Clinical significance of circulating immune cells in left- and right-sided colon cancer

Ethics statement

All procedures involving human participants were performed in accordance with the ethical standards of the research ethics committee of Peking University Cancer Hospital & Institute and with the 1964 Helsinki declaration and its later amendments (2015KT47). Written informed consent was obtained from all patients.

Patient specimens

From 2015 to 2016, 94 colon cancer patients who underwent primary tumor resection at the Department of Gastrointestinal Surgery IV, Peking University Cancer Hospital were enrolled in this prospective study, and around 8 mL peripheral blood samples were obtained from each patient pre-operatively. Patients’ clinicopathological characteristics were determined according to the National Comprehensive Cancer Network (NCCN) guidelines for colon cancer (2016, Version 2).

Microsatellite instability testing

For immunohistochemical staining, tissue slides (4 µm) were first deparaffinized in xylene, ethanol, and water, followed by treatment in 0.01 M citrate buffer (pH 6.0, 95 °C) for 10 min, and cooled down to RT. Slides were then steam treated (92−100 °C) in EDTA for 15 min. After cooling at RT and washing by PBS, to eliminate nonspecific binding of antibodies, endogenous peroxidase activity was quenched by immersing in 3% H₂O₂ for 15 min. The slides were then blocked with 5% milk for 1 h at RT. The sections were incubated with primary antibodies for MLH1, MSH2, MSH6 and PMS2 (1:500 diluted, Abcam, Cambridge, MA, USA) overnight at 4 °C, and then incubated with the corresponding secondary antibody labelled with HRP (1:250 diluted, Solarbio Life Sciences, Beijing, China) for 30 min at RT. The reaction products were visualized with DAB (Solarbio Life Sciences, Beijing, China) according to the manufacturer’s instructions, counterstained with hematoxylin, and differentiated by hydrochloric acid alcohol.

Microsatellite status was determined by two experienced pathologists according to immunohistological staining of the tumor tissue. Tumors expressing MLH1, MSH2, MSH6 and PMS2 were considered microsatellite stable (MSS) and patients lacking the expression of any of these markers were considered microsatellite instable (MSI) (Andersen et al., 2016).

Isolation of peripheral blood mononuclear cells

To isolate peripheral blood mononuclear cells (PBMC) from the fresh blood samples, the blood was first diluted 1:1 with Iscove’s Modified Dulbecco’s Medium (IMDM containing GlutaMAX; Gibco-BRL, Paisley, Scotland), followed by density gradient centrifugation with Human PBMC isolation buffer (TBD Science, Tianjin, China) for 20 min at room temperature (RT). After separation, the PBMC layer was transferred to a new tube and washed with PBS (Thermo Fisher Scientific, Waltham, MA, USA) for 10 min at RT. Then cells were washed twice with Hanks (with calcium, magnesium, phenol red, Solarbio Life Sciences, Beijing, China) for 5 min at 4 °C and cryopreserved in liquid nitrogen.

Cell labelling and flow cytometric analysis

To determine the cell phenotypes within the purified samples, we purchased anti-human CD1c-APC, CD127-APC, CD25-PE, CD45RO-FITC, CD123-FITC and CD303a-APC monoclonal antibodies from eBioscience (San Diego, CA, USA); anti-human CD45-PE, CD141-PE, CD11c-FITC, CD11c-PE-Cy7, CD8-PE, CD4 Alexa Fluor 488, CD4-APC and HLA-DR-PerCP-Cy5.5 antibodies from BD Pharmingen™ (San Diego, CA, USA); and anti-human CD16-BV421 and CD45RA-BV421 from BD Horizon™ (San Jose, CA, USA). To detect the various immune cells, PBMCs were stained with 3 µL of the above mentioned monoclonal antibodies for 30 min at 4 °C, washed twice in PBS containing 0.5% BSA and fixed in 1% paraformaldehyde (PFA, Sigma-Aldrich, St Louis, MO, USA). For quantification purpose, in each test 20000 PBMCs were counted by FACS.

Quantification of circulating immune cells

The number of cells in each subpopulation was measured using a BD FACS Aria SORP analyser (BD Biosciences, San Jose, CA, USA) equipped with 405, 488, 561 and 633 nm lasers, and the data were analysed with FlowJo version 7.6 (TreeStar, Ashland, OR, USA). The marker expression of different cells was determined by representative flow cytometry analysis in Fig. 1.

Statistical analysis

Fisher’s Exact Test using R version 3.2.2 (R Foundation for Statistical Computing, Vienna, Austria, 2013) was performed to determine the correlations between clinicopathological features and tumor location. Student’s t-test (unpaired) was carried out using GraphPad Prism 5 software (GraphPad Prism Software Inc.; La Jolla, CA, USA) to examine the differences of circulating immune cells in patients with LCC or RCC, and results were shown as mean ± standard error of the mean (SEM). P < 0.05 was considered statistically significant.

Patients with LCC or RCC have distinct clinicopathological characteristics

Of the 94 patients involved in this study, 55 had LCC and 39 had RCC. Patient clinicopathological parameters based on tumor location are shown in Table 1. Compared to patients with LCC, patients with RCC had significantly more poorly differentiated tumors (P = 0.002), more MSI tumors (P = 0.003), and larger tumors (P = 0.002). These data demonstrated that patients with LCC or RCC had different clinicopathological features.

Correlation between circulating immune cells and tumor stages

pDCs are CD45⁺HLA-DR^lowCD123⁺CD303a⁺ (Piccioli et al., 2007; Tel et al., 2013). mDCs are CD3⁻HLA-DR⁺CD11c⁺ and can be further subdivided based on the expression of CD1c, CD16 and CD141 (Piccioli et al., 2007). CD45RA expression is indicative of Tn cells whereas CD45RO is a well-known surface marker that defines Tm cells (Sallusto et al., 1999). To assess the frequency of Tn and Tm cells in PBMCs from colon cancer patients, we quantified the CD4⁺CD45RA⁺ T cells, CD4⁺CD45RO⁺ T cells, CD8⁺CD45RA⁺ T cells and CD8⁺CD45RO⁺ T cells. Treg cells were determined by the presence of CD4⁺CD25⁺CD127^low (Venken et al., 2008) (Fig. 1).

To determine whether LCC and RCC patients have different levels of circulating immune cells, patients were divided into two groups according to their TNM stage—stages I + II and stages III + IV—as shown in Table 2. LCC patients in stage III + IV had significantly more CD16⁺ mDCs (P = 0.013) than RCC patients in stage III + IV. Moreover, LCC patients in stages III + IV showed a significantly higher quantity of total circulating mDCs and pDCs (P = 0.049 and 0.018, respectively) than RCC patient in stage III + IV.

To identify any potential relation between the amount of circulating immune cells and disease severity, we compared those cells at different tumor stages. Patients with early stage (I + II) tumors and patients with later stage (III + IV) tumors had similar amount of DCs. However, in LCC, patients with early stage tumors had significantly lower levels of CD4⁺ T cells (P = 0.018), Tm cells (P = 0.039) and CD4⁺ Tm cells (P = 0.005) than patients with late stage tumors (Fig. 2A). Similarly in RCC, patients with early stage tumors had significantly fewer CD4⁺ T cells (P = 0.049), Tm cells (P = 0.027) and CD4⁺ Tm cells (P = 0.018) than patients with late stage tumors (Fig. 2A). Collectively, these results suggest that the circulating DC levels differed between LCC and RCC patients, and circulating T cells levels were elevated during tumor progression to later stages.

Circulating immune cells were correlated with tumor locations

In this cohort, the main population was patients with stage II or III diseases (n = 68). There were no significant differences in age, gender, TNM stage, and histological subtypes between stage II and III patients (Table S1). And no significant differences were observed between patients with stage II and stage III tumors in terms of DCs and T cell subsets (Table S2). Therefore, we considered that in patients with stage II and III colon cancer, tumor stage does not associate with the level of circulating immune cells.

Accordingly, we compared the circulating immune cells in patients with stage II and stage III colon cancer to investigate their relation with tumor location, and ruled out the effect by tumor stages. As shown in Table 3, our results show that patients with LCC had significantly more CD16⁺ mDC (P = 0.041), CD8⁺ Tm (P = 0.008), CD8⁺ T cells (P = 0.043), Tm cells (P = 0.011) and Treg cells (P = 0.009) than patients with RCC. These results indicate that circulating immune cell count was indeed associated with tumor location.

Circulating immune cells were associated with different differentiation status

It has been reported that patients with well differentiated colon cancer have a lower risk of lymph node metastasis than patients with poorly differentiated colon cancer (Derwinger et al., 2010). Moreover, patients with well/moderately differentiated colon cancer have a better prognosis than patients with poorly differentiated colon cancer. To understand whether this was associated to differences in the circulating immune cells, we analysed the circulating immune components of LCC and RCC patients based on the degree of tumor differentiation (Table 4). This analysis was also performed for patients with stage II and III diseases to reduce the effect by tumor stage. In patients with well/moderately differentiated tumors, patients with LCC had remarkably more CD8⁺Tm cells (P = 0.030) than patients with RCC. While in patients with poorly differentiated tumors, patients with LCC had significantly higher amount of pDCs (P = 0.036), CD4⁺ Tm cells (P = 0.012), CD4⁺ T cells (P = 0.028), Tm cells (P = 0.014) and Treg cells (P = 0.001) than patients with RCC. These results strongly suggest that in patients with poorly differentiated tumors, LCC patients had more circulating immune cells, which may indicate a better systemic immunity than RCC.

Moreover, to access whether circulating immune cells varies when the tumor differentiate to a more malignant status, we next compared immune cells at different differentiation grades. Patients with well/moderately differentiated tumors and patients with poorly differentiated tumors had similar amount of DCs. In LCC, patients with well/moderately differentiated or poorly differentiated tumors had similar amount of circulating T cells, as shown in Fig. 2B. In contrast, in RCC, patients with poorly differentiated tumors had borderline significantly less CD4⁺ T cells (P = 0.053), significantly less Tm cells (P = 0.048) and CD4⁺ Tm cells (P = 0.016, Fig. 2B). Taken together, these data suggest that tumor differentiation status was associated with circulating T cell levels in patients with RCC.

Circulating immune cells are associated with lymph node metastasis

Lymph node metastasis requires breakdown of the host immune response in addition to escape of cancer cells from the tumor (Zuckerman et al., 2013). Therefore, we wondered whether the amount of different circulating immune cells was also correlated with lymph node metastasis in colon cancer. Indeed, we found that in RCC, patients without nodal metastasis had significantly more CD1c⁺ mDCs and pDCs than patients with nodal metastasis (P = 0.038 and P = 0.044, respectively, Table 5), while patients without nodal metastasis had significantly less CD4⁺ Tm cells, CD4⁺ T cells, and Tm cells (P = 0.018, P = 0.049, and P = 0.027, respectively) than patients with nodal metastasis. Moreover, in LCC, patients without nodal metastasis, CD4⁺ Tm cells and CD4⁺ T cells were also significantly lower than patients with nodal metastasis (P = 0.004 and P = 0.014, respectively, Table 5). These results indicate that the amount of certain circulating T cells increases whereas DCs decrease when lymph node metastasis occurs, and circulating CD4⁺ Tm and CD4⁺ T cell counts may serve as a predictor for lymph node metastasis in patients with colon cancer.