Increased Levels of Circulating and Tumor-Infiltrating Granulocytic Myeloid Cells in Colorectal Cancer Patients

Abstract

Increased levels of myeloid cells, especially myeloid-derived suppressor cells (MDSCs), have been reported to correlate with bad prognosis and reduced survival in cancer patients. However, limited data are available on their conclusive phenotypes and their correlation with clinical settings. The aim of this study was to investigate levels and phenotype of myeloid cells in peripheral blood and tumor microenvironment (TME) of colorectal cancer (CRC) patients, compared to blood from healthy donors (HDs) and paired, adjacent non-tumor colon tissue. Flow cytometric analysis was performed to examine the expression of different myeloid markers in fresh peripheral blood samples from CRC patients and HDs, and tissue-infiltrating immune cells from CRC patients. We found significantly higher levels of cells expressing myeloid markers and lacking the expression of major histocompatibility complex class II molecule HLA-DR in blood and tumor of CRC patients. Further analysis revealed that these cells were granulocytic and expressed Arginase 1 indicative of their suppressive phenotype. These expanded cells could be neutrophils or granulocytic MDSCs, and we refer to them as granulocytic myeloid cells (GMCs) due to the phenotypical and functional overlap between these cell subsets. Interestingly, the expansion of peripheral GMCs correlated with higher stage and histological grade of cancer, thereby suggesting their role in cancer progression. Furthermore, an increase in CD33⁺CD11b⁺HLA-DR^-CD14^-CD15^- immature myeloid cells was also observed in CRC tumor tissue. Our work shows that GMCs are expanded in circulation and TME of CRC patients, which provides further insights for developing immunotherapeutic approaches targeting these cell subsets to enhance antitumor immune and clinical responses.

Keywords: circulation; colorectal cancer; myeloid cells; neutrophils; tumor microenvironment.

Figure 1

Gating strategy of myeloid cells and comparisons of levels of different subsets of circulating myeloid cells between HDs and CRC patients. Representative flow cytometric plots showing the gating strategy used to identify myeloid cells in peripheral blood of HDs and CRC patients (A). CD33⁺ cells were gated first from all cells, followed by gating CD11b⁺ cells and HLA-DR^−/low cells from parent populations. MMCs were then identified as CD14⁺ cells, while GMCs were identified based on the expression of CD15. Fresh whole blood from 21 HDs and 20 CRC patients was stained for these markers. (B) Shows scatter plots of the means of calculated percentages ± SEM of each myeloid subpopulation including CD33⁺ cells, CD33⁺CD11b⁺ cells, CD33⁺CD11b⁺HLA-DR^−/low cells, CD33⁺CD11b⁺HLA-DR^−/lowCD14⁺ cells, and CD33⁺CD11b⁺ HLA-DR^−/lowCD15⁺ cells.

Figure 2

Arginase 1 expression by MMCs and GMCs in peripheral blood of HDs and CRC patients. Fresh blood from HD (n = 21) and CRC patients (n = 20) was stained for different MDSC markers to identify MMCs and GMCs cells, followed by intracellular staining for the expression of ARG1. Representative flow cytometric plots showing expression of ARG1 by CD33⁺CD11b⁺HLA-DR^−/lowCD14⁺ MMCs and CD33⁺CD11b⁺HLA-DR^−/lowCD15⁺ GMCs cells in a HD (A) and a CRC patient (B). Scatter plots comparing calculated percentages ± SEM of ARG1 level in CD33⁺CD11b⁺HLA-DR^−/lowCD15⁺ (C) and CD33⁺CD11b⁺HLA-DR^−/lowCD14⁺ (D) subsets.

Figure 3

Myeloid cells and arginase 1 expression in tissue-infiltrating immune cells. (A) Representative flow cytometric plots showing levels of different subsets of myeloid cells in normal tissue (NT) and tumor tissue (TT) of nine CRC patients. Doublets were excluded and live cells were gated. The frequency of each population was calculated out of the parent population. Scatter plots showing mean of relative percentages ± SEM (B), and calculated percentages ± SEM, calculated by multiplying the relative percentages of each subpopulation with their respective parent populations, of CD33⁺CD11b⁺HLA-DR^−/low CD14⁺ MMCs and CD33⁺CD11b⁺HLA-DR^−/low CD15⁺ GMCs in NT and TT of CRC patients (C); inset showing the expansion of GMCs in TT compared with adjacent NT. (D) Means of calculated percentage ± SEM of CD33⁺CD11b⁺HLA-DR⁻CD14⁻CD15⁻ IMCs in NT and TT. Representative flow cytometric plots show the expression of ARG1 by tumor-infiltrating CD33⁺CD11b⁺HLA-DR^−/lowCD14⁺ MMCs (E) and CD33⁺CD11b⁺HLA-DR^−/lowCD15⁺ GMCs (F) in tumor tissue of CRC patients.

Figure 4

Comparisons of levels of circulating GMCs between HDs and different TNM stages and histological grades of colorectal cancer. Scatter plots comparing calculated percentages ± SEM of CD33⁺CD11b⁺HLA-DR^−/lowCD15⁺ GMCs (A) and their ARG1 level (B) between HDs and different TNM stages of CRC patients. Scatter plots comparing calculated percentages ± SEM of CD33⁺CD11b⁺HLA-DR^−/lowCD15⁺ GMCs (C) and their ARG1 level (D) between HDs and different histological grades of CRC patients.