Virchows Archiv

, Volume 471, Issue 3, pp 329–336 | Cite as

Intratumoral Fusobacterium nucleatum abundance correlates with macrophage infiltration and CDKN2A methylation in microsatellite-unstable colorectal carcinoma

  • Hye Eun Park
  • Jung Ho KimEmail author
  • Nam-Yun Cho
  • Hye Seung Lee
  • Gyeong Hoon KangEmail author
Original Article


Fusobacterium nucleatum (Fn), a specific species of gut microbiota, has been suggested to be enriched in the microsatellite instability-high (MSI-H) molecular subtype of colorectal carcinomas (CRCs). However, the clinicopathologic and molecular factors that interact with Fn in MSI-H CRCs are poorly understood. In this study, 16S ribosomal RNA gene DNA sequence of Fn was quantitatively measured by real-time polymerase chain reaction in tumor DNA samples from a total of 160 surgically resected MSI-H CRC tissues. Each case was classified into one of the three categories based on the Fn DNA amount: Fn-high, Fn-low, and Fn-negative. The clinicopathologic and molecular associations of Fn in MSI-H CRCs were statistically analyzed. Among the 160 MSI-H CRC samples, 15 (9%), 92 (58%), and 53 (33%) cases were Fn-high, Fn-low, and Fn-negative, respectively. Compared with Fn-low/negative tumors, Fn-high MSI-H CRCs were significantly associated with a high density of CD68+ tumor-infiltrating macrophages (P = 0.019) and promoter CpG island hypermethylation of the CDKN2A (p16) gene (P = 0.008). There were also tendencies toward associations of Fn-high with the BRAF V600E mutation (P = 0.047) and active Crohn-like lymphoid reactions (P = 0.052) in MSI-H CRCs. However, Fn-high was not significantly associated with CD3+ T cell density, CD163+ M2 macrophage density or PD-L1 expression status. In conclusion, high amounts of intratumoral Fn are correlated with increased macrophage infiltration and CDKN2A promoter methylation in MSI-H CRCs.


Colorectal cancer Fusobacterium nucleatum Gut microbiota Tumor-associated macrophage Microsatellite instability 



We thank Professor Katsuhiko Nosho, from the Department of Gastroenterology, Rheumatology and Clinical Immunology, Sapporo Medical University School of Medicine, Japan, for his kind instruction regarding the protocol of real-time PCR for Fusobacterium nucleatum. This study was supported by the National Research Foundation of Korea grant funded by the Korea government (Ministry of Science, ICT and Future Planning) (2016R1C1B2010627), a grant from the Basic Science Research Program through the National Research Foundation of Korea funded by the Korea government (Ministry of Education) (2016R1D1A1A02937130), National Research Foundation of Korea grants funded by the Korea government (Ministry of Science, ICT and Future Planning) (2011-0030049 and 2016M3A9B6026921), and a grant from the Korea Health Technology R&D Project through the Korea Health Industry Development Institute funded by the Korea government (Ministry of Health and Welfare) (HI14C1277).

Compliance with ethical standards

This study was approved by the institutional review board of Seoul National University Hospital (IRB No. 1203-072-402). Under the condition of retrospective archival tissue collection and patient data anonymization, our study was exempted from the acquisition of informed consent from patients.

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

428_2017_2171_MOESM1_ESM.pdf (32 kb)
Supplementary Fig. S1 (PDF 32 kb)
428_2017_2171_MOESM2_ESM.pdf (205 kb)
Supplementary Table S1 (PDF 205 kb)


  1. 1.
    Marchesi JR, Adams DH, Fava F, Hermes GD, Hirschfield GM, Hold G, Quraishi MN, Kinross J, Smidt H, Tuohy KM, Thomas LV, Zoetendal EG, Hart A (2016) The gut microbiota and host health: a new clinical frontier. Gut 65(2):330–339. doi: 10.1136/gutjnl-2015-309990 CrossRefPubMedGoogle Scholar
  2. 2.
    Garrett WS (2015) Cancer and the microbiota. Science 348(6230):80–86. doi: 10.1126/science.aaa4972 CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Louis P, Hold GL, Flint HJ (2014) The gut microbiota, bacterial metabolites and colorectal cancer. Nat Rev Microbiol 12(10):661–672. doi: 10.1038/nrmicro3344 CrossRefPubMedGoogle Scholar
  4. 4.
    Rubinstein MR, Wang X, Liu W, Hao Y, Cai G, Han YW (2013) Fusobacterium nucleatum promotes colorectal carcinogenesis by modulating E-cadherin/beta-catenin signaling via its FadA adhesin. Cell Host Microbe 14(2):195–206. doi: 10.1016/j.chom.2013.07.012 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Kostic AD, Chun E, Robertson L, Glickman JN, Gallini CA, Michaud M, Clancy TE, Chung DC, Lochhead P, Hold GL, El-Omar EM, Brenner D, Fuchs CS, Meyerson M, Garrett WS (2013) Fusobacterium nucleatum potentiates intestinal tumorigenesis and modulates the tumor-immune microenvironment. Cell Host Microbe 14(2):207–215. doi: 10.1016/j.chom.2013.07.007 CrossRefPubMedPubMedCentralGoogle Scholar
  6. 6.
    Gur C, Ibrahim Y, Isaacson B, Yamin R, Abed J, Gamliel M, Enk J, Bar-On Y, Stanietsky-Kaynan N, Coppenhagen-Glazer S, Shussman N, Almogy G, Cuapio A, Hofer E, Mevorach D, Tabib A, Ortenberg R, Markel G, Miklic K, Jonjic S, Brennan CA, Garrett WS, Bachrach G, Mandelboim O (2015) Binding of the Fap2 protein of Fusobacterium nucleatum to human inhibitory receptor TIGIT protects tumors from immune cell attack. Immunity 42(2):344–355. doi: 10.1016/j.immuni.2015.01.010 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Tian X, Liu Z, Niu B, Zhang J, Tan TK, Lee SR, Zhao Y, Harris DC, Zheng G (2011) E-cadherin/beta-catenin complex and the epithelial barrier. J Biomed Biotechnol 2011:567305. doi: 10.1155/2011/567305 PubMedPubMedCentralGoogle Scholar
  8. 8.
    Tahara T, Yamamoto E, Suzuki H, Maruyama R, Chung W, Garriga J, Jelinek J, Yamano HO, Sugai T, An B, Shureiqi I, Toyota M, Kondo Y, Estecio MR, Issa JP (2014) Fusobacterium in colonic flora and molecular features of colorectal carcinoma. Cancer Res 74(5):1311–1318. doi: 10.1158/0008-5472.CAN-13-1865 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Mima K, Nishihara R, Qian ZR, Cao Y, Sukawa Y, Nowak JA, Yang J, Dou R, Masugi Y, Song M, Kostic AD, Giannakis M, Bullman S, Milner DA, Baba H, Giovannucci EL, Garraway LA, Freeman GJ, Dranoff G, Garrett WS, Huttenhower C, Meyerson M, Meyerhardt JA, Chan AT, Fuchs CS, Ogino S (2016) Fusobacterium nucleatum in colorectal carcinoma tissue and patient prognosis. Gut 65(12):1973–1980. doi: 10.1136/gutjnl-2015-310101 CrossRefPubMedGoogle Scholar
  10. 10.
    Mima K, Sukawa Y, Nishihara R, Qian ZR, Yamauchi M, Inamura K, Kim SA, Masuda A, Nowak JA, Nosho K, Kostic AD, Giannakis M, Watanabe H, Bullman S, Milner DA, Harris CC, Giovannucci E, Garraway LA, Freeman GJ, Dranoff G, Chan AT, Garrett WS, Huttenhower C, Fuchs CS, Ogino S (2015) Fusobacterium nucleatum and T cells in colorectal carcinoma. JAMA Oncol 1(5):653–661. doi: 10.1001/jamaoncol.2015.1377 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Ito M, Kanno S, Nosho K, Sukawa Y, Mitsuhashi K, Kurihara H, Igarashi H, Takahashi T, Tachibana M, Takahashi H, Yoshii S, Takenouchi T, Hasegawa T, Okita K, Hirata K, Maruyama R, Suzuki H, Imai K, Yamamoto H, Shinomura Y (2015) Association of Fusobacterium nucleatum with clinical and molecular features in colorectal serrated pathway. Int J Cancer 137(6):1258–1268. doi: 10.1002/ijc.29488 CrossRefPubMedGoogle Scholar
  12. 12.
    Yu J, Chen Y, Fu X, Zhou X, Peng Y, Shi L, Chen T, Wu Y (2016) Invasive Fusobacterium nucleatum may play a role in the carcinogenesis of proximal colon cancer through the serrated neoplasia pathway. Int J Cancer 139(6):1318–1326. doi: 10.1002/ijc.30168 CrossRefPubMedGoogle Scholar
  13. 13.
    Kim JH, Bae JM, Song YS, Cho NY, Lee HS, Kang GH (2016) Clinicopathologic, molecular, and prognostic implications of the loss of EPCAM expression in colorectal carcinoma. Oncotarget 7(12):13372–13387. doi: 10.18632/oncotarget.5618 CrossRefPubMedGoogle Scholar
  14. 14.
    Boland CR, Thibodeau SN, Hamilton SR, Sidransky D, Eshleman JR, Burt RW, Meltzer SJ, Rodriguez-Bigas MA, Fodde R, Ranzani GN, Srivastava S (1998) A National Cancer Institute Workshop on Microsatellite Instability for cancer detection and familial predisposition: development of international criteria for the determination of microsatellite instability in colorectal cancer. Cancer Res 58(22):5248–5257PubMedGoogle Scholar
  15. 15.
    Kim JH, Park HE, Cho NY, Lee HS, Kang GH (2016) Characterisation of PD-L1-positive subsets of microsatellite-unstable colorectal cancers. Br J Cancer 115(4):490–496. doi: 10.1038/bjc.2016.211 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Kim JH, Bae JM, Oh HJ, Lee HS, Kang GH (2015) Pathologic factors associated with prognosis after adjuvant chemotherapy in stage II/III microsatellite-unstable colorectal cancers. J Pathol Transl Med 49(2):118–128. doi: 10.4132/jptm.2015.02.05 CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Kim JH, Kim KJ, Bae JM, Rhee YY, Cho NY, Lee HS, Kang GH (2015) Comparative validation of assessment criteria for Crohn-like lymphoid reaction in colorectal carcinoma. J Clin Pathol 68(1):22–28. doi: 10.1136/jclinpath-2014-202603 CrossRefPubMedGoogle Scholar
  18. 18.
    Mitsuhashi K, Nosho K, Sukawa Y, Matsunaga Y, Ito M, Kurihara H, Kanno S, Igarashi H, Naito T, Adachi Y, Tachibana M, Tanuma T, Maguchi H, Shinohara T, Hasegawa T, Imamura M, Kimura Y, Hirata K, Maruyama R, Suzuki H, Imai K, Yamamoto H, Shinomura Y (2015) Association of Fusobacterium species in pancreatic cancer tissues with molecular features and prognosis. Oncotarget 6(9):7209–7220. doi: 10.18632/oncotarget.3109 CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    Castellarin M, Warren RL, Freeman JD, Dreolini L, Krzywinski M, Strauss J, Barnes R, Watson P, Allen-Vercoe E, Moore RA, Holt RA (2012) Fusobacterium nucleatum infection is prevalent in human colorectal carcinoma. Genome Res 22(2):299–306. doi: 10.1101/gr.126516.111 CrossRefPubMedPubMedCentralGoogle Scholar
  20. 20.
    Kostic AD, Gevers D, Pedamallu CS, Michaud M, Duke F, Earl AM, Ojesina AI, Jung J, Bass AJ, Tabernero J, Baselga J, Liu C, Shivdasani RA, Ogino S, Birren BW, Huttenhower C, Garrett WS, Meyerson M (2012) Genomic analysis identifies association of Fusobacterium with colorectal carcinoma. Genome Res 22(2):292–298. doi: 10.1101/gr.126573.111 CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Liu P, Liu Y, Wang J, Guo Y, Zhang Y, Xiao S (2014) Detection of fusobacterium nucleatum and fadA adhesin gene in patients with orthodontic gingivitis and non-orthodontic periodontal inflammation. PLoS One 9(1):e85280. doi: 10.1371/journal.pone.0085280 CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Dejea CM, Wick EC, Hechenbleikner EM, White JR, Mark Welch JL, Rossetti BJ, Peterson SN, Snesrud EC, Borisy GG, Lazarev M, Stein E, Vadivelu J, Roslani AC, Malik AA, Wanyiri JW, Goh KL, Thevambiga I, Fu K, Wan F, Llosa N, Housseau F, Romans K, Wu X, McAllister FM, Wu S, Vogelstein B, Kinzler KW, Pardoll DM, Sears CL (2014) Microbiota organization is a distinct feature of proximal colorectal cancers. Proc Natl Acad Sci U S A 111(51):18321–18326. doi: 10.1073/pnas.1406199111 CrossRefPubMedPubMedCentralGoogle Scholar
  23. 23.
    Chen Y, Peng Y, Yu J, Chen T, Wu Y, Shi L, Li Q, Wu J, Fu X (2017) Invasive Fusobacterium nucleatum activates beta-catenin signaling in colorectal cancer via a TLR4/P-PAK1 cascade. Oncotarget. doi: 10.18632/oncotarget.15992
  24. 24.
    Mima K, Cao Y, Chan AT, Qian ZR, Nowak JA, Masugi Y, Shi Y, Song M, da Silva A, Gu M, Li W, Hamada T, Kosumi K, Hanyuda A, Liu L, Kostic AD, Giannakis M, Bullman S, Brennan CA, Milner DA, Baba H, Garraway LA, Meyerhardt JA, Garrett WS, Huttenhower C, Meyerson M, Giovannucci EL, Fuchs CS, Nishihara R, Ogino S (2016) Fusobacterium nucleatum in colorectal carcinoma tissue according to tumor location. Clin Transl Gastroenterol 7(11):e200. doi: 10.1038/ctg.2016.53 CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Yamauchi M, Morikawa T, Kuchiba A, Imamura Y, Qian ZR, Nishihara R, Liao X, Waldron L, Hoshida Y, Huttenhower C, Chan AT, Giovannucci E, Fuchs C, Ogino S (2012) Assessment of colorectal cancer molecular features along bowel subsites challenges the conception of distinct dichotomy of proximal versus distal colorectum. Gut 61(6):847–854. doi: 10.1136/gutjnl-2011-300865 CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Mehta RS, Nishihara R, Cao Y, Song M, Mima K, Qian ZR, Nowak JA, Kosumi K, Hamada T, Masugi Y, Bullman S, Drew DA, Kostic AD, Fung TT, Garrett WS, Huttenhower C, Wu K, Meyerhardt JA, Zhang X, Willett WC, Giovannucci EL, Fuchs CS, Chan AT, Ogino S (2017) Association of dietary patterns with risk of colorectal cancer subtypes classified by Fusobacterium Nucleatum in tumor tissue. JAMA Oncol. doi: 10.1001/jamaoncol.2016.6374
  27. 27.
    Llosa NJ, Cruise M, Tam A, Wicks EC, Hechenbleikner EM, Taube JM, Blosser RL, Fan H, Wang H, Luber BS, Zhang M, Papadopoulos N, Kinzler KW, Vogelstein B, Sears CL, Anders RA, Pardoll DM, Housseau F (2015) The vigorous immune microenvironment of microsatellite instable colon cancer is balanced by multiple counter-inhibitory checkpoints. Cancer Discov 5(1):43–51. doi: 10.1158/2159-8290.CD-14-0863 CrossRefPubMedGoogle Scholar
  28. 28.
    Xiao Y, Freeman GJ (2015) The microsatellite instable subset of colorectal cancer is a particularly good candidate for checkpoint blockade immunotherapy. Cancer Discov 5(1):16–18. doi: 10.1158/2159-8290.CD-14-1397 CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Le DT, Uram JN, Wang H, Bartlett BR, Kemberling H, Eyring AD, Skora AD, Luber BS, Azad NS, Laheru D, Biedrzycki B, Donehower RC, Zaheer A, Fisher GA, Crocenzi TS, Lee JJ, Duffy SM, Goldberg RM, de la Chapelle A, Koshiji M, Bhaijee F, Huebner T, Hruban RH, Wood LD, Cuka N, Pardoll DM, Papadopoulos N, Kinzler KW, Zhou S, Cornish TC, Taube JM, Anders RA, Eshleman JR, Vogelstein B, Diaz LA Jr (2015) PD-1 blockade in tumors with mismatch-repair deficiency. N Engl J Med 372(26):2509–2520. doi: 10.1056/NEJMoa1500596 CrossRefPubMedPubMedCentralGoogle Scholar
  30. 30.
    Traves PG, Luque A, Hortelano S (2012) Macrophages, inflammation, and tumor suppressors: ARF, a new player in the game. Mediat Inflamm 2012:568783. doi: 10.1155/2012/568783 CrossRefGoogle Scholar
  31. 31.
    Cudejko C, Wouters K, Fuentes L, Hannou SA, Paquet C, Bantubungi K, Bouchaert E, Vanhoutte J, Fleury S, Remy P, Tailleux A, Chinetti-Gbaguidi G, Dombrowicz D, Staels B, Paumelle R (2011) p16INK4a deficiency promotes IL-4-induced polarization and inhibits proinflammatory signaling in macrophages. Blood 118(9):2556–2566. doi: 10.1182/blood-2010-10-313106 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Deutschland 2017

Authors and Affiliations

  1. 1.Department of Pathology, Seoul National University HospitalSeoul National University College of MedicineSeoulRepublic of Korea
  2. 2.Laboratory of Epigenetics, Cancer Research InstituteSeoul National University College of MedicineSeoulRepublic of Korea
  3. 3.Department of PathologySeoul National University Bundang HospitalSeongnamRepublic of Korea
  4. 4.Department of PathologySeoul National University College of MedicineSeoulRepublic of Korea

Personalised recommendations