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. 2011 Aug 15;71(16):5522-5534.
doi: 10.1158/0008-5472.CAN-10-3143. Epub 2011 Jul 8.

CXCL12/CXCR4 blockade induces multimodal antitumor effects that prolong survival in an immunocompetent mouse model of ovarian cancer

Elda Righi #  1   2 , Satoshi Kashiwagi #  1 , Jianping Yuan  1 , Michael Santosuosso  1 , Pierre Leblanc  1 , Rachel Ingraham  1 , Benjamin Forbes  1 , Beth Edelblute  1 , Brian Collette  1 , Deyin Xing  3 , Magdalena Kowalski  1   4 , Maria Cristina Mingari  2 , Fabrizio Vianello  5 , Michael Birrer  6 , Sandra Orsulic  3   7 , Glenn Dranoff  8 , Mark C Poznansky  1
Affiliations

CXCL12/CXCR4 blockade induces multimodal antitumor effects that prolong survival in an immunocompetent mouse model of ovarian cancer

Elda Righi et al. Cancer Res. .

Abstract

The chemokine CXCL12 and its receptor CXCR4 are expressed widely in human cancers, including ovarian cancer, in which they are associated with disease progression at the levels of tumor cell proliferation, invasion, and angiogenesis. Here, we used an immunocompetent mouse model of intraperitoneal papillary epithelial ovarian cancer to show that modulation of the CXCL12/CXCR4 axis in ovarian cancer has multimodal effects on tumor pathogenesis associated with induction of antitumor immunity. siRNA-mediated knockdown of CXCL12 in BR5-1 cells that constitutively express CXCL12 and CXCR4 reduced cell proliferation in vitro, and tumor growth in vivo. Similarly, treatment of BR5-1-derived tumors with AMD3100, a selective CXCR4 antagonist, resulted in increased tumor apoptosis and necrosis, reduction in intraperitoneal dissemination, and selective reduction of intratumoral FoxP3(+) regulatory T cells (Treg). Compared with controls, CXCR4 blockade greatly increased T-cell-mediated antitumor immune responses, conferring a significant survival advantage to AMD3100-treated mice. In addition, the selective effect of CXCR4 antagonism on intratumoral Tregs was associated with both higher CXCR4 expression and increased chemotactic responses to CXCL12, a finding that was also confirmed in a melanoma model. Together, our findings reinforce the concept of a critical role for the CXCL12/CXCR4 axis in ovarian cancer pathogenesis, and they offer a definitive preclinical validation of CXCR4 as a therapeutic target in this disease.

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Figures

Figure 1
Figure 1
Effect of CXCL12 knockdown on cell proliferation in vitro and in vivo (A) CXCL12 ELISA results from cell supernatants of wtBR5-1, kdBR5-1, and scrBR5-1(mean ± SEM, n = 3). (B) Cell surface expression of CXCR4 on wtBR5-1, kdBR5-1, and scrBR5-1 cells (n=3). (C) Proliferation curves comparing CXCL12-producing cells, wtBR5-1 and scrBR5-1, kdBR5-1, **P<0.001(mean ± SEM, n=3). (D)Proliferation curves of wtBR5-1 and kdBR5-1 cells in standard media(DMEM with 10% FBS) and RNAi cells in media containing 100ng/ml of exogenous recombinant CXCL12(Exo CXCL12) *P < 0.005. (E)Annexin V staining of wtBR5-1 and kdBR5-1 cells(n=3). (F)Confocal imaging of CXCL12 expression(red) in WT vs. RNAi tumors in vivo. Perfused vessels(green)(fluorescein-labeled lectin) and nuclear counterstaining(blue)(To-Pro-3). In the RNAi image CXCL12 stromal expression indicated by arrows. Scale bar:50 μm. (G)Kaplan-Meier survival analysis. FBV/NJ mice were followed up for appearance of ascites and tumor progression to endpoint described in Methods section for wtBR5-1(WT) vs. kdBR5-1(RNAi) tumor(four to six mice, two independent experiments, *P< 0.05).
Figure 2
Figure 2
Effect of AMD3100 treatment on tumor progression and mice survival. (A-G)AMD3100 or PBS was delivered into FVB/NJ mice with wtBR5-1 tumors at onset of ascites(schedule A). (A-B) Survival curves (A) from the onset of ascites and (B) from the time of tumor cell injection. (n = 10 per group,*P<0.05). (C-E)Effect of AMD3100 treatment on tumor weight and ascites formation. Tumor bearing mice were treated with AMD3100 or PBS under schedule A (n = 8 from three experiments) and euthanized at day 3 post ascites. (C)Representative mice from AMD3100 and PBS groups are shown. (D)Total tumor weight and (E)ascites volumes were measured. (F)Bar graph representing the percentage of mice with visible tumor deposits on organs at necropsy (n=10 per group;*P<0.01). (G) Tumor dissemination. Classification: localized (localizations≤3), limited (6<localizations>3, with no visible masses on mesentery and diaphragm), and disseminated (localizations≥6 including mesentery and diaphragm localization), *P<0.01. (H-I)AMD3100 or control PBS delivered for 15 days after tumor cell injection (schedule B). (H)Survival curves of the mice treated under schedule B from the onset of ascites showing that AMD3100 treated mice increased survival window(n=6, *P<0.01) and (I)reduced tumor dissemination (n=6, P<0.05).
Figure 3
Figure 3
Effect of CXCL12/CXCR4 manipulation on tumor angiogenesis. (A)Confocal imaging of tumor angiogenesis from PBS(n = 8), AMD3100(n = 8), and RNAi(n = 6) tumors. Functional vessels are visible in green, and blue represents nuclear counterstaining (To-Pro-3). Scale bars: 50μm. (B)Vessel quantitation expressed as density per mm2. Microvascular density was quantified from five randomized fields using Image J, and expressed as average±SEM, *P<0.05.
Figure 4
Figure 4
(A)Confocal imaging of tumor apoptosis in tumor bearing mice treated with AMD3100(n = 8) or PBS(n = 7)(schedule A). Vessels(green) and nuclei(blue) were visualized(To-Pro-3). Apoptotic cells(red) were identified using TUNEL staining. (B)quantified from five randomized fields using Image J, *P<0.05. (C)H&E staining of PBS and AMD3100 tumor sections demonstrating necrosis in AMD3100 tumors. Scale bar:50μm.
Figure 5
Figure 5
(A-C)Effect of CXCL12/CXCR4 manipulation on intratumoral FoxP3+ and CD8+ lymphocyte infiltration.(A), upper panels; confocal imaging of intratumoral CD8+ cells(red) from PBS(n=7), AMD3100(n=7), and RNAi(n=5) tumors. (A), lower panels; confocal imaging of intratumoral FoxP3+ cells(red) from PBS(n= 3), AMD3100(n = 11), and RNAi(n=7) tumors vessels(green) and nuclei(blue). Scale bars: 50μm. The number of CD8+ and FoxP3+ cells was quantified from five randomized fields, *P<0.05. (B)CD8+ cell, (C)FoxP3+ count. (D) Representative results of three independent flow analyses on four to six mice per group and reported as percentage of CD4+CD25+FoxP3+ TILs(450,000 events collected). TILs were stained with anti-CD4, anti-CD25, and anti-FoxP3. (E) FoxP3+ cell count per mm2 from thymus and spleen(n = 3). (F)CD8+/FoxP3+ ratios, **P<0.01.
Figure 6
Figure 6
Effect of tumor-specific T-cell function and migration after AMD3100 treatment. (A-B) TILs from AMD3100 and PBS groups were stimulated with tumor lysates(Lysed) or Her2/neu peptide(Her2/neu)(n=3 per group, 450,000 flow events collected). Results are reported as difference between non-stimulated(media alone) and stimulated cells and expressed as frequency of parent CD4+(A) or CD8+ cells(B), *P<0.05. (C)TILs were depleted of T-regs and stimulated for 24hr. Granzyme B production was measured(n = 6 per group, 450,000 flow events collected). Results are reported as difference between non-stimulated(media) and stimulated, adjusted for the number of CD4+ cells in depleted and non-depleted groups, and reported as percentage of total CD4+ cells, *P<0.05. (D)Representative flow data relating to T-cell function in splenocytes from PBS and AMD3100 groups showing no difference in Granzyme B production in CD8+ cells per each stimulation condition(350,000 events per condition). (E) Expression of CXCR4 on intratumoral non-depleted(CD4+CD25+FoxP3+), depleted cells (CD4+CD25−) or (F) on CD8+ cells. CXCL12 induced transmigration of tumor derived T-regs(G and H) and intratumoral CD8+ cells(I). T-regs migration to CXCL12 was tested in a transwell assay. Mean ± SEM of three wells from five experiments are shown. Transmigration index was calculated as the ratio between migrated cells in the lower chambers and the average of the random migrated cells (media alone in the upper and lower chambers) ± SEM. *P<0.05, **P<0.005.
Figure 7
Figure 7
Effect of AMD3100 on B16F10 subcutaneous tumors in C57/BL6 mice. (A)Intratumoral CD8+ cells(n=6) and T-regs infiltration(n=6) was assessed by flow cytometry(750,000 events) and expressed as number of cells/gram of tumor. (B)Lower CXCR4 expression was shown on intratumoral T-regs cells (left panel) compared to T-effector cells (right panel)(1×106 events recorded)(representative histograms shown).
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