Genesis through STAT3-regulated pro-angiogenic genes in human tumors, we first stained human prostate tumor tissues for pSTAT3, CD19 and CD31. We then prepared RNAs from the tumor tissues with differential numbers of p-STAT3-positive B cells. Results from the analysis indicated that King the top 100 proteins identified in the first step of analysis expression levels of STAT3-regulated pro-angiogenic genes, such as S1PR1, MMP9 and HIF1a, correlated with the density of tumor-infiltrating B cells in human prostate cancers (Fig. 5A). On the other hand, anelevated expression of p53 was associated with lack of B cells in the tumors (Fig. 5A). The important role of p53 in inhibiting tumor angiogenesis and the inhibitory effect of STAT3 on p53 expression has been documented [39?1]. By co-staining tumor tissues with antibodies recognizing B cells and blood vessels, we observed that CD19+ B cells had a tendency to accumulate around microvessels rather than distribute evenly throughout human tumor tissues (Fig. 5B and Figure S5). Moreover, B cells around tumor vasculature exhibited persistently activated STAT3 (Fig. 5C).DiscussionA crucial role for tumor STAT3 in upregulating proliferation/ survival of tumor cells as well as dampening proper function of immune cells such as myeloid cells and T cells has been well characterized [37,38,42]. Our study further reveals a previously unrecognized role of 16985061 B cell STAT3 in accelerating tumor progression through increasing angiogenesis. Since B cells areFigure 4. B cells with activated STAT3 accumulate in human tumors. (A) Immunofluorescent staining of human melanoma and normal human skin tissue sections; anti-CD19 (red; B cell marker) and anti-p-STAT3 (green). Scale bars, 20 mm. (B) B cells in primary tumor sites impact overall tumor STAT3 activity. Confocal microscopic get KS 176 images showing primary melanoma tumor tissue staining of B cells and p-STAT3 (left), with quantification of CD19 and p-STAT3 positive cells (right). Scale bars, 20 mm. Total ten microscopic fields (10 X) were examined for each tumor section; n = 2. doi:10.1371/journal.pone.0064159.gSTAT3-High B Cells Crucial for Tumor AngiogenesisFigure 5. B cells with activated STAT3 express pro-angiogenic genes and accumulate around microvessels in human tumors. (A) B cells are important for expression of pro-angiogenic genes within human prostate tumor tissues. The density of B cells around tumor vasculature in prostate tumor tissue was determined by immunofluorescent staining using anti-CD19 and anti-CD31 antibodies (top); scale bars, 20 mm. Real-time RT-PCR measuring RNA expression levels of pro-angiogenic genes in the consecutive human prostate tumor tissue sections (bottom). The relative amount of mRNA is normalized to 18S and compared to RNA levels in tumor tissues with high p-STAT3, which is designated as 1; means 6 SD, n = 2.STAT3-High B Cells Crucial for Tumor Angiogenesis(B) Immunofluorescent staining of human prostate tumor tissue sections showing B cells and microvessels with CD31+ endothelial cells (green). Scale bars, 100 mm in the original (top) and 20 mm in the enlarged (bottom). (C) B cells around the microvessels display persistently activated STAT3. IHC showing B cells and p-STAT3-positive cells in the same area of human prostate tumor tissues; scale bars, 200 mm. H E staining of the consecutive tissue sections. Microvessel-like structures are marked by red dots; scale bars, 200 mm in the original and 50 mm in the enlarged. doi:10.1371/journal.pone.0064159.gcommonly present as aggregates with other immu.Genesis through STAT3-regulated pro-angiogenic genes in human tumors, we first stained human prostate tumor tissues for pSTAT3, CD19 and CD31. We then prepared RNAs from the tumor tissues with differential numbers of p-STAT3-positive B cells. Results from the analysis indicated that expression levels of STAT3-regulated pro-angiogenic genes, such as S1PR1, MMP9 and HIF1a, correlated with the density of tumor-infiltrating B cells in human prostate cancers (Fig. 5A). On the other hand, anelevated expression of p53 was associated with lack of B cells in the tumors (Fig. 5A). The important role of p53 in inhibiting tumor angiogenesis and the inhibitory effect of STAT3 on p53 expression has been documented [39?1]. By co-staining tumor tissues with antibodies recognizing B cells and blood vessels, we observed that CD19+ B cells had a tendency to accumulate around microvessels rather than distribute evenly throughout human tumor tissues (Fig. 5B and Figure S5). Moreover, B cells around tumor vasculature exhibited persistently activated STAT3 (Fig. 5C).DiscussionA crucial role for tumor STAT3 in upregulating proliferation/ survival of tumor cells as well as dampening proper function of immune cells such as myeloid cells and T cells has been well characterized [37,38,42]. Our study further reveals a previously unrecognized role of 16985061 B cell STAT3 in accelerating tumor progression through increasing angiogenesis. Since B cells areFigure 4. B cells with activated STAT3 accumulate in human tumors. (A) Immunofluorescent staining of human melanoma and normal human skin tissue sections; anti-CD19 (red; B cell marker) and anti-p-STAT3 (green). Scale bars, 20 mm. (B) B cells in primary tumor sites impact overall tumor STAT3 activity. Confocal microscopic images showing primary melanoma tumor tissue staining of B cells and p-STAT3 (left), with quantification of CD19 and p-STAT3 positive cells (right). Scale bars, 20 mm. Total ten microscopic fields (10 X) were examined for each tumor section; n = 2. doi:10.1371/journal.pone.0064159.gSTAT3-High B Cells Crucial for Tumor AngiogenesisFigure 5. B cells with activated STAT3 express pro-angiogenic genes and accumulate around microvessels in human tumors. (A) B cells are important for expression of pro-angiogenic genes within human prostate tumor tissues. The density of B cells around tumor vasculature in prostate tumor tissue was determined by immunofluorescent staining using anti-CD19 and anti-CD31 antibodies (top); scale bars, 20 mm. Real-time RT-PCR measuring RNA expression levels of pro-angiogenic genes in the consecutive human prostate tumor tissue sections (bottom). The relative amount of mRNA is normalized to 18S and compared to RNA levels in tumor tissues with high p-STAT3, which is designated as 1; means 6 SD, n = 2.STAT3-High B Cells Crucial for Tumor Angiogenesis(B) Immunofluorescent staining of human prostate tumor tissue sections showing B cells and microvessels with CD31+ endothelial cells (green). Scale bars, 100 mm in the original (top) and 20 mm in the enlarged (bottom). (C) B cells around the microvessels display persistently activated STAT3. IHC showing B cells and p-STAT3-positive cells in the same area of human prostate tumor tissues; scale bars, 200 mm. H E staining of the consecutive tissue sections. Microvessel-like structures are marked by red dots; scale bars, 200 mm in the original and 50 mm in the enlarged. doi:10.1371/journal.pone.0064159.gcommonly present as aggregates with other immu.
