots were stripped and reblotted for tubulin as a loading control. (<b>E</b>) MDA-MB-231 cells were treated with two different CCL2-neutralizing antibodies, and cell proliferation was measured by ³H-thymidine incorporation. (<b>F</b>) Luciferase-expressing MDA-MB-231 cells were injected into the mammary fat pads of SCID mice. Mice were treated with ABN912, a neutralizing anti-human CCL2 antibody, or isotype control. Arrows indicate time of antibody treatment. Tumor growth was followed by bioluminescence imaging.</p

Effect of mammary tumors, <i>Ccl2</i> disruption, and <i>Ccr2</i> disruption on monocyte accumulation in spleen, bone marrow, and tumors.

<p>(<b>A</b> and <b>B</b>) Proportions of monocytes in the spleen (<b>A</b>) and bone marrow (<b>B</b>) of tumor-free and tumor-bearing wild type, <i>Ccl2</i>^<i>-/-</i> and <i>Ccr2</i>^<i>-/-</i> mice were measured by flow cytometry. Comparisons were made by one-way ANOVA and Bonferroni’s multiple comparison test. #, p < 0.05; ##, p < 0.01 wild type <i>versus Ccl2</i>^<i>-/-</i> or <i>Ccr2</i>^<i>-/-</i> mice. (<b>C</b>) Total leukocyte content of tumors was determined as the percentage of CD45⁺ cells. (<b>D</b>) Proportions of tumor-infiltrating T cells, B cells, and monocytes/macrophages were determined by flow cytometry using antibodies against CD90⁺, B220⁺, and CD11b⁺, respectively.</p

Gene sets enrichment analysis of genes enriched in wild type compared to <i>Ccr2</i>^<i>-/-</i> monocytes.

<p>(A) Enrichment plots of wound healing, inflammation and locomotory behavior pathways. (B) Common genes enriched in similar pathways.</p

Effect of pharmacologic inhibition of CCR2 on mouse mammary carcinoma development.

<p>(<b>A</b> and <b>B</b>) Overall survival and tumor-free survival of <i>neu</i>^<i>+</i> mice treated with the CCR2 antagonist CCX872 (n = 26) or vehicle (n = 25). Median survivals were compared using Log-rank (Mantel-Cox) test. Mice were sacrificed when the diameter of any single tumor reached 2 cm, if a tumor became necrotic, or if mice were unable to reach food or water (<b>C</b> and <b>D</b>) Tumor growth in mice treated with CCX872 or vehicle measured as total tumor volume in a mouse <b>(C)</b> or as the volume of the single largest tumor mass in a mouse <b>(D)</b>. Tumors were measured twice weekly and averaged. Comparisons were made by two-way ANOVA.</p

CCL2 production associated with MMTV-neu-driven tumors.

<p>(<b>A</b>) CCL2 concentrations were measured by ELISA in sera from 4 wild type and 6 <i>neu</i>^<i>+</i> mice at the indicated ages. The difference between wild type and tumor bearing mice was significant by two-way ANOVA (p < 0.05). (<b>B</b>) Cell lines were developed from <i>neu</i>^<i>+</i>, <i>neu</i>^<i>+</i>/<i>CCL2</i>^<i>-/-</i>, and <i>neu</i>^<i>+</i>/<i>CCR2</i>^<i>-/-</i> mice. CCL2 released into the culture medium by 10⁶ cells over a 72 hr period was measured by ELISA.</p

<i>In vivo</i> anti-sarcoma response by IGF1R and ROR1 CAR T cells in a localized tumor mouse model.

<p>(A) The experimental schedule of tumor cell injection, CAR T cell infusion and BLI monitoring. Prior to testing, all mice displayed normal healthy status. (B) Bioluminescent imaging (BLI) of tumor growth in NOD/SCID mice (four groups, n = 12–13 each) treated with a sarcoma patient derived T cells expressing IGF1R CAR (IGZ), ROR1 CAR (RGZ) or mock T cells. One group mice were untreated. Two mice in the untreated group died of tumor progression on day 13 and 20 and the other two mice died of unknown causes on day 12 and 13. Three mice in the mock group died of tumor progression on day 7, 12 and 19 andone died of unknown causes on day 13. Three mice in the IGZ group died of unknown causes on day 10 and two died of unknown courses on day 17 and 18. Two mice in the RZG group died of tumor progression on day 12 and 50 andone died of unknown causes on day 13. (C) Bioluminescent intensity of the mice treated with the T cells. All <i>p</i> values were shown in the right panel table and were verified independently. (D) Animal survival after T-cell therapy. All <i>p</i> values were determined by the Mantel-Haenszel logrank test and are shown in the right panel table.</p

<i>In vitro</i> anti-sarcoma cytotoxicity of IGF1R and ROR1 CAR T cells.

<p>(A) Cytotoxicity against IGF1R⁺ target cells including sarcoma cell lines by SB modified IGF1R CAR T cells derived from two healthy donors. PBL1-IGZ, PBL1-IG, PBL2-IGZ and PBL2-mock T cells were generated by transfection of PBMCs derived from two healthy donors (PBL1 and PBL2) using pKT2-CaIG:Z or pKT2-CaIG plus pCMV-SB100X plasmids or without DNA. PBL-CD19CAR T cells were used as control. PBL1-IGZ: Rh1 vs K562 and OS17 vs K562, <i>p</i> < 0.001 at all 3 E/T ratios. PBL1-IG: Rh1 vs K562 and OS17 vs K562, <i>p</i> < 0.01. PBL2-IGZ: Rh1 vs K562, <i>p</i> < 0.001, OS17 vs K562, <i>p</i> < 0.01. Killing of Rh1 and OS17 in each CAR T cell group was also significant compared to the corresponding tumor cells in mock T cell group (<i>p</i> < 0.05). (B) Cytotoxicity of IGF1R CAR T cells against a panel of sarcoma cell lines. Comparisons in PBL1-IGZ and PBL2-IGZ were conducted between each sarcoma line and K562, <i>P</i> = 0.0001 at all E/T ratios. (C) Cytotoxicity against sarcoma cells by IGF1R CAR T cells derived from four more healthy donors but not by mock T cells. Comparisons in PBL3-IGZ, PBL4-IGZ, PBL5-IGZ and PBL6-IGZ were conducted between Rh30 vs K562 and SaOS2 vs K562, <i>P</i> = 0.0001 at all E/T ratios.(D) Expression of human IGF1R in R- transfected cell line confirmed by flow cytometry. (E) Specific cytotoxicity against human IGF1R transfected cell line by IGF1R CAR T cells. Comparisons in PBL3-IGZ, PBL4-IGZ, PBL5-IGZ and PBL6-IGZ were conducted between R- and R-/IGF1R, <i>P</i> = 0.0001 at all E/T ratios. (F) Expression of ROR1 in DB and RBV-LCL cell lines. ROR1^- K562 and ROR1⁺ RPMI8226 were used as control (data not shown). (G) Cytotoxicity against ROR1⁺ target cells including a sarcoma cell line by ROR1 CAR T cells derived from two healthy donors. Comparisons in PBL7-RGZ, PBL7-GZ, PBL8-RGZ and PBL8-GZ were conducted between each target cell and K562 at all E/T ratios. PBL7-RGZ: SaOS2 vs K562, <i>p</i> = 0.0001, 0.0008, 0.0001 (E/T of 60:1, 20:1 and 6:1). PBL7-GZ: SaOS2 vs K562, <i>p</i> = 0.0221, 0.0762, 0.1121. PBL8-RGZ: SaOS2 vs K562, <i>p</i> = 0.0001, 0.0009, 0.0041. PBL8-GZ: SAOS2 vs K562, <i>p</i> = 0.2585, 0.8439, 0.9298. <i>P</i> values for DB vs K562 and EBV-LCL vs K562 were not shown. All cytotoxicity data shown are mean ± S.E. of triplicates.</p

<i>In vivo</i> anti-sarcoma activity of IGF1R and ROR1 CAR T cells in a disseminated mouse model.

<p>(A) The experimental schedule of tumor cell injection, CAR T cell infusion and BLI monitoring. Prior to testing, all mice displayed normal healthy status. B) Bioluminescent imaging (BLI) of tumor growth in NSG mice (three groups, n = 6–8 each) treated with a sarcoma patient derived T cells expressing IGF1R CAR (IGZ), ROR1 CAR (RGZ) or mock T cells. SaOS2-fflucN cells were transduced with a lentiviral vector expressing humanized firefly luciferase and truncated nerve growth factor receptor (NGFR). Two mice in the mock group died of tumor progression on day 8. Four mice in IGZ group died of unknown causes on day 8, 9, 16 and 24, probably due to cytokine storms. (C) Bioluminescent intensity of the mice treated with the T cells. All <i>p</i> values were shown in the right panel table and were verified independently. (D) Animal survival after T-cell therapy. All <i>p</i> values were determined using Mantel-Haenszel logrank test and shown in the right panel table. The <i>p</i> values were independently confirmed. Note that <i>p</i> > 0.05 between mock vs IGZ was likely due to a small sample size.</p