Cancer Immunology, Immunotherapy

, Volume 68, Issue 12, pp 2067–2080 | Cite as

Tumor-associated macrophages expressing galectin-9 identify immunoevasive subtype muscle-invasive bladder cancer with poor prognosis but favorable adjuvant chemotherapeutic response

  • Yangyang Qi
  • Yuan Chang
  • Zewei Wang
  • Lingli Chen
  • Yunyi Kong
  • Peipei Zhang
  • Zheng Liu
  • Quan Zhou
  • Yifan Chen
  • Jiajun Wang
  • Qi Bai
  • Yu Xia
  • Li Liu
  • Yu Zhu
  • Le Xu
  • Bo Dai
  • Jianming Guo
  • Yiwei WangEmail author
  • Jiejie XuEmail author
  • Weijuan ZhangEmail author
Original Article



Tumor-associated macrophages (TAMs) exist as heterogeneous subsets and have dichotomous roles in cancer-immune evasion. This study aims to assess the clinical effects of Galectin-9+ tumor-associated macrophages (Gal-9+TAMs) in muscle-invasive bladder cancer (MIBC).

Experimental design

We identified Gal-9+TAMs by immunohistochemistry (IHC) analysis of a tumor microarray (TMA) (n = 141) from the Zhongshan Hospital and by flow cytometric analysis of tumor specimens (n = 20) from the Shanghai Cancer Center. The survival benefit of platinum-based chemotherapy in this subpopulation was evaluated. The effect of the tumor-immune microenvironment with different percentages of Gal-9+TAMs was explored.


The frequency of Gal-9+TAMs increased with tumor stage and grade. Gal-9+TAMs predicted poor overall survival (OS) and recurrence-free survival (RFS) and were better than Gal-9TAMs and TAMs to discriminate prognostic groups. In univariate and multivariate Cox regression analyses, patients with high percentages of Gal-9+TAMs showed the prominent survival benefit after receiving adjuvant chemotherapy (ACT). High Gal-9+TAM infiltration correlated with increasing numbers of regulatory T cells (Tregs) and mast cells and decreasing numbers of CD8+T and dendritic cells (DCs). Dense infiltration of Gal-9+TAMs was related to reduced cytotoxic molecules, enhanced immune checkpoints or immunosuppressive cytokines expressed by immune cells, as well as active proliferation of tumor cells. Additionally, the subpopulation accumulated was strongly associated with PD-1+TIM-3+CD8+T cells.


Gal-9+TAMs predicted OS and RFS and response to ACT in MIBC patients. High Gal-9+TAMs were associated with a pro-tumor immune contexture concomitant with T cell exhaustion.


Galectin-9+ tumor-associated macrophages Muscle-invasive bladder cancer Adjuvant chemotherapy Immune contexture 



Adjuvant chemotherapy


American Joint Committee on Cancer


Alkaline phosphatase




Dulbecco’s balanced salt solution


Flow cytometry


Galectin-9+ tumor-associated macrophages


Galectin-9 tumor-associated macrophages


Granzyme B


Hydrochloric acid


Hazard ratio


Horseradish peroxidase


Immune checkpoint inhibitors


Lymphovascular invasion


Muscle-invasive bladder cancer


Perforin 1


Recurrence-free survival


Tumor-associated macrophages


T-cell Ig and ITIM domain


Tissue microarray


Author contributions

YQ, YC, ZW and LC contributed to the acquisition, analysis and interpretation of data, statistical analysis and drafting of the manuscript. YK, PZ, ZL, QZ, YC, JW, QB, YX, LL, YZ, LX, BD and JG provided technical and material support; YW, WZ and JX were responsible for the study concept and design, analysis and interpretation of data, drafting of the manuscript, obtaining funding and study supervision. All authors read and approved the final manuscript.


This study was funded by grants from the National Natural Science Foundation of China (81671628, 31770851, 81702496, 81702497, 81702805, 81772696, 81871306, 81872082, 81902556, 81902563, 81902898, 81974393), the National Key R&D Program of China (2017YFC0114303), the Shanghai Municipal Natural Science Foundation (16ZR1406500, 17ZR1405100, 19ZR1431800), the Guide Project of Science and Technology Commission of Shanghai Municipality (17411963100), the Shanghai Sailing Program (18YF1404500, 19YF1407900, 19YF1427200), the Shanghai Municipal Commission of Health and Family Planning Program (20174Y0042, 201840168, 20184Y0151), the Fudan University Shanghai Cancer Center for Outstanding Youth Scholars Foundation (YJYQ201802) and a grant from the Shanghai Cancer Research Charity Center. None of the study sponsors contributed to the study design, or the collection, analysis or interpretation of data.

Compliance with ethical standards

Conflict of interest

The authors declare they have no conflict of interest.

Ethics approval and ethical standards

This study was approved by the Clinical Research Ethics Committee of Zhongshan Hospital, Fudan University (No. B2015-030) and the institutional review board and the ethics committee of Shanghai Cancer Center, Fudan University (No. 050432-4-1212B). This study was performed following the ethical principles of the Helsinki Declaration.

Informed consent

Patients from the Zhongshan hospital and the Shanghai Cancer Center signed informed consent forms before surgery that permitted the usage of specimens and clinical data for research and publication under the condition of anonymity.

Supplementary material

262_2019_2429_MOESM1_ESM.pdf (487 kb)
Supplementary material 1 (PDF 486 kb)


  1. 1.
    (2019) 17th International Congress of Immunology (2019) Beijing, China. Eur J Immunol 49:1–2223. CrossRefGoogle Scholar
  2. 2.
    Sanli O, Dobruch J, Knowles MA, Burger M, Alemozaffar M, Nielsen ME, Lotan Y (2017) Bladder cancer. Nat Rev Dis Primers 3:17022. CrossRefPubMedGoogle Scholar
  3. 3.
    Alfred Witjes J, Lebret T, Comperat EM et al (2017) Updated 2016 EAU guidelines on muscle-invasive and metastatic bladder cancer. Eur Urol 71:462–475. CrossRefPubMedGoogle Scholar
  4. 4.
    Chen DS, Mellman I (2017) Elements of cancer immunity and the cancer-immune set point. Nature 541:321–330. CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Spiess PE, Agarwal N, Bangs R et al (2017) Bladder cancer, version 5.2017, NCCN clinical practice guidelines in oncology. J Natl Compr Cancer Netw 15:1240–1267. CrossRefGoogle Scholar
  6. 6.
    Zibelman M, Ramamurthy C, Plimack ER (2016) Emerging role of immunotherapy in urothelial carcinoma-advanced disease. Urol Oncol 34:538–547. CrossRefPubMedGoogle Scholar
  7. 7.
    Sjodahl G, Lovgren K, Lauss M et al (2014) Infiltration of CD3(+) and CD68(+) cells in bladder cancer is subtype specific and affects the outcome of patients with muscle-invasive tumors. Urol Oncol 32:791–797. CrossRefPubMedGoogle Scholar
  8. 8.
    Sica A, Schioppa T, Mantovani A, Allavena P (2006) Tumour-associated macrophages are a distinct M2 polarised population promoting tumour progression: potential targets of anti-cancer therapy. Eur J Cancer 42:717–727. CrossRefPubMedGoogle Scholar
  9. 9.
    Ichimura T, Morikawa T, Kawai T et al (2014) Prognostic significance of CD204-positive macrophages in upper urinary tract cancer. Ann Surg Oncol 21:2105–2112. CrossRefPubMedGoogle Scholar
  10. 10.
    Fridman WH, Zitvogel L, Sautes-Fridman C, Kroemer G (2017) The immune contexture in cancer prognosis and treatment. Nat Rev Clin Oncol 14:717–734. CrossRefPubMedGoogle Scholar
  11. 11.
    Helm O, Held-Feindt J, Grage-Griebenow E et al (2014) Tumor-associated macrophages exhibit pro- and anti-inflammatory properties by which they impact on pancreatic tumorigenesis. Int J Cancer 135:843–861. CrossRefPubMedGoogle Scholar
  12. 12.
    Fridman WH, Pages F, Sautes-Fridman C, Galon J (2012) The immune contexture in human tumours: impact on clinical outcome. Nat Rev Cancer 12:298–306. CrossRefPubMedGoogle Scholar
  13. 13.
    Irie A, Yamauchi A, Kontani K et al (2005) Galectin-9 as a prognostic factor with antimetastatic potential in breast cancer. Clin Cancer Res 11:2962–2968. CrossRefPubMedGoogle Scholar
  14. 14.
    Choi SI, Seo KW, Kook MC, Kim CG, Kim YW, Cho SJ (2017) Prognostic value of tumoral expression of galectin-9 in gastric cancer. Turk J Gastroenterol 28:166–170. CrossRefPubMedGoogle Scholar
  15. 15.
    Wang Y, Sun J, Ma C et al (2016) Reduced expression of Galectin-9 contributes to a poor outcome in colon cancer by inhibiting NK cell chemotaxis partially through the Rho/ROCK1 signaling pathway. PLoS One 11:e0152599. CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Sideras K, Biermann K, Verheij J et al (2017) PD-L1, Galectin-9 and CD8 + tumor-infiltrating lymphocytes are associated with survival in hepatocellular carcinoma. OncoImmunology 6:e1273309. CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Liu Y, Liu Z, Fu Q, Wang Z, Fu H, Liu W, Wang Y, Xu J (2017) Galectin-9 as a prognostic and predictive biomarker in bladder urothelial carcinoma. Urol Oncol 35:349–355. CrossRefPubMedGoogle Scholar
  18. 18.
    Li H, Wu K, Tao K et al (2012) Tim-3/galectin-9 signaling pathway mediates T-cell dysfunction and predicts poor prognosis in patients with hepatitis B virus-associated hepatocellular carcinoma. Hepatology 56:1342–1351. CrossRefPubMedGoogle Scholar
  19. 19.
    Kratochvill F, Neale G, Haverkamp JM et al (2015) TNF counterbalances the emergence of M2 tumor macrophages. Cell Rep 12:1902–1914. CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Melief SM, Visconti VV, Visser M et al (2017) Long-term survival and clinical benefit from adoptive T-cell transfer in Stage IV melanoma patients is determined by a four-parameter tumor immune signature. Cancer Immunol Res 5:170–179. CrossRefPubMedGoogle Scholar
  21. 21.
    Fu H, Zhu Y, Wang Y et al (2018) Identification and validation of stromal immunotype predict survival and benefit from adjuvant chemotherapy in patients with muscle invasive bladder cancer. Clin Cancer Res. CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Liu Z, Zhu Y, Xu L et al (2018) Tumor stroma-infiltrating mast cells predict prognosis and adjuvant chemotherapeutic benefits in patients with muscle invasive bladder cancer. OncoImmunology 7:e1474317. CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Becht E, Giraldo NA, Dieu-Nosjean MC, Sautes-Fridman C, Fridman WH (2016) Cancer immune contexture and immunotherapy. Curr Opin Immunol 39:7–13. CrossRefPubMedGoogle Scholar
  24. 24.
    Heusschen R, Griffioen AW, Thijssen VL (2013) Galectin-9 in tumor biology: a jack of multiple trades. Biochim Biophys Acta 1836:177–185. CrossRefPubMedGoogle Scholar
  25. 25.
    Ohue Y, Kurose K, Nozawa R et al (2016) Survival of lung adenocarcinoma patients predicted from expression of PD-L1, Galectin-9, and XAGE1 (GAGED2a) on tumor cells and tumor-infiltrating T cells. Cancer Immunol Res 4:1049–1060. CrossRefPubMedGoogle Scholar
  26. 26.
    Enninga EA, Nevala WK, Holtan SG, Leontovich AA, Markovic SN (2016) Galectin-9 modulates immunity by promoting Th2/M2 differentiation and impacts survival in patients with metastatic melanoma. Melanoma Res 26:429–441. CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Enninga EAL, Chatzopoulos K, Butterfield JT, Sutor SL, Leontovich AA, Nevala WK, Flotte TJ, Markovic SN (2018) CD206-positive myeloid cells bind galectin-9 and promote a tumor-supportive microenvironment. J Pathol 245:468–477. CrossRefPubMedGoogle Scholar
  28. 28.
    Sotiriou C, Pusztai L (2009) Gene-expression signatures in breast cancer. N Engl J Med 360:790–800. CrossRefPubMedGoogle Scholar
  29. 29.
    Keren L, Bosse M, Marquez D et al (2018) A Structured tumor-immune microenvironment in triple negative breast cancer revealed by multiplexed ion beam imaging. Cell 174(1373–87):e19. CrossRefGoogle Scholar
  30. 30.
    Galluzzi L, Buque A, Kepp O, Zitvogel L, Kroemer G (2015) Immunological effects of conventional chemotherapy and targeted anticancer agents. Cancer Cell 28:690–714. CrossRefPubMedGoogle Scholar
  31. 31.
    Iwamoto T, Bianchini G, Booser D et al (2011) Gene pathways associated with prognosis and chemotherapy sensitivity in molecular subtypes of breast cancer. J Natl Cancer Inst 103:264–272. CrossRefPubMedGoogle Scholar
  32. 32.
    Ayari C, LaRue H, Hovington H, Decobert M, Harel F, Bergeron A, Tetu B, Lacombe L, Fradet Y (2009) Bladder tumor infiltrating mature dendritic cells and macrophages as predictors of response to bacillus Calmette-Guerin immunotherapy. Eur Urol 55:1386–1395. CrossRefPubMedGoogle Scholar
  33. 33.
    Ayari C, LaRue H, Hovington H, Caron A, Bergeron A, Tetu B, Fradet V, Fradet Y (2013) High level of mature tumor-infiltrating dendritic cells predicts progression to muscle invasion in bladder cancer. Hum Pathol 44:1630–1637. CrossRefPubMedGoogle Scholar
  34. 34.
    Winerdal ME, Marits P, Winerdal M, Hasan M, Rosenblatt R, Tolf A, Selling K, Sherif A, Winqvist O (2011) FOXP3 and survival in urinary bladder cancer. BJU Int 108:1672–1678. CrossRefPubMedGoogle Scholar
  35. 35.
    Di Caro G, Cortese N, Castino GF et al (2016) Dual prognostic significance of tumour-associated macrophages in human pancreatic adenocarcinoma treated or untreated with chemotherapy. Gut 65:1710–1720. CrossRefPubMedGoogle Scholar
  36. 36.
    Golden-Mason L, Rosen HR (2017) Galectin-9: diverse roles in hepatic immune homeostasis and inflammation. Hepatology 66:271–279. CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Apetoh L, Ghiringhelli F, Tesniere A et al (2007) Toll-like receptor 4-dependent contribution of the immune system to anticancer chemotherapy and radiotherapy. Nat Med 13:1050–1059. CrossRefPubMedGoogle Scholar
  38. 38.
    Topalian SL, Hodi FS, Brahmer JR et al (2012) Safety, activity, and immune correlates of anti–PD-1 antibody in cancer. N Engl J Med 366:2443–2454. CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Sharma P, Callahan MK, Bono P et al (2016) Nivolumab monotherapy in recurrent metastatic urothelial carcinoma (CheckMate 032): a multicentre, open-label, two-stage, multi-arm, phase 1/2 trial. Lancet Oncol 17:1590–1598. CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Wherry EJ, Kurachi M (2015) Molecular and cellular insights into T cell exhaustion. Nat Rev Immunol 15:486. CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Aitken M, Kleinrock M, Simorellis A, Nass D (2018) Global oncology trends 2018. Innovation, expansion and disruption. IQVIA Institute for Human Data Science. Parsippany Google Scholar. Accessed 24 May 2018
  42. 42.
    Galsky MD, Wang H, Hahn NM et al (2018) Phase 2 trial of gemcitabine, cisplatin, plus ipilimumab in patients with metastatic urothelial cancer and impact of DNA damage response gene mutations on outcomes. Eur Urol 73:751–759CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Immunology, School of Basic Medical SciencesFudan UniversityShanghaiChina
  2. 2.Department of UrologyFudan University Shanghai Cancer CenterShanghaiChina
  3. 3.Department of UrologyZhongshan Hospital, Fudan UniversityShanghaiChina
  4. 4.Department of PathologyZhongshan Hospital, Fudan UniversityShanghaiChina
  5. 5.Department of PathologyFudan University Shanghai Cancer CenterShanghaiChina
  6. 6.Department of PathologyRuijin Hospital, Shanghai Jiao Tong University School of MedicineShanghaiChina
  7. 7.Department of Biochemistry and Molecular Biology, School of Basic Medical SciencesFudan UniversityShanghaiChina
  8. 8.Department of UrologyRuijin Hospital, Shanghai Jiao Tong University School of MedicineShanghaiChina
  9. 9.Department of Urology, Shanghai Ninth People’s HospitalShanghai Jiao Tong University School of MedicineShanghaiChina

Personalised recommendations