Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Chemotherapy induces breast cancer stemness in association with dysregulated monocytosis

doi: 10.1158/1078-0432.CCR-17-2545

Figure Lengend Snippet: (A) Twenty pairs of pre- and post-NT sera from TNBC (n=8; black) or HER2+ BC (n=12; blue) patients were analyzed for the activity to stimulate the ALDEFLUORbright population of BC cells. MDA-MB-231 cells were cultured for 48 h in base medium supplemented with 10% human serum before ALDEFLUOR assays by flow cytometry. Wilcoxon test was performed. (B) ELISA to determine the levels of human CCL2/7/8 in the 20 pairs of sera. Wilcoxon tests were performed. (C) ALDEFLUOR assays using 6 pairs of sera (3 cases for each BC subtype) were performed as in A except that CCL2/7/8 neutralizing antibodies (NAb; 30 ng/ml; alone or all 3 combined) or control IgG were added during serum treatment. (D) ALDEFLUOR assays of MDA-MB-231 cells treated with patient sera in the presence of a CCR2 inhibitor (MK-0812; 600 nM) or vehicle. (E) NSG mice with or without MDA-MB-231 xenograft tumors and tumor-free BALB/c mice received 3 weekly injections with doxorubicin (DOXO; 4 mg/kg) or docetaxel (DTX; 25 mg/kg) (n=3). Six days later, serum was collected to treat MDA-MB-231 cells for ALDEFLUOR assays as in A. (F) ELISA to determine the serum levels of mouse CCL2/7/8. (G) ALDEFLUOR assays of MDA-MB-231 cells treated with mouse sera in the presence of the CCR2 inhibitor MK-0812 or vehicle. (H) Sera from chemotherapy-treated patients and mice induce tumorigenicity. MDA-MB-231 cells pre-treated with human (case T1) or mouse (tumor-free BALB/c) sera for 48 h were injected into the mammary fat pad of NSG mice (n=10) at the indicated numbers. Tumor incidence after 4 weeks was shown. The estimated TIC frequency was estimated by ELDA. *P<0.05, **P<0.01, ***P<0.001 (compared to the corresponding IgG group in C or as indicated).

Article Snippet: The CCR2 inhibitor MK-0812 was purchased from Cayman Chemical (Ann Arbor, MI).

Techniques: Activity Assay, Cell Culture, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Injection

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Chemotherapy induces breast cancer stemness in association with dysregulated monocytosis

doi: 10.1158/1078-0432.CCR-17-2545

Figure Lengend Snippet: (A) Complete blood count showing changes of various cell populations upon NT in a total of 13 patients including 8 cases of TNBC (black) and 5 cases of HER2+ BC (blue) that were treated at the City of Hope National Medical Center. The green boxes in the background indicate normal ranges. Wilcoxon tests were performed. (B) Complete blood count showing the dynamics of various cell populations during 4 cycles of NT in 19 BC patients treated at the Tianjin Medical University Cancer Institute and Hospital. The start of each cycle is indicated by a blue arrowhead. The green boxes in the background indicate normal ranges. (C) Eighteen pairs of pre- and post-NT breast tumors from the City of Hope patients were analyzed by immunohistochemistry (IHC) to show the percentages of tumor cells positive for Ki-67 or ALDH1, as well as the staining scores for nuclear NOTCH1 in tumor cells. Wilcoxon tests were performed. (D) Representative IHC images from two cases. (E) Pre-NT breast tumors were analyzed by IHC for the expression of CCR2 in tumor cells. Tumors with pCR (n=15) were compared to those with non-pCR (n=20). Mann–Whitney test was performed. Representative IHC images are shown.

Article Snippet: The CCR2 inhibitor MK-0812 was purchased from Cayman Chemical (Ann Arbor, MI).

Techniques: Immunohistochemistry, Staining, Expressing, MANN-WHITNEY

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Chemotherapy induces breast cancer stemness in association with dysregulated monocytosis

doi: 10.1158/1078-0432.CCR-17-2545

Figure Lengend Snippet: (A) Schema of the mouse model used to examine if CCR2 intervention suppresses post-chemotherapy tumor progression. One million of MDA-MB-231 cells with stable knockdown of CCR2 (shCCR2) or those expressing control shRNA (shCTRL) were injected into the #4 mammary fat pad of NSG mice. When tumor size reached ~250 mm3, mice were treated with DTX for 3 weeks, and then left free of chemotherapy until the end of experiment. One group with MDA-MB-231-shCTRL tumors also received the CCR2 inhibitor MK-0812 starting with the chemotherapy and continuing for a total of 30 days, whereas the other two groups received the vehicle. (B) Tumor onset and volume (n=8). The two groups with MDA-MB-231-shCTRL tumors received 3 times of treatment with DTX on days 24, 31, and 38 (blue arrowheads); the group with MDA-MB-231-shCCR2 tumors received DTX on days 32, 39, and 46 (blue arrows). (C) ALDEFLUOR assay of dissociated MDA-MB-231 tumor cells. (D) Schema of the mouse models used to examine if chemotherapy prior to BC cell engraftment enhances tumor formation. BALB/c and NSG mice were treated with DOXO, DTX, or PBS for 3 weeks and then left free of chemotherapy for 1 week, before 1000 FACS-isolated ALDEFLUORbright 4T1 or PDX265922 cells were injected into the #4 mammary fat pad to assess tumor development. (E) Tumor onset and volume for the 4T1 model in BALB/c mice (n=8). Inset shows numbers of mice with palpable tumors on day 14. (F) ALDEFLUOR assay of dissociated 4T1 tumor cells. (G) Relative RNA levels of indicated genes (normalized to Gapdh) in 4T1 tumor tissue determined by quantitative RT-PCR assay. (H) Tumor onset and volume for the PDX265922 model in NSG mice (n=8). CCR2 inhibitor MK-0812 or vehicle was orally administered at 30 mg/kg twice a day starting with the chemotherapy and continuing for 30 days after BC cell engraftment. Inset shows numbers of mice with palpable tumors on day 18. (I) ALDEFLUOR assay of dissociated PDX265922 tumor cells. (J) Western analysis showing indicated protein levels in PDX265922 tumor tissue. *P<0.05, **P<0.01, ***P<0.001 (compared to the corresponding PBS group in E–G or as indicated).

Article Snippet: The CCR2 inhibitor MK-0812 was purchased from Cayman Chemical (Ann Arbor, MI).

Techniques: Expressing, shRNA, Injection, Isolation, Quantitative RT-PCR, Western Blot

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Chemotherapy induces breast cancer stemness in association with dysregulated monocytosis

doi: 10.1158/1078-0432.CCR-17-2545

Figure Lengend Snippet: (A & B) Co-cultures were set up using MDA-MB-231 or BT474 BC cells with monocytes isolated from C57BL/6 mice treated with DOXO, DTX, or PBS, or with DOXO/DTX/PBS-pretreated THP-1 cells, in the presence or absence of the CCR2 inhibitor MK-0812 (A) or CCL2/7/8 NAb (B) as described in Materials and Methods. BC cells and monocytes were seeded at a ratio of 1:1 or 1:3. ALDEFLUOR assays (for MDA-MB-231) and ESA+CD44+CD24−/low flow cytometry (for BT474) were performed using BC cells harvested after 48 h of co-culture. (C) MCP secretion by THP-1 and CAF was determined by ELISA of the conditioned media (CM) of 105 THP-1 cells or cancer-activated CAF (pre-treated with the CM from PDX265922 cancer cells) that had been treated with DOXO (125 nM), DTX (4 nM), or PBS for 48 h. (D) THP-1 cells under DOXO/DTX/PBS treatment and mouse monocytes isolated as in A were cultured in the presence or absence of a JNK inhibitor SP600125 (1 μM) for 48 h and analyzed by quantitative RT-PCR using GAPDH/Gapdh for normalization. (E) MDA-MB-231 cells were treated with CCL2/7/8 at the indicated concentrations for 48 h and analyzed by sphere formation assay or ALDEFLUOR assay. (F) PDX265922 cells derived from a primary TNBC were treated with CCL2/7/8 (1 ng/ml) or PBS for 48 h and analyzed by sphere formation assay or ALDEFLUOR assay. (G) CCL2/7/8-treated BT474 cells were analyzed by sphere formation assay or flow cytometry for the ESA+CD44+CD24−/low population. (H) Indicated BC cells were treated with CCL2/7/8 (1 ng/ml) or PBS for 24 h and analyzed by quantitative RT-PCR for indicated stemness-related genes. Data are normalized to GAPDH and compared to the PBS group. (I) Western blot analysis showing stemness-associated gene expression in BC cells treated with CCL2/7/8 or PBS for 24 h. (J) Western blot analysis of MDA-MB-231 cells treated with CCL2/7/8 or PBS in the presence or absence of a γ-secretase inhibitor (GSI) RO4929097 (10 μM). *P<0.05, **P<0.01, ***P<0.001 (compared to the control group in the first column of each group or as indicated).

Article Snippet: The CCR2 inhibitor MK-0812 was purchased from Cayman Chemical (Ann Arbor, MI).

Techniques: Isolation, Flow Cytometry, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, Cell Culture, Quantitative RT-PCR, Tube Formation Assay, Derivative Assay, Western Blot, Expressing

Journal: Journal of biochemistry

Article Title: Identification of effective CCR2 inhibitors for cancer therapy using humanized mice.

doi: 10.1093/jb/mvad086

Figure Lengend Snippet: Fig. 1. CCR2 inhibition suppresses lung metastasis of breast cancer cell line. (A) Strategy for generating mouse CCR2 knockout mice by CRISPR/Cas9 electroporation. The target sequence of the guide RNA was designed on the 5′ end side of the mouse CCR2 ORF. RNP was prepared by mixing the CRISPR RNA (Table 1) and transactivating crRNA with recombinant Cas9 protein. Mouse zygotes of the C57BL/6 J strain were subjected to electroporation with RNP. (B) T7E1 assay of mosaic mice obtained by CRISPR/Cas9 electroporation in Figure 1A. The CCR2 ORF regions of mosaic and wild-type mice were amplified by PCR using DNA obtained from the tails, and the PCR products were denatured, reannealed and treated with T7E1 endonuclease I. CCR2 mutations were found in mouse no. 6. (C) Sanger sequencing the mouse CCR2 ORF region in mouse no. 6. (D) Flow cytometry analysis of bone marrow cells recovered from wild-type and CCR2 knockout (KO) mice using anti-mouse CCR2 (Anti-mCCR2) antibody. (E) Flow cytometry analysis of lung M-MDSCs in wild-type and CCR2 KO mice. ∗∗∗P < 0.001 (unpaired two-sided Student’s t-test). (N = 4). (F) Schematic representation of breast cancer cells’ lung metastasis evaluation assay. Wild-type or CCR2 KO mice were injected with tdTomato-labeled E0771 cancer cell line into the tail vein, and 14 days later, lungs were removed and evaluated for tdTomato-positive lung metastases. (G) Lung metastases were evaluated 14 days after tail vein injection of tdTomato-labeled E0771 cancer cell line. Lungs were photographed under blue light irradiation (left). The percentage of tdTomato-positive area in the lung was quantitated with ImageJ software. (right). ∗∗∗P < 0.001 (unpaired two-sided Student’s t-test). Scale bars: 5 mm. (N = 15 for wild-type; N = 9 for CCR2 KO).

Article Snippet: THP-1 cell line (in HBSS with 0.05% BSA) was incubated with Fure 2-AM (final concentration 5.0 μM) for 30 min at 37◦C in the dark, washed twice with HBSS with 0.05% BSA and added each CCR2 inhibitor (MK0812 (Cayman), PF04634817 (Sigma-Aldrich), PF04136309 (PharmaBlock), INCB3344 (Chemscene), cenicriviroc (Axon), JNJ2714149 (R&D systems), CCR2RA-[R] (MedChemExpress), CAS-445479-97-0 (Santa Cruz Biotechnology), RS504393 (Sigma-Aldrich) and RS102895 (Sigma-Aldrich)).

Techniques: Inhibition, Knock-Out, CRISPR, Electroporation, Sequencing, Recombinant, Amplification, Flow Cytometry, Injection, Labeling, Irradiation, Software

Journal: Journal of biochemistry

Article Title: Identification of effective CCR2 inhibitors for cancer therapy using humanized mice.

doi: 10.1093/jb/mvad086

Figure Lengend Snippet: Fig. 3. Generation of human CCR2B knock-in mice. (A) Strategy for generating human CCR2B knock-in ES cells by homologous recombination. The human CCR2B knock-in construct consists of human CCR2B ORF and HA (homologous arm). (B) Genotyping of cloned ES cells by PCR. Knock-in of the human CCR2B allele was confirmed in clone no. 29. (C) Southern blot analysis of DNA from human CCR2B knock-in ES cells (clone no. 29). (D) F0 generation chimeric mice were obtained by microinjection of clone no. 29 into blastocysts. (E) F1 generation mice were obtained by crossing F0 generation chimeric mice with C57BL/6 J mice. (F) Genotyping of F1 generation mice by PCR. N.C., negative control, P.C., positive control. DNA extracted from wild-type C57BL/6 mice was used as N.C. DNA extracted from chimeric mice in Figure 4D as P.C. (G) Flow cytometry analysis of bone marrow cells recovered from wild-type and human CCR2B knock-in (indicated as hCCR2B KI) mice using anti-mouse CCR2 (shown as Anti-mCCR2) or anti-human CCR2 (shown as Anti-hCCR2) antibodies.

Article Snippet: THP-1 cell line (in HBSS with 0.05% BSA) was incubated with Fure 2-AM (final concentration 5.0 μM) for 30 min at 37◦C in the dark, washed twice with HBSS with 0.05% BSA and added each CCR2 inhibitor (MK0812 (Cayman), PF04634817 (Sigma-Aldrich), PF04136309 (PharmaBlock), INCB3344 (Chemscene), cenicriviroc (Axon), JNJ2714149 (R&D systems), CCR2RA-[R] (MedChemExpress), CAS-445479-97-0 (Santa Cruz Biotechnology), RS504393 (Sigma-Aldrich) and RS102895 (Sigma-Aldrich)).

Techniques: Knock-In, Homologous Recombination, Construct, Clone Assay, Southern Blot, Microinjection, Negative Control, Positive Control, Flow Cytometry