Exploring Potential Epigenetic Biomarkers for Colorectal Cancer Metastasis.

Metastatic progression is a complex, multistep process and the leading cause of cancer mortality. There is growing evidence that emphasises the significance of epigenetic modification, specifically DNA methylation and histone modifications, in influencing colorectal (CRC) metastasis. Epigenetic modifications influence the expression of genes involved in various cellular processes, including the pathways associated with metastasis. These modifications could contribute to metastatic progression by enhancing oncogenes and silencing tumour suppressor genes. Moreover, specific epigenetic alterations enable cancer cells to acquire invasive and metastatic characteristics by altering cell adhesion, migration, and invasion-related pathways. Exploring the involvement of DNA methylation and histone modification is crucial for identifying biomarkers that impact cancer prediction for metastasis in CRC. This review provides a summary of the potential epigenetic biomarkers associated with metastasis in CRC, particularly DNA methylation and histone modifications, and examines the pathways associated with these biomarkers.

An epigenetic signature of advanced colorectal cancer metastasis.

Colorectal cancer (CRC) is a leading cause of morbidity and mortality worldwide. The majority of CRC deaths are caused by tumor metastasis, even following treatment. There is strong evidence for epigenetic changes, such as DNA methylation, accompanying CRC metastasis and poorer patient survival. Earlier detection and a better understanding of molecular drivers for CRC metastasis are of critical clinical importance. Here, we identify a signature of advanced CRC metastasis by performing whole genome-scale DNA methylation and full transcriptome analyses of paired primary cancers and liver metastases from CRC patients. We observed striking methylation differences between primary and metastatic pairs. A subset of loci showed coordinated methylation-expression changes, suggesting these are potentially epigenetic drivers that control the expression of critical genes in the metastatic cascade. The identification of CRC epigenomic markers of metastasis has the potential to enable better outcome prediction and lead to the discovery of new therapeutic targets.

Assessment of a Size-Based Method for Enriching Circulating Tumour Cells in Colorectal Cancer.

Circulating tumour cells (CTC) from solid tumours are a prerequisite for metastasis. Isolating CTCs and understanding their biology is essential for developing new clinical tests and precision oncology. Currently, CellSearch is the only FDA (U.S. Food and Drug Administration)-approved method for CTC enrichment but possesses several drawbacks owing to a reliance on the epithelial cell adhesion molecule (EpCAM) and a resource-intensive nature. Addressing these shortcomings, we optimised an existing size-based method, MetaCell, to enrich CTCs from blood of colorectal cancer (CRC) patients. We evaluated the ability of MetaCell to enrich CTCs by spiking blood with CRC cell lines and assessing the cell recovery rates and WBC depletion via immunostaining and gene expression. We then applied MetaCell to samples from 17 CRC patients and seven controls. Recovery rates were >85% in cell lines, with >95% depletion in WBCs. MetaCell yielded CTCs and CTC clusters in 52.9% and 23.5% of the patients, respectively, without false positives in control patients. CTCs and cluster detection did not correlate with histopathological parameters. Overall, we demonstrated that the MetaCell platform enriched CRC cells with high recovery rates and high purity. Our pilot study also demonstrated the ability of MetaCell to detect CTCs in CRC patients.

Portal vein embolization with absolute ethanol to induce hypertrophy of the future liver remnant.

BACKGROUND: Preoperative portal vein embolization (PVE) is widely used prior to major liver resection to reduce the risk of post-hepatectomy liver failure (PHLF). We evaluated the efficacy and safety of PVE using absolute ethanol. METHODS: Consecutive patients undergoing preoperative PVE between February 2003 and February 2020 at a high-volume tertiary institution were retrospectively reviewed. Hypertrophy of the future liver remnant (FLR) was determined by comparing volumetric data using semi-automated software on computed tomography or magnetic resonance imaging before and after PVE. Efficacy of absolute ethanol was evaluated by the percentage increase in the FLR volume and the ratio of the FLR to the total liver volume (TLV). Technical success and complications following PVE were evaluated. Feasibility of hepatectomy following PVE and the incidence of PHLF were determined. RESULTS: Sixty-two patients underwent preoperative PVE using absolute ethanol. The technical success rate was 95.2%. Median time interval between PVE and follow-up imaging was 34 days (range 6-144 days). The mean increase in FLR volume and ratio of the FLR to TLV were 43.6 ± 34.4% and 12.3 ± 7.7% respectively. Major adverse events occurred in 3 cases (4.8%) and did not preclude consideration of surgery. Forty-two patients (67.8%) proceeded to surgery for intended hepatectomy of which 36 patients (58.1%) underwent liver resection. Major post-operative complications occurred in 4 patients (11.1%) and there were no cases of PHLF. CONCLUSION: Preoperative PVE with absolute ethanol is effective and safe in inducing hypertrophy of the FLR before partial hepatectomy to prevent PHLF.

Strategies for Antibiotic Administration for Bowel Preparation Among Patients Undergoing Elective Colorectal Surgery: A Network Meta-analysis.

Importance: There are discrepancies in guidelines on preparation for colorectal surgery. While intravenous (IV) antibiotics are usually administered, the use of mechanical bowel preparation (MBP), enemas, and/or oral antibiotics (OA) is controversial. Objective: To summarize all data from randomized clinical trials (RCTs) that met selection criteria using network meta-analysis (NMA) to determine the ranking of different bowel preparation treatment strategies for their associations with postoperative outcomes. Data Sources: Data sources included MEDLINE, Embase, Cochrane, and Scopus databases with no language constraints, including abstracts and articles published prior to 2021. Study Selection: Randomized studies of adults undergoing elective colorectal surgery with appropriate aerobic and anaerobic antibiotic cover that reported on incisional surgical site infection (SSI) or anastomotic leak were selected for inclusion in the analysis. These were selected by multiple reviewers and adjudicated by a separate lead investigator. A total of 167 of 6833 screened studies met initial selection criteria. Data Extraction and Synthesis: NMA was performed according to Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) reporting guidelines. Data were extracted by multiple independent observers and pooled in a random-effects model. Main Outcomes and Measures: Primary outcomes were incisional SSI and anastomotic leak. Secondary outcomes included other infections, mortality, ileus, and adverse effects of preparation. Results: A total of 35 RCTs that included 8377 patients were identified. Treatments compared IV antibiotics (2762 patients [33%]), IV antibiotics with enema (222 patients [3%]), IV antibiotics with OA with or without enema (628 patients [7%]), MBP with IV antibiotics (2712 patients [32%]), MBP with IV antibiotics with OA (with good IV antibiotic cover in 925 patients [11%] and with good overall antibiotic cover in 375 patients [4%]), MBP with OA (267 patients [3%]), and OA (486 patients [6%]). The likelihood of incisional SSI was significantly lower for those receiving IV antibiotics with OA with or without enema (rank 1) and MBP with adequate IV antibiotics with OA (rank 2) compared with all other treatment options. The addition of OA to IV antibiotics, both with and without MBP, was associated with a reduction in incisional SSI by greater than 50%. There were minimal differences between treatments in anastomotic leak and in any of the secondary outcomes. Conclusions and Relevance: This NMA demonstrated that the addition of OA to IV antibiotics were associated with a reduction in incisional SSI by greater than 50%. The results support the addition of OA to IV antibiotics to reduce incisional SSI among patients undergoing elective colorectal surgery.

The Epigenetic landscape of Circulating tumour cells.

Cancer metastasis is the main reason for the high mortality in patients, contributing to 90% of cancer-related deaths. Biomarkers for early detection and therapeutic monitoring are essential to improve cancer outcomes. Circulating tumour cells (CTCs) arise from solid tumours and are capable of metastatic dissemination via the bloodstream or lymphatic system. Thus, CTCs can potentially be developed as a minimally invasive biomarker for early detection and therapeutic monitoring. Despite its clinical potential, research on CTCs remains limited, and this is likely due to their low numbers, short half-life, and the lack of robust methods for their isolation. There is also a need for molecular characterisation of CTCs to identify tumour-specific features, such as epigenetic signatures of metastasis. This review provides an overview of the epigenetic landscape of CTCs. We discuss the role of epigenetic modifications in CTC dissemination,metastatic tumour formation and progression and highlight its clinical implications.

Human liver-derived MAIT cells differ from blood MAIT cells in their metabolism and response to TCR-independent activation.

Mucosal associated invariant T (MAIT) cells are anti-microbial innate-like T cells that are abundant in blood and liver. MAIT cells express a semi-invariant T-cell receptor (TCR) that recognizes a pyrimidine ligand, derived from microbial riboflavin synthesis, bound to MR1. Both blood and liver derived (ld)-MAIT cells can be robustly stimulated via TCR or by cytokines produced during bacterial or viral infection. In this study, we compared the functional and transcriptomic response of human blood and ld-MAIT cells to TCR signals (Escherichia coli or the pyrimidine ligand) and cytokines (IL-12 + IL-18). While the response of blood and ld-MAIT cells to TCR signals were comparable, following cytokine stimulation ld-MAIT cells were more polyfunctional than blood MAIT cells. Transcriptomic analysis demonstrated different effector programmes of ld-MAIT cells with the two modes of activation, including the enrichment of a tissue repair signature in TCR-stimulated MAIT cells. Interestingly, we observed enhancement of IL-12 signaling and fatty acid metabolism in untreated ld-MAIT cells compared with blood MAIT cells. Additionally, MAIT cells from blood and liver were modulated similarly by TCR and cytokine signals. Therefore, we report that blood and ld-MAIT cells are fundamentally different but undergo conserved changes following activation via TCR or by cytokines.

Type I interferons are important co-stimulatory signals during T cell receptor mediated human MAIT cell activation.

Mucosal associated invariant T (MAIT) cells are abundant unconventional T cells that can be stimulated either via their TCR or by innate cytokines. The MAIT cell TCR recognises a pyrimidine ligand, derived from riboflavin synthesising bacteria, bound to MR1. In infection, bacteria not only provide the pyrimidine ligand but also co-stimulatory signals, such as TLR agonists, that can modulate TCR-mediated activation. Recently, type I interferons (T1-IFNs) have been identified as contributing to cytokine-mediated MAIT cell activation. However, it is unknown whether T1-IFNs also have a role during TCR-mediated MAIT cell activation. In this study, we investigated the co-stimulatory role of T1-IFNs during TCR-mediated activation of MAIT cells by the MR1 ligand 5-amino-6-d-ribitylaminouracil/methylglyoxal. We found that T1-IFNs were able to boost interferon-γ and granzyme B production in 5-amino-6-d-ribitylaminouracil/methylglyoxal-stimulated MAIT cells. Similarly, influenza virus-induced T1-IFNs enhanced TCR-mediated MAIT cell activation. An essential role of T1-IFNs in regulating MAIT cell activation by riboflavin synthesising bacteria was also demonstrated. The co-stimulatory role of T1-IFNs was also evident in liver-derived MAIT cells. T1-IFNs acted directly on MAIT cells to enhance their response to TCR stimulation. Overall, our findings establish an important immunomodulatory role of T1-IFNs during TCR-mediated MAIT cell activation.

Prevalence of diabetes in liver cirrhosis: A systematic review and meta-analysis.

An association between diabetes mellitus (DM) and liver cirrhosis is well-known, but estimates of the prevalence of DM in patients with liver cirrhosis vary widely. A systematic review was undertaken to determine the prevalence of DM in adult patients with liver cirrhosis. The Medline, EMBASE, and Cochrane Library databases were searched for peer-reviewed studies published in English (1979-2017) that investigated the prevalence of diabetes in adult patients with cirrhosis. Pooled estimates of prevalence of DM were determined for all eligible patients and according to aetiology and severity of liver disease. Fifty-eight studies satisfied criteria for inclusion, with 9705 patients included in the pooled prevalence analysis. The overall prevalence of DM was 31%. The prevalence of DM was highest in patients with nonalcoholic fatty liver disease (56%), cryptogenic (51%), hepatitis C (32%), or alcoholic (27%) cirrhosis. For assessing prevalence of DM as a function of severity of liver disease, evaluable data were available only for hepatitis C and hepatitis B cirrhosis. DM may be more prevalent in cirrhosis than previously thought. This has implications for prognosis and treatment in these patients.

High-Dimensional Mass Cytometric Analysis Reveals an Increase in Effector Regulatory T Cells as a Distinguishing Feature of Colorectal Tumors.

T cell infiltration of tumors plays an important role in determining colorectal cancer disease progression and has been incorporated into the Immunoscore prognostic tool. In this study, mass cytometry was used to demonstrate a significant increase in the frequency of both conventional CD25+FOXP3+CD127lo regulatory T cells (Tregs) as well as BLIMP-1+ Tregs in the tumor compared with nontumor bowel (NTB) of the same patients. Network cluster analyses using SCAFFoLD, VorteX, and CITRUS revealed that an increase in BLIMP-1+ Tregs was a single distinguishing feature of the tumor tissue compared with NTB. BLIMP-1+ Tregs represented the most significantly enriched T cell population in the tumor compared with NTB. The enrichment of ICOS, CD45RO, PD-1, PDL-1, LAG-3, CTLA-4, and TIM-3 on BLIMP-1+ Tregs suggests that BLIMP-1+ Tregs have a more activated phenotype than conventional Tregs and may play a role in antitumor immune responses.

Relationship Between Total Body Protein and Cross-Sectional Skeletal Muscle Area in Liver Cirrhosis Is Influenced by Overhydration.

Sarcopenia as defined by reduced skeletal muscle area (SMA) on cross-sectional abdominal imaging has been proposed as an objective measure of malnutrition, and it is associated with both wait-list mortality and posttransplant complications in patients with cirrhosis. SMA, however, has never been validated against the gold standard measurement of total body protein (TBP) by in vivo neutron activation analysis (IVNAA). Furthermore, overhydration is common in cirrhosis, and its effect on muscle area measurement remains unknown. We aimed to examine the relationship between SMA and TBP in patients with cirrhosis and to assess the impact of overhydration on this relationship. Patients with cirrhosis who had undergone IVNAA and cross-sectional imaging within 30 days were retrospectively identified. Patients with significant clinical events between measurements were excluded. Psoas muscle area (PMA) and SMA at the level of the third lumbar vertebrae were determined. Total body water was estimated from a multicompartment model and expressed as a fraction of fat-free mass (FFM), as determined by dual-energy X-ray absorptiometry, to provide an index of hydration status. In total, 107 patients underwent 109 cross-sectional imaging studies (87 computed tomography; 22 magnetic resonance imaging) within 30 days of IVNAA. Median time between measurements was 1 day (IQR, -1 to 3 days). Between 43% and 69% of the cohort was identified as sarcopenic, depending on muscle area cutoff values used. TBP was strongly correlated with SMA (r = 0.78; P < 0.001) and weakly correlated with PMA (r = 0.49; P < 0.001). Multiple linear regression showed SMA was significantly and positively associated with FFM hydration (P < 0.001) independently of TBP. In conclusion, SMA is more closely related to TBP than is PMA, and it should be preferred as a measure of sarcopenia. Overhydration significantly affects the measurement of cross-sectional muscle area.

Prognostic roles for IL-2-producing and CD69+ T cell subsets in colorectal cancer patients.

Tumor infiltrating T cells are a predictor of patient outcome in patients with colorectal cancer (CRC). However, many T cell populations have been associated with both poor and positive patient prognoses, indicating a need to further understand the role of different T cell subsets in CRC. In this study, the T cell infiltrate from the tumor and nontumor bowel (NTB) was examined in 95 CRC patients using flow cytometry and associations with cancer stage and disease recurrence made. Our findings showed that IFN-γ-producing T cells were associated with positive patient outcomes, and CD69+ T cells were associated with disease recurrence. Inflammatory (IL-17) and regulatory T cells were not associated with disease recurrence. Surprisingly, in a second cohort of 32 patients with long-term clinical follow up data, tumor infiltrating IL-2-producing T cells correlated negatively with disease free survival (DFS) and a higher frequency of IL-2-producing T cells was found in the NTB of patients with poorly differentiated tumors. These results point toward the possibility of a negative impact of IL-2 in tumor immune responses, which may influence future immunotherapy treatments in CRC patients.

Genome-wide methylation analysis identifies a core set of hypermethylated genes in CIMP-H colorectal cancer.

BACKGROUND: Aberrant DNA methylation profiles are a characteristic of all known cancer types, epitomized by the CpG island methylator phenotype (CIMP) in colorectal cancer (CRC). Hypermethylation has been observed at CpG islands throughout the genome, but it is unclear which factors determine whether an individual island becomes methylated in cancer. METHODS: DNA methylation in CRC was analysed using the Illumina HumanMethylation450K array. Differentially methylated loci were identified using Significance Analysis of Microarrays (SAM) and the Wilcoxon Signed Rank (WSR) test. Unsupervised hierarchical clustering was used to identify methylation subtypes in CRC. RESULTS: In this study we characterized the DNA methylation profiles of 94 CRC tissues and their matched normal counterparts. Consistent with previous studies, unsupervized hierarchical clustering of genome-wide methylation data identified three subtypes within the tumour samples, designated CIMP-H, CIMP-L and CIMP-N, that showed high, low and very low methylation levels, respectively. Differential methylation between normal and tumour samples was analysed at the individual CpG level, and at the gene level. The distribution of hypermethylation in CIMP-N tumours showed high inter-tumour variability and appeared to be highly stochastic in nature, whereas CIMP-H tumours exhibited consistent hypermethylation at a subset of genes, in addition to a highly variable background of hypermethylated genes. EYA4, TFPI2 and TLX1 were hypermethylated in more than 90% of all tumours examined. One-hundred thirty-two genes were hypermethylated in 100% of CIMP-H tumours studied and these were highly enriched for functions relating to skeletal system development (Bonferroni adjusted p value =2.88E-15), segment specification (adjusted p value =9.62E-11), embryonic development (adjusted p value =1.52E-04), mesoderm development (adjusted p value =1.14E-20), and ectoderm development (adjusted p value =7.94E-16). CONCLUSIONS: Our genome-wide characterization of DNA methylation in colorectal cancer has identified 132 genes hypermethylated in 100% of CIMP-H samples. Three genes, EYA4, TLX1 and TFPI2 are hypermethylated in >90% of all tumour samples, regardless of CIMP subtype.

Inclusion of BLIMP-1+ effector regulatory T cells improves the Immunoscore in a cohort of New Zealand colorectal cancer patients: a pilot study.

Analysis of tumour-infiltrating T cells in colorectal cancer can predict disease-free survival. The Immunoscore, obtained by quantifying tumour-infiltrating CD3+ and CD8+ T cells, may improve current staging. Effector regulatory T cells are a potently suppressive subset in mice and, while present in human colorectal cancer, their role in patient outcome is unknown. Immunofluorescence was used to analyse immune cell infiltrates in patients with early (stage II) colorectal cancer with (n = 13) and without (n = 19) recurrent disease. CD3 and CD8 were used for the Immunoscore; FOXP3, BLIMP-1 and CD3 to identify effector regulatory T cells. Patients with high Immunoscores had increased disease-free survival compared to patients with low Immunoscores (Log-rank test p < 0.01). Prediction of outcome was further improved by stratifying patients with a low Immunoscore according to CD3+FOXP3+BLIMP-1+ cell infiltration at the invasive margin. Patients with a low Immunoscore and high infiltrate of CD3+FOXP3+BLIMP-1+ cells tended to have better disease-free survival than patients with low Immunoscore and low infiltrate of CD3+FOXP3+BLIMP-1+ cells. Patients with a high Immunoscore had better disease-free survival than patients with a low Immunoscore and low infiltrate of CD3+ FOXP3+ BLIMP-1+ cells (Log-rank test p < 0.001). These results indicate that tumour infiltration with effector regulatory T cells improves the prognostic value of the Immunoscore and implies that these cells may play a role in colorectal cancer patient outcome.

Low Versus Standard Urine Output Targets in Patients Undergoing Major Abdominal Surgery: A Randomized Noninferiority Trial.

OBJECTIVE: To determine whether a low perioperative minimum urine output target is safe and fluid sparing when compared with the standard target. BACKGROUND: A minimum hourly urine output of 0.5 mL/kg is a key target guiding perioperative fluid therapy. Few data support this standard practice, which may contribute to perioperative fluid overloading. METHODS: We randomized patients without significant risk factors for acute kidney injury undergoing elective colectomy to a minimum urine output target of 0.2 mL/kg/h (low group) or 0.5 mL/kg/h (standard group) from induction of anesthesia until 8 AM 2 days after surgery. Maintenance fluids were standardized and additional fluids administered to achieve the targets. Primary outcome was noninferiority for urine neutrophil gelatinase-associated lipocalin on the day after surgery. RESULTS: Between November 21, 2011 and July 11, 2013, 40 participants completed the study. The low group received 3170 mL (95% confidence interval 2380-3960) intravenous fluids versus 5490 mL (95% confidence interval 4570-6410) in the standard group (P = 0.0004), and was noninferior for neutrophil gelatinase-associated lipocalin [14.7 µg/L (interquartile range 7.60-28.9) vs 18.4 µg/L (interquartile range 8.30-21.2); Pnoninferiority = 0.0011], serum cystatin C (Pnoninferiority < 0.0001), serum creatinine (Pnoninferiority = 0.0004), and measured glomerular filtration (Pnoninferiority = 0.0003). Effective renal plasma flow increased in both groups after surgery, and more in the standard group (Pnoninferiority = 0.125). CONCLUSIONS: A perioperative urine output target of 0.2 mL/kg/h is noninferior to the standard target of 0.5 mL/kg/h and results in a large intravenous fluid sparing. This target should be adopted in surgical patients without significant kidney injury risk factors.

Nadolol reduces insulin sensitivity in liver cirrhosis: a randomized double-blind crossover trial.

BACKGROUND: Liver cirrhosis is frequently complicated by portal hypertension leading to increased mortality from variceal bleeding and hepatic decompensation. Noncardioselective ß-blockers not only reduce portal hypertension and prevent variceal bleeding in cirrhosis but also impair glucose tolerance and insulin sensitivity in other settings. This study aimed to determine whether nonselective ß-blockade with nadolol impairs glucose metabolism in liver cirrhosis. METHODS: A randomized, double-blind, placebo-controlled crossover trial of nadolol in cirrhotic patients examined insulin sensitivity, disposition index, and glucose tolerance. Stable cirrhotic patients of mixed etiology underwent an intravenous glucose tolerance test and hyperinsulinemic-euglycemic clamp for the measurement of insulin secretion and insulin sensitivity (n = 16) and a 75-g oral glucose tolerance test (n = 17). These measurements were conducted twice (after 3 months of treatment with nadolol or placebo and, after a 1-month washout period, after 3 months on the alternative treatment). Total body fat and plasma catecholamines were measured at the end of each 3-month treatment. RESULTS: Compared with placebo, nadolol treatment reduced insulin sensitivity (79.7 ± 10.1 vs 99.6 ± 10.3 µL/kg fat-free mass·min-1 ·(mU/L)-1 , P = .005). Insulin secretion was unchanged (P = .24), yielding a lower disposition index with nadolol (6083 ± 2007 vs 8692 ± 2036, P = .050). There was no change in total body fat or plasma catecholamines. A 2-hour plasma glucose concentration from the oral glucose tolerance test was higher on nadolol than placebo (10.8 ± 0.9 vs 9.9 ± 0.9 mmol/L, P = .035). CONCLUSIONS: Nadolol significantly worsened insulin sensitivity, glycemia, and disposition index in patients with liver cirrhosis. These findings may have significant clinical implications because cirrhosis is already associated with an increased prevalence of diabetes.

Functional impairment of infiltrating T cells in human colorectal cancer.

T cells play a crucial role in preventing the growth and spread of colorectal cancer (CRC). However, immunotherapies against CRC have only shown limited success, which may be due to lack of understanding about the effect of the local tumor microenvironment (TME) on T cell function. The goal of this study was to determine whether T cells in tumor tissue were functionally impaired compared to T cells in non-tumor bowel (NTB) tissue from the same patients. We showed that T cell populations are affected differently by the TME. In the tumor, T cells produced more IL-17 and less IL-2 per cell than their counterparts from NTB tissue. T cells from tumor tissue also had impaired proliferative ability compared to T cells in NTB tissue. This impairment was not related to the frequency of IL-2 producing T cells or regulatory T cells, but T cells from the TME had a higher co-expression of inhibitory receptors than T cells from NTB. Overall, our data indicate that T cells in tumor tissue are functionally altered by the CRC TME, which is likely due to cell intrinsic factors. The TME is therefore an important consideration in predicting the effect of immune modulatory therapies.

Gut macrophage phenotype is dependent on the tumor microenvironment in colorectal cancer.

In contrast to many cancers, a high infiltration of macrophages in colorectal cancer (CRC) has been associated with improved prognosis for patients. Cytokines and other stimuli from the tumor microenvironment affect monocyte to macrophage maturation and subsequent phenotype and function. Heterogeneous myeloid populations were identified using a novel flow cytometry panel in both tumor and paired non-tumor bowel (NTB) from CRC patients. The frequency of macrophage subsets with a gut-conditioned phenotype was lower in tumor compared with NTB. We used an in vitro system to show that two of the macrophage populations represented pro-inflammatory and anti-inflammatory phenotypes. Conditioned media that contained high levels of interleukin-6 promoted and maintained an anti-inflammatory phenotype in vitro. This study demonstrates the plasticity and heterogeneity of macrophage subtypes in human CRC, and the feasibility of studying complex populations. Ex vivo experiments demonstrate that macrophage subsets are influenced by the tumor microenvironment.