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Characterization of tumor-infiltrating immune cells in relation to microbiota in colorectal cancers

  • Tomohiro Kikuchi
  • Kosaku MimuraEmail author
  • Mai Ashizawa
  • Hirokazu Okayama
  • Eisei Endo
  • Katsuharu Saito
  • Wataru Sakamoto
  • Shotaro Fujita
  • Hisahito Endo
  • Motonobu Saito
  • Tomoyuki Momma
  • Zenichiro Saze
  • Shinji Ohki
  • Kazunori Shimada
  • Kiyoshi Yoshimura
  • Takuya Tsunoda
  • Koji Kono
Original Article

Abstract

Background

Several articles have recently reported that certain colon microbiota can improve the efficacy of cancer immunotherapy. To develop new treatment strategies, including immunotherapy for colorectal cancer (CRC), we evaluated the correlations between subpopulations of tumor-infiltrating immune cells (TIICs) and intestinal microbiota in CRC.

Methods

Fresh surgically resected specimens, formalin-fixed paraffin-embedded whole tissue samples, and stool samples were collected. TIICs including Tregs, Th17 cells and tumor-associated macrophages (TAMs) in the surgically resected specimens were analyzed using flow cytometry. FOXp3, CD8, CD163, and phosphorylated-STAT1-positive TIICs in the whole tissue samples were analyzed using IHC, and intestinal microbiota in the stool samples was analyzed using 16S metagenome sequencing. TIICs subpopulations in the normal mucosa and tumor samples were evaluated, and the correlations between the TIIC subpopulations and intestinal microbiota were analyzed.

Results

FOXp3lowCD45RA+ Tregs were significantly reduced (p = 0.02), FOXp3lowCD45RA Tregs were significantly increased (p = 0.006), and M1 TAMs were significantly reduced in the tumor samples (p = 0.03). Bacteroides (phylum Bacteroidetes) and Faecalibacterium (phylum Firmicutes) were increased in the patients with high numbers of Tregs and clearly high distribution of FOXp3highCD45RA Tregs, which are the effector Tregs. Faecalibacterium, Ruminococcaceae, Eubacterium (phylum Firmicutes), and Bacteroides were increased in patients with a high distribution of M1 TAMs.

Conclusions

The findings of the present study indicate that immune responses to tumors are suppressed in the tumor microenvironment of CRC depending on the increment of Tregs and the reduction of M1 TAMs and that intestinal microbiota might be involved in immunosuppression.

Keywords

Colorectal cancer Microbiota Tumor-associated macrophage Treg 

Abbreviations

CRC

Colorectal cancer

p-STAT1

Phosphorylated-STAT1

PCoA

Principal coordinates analysis

TAMs

Tumor-associated macrophages

TIICs

Tumor-infiltrating immune cells

Notes

Author contributions

TK, KM, TT, and KK contributed to the study conception and design. TK, MA, HO, EE, KS, WS, SF, HE, MS, TM, ZS, and SO contributed to the acquisition of patient samples. TK, KM, MA, HO, WS, SF, HE, MS, TM, ZS, and SO performed flow cytometry and analyzed the flow cytometry data. KS, KY, and TT performed the 16S metagenome sequencing and analyzed the microbiota data. EE and KS performed IHC and evaluated the IHC staining. TK, KM, KS, KY, TT, and KK drafted the manuscript.

Funding

No relevant funding.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval and standards

This study was conducted in accordance with the ethical principles that have their origin in the Declaration of Helsinki and was approved by the Fukushima Medical University Research Ethics Committee (Receipt No. 29020).

Informed consent

Written informed consent was obtained from all patients included in the study for the use of their specimens and clinical data for research and publication prior to collecting the specimen at Fukushima Medical University Hospital.

Supplementary material

262_2019_2433_MOESM1_ESM.pdf (889 kb)
Supplementary material 1 (PDF 889 kb)

References

  1. 1.
    Siegel RL, Miller KD, Jemal A (2015) Cancer statistics. CA Cancer J Clin 65(1):5–29.  https://doi.org/10.3322/caac.21254 CrossRefPubMedGoogle Scholar
  2. 2.
    Torre LA, Bray F, Siegel RL, Ferlay J, Lortet-Tieulent J, Jemal A (2015) Global cancer statistics, 2012. CA Cancer J Clin 65(2):87–108.  https://doi.org/10.3322/caac.21262 CrossRefGoogle Scholar
  3. 3.
    Brenner H, Kloor M, Pox CP (2014) Colorectal cancer. Lancet 383(9927):1490–1502.  https://doi.org/10.1016/s0140-6736(13)61649-9 CrossRefPubMedGoogle Scholar
  4. 4.
    Cercek A, Roxburgh CSD, Strombom P, Smith JJ, Temple LKF, Nash GM, Guillem JG, Paty PB, Yaeger R, Stadler ZK, Seier K, Gonen M, Segal NH, Reidy DL, Varghese A, Shia J, Vakiani E, Wu AJ, Crane CH, Gollub MJ, Garcia-Aguilar J, Saltz LB, Weiser MR (2018) Adoption of total neoadjuvant therapy for locally advanced rectal cancer. JAMA Oncol.  https://doi.org/10.1001/jamaoncol.2018.0071 CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Gollins S, Sebag-Montefiore D (2016) Neoadjuvant treatment strategies for locally advanced rectal cancer. Clin Oncol 28(2):146–151.  https://doi.org/10.1016/j.clon.2015.11.003 CrossRefGoogle Scholar
  6. 6.
    Mellman I, Coukos G, Dranoff G (2011) Cancer immunotherapy comes of age. Nature 480(7378):480–489.  https://doi.org/10.1038/nature10673 CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Afreen S, Dermime S (2014) The immunoinhibitory B7-H1 molecule as a potential target in cancer: killing many birds with one stone. Hematol Oncol stem cell Ther 7(1):1–17.  https://doi.org/10.1016/j.hemonc.2013.09.005 CrossRefPubMedGoogle Scholar
  8. 8.
    Shibuya KC, Goel VK, Xiong W, Sham JG, Pollack SM, Leahy AM, Whiting SH, Yeh MM, Yee C, Riddell SR, Pillarisetty VG (2014) Pancreatic ductal adenocarcinoma contains an effector and regulatory immune cell infiltrate that is altered by multimodal neoadjuvant treatment. PLoS One 9(5):e96565.  https://doi.org/10.1371/journal.pone.0096565 CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Tassi E, Gavazzi F, Albarello L, Senyukov V, Longhi R, Dellabona P, Doglioni C, Braga M, Di Carlo V, Protti MP (2008) Carcinoembryonic antigen-specific but not antiviral CD4 + T cell immunity is impaired in pancreatic carcinoma patients. J Immunol (Baltimore, Md: 1950) 181(9):6595–6603CrossRefGoogle Scholar
  10. 10.
    De Monte L, Reni M, Tassi E, Clavenna D, Papa I, Recalde H, Braga M, Di Carlo V, Doglioni C, Protti MP (2011) Intratumor T helper type 2 cell infiltrate correlates with cancer-associated fibroblast thymic stromal lymphopoietin production and reduced survival in pancreatic cancer. J Exp Med 208(3):469–478.  https://doi.org/10.1084/jem.20101876 CrossRefPubMedPubMedCentralGoogle Scholar
  11. 11.
    Ozdemir BC, Pentcheva-Hoang T, Carstens JL, Zheng X, Wu CC, Simpson TR, Laklai H, Sugimoto H, Kahlert C, Novitskiy SV, De Jesus-Acosta A, Sharma P, Heidari P, Mahmood U, Chin L, Moses HL, Weaver VM, Maitra A, Allison JP, LeBleu VS, Kalluri R (2014) Depletion of carcinoma-associated fibroblasts and fibrosis induces immunosuppression and accelerates pancreas cancer with reduced survival. Cancer Cell 25(6):719–734.  https://doi.org/10.1016/j.ccr.2014.04.005 CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Soares KC, Rucki AA, Kim V, Foley K, Solt S, Wolfgang CL, Jaffee EM, Zheng L (2015) TGF-β blockade depletes T regulatory cells from metastatic pancreatic tumors in a vaccine dependent manner. Oncotarget 6(40):43005–43015.  https://doi.org/10.18632/oncotarget.5656 CrossRefPubMedPubMedCentralGoogle Scholar
  13. 13.
    Soares KC, Rucki AA, Wu AA, Olino K, Xiao Q, Chai Y, Wamwea A, Bigelow E, Lutz E, Liu L, Yao S, Anders RA, Laheru D, Wolfgang CL, Edil BH, Schulick RD, Jaffee EM, Zheng L (2015) PD-1/PD-L1 blockade together with vaccine therapy facilitates effector T-cell infiltration into pancreatic tumors. J Immunother (Hagerstown, Md: 1997) 38(1):1–11.  https://doi.org/10.1097/cji.0000000000000062 CrossRefGoogle Scholar
  14. 14.
    Saito T, Nishikawa H, Wada H, Nagano Y, Sugiyama D, Atarashi K, Maeda Y, Hamaguchi M, Ohkura N, Sato E, Nagase H, Nishimura J, Yamamoto H, Takiguchi S, Tanoue T, Suda W, Morita H, Hattori M, Honda K, Mori M, Doki Y, Sakaguchi S (2016) Two FOXP3(+)CD4(+) T cell subpopulations distinctly control the prognosis of colorectal cancers. Nat Med 22(6):679–684.  https://doi.org/10.1038/nm.4086 CrossRefPubMedGoogle Scholar
  15. 15.
    Qian BZ, Pollard JW (2010) Macrophage diversity enhances tumor progression and metastasis. Cell 141(1):39–51.  https://doi.org/10.1016/j.cell.2010.03.014 CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Condeelis J, Pollard JW (2006) Macrophages: obligate partners for tumor cell migration, invasion, and metastasis. Cell 124(2):263–266.  https://doi.org/10.1016/j.cell.2006.01.007 CrossRefPubMedGoogle Scholar
  17. 17.
    Pollard JW (2004) Tumour-educated macrophages promote tumour progression and metastasis. Nat Rev Cancer 4(1):71–78.  https://doi.org/10.1038/nrc1256 CrossRefPubMedGoogle Scholar
  18. 18.
    Gordon S (2003) Alternative activation of macrophages. Nat Rev Immunol 3(1):23–35.  https://doi.org/10.1038/nri978 CrossRefPubMedGoogle Scholar
  19. 19.
    Mantovani A, Sica A (2010) Macrophages, innate immunity and cancer: balance, tolerance, and diversity. Curr Opin Immunol 22(2):231–237.  https://doi.org/10.1016/j.coi.2010.01.009 CrossRefPubMedGoogle Scholar
  20. 20.
    Biswas SK, Gangi L, Paul S, Schioppa T, Saccani A, Sironi M, Bottazzi B, Doni A, Vincenzo B, Pasqualini F, Vago L, Nebuloni M, Mantovani A, Sica A (2006) A distinct and unique transcriptional program expressed by tumor-associated macrophages (defective NF-kappaB and enhanced IRF-3/STAT1 activation). Blood 107(5):2112–2122.  https://doi.org/10.1182/blood-2005-01-0428 CrossRefPubMedGoogle Scholar
  21. 21.
    Ojalvo LS, King W, Cox D, Pollard JW (2009) High-density gene expression analysis of tumor-associated macrophages from mouse mammary tumors. Am J Pathol 174(3):1048–1064.  https://doi.org/10.2353/ajpath.2009.080676 CrossRefPubMedPubMedCentralGoogle Scholar
  22. 22.
    Pucci F, Venneri MA, Biziato D, Nonis A, Moi D, Sica A, Di Serio C, Naldini L, De Palma M (2009) A distinguishing gene signature shared by tumor-infiltrating Tie2-expressing monocytes, blood “resident” monocytes, and embryonic macrophages suggests common functions and developmental relationships. Blood 114(4):901–914.  https://doi.org/10.1182/blood-2009-01-200931 CrossRefPubMedGoogle Scholar
  23. 23.
    Nakajima S, Koh V, Kua LF, So J, Davide L, Lim KS, Petersen SH, Yong WP, Shabbir A, Kono K (2016) Accumulation of CD11c + CD163 + adipose tissue macrophages through upregulation of intracellular 11beta-HSD1 in human obesity. J Immunol (Baltimore, Md: 1950) 197(9):3735–3745.  https://doi.org/10.4049/jimmunol.1600895 CrossRefGoogle Scholar
  24. 24.
    Dalmas E, Clement K, Guerre-Millo M (2011) Defining macrophage phenotype and function in adipose tissue. Trends Immunol 32(7):307–314.  https://doi.org/10.1016/j.it.2011.04.008 CrossRefPubMedGoogle Scholar
  25. 25.
    Hill AA, Reid Bolus W, Hasty AH (2014) A decade of progress in adipose tissue macrophage biology. Immunol Rev 262(1):134–152.  https://doi.org/10.1111/imr.12216 CrossRefPubMedPubMedCentralGoogle Scholar
  26. 26.
    Komohara Y, Fujiwara Y, Ohnishi K, Shiraishi D, Takeya M (2016) Contribution of macrophage polarization to metabolic diseases. J Atheroscler Thromb 23(1):10–17.  https://doi.org/10.5551/jat.32359 CrossRefPubMedGoogle Scholar
  27. 27.
    Deiuliis J, Shah Z, Shah N, Needleman B, Mikami D, Narula V, Perry K, Hazey J, Kampfrath T, Kollengode M, Sun Q, Satoskar AR, Lumeng C, Moffatt-Bruce S, Rajagopalan S (2011) Visceral adipose inflammation in obesity is associated with critical alterations in tregulatory cell numbers. PLoS One 6(1):e16376.  https://doi.org/10.1371/journal.pone.0016376 CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Michaud A, Pelletier M, Noel S, Bouchard C, Tchernof A (2013) Markers of macrophage infiltration and measures of lipolysis in human abdominal adipose tissues. Obesity (Silver Spring, Md) 21(11):2342–2349.  https://doi.org/10.1002/oby.20341 CrossRefGoogle Scholar
  29. 29.
    Boon MR, Bakker LE, Haks MC, Quinten E, Schaart G, Van Beek L, Wang Y, Van Schinkel L, Van Harmelen V, Meinders AE, Ottenhoff TH, Van Dijk KW, Guigas B, Jazet IM, Rensen PC (2015) Short-term high-fat diet increases macrophage markers in skeletal muscle accompanied by impaired insulin signalling in healthy male subjects. Clin Sci (London, England: 1979) 128(2):143–151.  https://doi.org/10.1042/cs20140179 CrossRefGoogle Scholar
  30. 30.
    Consortium HMP (2012) Structure, function and diversity of the healthy human microbiome. Nature 486(7402):207–214.  https://doi.org/10.1038/nature11234 CrossRefGoogle Scholar
  31. 31.
    Sears CL, Pardoll DM (2018) The intestinal microbiome influences checkpoint blockade. Nat Med 24(3):254–255.  https://doi.org/10.1038/nm.4511 CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    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.  https://doi.org/10.1101/gr.126516.111 CrossRefPubMedPubMedCentralGoogle Scholar
  33. 33.
    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.  https://doi.org/10.1158/0008-5472.Can-13-1865 CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    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.  https://doi.org/10.1101/gr.126573.111 CrossRefPubMedPubMedCentralGoogle Scholar
  35. 35.
    Marchesi JR, Dutilh BE, Hall N, Peters WH, Roelofs R, Boleij A, Tjalsma H (2011) Towards the human colorectal cancer microbiome. PLoS One 6(5):e20447.  https://doi.org/10.1371/journal.pone.0020447 CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Burns MB, Lynch J, Starr TK, Knights D, Blekhman R (2015) Virulence genes are a signature of the microbiome in the colorectal tumor microenvironment. Genome Med 7(1):55.  https://doi.org/10.1186/s13073-015-0177-8 CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    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.  https://doi.org/10.1016/j.chom.2013.07.007 CrossRefPubMedPubMedCentralGoogle Scholar
  38. 38.
    Warren RL, Freeman DJ, Pleasance S, Watson P, Moore RA, Cochrane K, Allen-Vercoe E, Holt RA (2013) Co-occurrence of anaerobic bacteria in colorectal carcinomas. Microbiome 1(1):16.  https://doi.org/10.1186/2049-2618-1-16 CrossRefPubMedPubMedCentralGoogle Scholar
  39. 39.
    Wei Z, Cao S, Liu S, Yao Z, Sun T, Li Y, Li J, Zhang D, Zhou Y (2016) Could gut microbiota serve as prognostic biomarker associated with colorectal cancer patients’ survival? A pilot study on relevant mechanism. Oncotarget 7(29):46158–46172.  https://doi.org/10.18632/oncotarget.10064 CrossRefPubMedPubMedCentralGoogle Scholar
  40. 40.
    Fjeldborg K, Pedersen SB, Møller HJ, Christiansen T, Bennetzen M, Richelsen B (2014) Human adipose tissue macrophages are enhanced but changed to an anti-inflammatory profile in obesity. J Immunol Res 2014:309548.  https://doi.org/10.1155/2014/309548 CrossRefPubMedPubMedCentralGoogle Scholar
  41. 41.
    Khodarev NN, Roizman B, Weichselbaum RR (2012) Molecular pathways: interferon/stat1 pathway: role in the tumor resistance to genotoxic stress and aggressive growth. Clin Cancer Res 18(11):3015–3021.  https://doi.org/10.1158/1078-0432.Ccr-11-3225 CrossRefPubMedGoogle Scholar
  42. 42.
    Edin S, Wikberg ML, Dahlin AM, Rutegård J, Öberg Å, Oldenborg PA, Palmqvist R (2012) The distribution of macrophages with a M1 or M2 phenotype in relation to prognosis and the molecular characteristics of colorectal cancer. PLoS One 7(10):e47045.  https://doi.org/10.1371/journal.pone.0047045 CrossRefPubMedPubMedCentralGoogle Scholar
  43. 43.
    Erreni M, Mantovani A, Allavena P (2011) Tumor-associated macrophages (TAM) and inflammation in colorectal cancer. Cancer Microenviron 4(2):141–154.  https://doi.org/10.1007/s12307-010-0052-5 CrossRefPubMedGoogle Scholar
  44. 44.
    Braster R, Bogels M, Beelen RH, van Egmond M (2017) The delicate balance of macrophages in colorectal cancer; their role in tumour development and therapeutic potential. Immunobiology 222(1):21–30.  https://doi.org/10.1016/j.imbio.2015.08.011 CrossRefPubMedGoogle Scholar
  45. 45.
    Norton SE, Ward-Hartstonge KA, Taylor ES, Kemp RA (2015) Immune cell interplay in colorectal cancer prognosis. World J Gastrointest Oncol 7(10):221–232.  https://doi.org/10.4251/wjgo.v7.i10.221 CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

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

Authors and Affiliations

  • Tomohiro Kikuchi
    • 1
  • Kosaku Mimura
    • 1
    • 2
    • 3
    • 4
    Email author
  • Mai Ashizawa
    • 1
  • Hirokazu Okayama
    • 1
  • Eisei Endo
    • 1
  • Katsuharu Saito
    • 1
  • Wataru Sakamoto
    • 1
  • Shotaro Fujita
    • 1
  • Hisahito Endo
    • 1
  • Motonobu Saito
    • 1
  • Tomoyuki Momma
    • 1
  • Zenichiro Saze
    • 1
  • Shinji Ohki
    • 1
  • Kazunori Shimada
    • 5
  • Kiyoshi Yoshimura
    • 5
  • Takuya Tsunoda
    • 6
  • Koji Kono
    • 1
  1. 1.Department of Gastrointestinal Tract SurgeryFukushima Medical University School of MedicineFukushima CityJapan
  2. 2.Department of Blood Transfusion and Transplantation ImmunologyFukushima Medical University School of MedicineFukushima CityJapan
  3. 3.Department of Advanced Cancer ImmunotherapyFukushima Medical University School of MedicineFukushima CityJapan
  4. 4.Department of Progressive DOHaD ResearchFukushima Medical University School of MedicineFukushima CityJapan
  5. 5.Department of Clinical Immunology and Oncology, Clinical Research Institute for Clinical Pharmacology and TherapeuticsShowa UniversityTokyoJapan
  6. 6.Department of Medical OncologyShowa UniversityTokyoJapan

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