F B cell infiltration is a predictor ofSTAT3-High B Cells Crucial for Tumor Angiogenesispatient survival and correlates highly with activated STAT3 [18]. However, the underlying molecular Fruquintinib web mechanisms on B cellmediated tumor development are unclear. Angiogenesis is a hallmark of cancer and anti-angiogenesis therapies have shown promise for treating cancer [19?2]. Tumor angiogenesis requires the interplay between tumor cells and tumor-infiltrating stromal cells [23?6]. Several reports show that signal transducer and activator of transcription 3 (STAT3) is crucial for tumor angiogenesis [27?9]. Our recent studies have also demonstrated that STAT3 mediates multidirectional crosstalk among tumor cells, endothelial cells and myeloid cells in promoting tumor angiogenesis [30]. In the current study, we define a crucial role of B cells as well as their STAT3 activity as important contributors for tumor progression and tumor angiogenesis.Materials and Methods Ethics StatementThe study on human tissue array slides and human prostate tumor tissues was approved by the City of Hope Institutional Review Board (COH IRB 09213). Human melanoma tumor and normal skin tissue sections were provided by John Wayne Cancer Research Institute (JWCI), with approval from JWCI and Western Institutional Review Board (WIRB 1095596). Informed consent was 16985061 waived by the IRB because the research was performed on deidentified archival tissues. Mouse care and experimental procedures were carried out under pathogen-free conditions in accordance with established institutional guidance and approved protocols from the Institutional Animal Care and Use Committee of Beckman Research Institute at City of Hope Medical Center.isolate B cell populations for RNA and SC 66 site protein extraction. For coimplanting tumor cells with B cells into Rag12/2 mice, B cells isolated from spleen of tumor-bearing mice (16106) were mixed 10:1 ratio with either B16 or LLC tumor cells then injected into Rag12/2 mice. Tumor size was measured every other day for the indicated time. Tumors were harvested then pooled to prepare frozen tissue sections for immunofluorescent staining. Tumorinfiltrating B cells were also isolated from pooled 23148522 tumors to prepare RNA and protein for real-time RT-PCR and western blotting, respectively. To generate experimental lung metastasis model, B16 tumor cells (56105) were injected intravenously into C57BL/6 mice with Stat3+/+ or Stat32/2 B cells, which is generated by crossing Stat3flox and CD19-Cre mice. After 15 d, lungs were removed and washed in Hank’s buffered salt solution (HBSS). Number of visible metastatic tumor nodules was enumerated by counting individual nodules. B16 tumor nodules were easily identifiable due to their pigmentation.B Cell PreparationTo isolate tumor-infiltrating B cells, tumors were gently minced and incubated (30 min, 37uC) with collagenase D and DNAse solution (Roche, 400 U/ml). Cells were resuspended by repeated pipetting and filtered through a mesh filter. Mononuclear cells were separated by gradient centrifugation using Histopaque (Sigma, 1.083 g/ml) and kept as tumor-infiltrating immune cells. Then tumor B cells were isolated from immune cell mixtures using the Mouse CD19 Positive Selection Kit (EasySep, StemCell Technologies) or MACS Cell Separation System Positive Selection Kit (Miltenyi Biotec). B cells from spleens and lymph nodes were prepared in the same manner.MaterialsThe B16 mouse melanoma cell line and MB49 mouse bladder cancer cell line wer.F B cell infiltration is a predictor ofSTAT3-High B Cells Crucial for Tumor Angiogenesispatient survival and correlates highly with activated STAT3 [18]. However, the underlying molecular mechanisms on B cellmediated tumor development are unclear. Angiogenesis is a hallmark of cancer and anti-angiogenesis therapies have shown promise for treating cancer [19?2]. Tumor angiogenesis requires the interplay between tumor cells and tumor-infiltrating stromal cells [23?6]. Several reports show that signal transducer and activator of transcription 3 (STAT3) is crucial for tumor angiogenesis [27?9]. Our recent studies have also demonstrated that STAT3 mediates multidirectional crosstalk among tumor cells, endothelial cells and myeloid cells in promoting tumor angiogenesis [30]. In the current study, we define a crucial role of B cells as well as their STAT3 activity as important contributors for tumor progression and tumor angiogenesis.Materials and Methods Ethics StatementThe study on human tissue array slides and human prostate tumor tissues was approved by the City of Hope Institutional Review Board (COH IRB 09213). Human melanoma tumor and normal skin tissue sections were provided by John Wayne Cancer Research Institute (JWCI), with approval from JWCI and Western Institutional Review Board (WIRB 1095596). Informed consent was 16985061 waived by the IRB because the research was performed on deidentified archival tissues. Mouse care and experimental procedures were carried out under pathogen-free conditions in accordance with established institutional guidance and approved protocols from the Institutional Animal Care and Use Committee of Beckman Research Institute at City of Hope Medical Center.isolate B cell populations for RNA and protein extraction. For coimplanting tumor cells with B cells into Rag12/2 mice, B cells isolated from spleen of tumor-bearing mice (16106) were mixed 10:1 ratio with either B16 or LLC tumor cells then injected into Rag12/2 mice. Tumor size was measured every other day for the indicated time. Tumors were harvested then pooled to prepare frozen tissue sections for immunofluorescent staining. Tumorinfiltrating B cells were also isolated from pooled 23148522 tumors to prepare RNA and protein for real-time RT-PCR and western blotting, respectively. To generate experimental lung metastasis model, B16 tumor cells (56105) were injected intravenously into C57BL/6 mice with Stat3+/+ or Stat32/2 B cells, which is generated by crossing Stat3flox and CD19-Cre mice. After 15 d, lungs were removed and washed in Hank’s buffered salt solution (HBSS). Number of visible metastatic tumor nodules was enumerated by counting individual nodules. B16 tumor nodules were easily identifiable due to their pigmentation.B Cell PreparationTo isolate tumor-infiltrating B cells, tumors were gently minced and incubated (30 min, 37uC) with collagenase D and DNAse solution (Roche, 400 U/ml). Cells were resuspended by repeated pipetting and filtered through a mesh filter. Mononuclear cells were separated by gradient centrifugation using Histopaque (Sigma, 1.083 g/ml) and kept as tumor-infiltrating immune cells. Then tumor B cells were isolated from immune cell mixtures using the Mouse CD19 Positive Selection Kit (EasySep, StemCell Technologies) or MACS Cell Separation System Positive Selection Kit (Miltenyi Biotec). B cells from spleens and lymph nodes were prepared in the same manner.MaterialsThe B16 mouse melanoma cell line and MB49 mouse bladder cancer cell line wer.
