Oncogene (2007) 26, 4679–4688 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc ORIGINAL ARTICLE CXCR3 promotes colon cancer metastasis to lymph nodes

K Kawada1,2,5, H Hosogi1,2,5, M Sonoshita1, H Sakashita3, T Manabe3, Y Shimahara2, Y Sakai2, A Takabayashi4, M Oshima1 and MM Taketo1

1Department of Pharmacology, Graduate School of Medicine, Kyoto University, Kyoto, Japan; 2Department of Surgery, Graduate School of Medicine, Kyoto University, Kyoto, Japan; 3Department of Clinical Pathology, Graduate School of Medicine, Kyoto University, Kyoto, Japan and 4Kitano Hospital Medical Institute, Osaka, Japan

Chemokines and their receptors are essential for leuko- inflammatory cytokines, growth factors and/or patho- cyte trafficking, and also implicated in cancer metastasis genic stimuli. Important roles of and their to specific organs. We have recently demonstrated that receptors have been demonstrated in inflammation, CXCR3 plays a critical role in metastasis of mouse infection, tissue injury, allergy and cardiovascular melanoma cells to lymph nodes. Here, we show that some diseases as well as in malignant tumors. Chemokine human colon cancer cell lines express CXCR3 constitu- receptor CXCR3 is essential for the physiologic and tively. We constructed cells that expressed CXCR3 cDNA pathologic recruitment of plasmacytoid dendritic cell (‘DLD-1-CXCR3’), and compared with nonexpressing precursors, monocytes and natural killer cells to controls by rectal transplantation in nude mice. Although inflamed lymph nodes (LNs) (Cella et al., 1999; both cell lines disseminated to lymph nodes at similar Janatpour et al., 2001; Martin-Fontecha et al., 2004), frequencies at 2 weeks, DLD-1-CXCR3 expanded more and for retention of Th1 within LNs rapidly than the control in 4 weeks. In 6 weeks, 59% of (Yoneyama et al., 2002). On the other hand, chemokine mice inoculated with DLD1-CXCR3 showed macroscopic receptor CCR7 plays a central role in the recruitment of metastasis in para-aortic lymph nodes, whereas only 14% naı¨ ve T cells, some memory T cells, and mature of those with the control (Po0.05). In contrast, dendritic cells to LNs (Cyster, 1999; von Andrian and metastasis to the liver or lung was rare, and unaffected Mempel, 2003). In contrast, the roles of chemokines in by CXCR3 expression. In clinical colon cancer samples, malignant tumors appear complex. Whereas many we found expression of CXCR3 in 34% cases, most of chemokines show antitumor activities by stimulating which had lymph node metastasis. Importantly, patients immune cells or by inhibiting tumor neovascularization with CXCR3-positive cancer showed significantly poorer (angiogenesis), other chemokines may promote tumor prognosis than those without CXCR3, or those expressing growth and metastasis by directly stimulating growth, and CXCR4 or CCR7. These results indicate that activation enhancing cell motility and/or angiogenesis (Balkwill, of CXCR3 with its ligands stimulates colon cancer 2004). Regarding the direct role of chemokines in LN metastasis preferentially to the draining lymph nodes with metastasis, recent reports suggest a critical role for poorer prognosis. chemokine receptors CXCR3 and CCR7 in metastasis Oncogene (2007) 26, 4679–4688; doi:10.1038/sj.onc.1210267; of melanoma and breast cancer (Mu¨ ller et al., 2001; published online 5 February 2007 Robledo et al., 2001; Kawada et al., 2004). In this study, we demonstrate that CXCR3 is Keywords: CXCR3; colon cancer; lymph node; meta- expressed in colon cancer cells, and plays a key role in stasis colon cancer metastasis to LNs, although CCR7 does not. In addition to CXCL9, 10and 11, CCR7 ligand CCL21 can also activate CXCR3 on colon cancer cells. These results also suggest that CXCR3 can be a novel therapeutic target to suppress LN metastasis in colon Introduction cancer. Chemokines are chemotactic polypeptides that cause directed migration of leukocytes, and are induced by Results Correspondence: Professor MM Taketo, Department of Pharmacol- ogy, Graduate School of Medicine, Kyoto University, Yoshida- Expression of chemokine receptors CXCR3, CXCR4 Konoe´ -cho, Sakyo, Kyoto 606-8501, Japan. and CCR7 in colon cancer cell lines E-mail: [email protected] To investigate CXCR3 expression in human colon 5These authors contributed equally to this paper. Received 25 September 2006; revised 14 November 2006; accepted 1 cancer cell lines, we first performed a Western blot December 2006; published online 5 February 2007 analysis. We found expression of CXCR3 at high levels CXCR3 in colon cancer metastasis to lymph nodes K Kawada et al 4680 in five (Colo205, HCT116, HT29, RKO and WiDr) to the draining para-aortic LNs (Kashtan et al., 1992; of 10colon cancer cell lines (Figure 1a). As none of Tsutsumi et al., 2001; Ninomiya et al., 2004). For CCR7 or CXCR4 antibodies commercially available example, it was reported that HT29 metastasized to LNs was suitable for Western-blot analysis, we resorted to in all tumor-bearing mice (10/10) (Tsutsumi et al., 2001), RT–PCR analysis. CCR7 mRNA was expressed only in whereas LS174T did not (0/5) (Kashtan et al., 1992). two (SW480and Caco2) cell lines, whereas CXCR4 Accordingly, we constructed five colon cancer cell lines mRNA was expressed in seven (Colo205, HCT116, that expressed green fluorescent (GFP), and HT29, WiDr, SW480, LS174T and Caco2) cell lines determined the metastatic frequencies to LNs by rectal (Figure 1b). We then confirmed expression of CXCR3, injection into nude mice. Six weeks after inoculations, CXCR4 and CCR7 using flow cytometry. we dissected para-aortic LNs, and examined metastatic Consistent with the Western-blot and RT–PCR data, foci using GFP fluorescence. Cell lines Colo205, Colo205, HCT116, HT29, RKO and WiDr expressed HCT116 and RKO produced metastatic foci in LNs at CXCR3 strongly, whereas DLD-1 or SW480did not at modest to high frequencies (8/10, 8/10 and 5/10, levels detectable by these methods (Figure 1c; data not respectively), whereas DLD-1 and SW480metastasized shown for HT29, RKO or WiDr). only at low frequencies (3/22 and 4/20, respectively) The rectal xenograft method offers a convenient (Table 1a). It was also reported that Caco2 metastasized animal model for colorectal cancer that can metastasize to LNs at a low frequency (1/10) with a cecal

Figure 1 Expression of chemokine receptors CXCR3, CCR7 and CXCR4 in human colon cancer cell lines. (a) Western-blot analysis of CXCR3 expression. A spleen extract was used as a positive control, whereas b-actin was used as the internal control. (b) An RT– PCR analysis for CCR7 and CXCR4 expression. RNA from spleen was used as a positive control, with b-actin as the internal control. (c) A flow cytometric analysis of Colo205, HCT116, DLD-1 and SW480 cells for CXCR3 (top), CCR7 (middle) and CXCR4 (bottom) expression. Shaded areas represent cells stained with anti-CXCR3, anti-CCR7 mAb or anti-CXCR4 mAb, whereas white areas show cells stained with the isotype-matched control mAb.

Oncogene CXCR3 in colon cancer metastasis to lymph nodes K Kawada et al 4681 Table 1 Metastasis frequency of colon cancer cells Cell line CXCR3 CCR7 CXCR4 Number of metastatic LNsa

n %

(a) Human colon cancer cell lines Colo205 + À + 8/1080 HCT116 + – + 8/1080 RKO + – – 5/1050 DLD-1 – – – 3/22 14 SW480 À + + 4/2020

Cell line Primary tumor Number of metastatic organsc

Number Volume (mm3)d LNs (%) Lungs (%) Liver (%)

(b) Human colon cancer DLD-1 cells to LNs, lungs and liverb DLD-1-CXCR3 22/22 9057238 13/22 (59)e 2/22 (9) 1/22 (5) DLD-1-EV 22/22 10287334 3/22 (14) 4/22 (18) 1/22 (5) aLN metastases were examined 6 weeks after inoculations of tumor cells (1 Â 106 cells). bMice were killed at 6 weeks after inoculation of tumor cells (1 Â 106 cells). cMetastatic foci were identified macroscopically with GFP fluorescence. dValues are mean7s.d. ePo0.01 compared with DLD-1-EV cells (Fisher’s exact test).

transplantation model (Flatmark et al., 2004). Recent in other colon cancer cell lines (HCT116 and HT29) that studies showed that CCR7 was involved in LN meta- expressed CXCR3, but not CCR7 (data not shown). stasis in breast and stomach cancers, and melanoma Consistent with the result, Colo205 cells exhibited the (Mu¨ ller et al., 2001; Wiley et al., 2001; Mashino et al., CXCL10-induced migration, and the neutralizing anti- 2002). Among the colon cancer cell lines tested by CXCR3 antibody significantly suppressed the migratory RT–PCR and flow cytometry, however, we found response in a dose-dependent manner (Figure 2b). This expression of CCR7 only in SW480and Caco2 that anti-CXCR3 antibody did not suppress the CXCL12- rarely metastasized to LNs (Figure 1b and c). These induced migration (Figure 2b), nor did it induce cell results suggest that metastasis of colon cancer to LNs is aggregation or toxicity (data not shown), which suggests associated with rather CXCR3 than CCR7. that the anti-CXCR3 antibody did not inhibit cell motility nonspecifically. As expected, Colo205 cells exhibited the CCL21-induced migration with a little CXCR3-mediated cellular responses of colon cancer cell lower efficiency than CXCL10(Figure 2c). Although line Colo205 in vitro the neutralizing antibodies against CXCR3 and CCL21, In addition to CXCL9, CXCL10and CXCL11, it has respectively, suppressed the migratory response in a been reported that one of the ligands for CCR7, CCL21/ dose-dependent manner, that against CCR7 showed no SLC can also bind to, and activate CXCR3 in mice suppressive effects (Figure 2c). These results indicate (Soto et al., 1998). Also in some human cells, CCL21 is a that CCL21 induces of human colon cancer functional ligand for endogenously expressed CXCR3 cell line Colo205 through CXCR3. Reorganization of (Dijkstra et al., 2004; Poggi et al., 2004). To investigate the actin cytoskeleton is a prerequisite for cell motility whether CXCR3 can transduce the ligand signals in and migration in cultured leukocytes. With CXCL10at colon cancer cells, we first performed calcium mobiliza- 100 ng/ml, the cells showed intense F-actin staining in tion assays. Addition of CXCL10and CXCL12 the periphery, and redistribution toward the leading (CXCR4 ligand), respectively, to Colo205 that expressed edge (Figure 2d). These changes lasted for up to 20min both CXCR3 and CXCR4, but not CCR7, induced of incubation. B20% increase in the intracellular Ca2 þ concentration In addition to induction of chemotaxis, activation of a (Figure 2a and data not shown). Intracellular Ca2 þ flux by its ligand stimulates cell growth induced by CXCL10was suppressed by preincubation and survival in some cell types (Vlahakis et al., 2002). with a neutralizing anti-CXCR3 antibody, but that by Therefore, we determined the viability of Colo205 cells CXCL12 was not (data not shown). Next, we added exposed to CXCR3 ligands (Figure 3a). Neither CCL21 and found similar increase in the Ca2 þ CXCL10nor CCL21 had any effects on cell prolifera- concentration, although another ligand for CCR7, tion under the normal (10% fetal calf serum (FCS)) or CCL19/ELC did not show such an effect (Figure 2a). low (0.5% FCS) serum condition. Without serum, We also confirmed that intracellular Ca2 þ flux induced however, both ligands significantly enhanced cell survi- by CCL21 was suppressed by neutralizing antibodies val, compared with the untreated controls (Po0.05). against CXCR3 and CCL21, respectively (data not Regarding the molecular mechanism of CXCR3 signal- shown). We further confirmed that not only CXCL10, ing, phosphorylation of ERK1/2 took place shortly after but also CCL21 increased the Ca2 þ concentration also addition of CXCL10, lasted for B10min, and then

Oncogene CXCR3 in colon cancer metastasis to lymph nodes K Kawada et al 4682

Figure 2 Activation of CXCR3 on Colo205 cells with its ligands. (a) Intracellular calcium fluxes upon exposure to CXCL10, CCL21 and CCL19 (100 ng/ml, respectively), after preincubation with Fluo-3 AM. (b and c) Chemotaxis assays in Transwell chambers. Chemotactic responses to CXCL10and CXCL12, respectively ( b), or to CCL21 (c) are shown with or without neutralizing antibodies. Mean; bars,7s.d. (*,**Po0.01 by Student’s t-test). (d) Immunofluorescence staining for actin cytoskeleton with phalloidin, after incubation with or without CXCL10(100ng/ml) for 5 min. Representative photographs of diffusely stained ( ÀCXCL10) and polarized ( þ CXCL10) cells are shown.

decreased gradually (Figure 3b). Furthermore, CXCL10 suppressed by neutralization of CXCR3 (Figure 3d). caused phosphorylation of Akt/PKB at Ser473 These results suggest the important role of CXCR3 and (Figure 3b) that appears crucial for the micrometastatic its ligands in gelatinase induction in cancer. foci to survive and grow (Bao et al., 2004). Tumor cells often encounter stress conditions, hypoxia, immune responses or nutrient deprivation. Accordingly, Expression of CXCR3 ligands in various normal organs we investigated regulation of CXCR3 in colon cancer To examine CXCR3 ligands in various normal (i.e., cells under stress by serum deprivation. The total non-tumor) organs of wild-type mice, we first deter- cellular protein levels of CXCR3 were similar between mined by quantitative RT–PCR, the mRNA levels for high serum (10% FCS) and serum-starved cultures for 3 CXCL9, CXCL10, CXCL11, and CCL21 (Supplemen- days. However, expression of CXCR3 on the membrane tary Figure 1a). Consistent with earlier reports (Gattass changed dramatically upon serum starvation, as deter- et al., 1994; Amichay et al., 1996; Luther et al., 2000; mined by flow cytometry (Figure 3c). Namely, most cells Mu¨ ller et al., 2001), CXCL9, CXCL10 and CCL21 were in the population expressed CXCR3 in the absence of expressed preferentially in lymphoid organs such as LNs serum, whereas only approximately half did in its and spleen, but not in the liver, lung, large intestine or presence, although its total cellular content remained skin, although a low level of CXCL9 was found in the at similar levels (Figure 3c, right). Thus, it is possible liver. In contrast, CXCL11 mRNA was hardly expressed that expression of CXCR3 on the cell surface helps in any of these organs. We also determined the levels of tumor cells to survive under stress. these CXCR3 ligands within the draining LNs of tumor- Matrix metalloproteinases (MMPs), particularly, inoculated mice, but these expression levels were not gelatinases as MMP-2 and MMP-9 are implicated in altered significantly by tumor cell inoculation (data colon cancer progression and metastasis. Upon exposure not shown). We further examined expression and of Colo205 cells to CXCL10, expression and activities of localization of CXCL9, CXCL10and CCL21 proteins MMP-2 and MMP-9 increased significantly, which was within LNs by immunohistochemistry (Supplementary

Oncogene CXCR3 in colon cancer metastasis to lymph nodes K Kawada et al 4683

Figure 3 Effects of CXCR3 ligands on Colo205 cells. (a) Cell viability assays. Viable cell fractions (in percentage) are shown after cultured in serum-containing (10 and 0.5% FCS) or serum-free medium, with or without CXCL10 or CCL21 (100 ng/ml). Mean; bars,7s.d. (*Po0.01 by Student’s t-test). (b) A Western-blot analysis of Colo205 cells incubated with or without CXCL10 (100 ng/ml) for the levels of anti-phosho-ERK1/2, anti-ERK1/2, anti-phospho-Akt/PKB (Ser473) and anti-Akt/PKB. (c) Expression of CXCR3 in the serum containing (10% FCS) or serum-free medium. Total expression level was determined by a Western-blot analysis (right), whereas the cell surface expression was quantified by flow cytometry (left). (d) Effects of CXCL10(100ng/ml) on gelatinase secretion with or without neutralizing anti-CXCR3 antibody (10 mg/ml). Positions of the respective gelatinase species are indicated.

Figure 1b). As described previously (Luther et al., (‘DLD-1-CXCR3’ cells; Supplementary Figure 2b, 2000; Janatpour et al., 2001; Kawada et al., 2004), we right). As expected, these cells expressed high levels of found that both CXCL9 and CXCL10were expressed CXCR3 as assessed by RT–PCR (Supplementary Figure mainly in the subcapsular and cortical sinuses, whereas 2c). We also isolated cells expressing an empty vector CCL21 throughout the T-zone. that contained EGFP, but not CXCR3 (‘DLD-1-EV’ cells; Supplementary Figure 2c). To evaluate the responses to the CXCR3 ligands, we performed Expression of mouse CXCR3 in human colon cancer chemotaxis and cell viability assays using these DLD-1 cells DLD-1 sublines. Treatment with CXCL10induced migratory To investigate whether CXCR3 plays a role in colon responses in DLD-1-CXCR3 cells, but not in DLD-1- cancer metastasis, we introduced a retroviral expression EV (Supplementary Figure 2d, left). In addition, construct for CXCR3 into DLD-1 cells that expressed CXCL10significantly enhanced the survival of DLD- neither CXCR3 nor CCR7 (Supplementary Figure 2a). 1-CXCR3 cells upon serum starvation, but not of DLD- In this construct, CXCR3 mRNA was expressed from 1-EV (Supplementary Figure 2d, middle). In contrast, an long terminal repeat (LTR) promoter followed by an CXCL10did not affect the proliferation rate of DLD-1- internal ribosome entry site (IRES) and enhanced GFP EV or DLD-1-CXCR3 cells, in either normal (10% (EGFP) cDNA. Although the transduction efficiency FCS) or low (0.5% FCS) serum concentration (data not was B10% (Supplementary Figure 2b, left), we could shown). In the absence of ligands, the proliferation rates sort by fluorescence-activated cell sorting (FACS) the were also similar between the DLD-1-CXCR3 and cell population that expressed EGFP efficiently DLD-1-EV cells (Supplementary Figure 2d, right).

Oncogene CXCR3 in colon cancer metastasis to lymph nodes K Kawada et al 4684 Enhanced metastasis of DLD-1-CXCR3 cells to draining lymph nodes To assess the effect of CXCR3 expression on LN metastasis, we inoculated DLD-1-CXCR3 and DLD-1- EV cells, respectively, in the rectum of nude mice. One week later, transplanted rectal tumors became grossly visible, and their size increased rapidly with time. Six weeks later, we confirmed that CXCR3 protein con- tinued to be expressed in the transplanted cells by immunohistochemistry (Figure 4a), and examined macroscopically metastatic foci of the para-aortic LNs using GFP fluorescence. Interestingly, the mice inocu- lated not only with DLD-1-CXCR3 cells, but also with DLD-1-EV cells showed macroscopic enlargement of the para-aortic LNs, which reflects that sensitized immune cells can reach LNs and cause proliferative reactions (Ochsenbein et al., 2001). However, under a fluorescence dissection microscope, we found that DLD- 1-CXCR3 cells metastasized to LNs with a significantly higher frequency than DLD-1-EV (Figure 4b). Although 14% (3 of 22) of the mice inoculated with DLD-1-EV cells showed visible metastatic foci of relatively small size, 59% (13 of 22) of the mice with DLD-1-CXCR3 formed larger foci (Figure 4b and Table 1b; Po0.01). Regarding the transplanted primary rectal tumors, there was no significant difference in size between the two groups (Table 1b; Student’s t-test). As for metastasis to the lung or liver, there was no significant difference between the two groups (Table 1b; Student’s t-test). To quantify the metastasized DLD-1 cells to the para- aortic LNs, we determined the level of human b-globin- Figure 4 Stimulation of DLD-1 metastasis to LNs by expression related sequence (HBB) by quantitative PCR (Ninomiya of CXCR3. (a) Expression of CXCR3 and GFP proteins in the et al., 2004). One day after inoculation, we detected no primary rectal tumor sections determined by immunohistochem- amplified signals from LNs. Two weeks later, we found istry (6 weeks postinoculation). Tumors of DLD-1-CXCR3 (top) and DLD-1-EV (bottom) cells are shown. (Serial sections were DNA of DLD-1-EV and DLD-1-CXCR3 cells in LNs in analysed for hematoxylin and eosin stain (H&E), and immunos- five of 10, and six of 10 mice, respectively (Figure 4c). taining for CXCR3, GFP and IgG isotype, respectively.) Note that The number of cells spread to LNs were estimated to be DLD-1-CXCR3 cells expressed both CXCR3 and GFP proteins, p500 /mouse, except one mouse with DLD-1-CXCR3 whereas DLD-1-EV cells showed GFP protein only. Scale bars, 100 mm. (b) Representative photographs of the para-aortic LNs (Figure 4c). The result indicated that there was no from mice 6 weeks after inoculations with DLD-1-EV (left) and apparent difference in the number of DLD-1 cells DLD-1-CXCR3 (center) cells, respectively. Normal LNs from disseminated to LNs between the two groups at this uninoculated control mice are also shown (right). Metastatic foci stage (DLD-1-EV, 120790cells; and DLD-1-CXCR3, are visualized by GFP fluorescence. Scale in millimeters. (c) Time 51071200 cells; mean7SD of 10transplants/group). course of the tumor cell metastasis in LNs. Ten mice from each group were analysed at the indicated time after tumor inoculation. Four weeks after inoculations, we occasionally found The para-aortic LNs were pooled for each mouse, and extracted GFP fluorescence from DLD-1-CXCR3 foci in the LNs, DNA was analysed for the numbers of metastasized tumor cells although we never found such foci in mice inoculated using the human b-globin (HBB). The estimation was based with DLD-1-EV. Quantification of metastasized DLD-1 on a calibration curve of given numbers of cultured DLD-1 cells. cells showed that the number of DLD-1-CXCR3 cells in Mean; bars (*Po0.05 by Student’s t-test). LNs was significantly higher than that of DLD-1-EV (DLD-1-EV, B5007700 cells; and DLD-1-CXCR3, 20 000728 000 cells; mean7s.d. of 10transplants/ CCR7 as well as CXCR4. We found that 31 samples group) (Figure 4c; Po0.05). These results suggest that (33.7%) expressed CXCR3 in cancer epithelial cells, activation of CXCR3 is important for establishing whereas only 13 (14.1%) had CCR7, and 50(54.3%) metastatic foci of colon cancer cells in LNs. expressed CXCR4. The CXCR3, CXCR4 and CCR7 proteins were detected in the plasma membrane and cytoplasm of cancer cells (Figure 5a and Supplementary CXCR3 expression in clinical samples Figure 3a). The normal epithelial cells of the colon did To evaluate the clinical relevance of the above results, not express CXCR3, although some infiltrating inflam- we immunohistochemically examined specimens from 92 matory cells did. We then compared several clinico- colon cancer patients for expression of CXCR3 and pathological factors between the cases with and without

Oncogene CXCR3 in colon cancer metastasis to lymph nodes K Kawada et al 4685 positive cases, 20 (80.0%) of 25 cases with CXCR3 metastasized to LNs, whereas only six (24.0%) of 25 cases without CXCR3 did (Supplementary Table 4; Po0.01). We also determined independent risk factors for LN metastasis by a stepwise logistic regression analysis (Supplementary Table 2). According to the multivariate analysis of several variables, we found that CXCR3 expression and lymphatic invasion were sig- nificant independent risk factors. Finally, we examined the association of CXCR3 and/or CXCR4 expression with survival of the patients with colon cancers. Statistical analysis of the results by the log- (Mantel–Cox) test showed that the patients with CXCR3-positive tumors had a significantly poorer prognosis than those with CXCR3-negative tumors (Figure 5b; Po0.01). Importantly, the patients with CXCR4-positive tumors also had a significantly poorer prognosis, but its effect was less than CXCR3 (Figure 5b; Po0.05). In addition, the patients with tumors doubly positive for CXCR3 and CXCR4 had a significantly poorer prognosis than those with tumors positive only for CXCR4 or doubly negative (Figure 5c; Po0.01). In contrast, expression of CCR7 had little effects on the patient survival (Supplementary Figure 3b, P ¼ 0.46). These results collectively indicate that expression of CXCR3 plays a pivotal role in LN metastasis of colon cancer, and causes poorer prognosis.

Discussion

Metastasis is an inefficient process because it consists of multiple and complex steps, all of which must be successfully completed to give rise to the formation of Figure 5 Expression of CXCR3 and CXCR4 in the primary human colon cancer, and its relationship with prognosis. (a) Immuno- metastatic tumors (Fidler and Kripke, 1977). Recent histochemical evaluation of CXCR3 and CXCR4 expression studies suggest that the least efficient steps in metastasis ( Â 100). Sections of the primary colon cancer tissues (cases 1 and are the survival and growth of the micrometastatic foci 2; CXCR3-positive cases) and the matched normal colonic mucosa and their persistence (Chambers et al., 2002; Bao et al., were immunostained with anti-CXCR3 mAb. Three primary colon cancer tissues (case 3; CXCR4-negative case, cases 4 and 5; 2004), which indicates the importance of interactions CXCR4-positive cases) were immunostained with anti-CXCR4 between the ‘seed’ (cancer cells) and ‘soil’ (microen- mAb. Insets show higher magnifications of the boxed areas vironment). Accumulating evidence suggests that meta- ( Â 400). Hematoxylin counterstaining. Scale bars, 100 mm. (b) stasis of tumors to specific organs can be aided by Survival curves (Kaplan and Meier estimates) by expression of interactions between chemokine receptors on cancer CXCR3 and CXCR4 in colon cancer. (c) Survival curves by combined expression of CXCR3 and CXCR4 in colon cancer. cells and their ligands in the target organs. For example, CXCR4 stimulates metastasis of breast cancer to the bone and lung where its ligand CXCL12 is abundant (Mu¨ ller et al., 2001; Kang et al., 2003). We have recently CXCR3 expression (Supplementary Table 1). We found shown that CXCR3 is responsible for LN metastasis of a significant difference between the two groups in LN mouse melanoma cells B16F10, without affecting that to metastasis, tumor, nodes, metastasis (TNM) stage, the lung (Kawada et al., 2004). Interestingly, CCR7 was lymphatic invasion and vascular invasion. Regarding also implicated in the LN metastasis of breast and the LN metastasis, 24 of 31 (77.4%) cases with CXCR3 stomach cancers (Mu¨ ller et al., 2001; Mashino et al., expression metastasized to LNs, whereas only 14 of 61 2002). Importantly, both CXCR3 and CCR7 are (22.9%) cases without CXCR3. On the other hand, involved in the recruitment and patterning of several there was no significant correlation between expression types of immune cells to the LNs, suggesting that tumor of CCR7 and these clinicopathological factors. Regard- cells have acquired these receptors and are controlled by ing CXCR4 expression, we found a significant difference their ligands in a manner analogous to the immune cell between expression of CXCR4 and distant metastasis, interactions. Here, we have demonstrated that CXCR3 vascular invasion, TNM stage and LN metastasis plays a critical role in the metastasis of colon cancer cells (Supplementary Table 3). Among the 50CXCR4- to LNs, whereas CCR7 or CXCR4 does less. Notably,

Oncogene CXCR3 in colon cancer metastasis to lymph nodes K Kawada et al 4686 forced expression of CXCR3 in DLD-1 cells enhanced A complex network of chemokines and their receptors LN metastasis, although that to the lung or liver was influences development of the primary tumors and their not affected. It has been reported that metastasis of metastatic foci. The biological role of chemokines has mouse colon cancer cell line CT-26 to the liver and lung two aspects in these processes; the role of chemokines in is greatly reduced if CXCR4 function is blocked controlling leukocyte infiltration in cancer, and the (Zeelenberg et al., 2003). We have also found that influence of chemokines on the metastatic potential and CXCR4 is expressed in some human colon cancer cell site-specific spread of tumor cells (Balkwill, 2004). It has lines (Figure 1b and c). It is also reported that been demonstrated that CXCL9, CXCL10and CCL21 expression of CXCR4 in the primary colon cancer is exert antitumor activities by inducing immune-stimulat- associated with metastasis to distant organs (Kim et al., ing and angiostatic effects (Tannenbaum et al., 1998; 2005; Schimanski et al., 2005). These results collectively Vicari et al., 2000). Notably, CXCL9, CXCL10 and suggest that distinct chemokine receptors control CCL21 play additional roles in the tumor microen- metastasis of colon cancer cells to specific organs, for vironment. For example, CXCL9 and CXCL10activate example, CXCR3 to the LNs, and CXCR4 mainly to the RhoA and Rac1, induce actin reorganization, and liver and lung. trigger migration and invasion of melanoma, malignant In the human colon, CXCR3 is expressed in mono- B-, and lung and breast cancers (Trentin nuclear cells in the lamina propria, but not in the colonic et al., 1999; Robledo et al., 2001; Soejima and Rollins, epithelial cells (Dwinell et al., 2001), whereas CXCR4 is 2001; Kawada et al., 2004; Walser et al., 2006). Here, we present in the normal colonic epithelium (Jordan et al., have demonstrated that CXCR3 plays a critical role in 1999; Supplementary Figure 3a). In the present study, colon cancer cell metastasis to LNs by inducing diverse we have demonstrated that CXCR3 is absent in the cellular effects such as cytoskeletal rearrangement, normal colonic epithelium, but is expressed strongly in migration, invasion, MMP-2/9 expression and cell the colon cancer epithelium in 31 (33.7%) of 92 primary survival through activation of ERK1/2 and Akt/PKB tumors. Interestingly, we have found that CXCL10 pathways. Pro-inflammatory cytokines TNF-a and enhances the cell survival and gelatinase expression in IFN-g strongly stimulate the production of CXCL9, culture, and that cell surface expression of CXCR3 is CXCL10and CXCL11 in human intestinal epithelial upregulated upon serum starvation. It is therefore cells (Dwinell et al., 2001). Moreover, CXCL10 has been possible that cell surface expression of CXCR3 is shown to be one of the Ras targets, and overexpressed in upregulated under various kinds of stress, which allows many cases of colorectal cancer (Zhang et al., 1997). the tumor cells to survive and grow in a less favorable Notably, a small molecular antagonist of CXCR3 has microenvironment as distant organs. We have also been recently reported to inhibit lung metastasis of demonstrated that DLD-1-CXCR3 cells disseminate to breast cancer in a murine model, although they did not LNs at a similar frequency to that of DLD-1-EV cells 2 examine the metastatic potential to LNs (Walser et al., weeks after inoculations. By 4 weeks, however, more 2006). Thus, our present results suggest that CXCR3 DLD-1-CXCR3 cells expanded rapidly, than DLD-1- can be a novel therapeutic target to suppress LN EV cells. It is conceivable that the presence of CXCR3 metastasis of colon and other CXCR3-expressing cancer not only helps the initial dissemination of the cancer cells. The efficiency of chemotherapy may be improved cells to the LNs, but also stimulates the growth and significantly, if chemokine receptors are examined in expansion of the metastatic foci in later stages by biopsy and surgical samples, and a new strategy is added enhancing cell survival and gelatinase expression. to inhibit the putative chemokine receptors responsible We have demonstrated here that a CCR7 ligand for metastasis. CCL21 is functional also for CXCR3 in human colon cancer cells, and that CCL21 is expressed constitutively at high levels within lymphoid organs (Supplementary Materials and methods Figure 1). It is worth noting that CCL21 is produced also by lymphatic endothelial cells (LECs) (Saeki et al., Cell lines, reagents and tissue samples 1999). In addition to CCL21, LECs derived from Colo205, WiDr, DLD-1, SW480, LS174T and HCT15 were inflammation-induced lymphangiomas produce a num- supplied from Cell Resource Center for Biomedical Research, ber of chemokines including CXCL9 and CXCL10 Tohoku University, and HCT116, HT29, RKO and Caco2 (Mancardi et al., 2003), although LECs isolated from were obtained from American Type Culture Collection. normal human skin do not appear to produce them Female BALB/c nude (nu/nu) mice and wild-type BALB/c (Petrova et al., 2002). Inflammation-induced chemo- mice, 6- to 8-week old, were obtained from CLEA Japan (Osaka, Japan). Recombinant chemokines were purchased kines including the CXCR3 ligands are transported via from Peprotech (Rocky Hill, NJ, USA). Surgical specimens of lymphatic vessels to the draining LNs (Palframan et al., the primary colon cancer were collected, with informed 2001). Because inflammation is often a critical compo- consents, upon surgery at Kyoto University Hospital. Histo- nent of tumor progression, it is possible that CXCR3 pathological diagnosis was confirmed for each specimen. mediates migration of tumor cells to the draining lymphatic vessels through interaction with its ligands. Immunohistochemistry and immunofluorescence microscopy Consistently, our data show that expression of CXCR3 Formalin-fixed, paraffin-embedded sections were stained with in the primary colon tumors significantly correlates with anti-human CXCR3 mAb (R&D Systems, Minneapolis, MN, lymphatic invasion as well as LN metastasis. USA and BD PharMingen, San Diego, CA, USA), anti-human

Oncogene CXCR3 in colon cancer metastasis to lymph nodes K Kawada et al 4687 CXCR4 mAb (R&D Systems), anti-human CCR7 mAb (BD mouse to quantify the metastasized cells by quantitative PCR PharMingen), anti-GFP mAb (Molecular Probes, Eugene, (Ninomiya et al., 2004). All animal experiments were approved OR, USA), or anti-mouse CXCR3 (Santa Cruz Biotechnology, by the Animal Care and Use Committee of Kyoto University. Santa Cruz, CA, USA) by the avidin–biotin immunoper- oxidase method. In the primary human colon cancer tissues, microwave antigen retrieval was performed. In some cases, we Clinicopathological data examined chemokine receptor expression in both frozen and All data including the age, sex, histology, depth of tumor paraffin-embedded specimens from the same tissues, and invasion, lymphatic invasion, vascular invasion, LN metastasis found similar levels of expression. We interpreted a sample and disease stage were obtained from the clinical and as positive for CXCR3, CXCR4 or CCR7, when >10% of pathological records. Disease stages were classified according the tumor cells were stained with either antibody. Frozen to the criteria proposed by Standard American Joint Commit- 22-oxacalcitriol (OCT) compound-embedded tissues were tee on Cancer (AJCC) (Fleming et al., 1997). sectioned at 4 mm, and stained with anti-CXCL9 (R&D Systems), anti-CXCL10(Santa Cruz Biotechnology), anti- Statistical analysis CCL21 (R&D Systems), anti-human CXCR3 mAb, or anti- The statistical significance of differences was determined by the human CXCR4 mAb (Santa Cruz Biotechnology) followed by w2-test or Student’s t-test. A stepwise logistic regression model a biotinylated anti-goat secondary antibody and Fluorescein was used for the multivariate analysis. Survival curves were Avidin DCS (Vector Laboratories, Burlingame, CA, USA). calculated according to the method of Kaplan and Meier, and analysed using the log-rank test. Differences with Po0.05 were Quantification of human tumor cell metastasis considered significant. Dissemination of the human cancer cells to LNs was quantified using the human b-globin-related sequence (HBB; GenBank accession no. NG 000007) as described by Ninomiya et al. Acknowledgements (2004). We thank Masahiro Aoki and Takanori Kitamura for fruitful In vivo metastasis studies discussion and Hiromi Kikuchi for technical assistance. We For the rectal xenograft model, colon cancer cells (1 Â 106 cells also thank Drs T Kitamura and M Onishi (University of in 50 ml of phosphate-buffered saline (PBS)) were injected Tokyo) for retroviral vector pMX. This work was supported submucosally into the rectum of nude mice at day 0. At day 14 by grants from the Ministry of Education, Culture, Sports, or 28, the para-aortic LNs were dissected and pooled for each Science and Technology of Japan.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc).

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