Phenotyping of tumor-associated macrophages in colorectal cancer: Impact on single cell invasion (tumor budding) and clinicopathological outcome

ABSTRACT

Tumor-associated macrophages (TAM) play a controversial role in epithelial–mesenchymal transition (EMT) and prognosis of colorectal cancer (CRC). In particular, the microlocalization, polarization and prognostic impact of TAM in the immediate environment of invading CRC cells has not yet been established. To address this clinically relevant question, intraepithelial (iCD68) and stromal macrophages (sCD68), M1-macrophages (iNOS), M2-macrophages (CD163), cytokeratin-positive cancer cells (tumor buds) and expression of the anti-phagocytic marker CD47 were investigated in primary tumors of 205 well-characterized CRC patients. Cell-to-cell contacts between tumor buds and TAM were detected using high-resolution digital scans. The composition of the tumor microenvironment was analyzed with clinicopathological and molecular features. High CD68 counts predicted long term overall survival independent of microlocalization (iCD68 p=0.0016; sCD68 p=0.03), pT, pN, pM and post-operative therapy. CD68 infiltration correlated with significantly less tumor budding (iCD68 p=0.0066; sCD68 p=0.0091) and absence of lymph node metastasis (sCD68 p=0.0286). Cell-to-cell contact of sCD68 and invading cancer cells was frequent and ameliorated the detrimental prognostic effect of the tumor budding phenotype. Subgroup analysis identified long-term survival with CD47 loss and predominance of CD163⁺ M2 macrophages (p = 0.0366). CD163⁺ macrophages represented 40% of the total population, and positively correlated with total CD68 macrophage numbers (r[CD68/CD163] = 0.32; p = 0.0001). Strong CD163 infiltration predicted lower tumor grade (p = 0.0026) and less lymph node metastasis (p = 0.0056). This study provides direct morphological evidence of an interaction between TAM and infiltrating cancer cells. The prognostic impact of TAM is modulated by phenotype, microlocalization and the expression of anti-phagocytic markers in CRC.

KEYWORDS:

• CD47
• CD163
• colorectal cancer
• epithelial–mesenchymal transition
• iNOS
• metastasis
• prognostic factor
• tumor-associated macrophages
• tumor budding

Disclosure of potential conflicts of interest

The authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. This includes employment, consultancies, honoraria, stock ownership or options, expert testimony, royalties or patents received or pending.

Acknowledgments

The authors would like to express their sincere appreciation to Dr José Galvan, Dr Irene Centeno and Caroline Hammer from the Translational Research Unit, Institute of Pathology, University of Bern, Switzerland for excellent technical support and to Dr Christian Schürch for helpful discussion regarding the use of CD47 as a prognostic biomarker.

Authors' contributions

VHK together with IZ conceived the study and study design, conducted the study, performed data interpretation and drafted the manuscript; IZ performed statistical analysis. KC scored immunohistochemistry, contributed to the study design and data interpretation, reviewed and approved the final manuscript. HD scored CD47 immunohistochemistry, critically reviewed and approved the final manuscript. LS performed digital image analysis, critically reviewed and approved the final manuscript. EK reviewed and categorized patient material and clinical data, reviewed and approved the final manuscript. AL contributed to the study design and data interpretation, critically reviewed and approved the final manuscript. All authors have reviewed and approved the final version to be published.

Funding

This study was kindly funded by the Bernese Cancer League and Oncosuisse (KFS 3294-08-2013).