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



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.


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.


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.


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.


Colorectal cancer Microbiota Tumor-associated macrophage Treg 



Colorectal cancer




Principal coordinates analysis


Tumor-associated macrophages


Tumor-infiltrating immune cells


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.


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)


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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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