B Cells Promote Tumor Progression via STAT3 Regulated-Angiogenesis

<div><p>The role of B cells in cancer and the underlying mechanisms remain to be further explored. Here, we show that tumor-associated B cells with activated STAT3 contribute to tumor development by promoting tumor angiogenesis. B cells with or without Stat3 have opposite effects on tumor growth and tumor angiogenesis in both B16 melanoma and Lewis Lung Cancer mouse models. <i>Ex vivo</i> angiogenesis assays show that B cell-mediated tumor angiogenesis is mainly dependent on the induction of pro-angiogenic gene expression, which requires Stat3 signaling in B cells. Furthermore, B cells with activated STAT3 are mainly found in or near tumor vasculature and correlate significantly with overall STAT3 activity in human tumors. Moreover, the density of B cells in human tumor tissues correlates significantly with expression levels of several STAT3-downstream pro-angiogenic genes, as well as the degree of tumor angiogenesis. Together, these findings define a novel role of B cells in promoting tumor progression through angiogenesis and identify STAT3 in B cells as potential therapeutic target for anti-angiogenesis therapy.</p></div

B cells with activated Stat3 accelerate tumor progression and increase blood vessel formation in tumors.

<p>(<b>A and B</b>) Left, Growth curve of B16 (<b>A</b>) or LLC (<b>B</b>) tumors in <i>Rag1^−/−</i> mice, without or with <i>Stat3^+/+</i>or <i>Stat3</i>^−/− B cells. B cells were enriched from splenocytes of B16 or LLC tumor-bearing mice with or without <i>Stat3</i> ablated in hematopoietic cells; <i>n</i> = 8; ***, <i>P</i><0.001. Middle, Immunofluorescent staining of tumors to detect microvessels (anti-CD31, red) and nuclei (Hoechst, blue); scale bars, 200 µm. Right, Graph showing the number of CD31⁺ vessels in the tumor tissues (3 microscopic fields/section); means ± SD, <i>n</i> = 2. (<b>C</b>) Representative images (left) and a graph (right) showing the number of spontaneous lung metastatic nodule in mice with <i>Stat3^+/+</i> or <i>Stat3^−/−</i> B cells receiving B16 tumor cells; means ± SEM, <i>n = </i>5.</p

B cells with activated STAT3 express pro-angiogenic genes and accumulate around microvessels in human tumors.

<p>(<b>A</b>) 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 µm. 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 <i>18S</i> and compared to RNA levels in tumor tissues with high p-STAT3, which is designated as 1; means ± SD, <i>n</i> = 2. (<b>B</b>) Immunofluorescent staining of human prostate tumor tissue sections showing B cells and microvessels with CD31⁺ endothelial cells (green). Scale bars, 100 µm in the original (top) and 20 µm in the enlarged (bottom). (<b>C</b>) 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 µm. H&E staining of the consecutive tissue sections. Microvessel-like structures are marked by red dots; scale bars, 200 µm in the original and 50 µm in the enlarged.</p

B cells with activated STAT3 accumulate in human tumors.

<p>(<b>A</b>) 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 µm. (<b>B</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 µm. Total ten microscopic fields (10 X) were examined for each tumor section; <i>n</i> = 2.</p

<i>Stat3</i> intrinsic to B cells is crucial for expression of pro-angiogenic genes and endothelial cell tube formation.

<p>(<b>A</b>) Expression levels of mRNA (left) or protein (right) of the indicated genes in B16 tumors. The tumors were formed by implanting B16 tumor cells with the indicated tumor-primed B cells in <i>Rag1^−/−</i> mice. (<b>B</b>) mRNA (left) or protein (right) expression levels of the indicated genes in tumor-primed B cells from splenocytes of B16-tumor bearing C57BL/6 mice with <i>Stat3^+/+</i> or <i>Stat3^−/−</i> hematopoietic cells. (<b>C</b> and <b>D</b>) mRNA and protein levels of the indicated pro-angiogenic genes in <i>Stat3^+/+</i> or <i>Stat3^−/−</i> B cells isolated from either B16 tumors (<b>C</b>) or B16 tumor-draining lymph nodes (TDLN) (<b>D</b>). In all cases, mRNA is determined by real-time RT-PCR. The relative amount of mRNA is compared to that of <i>Stat3^+/+</i> B cells, which is designated as 1 (means ± SEM, <i>n</i> = 3). Western blotting data represent two independent experiments. (<b>E</b>) Representative images of EC tube formation by co-culturing ECs with splenic <i>Stat3^+/+</i> B cells from B16 tumor-bearing mice in the presence of VEGF neutralizing antibodies. IgG antibody at the same concentration is used as control; <i>n</i> = 3 (top). Quantitative graph showing the average number of vessel-like structures formed by ECs upon addition of the indicated B cells; means ± SEM, <i>n</i> = 3; **, <i>P</i><0.001 (bottom).</p

B cells promote tumor angiogenesis by enhancing endothelial cell function in a Stat3-dependent manner.

<p>(<b>A</b>) Representative images of vessel formation in Matrigel plugs implanted in <i>Rag1^−/−</i> mice. The Matrigel plugs contain either B16 tumor cell alone, <i>Stat3^+/+</i> B cell alone or B16 tumor cells plus <i>Stat3^+/+</i> or <i>Stat3</i>^−/− B cells, which were isolated from splenocytes of tumor-bearing mice; <i>n</i> = 5; area indicated by blue dot showing level of blood vessel formation. (<b>B</b>) Hemoglobin contents in the pooled Matrigel plugs determined by colorimetric assay; means ± SEM, <i>n</i> = 5. (<b>C</b>) <i>In vitro</i> collagen tube formation assay showing the number of tubes formed by ECs with or without the indicated B cells. <i>Stat3^+/+</i> B cells were enriched from splenocytes of B16 tumor-bearing mice (left) or B16 tumors (right); means ± SEM, <i>n</i> = 3. (<b>D</b>) B cell-mediated endothelial cell tube formation requires Stat3 signaling in B cells. EC tube formation by co-culturing ECs with tumor-primed <i>Stat3^+/+</i> or <i>Stat3^−/−</i> splenic B cells. Tumor-primed B cells were enriched from splenocytes of MB49 tumor-bearing mice with <i>Stat3^+/+</i> or <i>Stat3^−/−</i> hematopoietic cells; means ± SEM, <i>n</i> = 3; ****, <i>P</i><0.0001.</p

pSTAT3-positive B cells are readily detectable in the omental tissues of ovarian cancer patients.

<p>(<b>A</b>) Immunofluorescent staining followed by confocal microscopy showing examples of representative specimens from a patient with low B-cell infiltration and low pSTAT3 expression (top left and right), and a separate patient with high B-cell infiltration and high pSTAT3 expression (bottom left and right); scale bars, 20 µm. (<b>B</b>) IHC images showing B cells and pSTAT3-positive cells in the same area of omental tissues; scale bars, 200 µm.</p

Results of univariate and multivariate analyses.

<p>Results of univariate and multivariate analyses.</p

B cell clustering and pSTAT3 in the omental tissues predict poor prognosis in patients with ovarian cancer.

<p>Kaplan-Meier curve illustrating the duration of survival in patients with ovarian cancer based on (<b>A</b>) degree of B-cell infiltration (P = 0.0015), (<b>B</b>) degree of pSTAT3 expression (P = 0.02), (<b>C</b>) histological staging system and (<b>D</b>) debulking status.</p

Icaritin inhibits 786-O, Renca cell proliferation.

<p>A. Analysis of RCC cell proliferation following treatment of Icaritin. Renca and 786-O cells were treated (24 and 48 hours) at increasing doses (1∼10 µM). Cell proliferation was evaluated by MTS assay. Columns, mean (n = 3, in triplicate); bars, SD. *, P<0.05; **, P<0.01. B. Icaritin treatment of 786-O cells reduces expression of proliferative proteins. Western blot analyses of 786-O cells treated (24 hours) with Icaritin to evaluate protein levels of cyclinD1, cyclinE and survivin. Bottom. The optical density of each protein was quantified by β-actin optical density.</p