{'fraction_non_alphanumeric': 0.033180719172796955, 'fraction_numerical': 0.019394001749061165, 'mean_word_length': 5.037267080745342, 'pattern_counts': {'":': 0, '<': 5, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Expression of Aggrus/podoplanin in bladder cancer and its role in pulmonary metastasis

Platelet aggregation-inducing factor Aggrus, also known as podoplanin, is associated with tumor malignancy by promoting hematogenous metastasis. Aggrus overexpression has been reported in some tumor tissues including lung, esophagus, head and neck and brain. We here found the frequent upregulation of aggrus mRNA in urinary bladder cancers using cancer tissue panels from various organs. Immunohistochemical analysis confirmed Aggrus protein expression in urinary bladder cancers and suggested a positive correlation between Aggrus expression and metastatic tendency in bladder cancers. Endogenous expression of Aggrus protein on the cell surface was found in the mouse bladder cancer MBT-2 cell line and human bladder cancer SCaBER cell lines. Knockdown of Aggrus expression in MBT-2 cells decreased their ability to induce platelet aggregation and form pulmonary metastasis in syngeneic mouse models. Knockdown of Aggrus expression in the human bladder cancer SCaBER cells also attenuated their ability to induce platelet aggregation and form pulmonary metastasis in mice. Moreover, pulmonary metastasis of SCaBER cells was prevented by prior administration of our generated anti-Aggrus neutralizing monoclonal antibodies by attenuating their retention in lung. These results indicate that Aggrus plays an important role in bladder cancer metastasis. Thus, anti-Aggrus neutralizing antibodies would be useful for the prevention of hematogenous metastasis of Aggrus-positive bladder cancer.

embolization and epithelial-mesenchymal transition in the microvasculature and protecting the tumor cells from shear stress or immunological assault. 12 In contrast, the risk of hematogenous metastasis is low in the patients with thrombocytopenia. 13,14 In addition, antiplatelet agents have been reported to reduce the number of experimental tumor metastasis. 15 Therefore, Aggrus with platelet aggregation-inducing ability would increase the risk of hematogenous metastasis with assistance from host platelets.
Aggrus is a type-I transmembrane sialoglycoprotein that is frequently upregulated in several cancers, including SCC, mesothelioma, Kaposi's sarcoma, testicular germ cell tumors and brain tumors. [16][17][18][19] Aggrus upregulation in cancer-associated fibroblasts has also been reported. Its expression in these cells is associated with poor prognosis in patients with lung ADC. 20 Recently, Aggrus expression was found in tumor-initiating cells, suggesting a role of Aggrus in cancer progression. 21 The C-type lectin-like receptor 2 (CLEC-2) expressed on platelets was recently identified as one of the counter receptors of Aggrus. 16,22,23 Aggrus binding to CLEC-2 transmits platelet activation signals through Src family kinases, Syk and phospholipase Cg2 in platelets. 24,25 CLEC-2 was previously identified as a platelet receptor for the snake venom toxin rhodocytin. 26 Because of the similarities in platelet-aggregation phenotypes induced by rhodocytin and Aggrus, CLEC-2 was identified as a platelet receptor of Aggrus. 22 In addition, similarities were observed in the phenotype of mice deficient in CLEC-2 to that of Aggrus/podoplanin knockout mice, 25,26 confirming that CLEC-2 functions as a platelet receptor of Aggrus in vivo.
In this study, we screened the aggrus mRNA expression in various cancers and found that some bladder cancers showed high aggrus mRNA levels. Tissue microarray analysis confirmed that Aggrus expression is frequently upregulated in metastatic bladder TCC and SCC. Because Aggrus knockdown in Aggrus-positive bladder cancer cell lines decreased the number of pulmonary metastatic foci, Aggrus expression was directly linked to the lung metastasis of bladder cancers. Moreover, we found that our generated anti-Aggrus neutralizing antibodies attenuated the pulmonary metastasis of bladder cancers suggesting the usefulness of the neutralizing antibodies as metastasis-inhibitory drugs.

What's new?
Expression of the platelet-aggregation-inducing factor Aggrus (podoplanin) by tumor cells is associated with metastasis via the bloodstream. In this study, the authors found that Aggrus is over-expressed in highly metastatic human bladder cancers, and that knockdown of this factor decreased metastasis of both mouse and human bladder-cancer cells in mice. Anti-Aggrus antibodies were also able to prevent this metastasis. This type of antibody may therefore be a promising adjunct to bladdercancer therapy, and Aggrus over-expression may be a useful biomarker for predicting metastasis.
intensity score (the average staining intensity) was defined as follows: 0: negative, 1: weakly positive, 2: moderately positive, 3: strongly positive. Scoring of immunohistochemical (IHC) analyzed slides was performed by two independent pathologists who were blind to diagnosis.

Platelet-aggregation assay
Murine whole blood was drawn by cardiac puncture from Jcl:ICR mice terminally anesthetized with chloroform and taken with heparin solution. Platelet aggregation was measured using the screen filtration pressure method and a WBA Carna aggregometer (IMI, Saitama, Japan). Whole blood samples (200 ml) were stirred in the reaction tubes at 1,000 rpm at 37 C and preincubated for 2 min, followed by cell addition (MBT-2 and its transfectants: 1.6 3 10 5 cells, SCaBER: 1.3 3 10 5 cells). Using a 3.7-mm diameter syringe containing screen microsieves made of nickel with 300 openings per 30 3 30 lm 2 in an area 1 mm in diameter, whole blood were sucked to detect aggregation pressure at 1-12 min later. The final plateletaggregation pressure in each reaction tube was determined depending on the pressure rate (%) detected by the pressure sensor. In some experiments, SCaBER cells were preincubated for 30 min on ice with 300 mg/ml of anti-human Aggrus neutralizing mAbs, previously established in our laboratory (clone: P2-0 and MS-1), 31,32 or control mouse IgG (Sigma-Aldrich).

Animals
Male C3H/HeNCrlCrlj and female CB17/Icr-Prkdc scid /CrlCrlj mice were purchased from Charles River Laboratories Japan, Inc. (Kanagawa, Japan). Jcl:ICR mice were purchased from Clea Japan. (Tokyo, Japan). All animal procedures were performed using protocols approved by the Japanese Foundation for Cancer Research Animal Care and Use Committee.

In vitro and in vivo proliferation assays
To assess in vitro cell proliferation, the CellTiter-Glo luminescent cell viability assay (Promega, Madison, WI) was used after 24, 48 or 72 h in cell culture. For the in vivo proliferation assay, SCaBER cells were harvested, washed and resuspended in HBSS. In total, 3.0 3 10 6 cells suspended in 50 ll HBSS were subcutaneously injected into the backs of 8-weekold male C3H/HeNCrlCrlj mice. Tumor volume was calculated using the following formula: volume 5 W 2 3 L/2, where W is width (the shortest dimension) and L is length (the longest dimension).

Statistical analysis
The Mann-Whitney U test was performed to determine the statistical significance of the results of the metastasis assays. Some results were compared using Student t-test. Significant p values are shown as *p < 0.05, **p < 0.01. All statistical tests were two-sided.

Aggrus expression in bladder cancers
To identify new tumors expressing Aggrus, qRT-PCR was initially performed using the TissueScan Cancer Survey Panel containing 381 human tissues from patients with 22 different forms of cancer. Upregulation of aggrus mRNA was detected in cancer tissues from esophagus, lung, testis and urinary bladder, which were then compared with normal tissues (Fig.  1). Because several groups have already reported Aggrus overexpression in esophageal, lung and testicular tumors, 18,33-35 we focused on aggrus expression in urinary bladder cancers in this study. Although we compared the aggrus mRNA level among histological types or differentiation degrees of urinary bladder cancers, no correlation could be found (Supporting Information Table S1).

Correlation of Aggrus expression with metastatic tendency in bladder cancers
We next examined Aggrus protein expression in 135 different cases of human bladder cancer by IHC staining of tissue arrays with anti-human Aggrus/podoplanin mAb D2-40. Although most G1, G2 and G3 TCCs received scores of 0-3, approximately half of the metastatic TCC tissues received scores 4 ( Fig. 2a and Supporting Information Table S2). Moreover, clear staining of SCC (but not ADC) of bladder was observed (Fig. 2b). Because distant metastasis is more common in TCC and SCC than in ADC, 6,7 these results suggest that Aggrus would be useful as a biomarker for prediction of metastatic TCC and SCC of bladder.

Aggrus expression in mouse bladder cancer cell line MBT-2
Aggrus is known to enhance hematogenous metastasis of various cancers by interacting with and activating platelet receptor CLEC-2. 16 To investigate the effects of Aggrus expression in bladder cancer metastasis, the chemically induced mouse bladder TCC cell line MBT-2 was initially utilized because of its applicability to syngeneic tumor models. Expression of mouse Aggrus protein has been reported in NL-17 cells, a highly metastatic subclone of the mouse colon ADC 26 cell line. 28 Therefore, NL-17 cells were used as a positive control in this study. Aggrus protein and mRNA expression was detected in MBT-2 cells (Figs. 3a and 3b). Aggrusknockdown MBT-2 cells were then established and designated as MBT-2/shAgg cells (Figs. 3c and 3d). These cells were examined for their binding capacity against the recombinant CLEC-2 protein. Although cell surface binding of CLEC-2 was detected by flow cytometry in a control cell line (MBT-2/shCont); the CLEC-2 binding was dramatically attenuated in MBT-2/shAgg cells (Fig. 3e). These results indicated an interaction of MBT-2 cells with the CLEC-2 protein through Aggrus expression on the cell surface.

Involvement of Aggrus in pulmonary metastasis of MBT-2 cells
Aggrus-CLEC-2 interaction is critical for Aggrus-induced platelet aggregation and hematogenous metastasis. 16 Thus, platelet aggregation-inducing ability and lung metastasizing ability of Aggrus-knockdown cells were measured. It was observed that platelet-aggregation rate and the number of lung surface metastatic foci were attenuated in MBT-2/shAgg cells, which is consistent with the results shown in Figure 3e, but the attenuation was not observed in MBT-2/shCont cells (Figs. 4a-4c). The in vivo growth rate was similar among the established MBT-2 clones (Supporting Information Fig. S1). These results indicated that Aggrus plays an important role in pulmonary metastasis of MBT-2 cells.

Aggrus expression in human bladder cancer cells and its role in pulmonary metastasis
By collecting several human bladder cancer cell lines, we screened the expression of human Aggrus protein in these cells. Our previously established Aggrus-transfected CHO

Cancer Cell Biology
Takagi et al.
(CHO/Aggrus) cells were used as a positive controls. 15 Aggrus expression was detected in SCC cell lines such as UM-UC-5 and SCaBER, and a transitional cell papilloma cell line, RT4 (Figs. 5a and 5b). No Aggrus expression was detected in the collected TCC cell lines (J82, UM-UC-3 and T24). To investigate the role of Aggrus expression in hematogenous metastasis in human bladder cancer cells, Aggrus-knockdown SCaBER cells were established (SCaBER/shAgg1h and SCaBER/ shAgg2h; Figs. 5c and 5d) and their role in pulmonary metastasis was examined. Metastatic ability was attenuated in SCaBER/shAgg1h and SCaBER/shAgg2h cells consistent with Aggrus expression levels (Figs. 5e and 5f). These results indicate that human Aggrus also plays an important role in pulmonary metastasis of human bladder cancer cells.
Efficacy and mechanism of anti-Aggrus neutralizing antibodies on pulmonary metastasis of human bladder cancer cells We have previously established anti-human Aggrus neutralizing antibodies designated as P2-0 and MS-1, and clarified their efficacy as hematogenous metastasis inhibitors using Aggrus-overexpressing cell lines. 31,32 Thus, we attempted to examine the efficacy of anti-human Aggrus neutralizing antibodies on SCaBER cell-mediated platelet aggregation. Platelet aggregation induced by SCaBER cells was significantly suppressed by the addition of P2-0 or MS-1 mAb (Fig. 6a). The effects of these antibodies on pulmonary metastasis of SCaBER cells were then investigated. Consistent with the earlier results, pulmonary metastasis of SCaBER cells was prevented by prior administration of P2-0 or MS-1 mAb into mice (Figs. 6b and 6c). To clarify how these mAbs suppress lung metastasis, calcein-AM-labeled SCaBER cells were intravenously injected into nude mice and fluorescent cells in lung microvessels were counted after 30 min. Fluorescent cells were detected in lungs of control IgG-administrated mice, indicating that several SCaBER cells were trapped in lung (Figs. 6d and 6e). In contrast, prior administration of P2-0 or MS-1 mAbs dramatically decreased the number of cells trapped in lung. These results indicated that P2-0 and MS-1 mAbs suppressed pulmonary metastasis of SCaBER cells by inhibiting cell retention in lung. Therefore, anti-Aggrus neutralizing antibodies would be useful for the prevention of pulmonary metastasis of Aggrus-positive bladder cancer cells.

Discussion
Although superficial tumors in patients with bladder cancer are generally not life-threatening, occult distant metastasis in recurrent cancer is associated with poor 5-year survival rates. 5 Molecular markers to identify localized tumors with high metastatic potential are currently lacking. 36 Thus, identification and inhibition of key molecules that induce distant metastasis is important for improving the 5-year survival rate of patients with bladder cancer. In this study, upregulation of the platelet aggregationinducing factor Aggrus was found in bladder TCC tissues from patients with distant metastasis (Fig. 2). Although Aggrus reportedly enhances hematogenous metastasis in experimental pulmonary metastasis models, 11,15 the correlation between Aggrus expression and distant metastasis in cancer patients is not so clear. This is the first study to report the Aggrus upregulation in bladder cancer patients with distant metastasis and to indicate the possibility of Aggrus as a biomarker for predicting distant metastasis of bladder cancer. Previous reports have examined ET-1, ET-1 receptor, and CD24 as possible diagnostic markers of distant metastasis in bladder cancer. 8,9 ET-1 is known to promote tumor colonization through ET-1 receptor activation without affecting primary tumor growth. 8 CD24 is reported to be involved in the progression of tumorigenesis and metastasis of male bladder cancer under androgen regulation. 9 Using existing diagnostic markers such as ET-1 or CD24 in combination with Aggrus would improve accuracy in identification of distant metastasis in bladder cancer patients.
Moreover, Aggrus expression was found in clinical specimens of bladder SCC by IHC analysis (Fig. 2) and in bladder SCC cell lines (SCaBER and UM-UC-5, Fig. 5). Several groups have reported Aggrus upregulation in SCC of lung, esophagus and head and neck. 18,34,35,37 Thus, our finding is consistent with those of the above mentioned reports.
An association of Aggrus with pulmonary metastasis of bladder cancer cell lines was also found in this study (Figs. 4 and 5). The results of this study indicate that pulmonary metastasis may be prevented by prior administration of anti-Aggrus neutralizing mAbs (Fig. 6). The two anti-Aggrus neutralizing mAbs that we established, P2-0 (mouse IgG1) and MS-1 (mouse IgG2a), possess similar affinity, epitope and platelet-neutralizing ability. However, only MS-1 exhibits Antibody-dependent cellular cytotoxic (ADCC) activity and complement-dependent cytotoxic (CDC) activity. 32 Because P2-0 and MS-1 mAbs suppressed lung retention of bladder Figure 6. Efficacy and mechanism of anti-Aggrus neutralizing mAbs on pulmonary metastasis of human bladder cancer cells. (a) SCaBER cells were incubated with 300 mg/ml of anti-human Aggrus mAbs (clone: P2-0 and MS-1) or control mouse IgG for 30 min on ice, followed by incubation with whole-blood samples. The platelet-aggregation rate was determined using WBA Carna aggregometer. *p < 0.05 by the Mann-Whitney U test. (b) to (e) CB17/Icr-Prkdc scid /CrlCrlj mice were intravenously injected with control mouse IgG, P2-0 mAb, or MS-1 mAb (30 mg/mouse). After 24 hr of mAb administration, SCaBER cells were intravenously inoculated into the mice. After 35 days of tumor inoculation, lung surface metastatic foci were counted. Bars, mean. n 5 8. **p < 0.01 by the Mann-Whitney U test (b). Representative pictures of the lung surface metastatic foci were shown (c). After 24 hr of mAb injection, calcein-AM-labeled SCaBER cells were intravenously inoculated into the mice (d and e). After 30 min of tumor inoculation, frozen section of lung was prepared and number of micro metastatic foci labeled by calcein-AM was counted (d). Representative pictures of the frozen section of lung are shown (e). Scale bar, 0.5 mm. Bars, mean. n 5 3. **p < 0.01 by the Mann-Whitney U test (d).
cancer cells, the anti-metastatic effects of these anti-Aggrus neutralizing antibodies would be platelet-aggregation inhibitory activity rather than effector activity. Although inhibitory effects of P2-0 and MS-1 mAbs are similar in the plateletaggregation assay (Fig. 6a), P2-0 mAb exhibited inhibitory activity higher than MS-1 mAb in the lung retention assay (Fig. 6d). We performed platelet-aggregation assay after mixing cancer cells with antibodies, while in the lung retention assay the cancer cells were intravenously inoculated on the day following administration of antibodies into mice. Because P2-0 and MS-1 mAbs are similar in the affinity (9.3 and 9.0 nM, respectively) but slightly different in the dissociation rate constant (8.5 3 10 23 and 1 3 10 22 sec 21 , respectively), 31,32 it might be appeared as a difference of the capability to block Aggurs-CLEC-2 interaction in a short period until the retention of cancer cells in lung microvessels. We have also confirmed that the administration of either P2-0 or MS-1 mAb on the day following intravenous inoculation of tumor cells significantly suppressed the number of lung metastatic foci (data not shown). Thus, anti-Aggrus neutralizing antibodies may be appropriate as both neo-adjuvant and adjuvant therapy in patients with highly metastatic bladder cancer. However, further studies are required to evaluate the function of anti-Aggrus neutralizing mAbs on Aggrus-positive cancer cells in the bloodstream.
Because systemic chemotherapy regimens used to treat bladder TCC are generally ineffective for treatment of pure bladder SCC, 38 effective molecular target drugs against bladder SCC are urgently needed. ADCC and CDC activities are effective modes of action for therapeutic antibodies. Drugs based on these activities have been approved for clinical usage (i.e., trastuzumab and rituximab). Abe et al. reported the efficacy of co-administration therapy of anti-Aggrus antibodies exhibiting ADCC activity with natural killer cells against Aggrus-positive malignant pleural mesothelioma, 39 suggesting that anti-Aggrus antibody-based immunotherapy may be a promising strategy for the treatment of bladder SCC.