Soichiro Sasaki

Chemokines in Cancer Development and Progression and Their Potential as Targeting Molecules for Cancer Treatment

Chemokines were initially identified as bioactive substances, which control the trafficking of inflammatory cells including granulocytes and monocytes/macrophages. Moreover, chemokines have profound impacts on other types of cells associated with inflammatory responses, such as endothelial cells and fibroblasts. These observations would implicate chemokines as master regulators in various inflammatory responses. Subsequent studies have further revealed that chemokines can regulate the movement of a wide variety of immune cells including lymphocytes, natural killer cells, and dendritic cells in both physiological and pathological conditions. These features endow chemokines with crucial roles in immune responses. Furthermore, increasing evidence points to the vital effects of several chemokines on the proliferative and invasive properties of cancer cells. It is widely acknowledged that cancer develops and progresses to invade and metastasize in continuous interaction with noncancerous cells present in cancer tissues, such as macrophages, lymphocytes, fibroblasts, and endothelial cells. The capacity of chemokines to regulate both cancerous and noncancerous cells highlights their crucial roles in cancer development and progression. Here, we will discuss the roles of chemokines in carcinogenesis and the possibility of chemokine targeting therapy for the treatment of cancer.

Effects of Kupffer cell-depletion on Concanavalin A-induced hepatitis

TNF-alpha, IFN-gamma, IL-4, and MIP-2 are known to be involved in Con A-induced hepatitis. Although Kupffer cells are reportedly involved in TNF-alpha production, it is largely unknown whether or not Kupffer cells also play a role in the production of other cytokines, such as IFN-gamma, IL-4, and MIP-2. In this study we examined the liver injury and the levels of plasma cytokines, including above four cytokines, KC, and IL-10 in Kupffer cell-depleted mice obtained through administration of liposome-encapsulated dichloromethylene bisphosphonate. The liver injury was significantly suppressed in Kupffer cell-depleted mice, as assessed as to the plasma ALT level and histochemistry. The cytokine levels were also significantly suppressed in such mice except for those of IFN-gamma, which was slightly suppressed at 12h, and IL-10, which was not significantly suppressed at any time. Apoptosis was also significantly suppressed in such mice, as found immunohistochemically with anti-ssDNA Ab. Taken together, these results suggest that Kupffer cells are involved in the production of MIP-2, KC, IL-4, and TNF-alpha in Con A-induced hepatitis, thereby contributing to the liver injury either directly or indirectly.

Crucial involvement of the CCL3‐CCR5 axis‐mediated fibroblast accumulation in colitis‐associated carcinogenesis in mice

Patients with inflammatory bowel diseases often develop colon carcinoma. Combined treatment of azoxymethane (AOM) and dextran sulfate sodium (DSS) recapitulates colitis‐associated cancer in mice. AOM/DSS‐induced tumor formation was reduced in CCL3‐ or its specific receptor, CCR5‐deficient mice despite the presence of a massive infiltration of inflammatory cells. However, AOM/DSS‐induced type I collagen‐positive fibroblast accumulation in the colon was reduced in CCL3‐ or CCR5‐deficient mice. This was associated with depressed expression of heparin‐binding epidermal growth factor‐like growth factor (HB‐EGF), which is expressed mainly by fibroblasts. Moreover in vitro, CCL3 induced fibroblasts to proliferate and to enhance HB‐EGF expression. Furthermore, CCR5 blockade reduced tumor formation together with reduced fibroblast accumulation and HB‐EGF expression, even when administered after the development of multiple colon tumors. Thus, CCL3‐CCR5‐mediated fibroblast accumulation may be required, in addition to leukocyte infiltration, to induce full‐blown colitis‐associated carcinogenesis. Our studies shed light on a therapeutic potential of CCR5 antagonist for patients with colitis‐associated cancer.

Regulation of the estrous cycle by neutrophil infiltration into the vagina

During metestrus of the estrous cycle, a number of neutrophils infiltrate into the vaginal vault, presumably due to a neutrophil-specific chemokine, MIP-2, in mice. The physiological role of the infiltrating neutrophils, however, remains largely obscure. In this study we examined the effects of neutrophil depletion on the estrous cycle and steroid hormone levels. When mice were treated with an anti-Gr-1 mAb, they became neutropenic, as assessed as to the number of neutrophils in the peripheral blood. The estrous cycle of such mice was specifically blocked at diestrus irrespective of the phase at which the anti-Gr-1 mAb was administered. The blockade was reversible, because restoration of neutrophils to a normal level caused a restart of the cycle. Immunohistochemical analyses revealed that neutrophils were present mainly on the luminal surface and in the lumen at metestrus and to a lesser extent at diestrus but scarcely in the uterine cervix at any phase, and that the anti-Gr-1 mAb depleted neutrophils but not eosinophils in the vagina. The treatment with the anti-Gr-1 mAb significantly affected the serum 17beta-estradiol and progesterone levels at diestrus after the estrous cycle was blocked. Together, these results suggest that neutrophil infiltration into the vagina is critical in maintaining the estrous cycle through control of steroid hormone levels.

Essential roles of the interaction between cancer cell-derived chemokine, CCL4, and intra-bone CCR5-expressing fibroblasts in breast cancer bone metastasis.

From a murine breast cancer cell line, 4T1, we established a subclone, 4T1.3, which consistently metastasizes to bone upon its injection into the mammary fat pad. 4T1.3 clone exhibited similar proliferation rate and migration capacity as the parental clone. However, the intra-bone injection of 4T1.3 clone caused larger tumors than that of the parental cells, accompanied with increases in fibroblast, but not osteoclast or osteoblast numbers. 4T1.3 clone displayed an enhanced expression of a chemokine, CCL4, but not its specific receptor, CCR5. CCL4 shRNA-transfection of 4T1.3 clone had few effects on its in vitro properties, but reduced the tumorigenicity arising from the intra-bone injection. Moreover, intra-bone injection of 4T1.3 clone caused smaller tumors in mice deficient in CCR5 or those receiving CCR5 antagonist than in wild-type mice. The reduced tumor formation was associated with attenuated accumulation of CCR5-positive fibroblasts expressing connective tissue growth factor (CTGF)/CCN2. Tumor cell-derived CCL4 could induce fibroblasts to express CTGF/CCN2, which could support 4T1.3 clone proliferation under hypoxic culture conditions. Thus, the CCL4-CCR5 axis can contribute to breast cancer metastasis to bone by mediating the interaction between cancer cells and fibroblasts in bone cavity.

Regulation of the estrous cycle by neutrophils via opioid peptides.

We found previously that neutrophil-depleted mice exhibited significant blockading of both the regular estrous cycle and cyclic changes of steroid hormone levels. In this study, we aimed at elucidation of the underlying mechanism. To examine the possibility that an increase in bacteria in the vaginal vault of neutrophil-depleted mice causes blockading of the estrous cycle, we treated neutrophil-depleted mice with antibiotics but failed to restore the estrous cycle. We then examined another possibility that neutrophils regulate the estrous cycle via opioid peptides, because opioid peptides regulate steroidogenesis in theca and granulosa cells in the ovaries, and because neutrophils contain opioid peptides. In support of this possibility, naloxone, an opioid antagonist, blocked the estrous cycle and a μ opioid receptor agonist restored the estrous cycle in neutrophil-depleted mice. Pro-opiomelanocortin was immunohistochemically detected in peripheral blood neutrophils but not in ones that had infiltrated into the ovaries. i.v. injection of anti–MIP-2 polyclonal Ab caused blockading of the estrous cycle, whereas MIP-2 was detected in the ovaries, suggesting a role of MIP-2 in the regulation of the estrous cycle. Moreover, i.v. injection of MIP-2 decreased the pro-opiomelanocortin signal in peripheral blood neutrophils and caused blockading of the estrous cycle. Together, these results suggest that neutrophils maintain the estrous cycle via opioid peptides.

Fibroblasts, an inconspicuous but essential player in colon cancer development and progression.

Tumor microenvironments have a crucial role in cancer initiation and progression, and share many molecular and pathological features with wound healing process. Unless treated, tumors, however, do not heal in contrast to wounds that heal within a limited time framework. Wounds heal in coordination of a myriad of types of cells, particularly endothelial cells, leukocytes, and fibroblasts. Similar sets of cells also contribute to cancer initiation and progression, and as a consequence, anti-cancer treatment strategies have been proposed and tested by targeting endothelial cells and/or leukocytes. Compared with endothelial cells and leukocytes, less attention has been paid to the roles of cancer-associated fibroblasts (CAFs), fibroblasts present in tumor tissues, because their heterogeneity hinders the elucidation on them at cellular and molecular levels. Here, we will discuss the origin of CAFs and their crucial roles in cancer initiation and progression, and the possibility to develop a novel type of anti-cancer treatment by manipulating the migration and functions of CAFs.

Suppressed rate of carcinogenesis and decreases in tumour volume and lung metastasis in CXCL14/BRAK transgenic mice

Cancer progression involves carcinogenesis, an increase in tumour size, and metastasis. Here, we investigated the effect of overexpressed CXC chemokine ligand 14 (CXCL14) on these processes by using CXCL14/BRAK (CXCL14) transgenic (Tg) mice. The rate of AOM/DSS-induced colorectal carcinogenesis in these mice was significantly lower compared with that for isogenic wild type C57BL/6 (Wt) mice. When tumour cells were injected into these mice, the size of the tumours that developed and the number of metastatic nodules in the lungs of the animals were always significantly lower in the Tg mice than in the Wt ones. Injection of anti-asialo-GM1 antibodies to the mice before and after injection of tumour cells attenuated the suppressing effects of CXCL14 on the tumor growth and metastasis, suggesting that NK cell activity played an important role during CXCL14-mediated suppression of tumour growth and metastasis. The importance of NK cells on the metastasis was also supported when CXCL14 was expressed in B16 melanoma cells. Further, the survival rates after tumour cell injection were significantly increased for the Tg mice. As these Tg mice showed no obvious abnormality, we propose that CXCL14 to be a promising molecular target for cancer suppression/prevention.

Ag and IL‐2 immune complexes efficiently expand Ag‐specific Treg cells that migrate in response to chemokines and reduce localized immune responses

An intravenous administration of a high‐dose antigen (Ag) can induce immune tolerance and suppress the immune response, but the mechanism remains unclear. We recently proved that a combined i.v. administration of OVA and IL‐2‐anti‐IL‐2 Ab immune complexes (IL‐2 ICs) efficiently expands OVA‐specific Treg cells in the thymus and induces their migration into peripheral blood, by using OVA‐specific TCR Tg‐expressing DO11.10 mice. Here, we demonstrate that the expanded OVA‐specific Treg cells rapidly move into the air pouch after OVA injection in DO11.10 mice. The migration was inhibited by blocking the axis of a chemokine receptor, CCR2. Moreover, prior treatment with OVA and IL‐2 ICs enhanced OVA‐specific Treg‐cell migration and inhibited OVA‐induced delayed‐type hypersensitivity (DTH) reactions in the skin of BM chimeric mice with 15% of T cells expressing OVA‐specific TCR. Blocking the CCR2 axis reversed this suppression of DTH in these mice. Furthermore, prior treatment with OVA and IL‐2 ICs effectively reduced DTH reactions even in WT mice possessing only a very small population of OVA‐specific T cells. Thus, the treatment with Ag and IL‐2 ICs can efficiently expand Ag‐specific Treg cells with the capacity to migrate and reduce localized immune responses.

Blockade of the chemokine receptor, CCR5, reduces the growth of orthotopically injected colon cancer cells via limiting cancer-associated fibroblast accumulation

We previously demonstrated that cancer-associated fibroblasts (CAFs) accumulate at tumor sites through the interaction between a chemokine, CCL3, and its receptor, CCR5, in the late phase of colitis-associated colon carcinogenesis. Here we examined the effect of a CCR5 antagonist, maraviroc, on tumor growth arising from the orthotopic injection of mouse or human colon cancer cell lines into the cecal wall by focusing on CAFs. Orthotopic injection of either cell line caused tumor formation together with leukocyte infiltration and fibroblast accumulation. Concomitant oral administration of maraviroc reduced tumor formation with few effects on leukocyte infiltration. In contrast, maraviroc reduced the intratumor number of α-smooth muscle actin-positive fibroblasts, which express epidermal growth factor, a crucial growth factor for colon cancer cell growth. These observations suggest that maraviroc or other CCR5 antagonists might act as novel anti-CRC drugs to dampen CAFs, an essential cell component for tumor progression.