Hormone refractory metastatic prostate cancer remains an incurable disease. We found that high expression levels of the chemokine receptor CXCR4 correlated with the presence of metastatic disease in prostate cancer patients. Positive staining for CXCL12, the ligand for CXCR4, was mainly present in the tumor-associated blood vessels and basal cell hyperplasia. Subcutaneous xenografts of PC3 and 22Rv1 prostate tumors that overexpressed CXCR4 in NOD/SCID mice were two- to threefold larger in volume and weight vs. controls. Moreover, blood vessel density, functionality, invasiveness of tumors into the surrounding tissues, and metastasis to the lymph node and lung were significantly increased in these tumors. Neutralizing the interactions of CXCL12/CXCR4 in vivo with CXCR4 specific antibodies inhibited the CXCR4-dependent tumor growth and vascularization. In vitro, CXCL12 induced the proliferation and VEGF secretion but not migration of PC3 and 22Rv1 cells overexpressing CXCR4. Similar effects of CXCR4 overexpression on tumor growth in vivo were also noted in two breast cancer lines, suggesting that the observed effect of CXCR4 is not unique to prostate tumor cells. Thus high levels of the chemokine receptor CXCR4 induce a more aggressive phenotype in prostate cancer cells and identify CXCR4 as a potential therapeutic target in advanced cases of metastatic prostate cancer.

Chronic hepatitis C virus (HCV) and hepatitis B virus (HBV) infection is accompanied by inflammation and fibrosis eventually leading to cirrhosis. The chemokine CXCL12 is involved in chronic inflammatory conditions. The role of the CXCL12/CXCR4 pathway in HCV-and HBV-associated liver inflammation and fibrosis was therefore studied. The levels and tissue localization of CXCL12 in liver and plasma of HCV and HBV patients were tested using immunohistochemistry and ELISA. The expression and function of CXCR4 on liver-infiltrating lymphocytes (LIL) were tested by FACS and transwell migration assays. We found that CXCL12 is expressed by bile duct epithelial cells in normal liver tissue. Bile duct proliferation and liver fibrosis in chronic HCV and HBV infection result in the anatomical re-distribution of CXCL12 in the liver. Moreover, CXCL12 is up-regulated in the endothelium of neo-bloodvessels formed in active inflammatory foci and is significantly elevated, compared with controls, in the plasma of patients with advanced liver fibrosis. Complementing these observations were others indicating that over 50% of LIL express CXCR4 and, in response to CXCL12, migrated and adhered to fibronectin. These observations suggest an important role for the CXCL12/CXCR4 pathway in recruitment and retention of immune cells in the liver during chronic HCV and HBV infection.

Multiple myeloma (MM) cells specifically attract peripheral-blood monocytes, while interaction of MM with bone marrow stromal cells (BMSCs) significantly increased monocyte recruitment (p<0.01). The CXCL12 chemokine, produced by both the MM and BMSCs, was found to be a critical regulator of monocyte migration. CXCL12 production was up-regulated under MM-BMSCs co-culture conditions, whereas blockage with anti-CXCR4 antibodies significantly abrogated monocyte recruitment toward a MM-derived conditioned medium (p<0.01). Furthermore, elevated levels of CXCL12 were detected in MM, but not in normal BM samples, whereas malignant MM cells often represented the source of increased CXCL12 in the BM. Blood-derived macrophages effectively supported MM cells proliferation and protected them from chemotherapy-induced apoptosis. Importantly, MM cells affected macrophage polarization, elevating the expression of M2-related scavenger receptor CD206 in macrophages and blocking LPS-induced TNFα secretion (a hallmark of M1 response). Of note, MM-educated macrophages suppressed T-cell proliferation and IFNγ production in response to activation. Finally, increased numbers of CXCR4-expressing CD163+CD206+ macrophages were detected in the BM of MM patients (n=25) in comparison to MGUS (n=11) and normal specimens (n=8).Taken together, these results identify macrophages as important players in MM tumorogenicity, and recognize the CXCR4/CXCL12 axis as a critical regulator of MM-stroma interactions and microenvironment formation.

IntroductionHuman natural killer (NK) cells are predominantly large granular lymphocytes (LGLs), the majority of which express CD16 and CD56 cell-surface antigens. A small subset of NK cells lacks CD16 and expresses high levels of CD56 and CD94. Another major CD56 ϩ population is composed of T cells that express the CD3 antigen. The human peripheral blood lymphocyte (PBL) NK T-cell population is enriched with CD8 ϩ T cells of effector phenotypes. NK and NK T cells can mediate cytolysis of tumor cells and virus-infected cells. 1,2 To improve responses against metastatic tumors, a variety of adoptive cellular strategies have been tested. Among the techniques most studied and developed is the use of lymphokine-activated killer (LAK) cells. LAK cells express surface markers characteristic of NK cells, including CD56 and CD16, and rarely express the common T-cell marker CD3. 3,4 The LAK activity is induced from a population of resting lymphoid cells by in vitro exposure of these cells to a supraphysiologic concentration of IL-2 (500-1000 IU/mL). 3,4 NK and NK T cells are also key effector cells mediating the graft-versus-leukemia (GVL) effect, which is used to control minimal residual disease (MRD) and for the reinduction of remission in chronic myelogenous leukemia patients who relapse after allogeneic stem cell transplantation. 1,[5][6][7][8] These cells are also important for low-intensity conditions and nonmyeloablative allogeneic stem cell transplantation, which are currently being performed not only in hematologic malignancies, but also in solid tumors such as renal cell carcinoma. 9 In hematogenic metastasis, such as that of prostate cancer, breast cancer, and neuroblastoma, the malignant cells home, arrest, and develop in the BM. 10,11 In order to extract their function, NK and NK T cells must reach the BM. However, the mechanism that regulates the trafficking of these cells to the BM is unknown.Chemokines and their receptors have an essential role in the recruitment and tissue localization of cells from the immune system. 12 NK cells have mainly been reported to express chemokine receptors from the CXC family, such as CXCR1-4 and CXC3R1. In contrast, CD56 ϩ , CD16 Ϫ , CD3 ϩ NK T cells have been shown to express the chemokine receptors CCR1, CCR2, CCR5, and CCR6 from the CC family and the chemokine receptors CXCR 3, CXCR4, and CXCR6 from the CXC family. 13,14 The chemokine receptor CXCR4 and its ligand, CXCL12, have been shown to play a crucial role in the trafficking and tissue localization of human hematopoietic stem cells and breast tumor cells to hematopoietic organs. 15,16 This makes the chemokine-receptor pair CXCL12/CXCR4 of particular interest in investigating their own role in the homing of NK and NK T cells to the BM microenvironment. Here we show that the homing of NK and NK T cells to the BM was dependent upon G protein signaling and CXCR4 and was inhibited by IL-2 treatment. Upon activation of these cells with IL-2, CXCR4 expression was reduced, whereas the expression of CXCR3 on the cell surface...

In order to explore the mechanism(s) underlying the pro-tumorigenic capacity of heparanase we established an inducible Tet-on system. Heparanase expression was markedly increased following addition of doxycycline (Dox) to the culture medium of CAG human myeloma cells infected with the inducible heparanase gene construct, resulting in increased colony number and size in soft agar. Moreover, tumor xenografts produced by CAG-heparanase cells were markedly increased in mice supplemented with Dox in their drinking water compared with control mice maintained without Dox. Consistently, we found that heparanase induction is associated with decreased levels of CXCL10, suggesting that this chemokine exerts tumor suppressor properties in myeloma. Indeed, recombinant CXCL10 attenuated the proliferation of CAG, U266 and RPMI-8266 myeloma cells. Similarly, CXCL10 attenuated the proliferation of human umbilical vein endothelial cells (HUVEC), implying that CXCL10 exhibits anti-angiogenic capacity. Strikingly, development of tumor xenografts produced by CAG-heparanase cells over expressing CXCL10 was markedly reduced compared with control cells. Moreover, tumor growth was significantly attenuated in mice inoculated with human or mouse myeloma cells and treated with CXCL10-Ig fusion protein, indicating that CXCL10 functions as a potent anti-myeloma cytokine.

CXCR4 is a key player in the retention and survival of human acute myeloid leukemia (AML) blasts in the bone marrow (BM) microenvironment. We studied the effects of the CXCR4 antagonist BL-8040 on the survival of AML blasts, and investigated the molecular mechanisms by which CXCR4 signaling inhibition leads to leukemic cell death. Treatment with BL-8040 induced the robust mobilization of AML blasts from the BM. In addition, AML cells exposed to BL-8040 underwent differentiation. Furthermore, BL-8040 induced the apoptosis of AML cells in vitro and in vivo. This apoptosis was mediated by the upregulation of miR-15a/miR-16-1, resulting in downregulation of the target genes BCL-2, MCL-1 and cyclin-D1. Overexpression of miR-15a/miR-16-1 directly induced leukemic cell death. BL-8040-induced apoptosis was also mediated by the inhibition of survival signals via the AKT/ERK pathways. Importantly, treatment with a BCL-2 inhibitor induced apoptosis and act together with BL-8040 to enhance cell death. BL-8040 also synergized with FLT3 inhibitors to induce AML cell death. Importantly, this combined treatment prolonged the survival of tumor-bearing mice and reduced minimal residual disease in vivo. Our results provide a rationale to test combination therapies employing BL-8040 and BCL-2 or FLT3 inhibitors to achieve increased efficacy of these agents.

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