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Original research
High-dimensional cytometric analysis of colorectal cancer reveals novel mediators of antitumour immunity
  1. Natasja L de Vries1,2,
  2. Vincent van Unen1,
  3. Marieke E Ijsselsteijn3,
  4. Tamim Abdelaal4,5,
  5. Ruud van der Breggen3,
  6. Arantza Farina Sarasqueta3,
  7. Ahmed Mahfouz4,5,
  8. Koen C M J Peeters6,
  9. Thomas Höllt5,7,
  10. Boudewijn P F Lelieveldt4,8,
  11. Frits Koning1,
  12. Noel F C C de Miranda3
  1. 1 Immunohematology and Blood Transfusion, Leiden University Medical Center, Leiden, Netherlands
  2. 2 TECObiosciences GmbH, Landshut, Germany
  3. 3 Pathology, Leiden University Medical Center, Leiden, The Netherlands
  4. 4 Pattern Recognition and Bioinformatics, Delft University of Technology, Delft, The Netherlands
  5. 5 Leiden Computational Biology Center, Leiden University Medical Center, Leiden, The Netherlands
  6. 6 Surgery, Leiden University Medical Center, Leiden, The Netherlands
  7. 7 Computer Graphics and Visualization, Delft University of Technology, Delft, The Netherlands
  8. 8 LKEB Radiology, Leiden University Medical Center, Leiden, The Netherlands
  1. Correspondence to Dr Noel F C C de Miranda, Pathology, Leiden University Medical Center, Leiden, The Netherlands; N.F.de_Miranda{at}lumc.nl

Abstract

Objective A comprehensive understanding of anticancer immune responses is paramount for the optimal application and development of cancer immunotherapies. We unravelled local and systemic immune profiles in patients with colorectal cancer (CRC) by high-dimensional analysis to provide an unbiased characterisation of the immune contexture of CRC.

Design Thirty-six immune cell markers were simultaneously assessed at the single-cell level by mass cytometry in 35 CRC tissues, 26 tumour-associated lymph nodes, 17 colorectal healthy mucosa and 19 peripheral blood samples from 31 patients with CRC. Additionally, functional, transcriptional and spatial analyses of tumour-infiltrating lymphocytes were performed by flow cytometry, single-cell RNA-sequencing and multispectral immunofluorescence.

Results We discovered that a previously unappreciated innate lymphocyte population (LinCD7+CD127CD56+CD45RO+) was enriched in CRC tissues and displayed cytotoxic activity. This subset demonstrated a tissue-resident (CD103+CD69+) phenotype and was most abundant in immunogenic mismatch repair (MMR)-deficient CRCs. Their presence in tumours was correlated with the infiltration of tumour-resident cytotoxic, helper and γδ T cells with highly similar activated (HLA-DR+CD38+PD-1+) phenotypes. Remarkably, activated γδ T cells were almost exclusively found in MMR-deficient cancers. Non-activated counterparts of tumour-resident cytotoxic and γδ T cells were present in CRC and healthy mucosa tissues, but not in lymph nodes, with the exception of tumour-positive lymph nodes.

Conclusion This work provides a blueprint for the understanding of the heterogeneous and intricate immune landscape of CRC, including the identification of previously unappreciated immune cell subsets. The concomitant presence of tumour-resident innate and adaptive immune cell populations suggests a multitargeted exploitation of their antitumour properties in a therapeutic setting.

  • colorectal cancer
  • single-cell immunophenotyping
  • mass cytometry
  • innate lymphoid cells
  • tissue-resident memory T cells
  • immune landscape
http://creativecommons.org/licenses/by-nc/4.0/

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Footnotes

  • VU, MEI and TA contributed equally.

  • FK and NFCCM contributed equally.

  • Contributors NLdV conceived the study, performed experiments and wrote the manuscript. NLdV, VvU, MEI, TA and AM analysed the data. AFS and KCMJP provided samples from patients. MEI, RvdB and NFCCdM processed the samples and performed experiments. TH, VvU and BPFL developed Cytosplore and HSNE applications. FK and NFCCdM initiated and led the project and wrote the manuscript. All authors discussed the results and commented on the manuscript.

  • Funding The authors acknowledge funding from the Fight Colorectal Cancer-Michael’s Mission-AACR Fellowship in Young Onset, Late-Stage Colorectal Cancer Research 2015 (15-40-1645-DEMI), the KWF Bas Mulder Award UL (2015-7664), the ZonMw Veni grant (016.176.l44), the NWO-AES grant (12720: VANPIRE) and the European Commission under a MSCA-ITN award (675743: ISPIC).

  • Competing interests None declared.

  • Ethics approval Medical Ethical Committee of the Leiden University Medical Centre (protocol P15.282).

  • Provenance and peer review Not commissioned; externally peer reviewed.

  • Patient consent for publication Not required.

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