Soluble CD163: a novel independent prognostic biomarker in patients with metastatic renal cell carcinoma.

The hemoglobin-haptoglobin scavenger receptor CD163 is present in both a membrane-bound form on monocytes and macrophages (mCD163) and a shed soluble circulating form (sCD163). CD163 is a well-described marker of M2-like tumor-associated macrophages, but in patients with metastatic renal cell carcinoma (mRCC), monocyte mCD163 and serum sCD163 levels have not previously been investigated and associated with patient overall survival (OS). Here, we report mCD163 expression on peripheral blood monocytes, as well as sCD163 serum levels, in samples from 89 patients newly diagnosed with mRCC and 20 healthy controls. We found that in mRCC patients, compared to healthy controls, monocyte mCD163 levels were reduced (P < 0.001) whereas serum sCD163 levels were increased (P = 0.004). Moreover, an inverse correlation between mCD163 and sCD163 levels (P = 0.04) was shown. In survival analyses, intermediary levels of monocyte mCD163 were associated with longest OS, compared to both lower and higher mCD163 levels, which were both associated with worse outcomes (P < 0.01). Further, higher levels of sCD163 at diagnosis were associated with poor OS in both univariate (P < 0.001) and multivariate analysis (HR = 1.28; 95%CI 1.09-1.50, P = 0.002). Importantly, stratification by low vs. high sCD163 was able to separate patients with International Metastatic RCC Database Consortium (IMDC) intermediate risk (IMDCINT) into two subgroups with different OS (P = 0.03): IMDCINT-sCD163LOW showed survival similar to IMDCFAV patients, and IMDCINT-sCD163HIGH showed survival similar to IMDCPOOR patients. Thus, baseline sCD163 is a novel independent biomarker of OS in mRCC, and using sCD163 as an add-on biomarker may improve prognostic value for patients in the heterogenous IMDC intermediate group.

STAT3 is over-activated within CD163pos bone marrow macrophages in both Multiple Myeloma and the benign pre-condition MGUS.

Tumour-associated macrophages (TAMs) support cancer cell survival and suppress anti-tumour immunity. Tumour infiltration by CD163pos TAMs is associated with poor outcome in several human malignancies, including multiple myeloma (MM). Signal transducer and activator of transcription 3 (STAT3) is over-activated in human cancers, and specifically within TAMs activation of STAT3 may induce an immunosuppressive (M2-like) phenotype. Therefore, STAT3-inhibition in TAMs may be a future therapeutic strategy.We investigated TAM markers CD163, CD206, and activated STAT3 (pSTAT3) in patients with MGUS (n = 32) and MM (n = 45), as well as healthy controls (HCs, n = 13).Blood levels of the macrophage biomarkers sCD163 and sCD206, and circulating cytokines, as well as bone marrow mRNA expression of CD163 and CD206, were generally increased in MGUS and MM patients, compared to HCs, but to highly similar levels. By immunohistochemistry, bone marrow levels of pSTAT3 were increased specifically within CD163pos cells in both MGUS and MM patients.In conclusion, macrophage-related inflammatory changes, including activation of STAT3, were present already at the MGUS stage, at similar levels as in MM. Specific increase in pSTAT3 levels within CD163pos cells supports that the CD163 scavenger receptor may be a useful target for future delivery of STAT3-inhibitory drugs to TAMs in MM patients.

Expression of NK cell receptor ligands in primary colorectal cancer tissue in relation to the phenotype of circulating NK- and NKT cells, and clinical outcome.

INTRODUCTION: Natural killer (NK) cells and natural killer T (NKT) cells are implicated in the development and progression of colorectal cancer (CRC). Tumor cells express NK cell receptor ligands that modulate their function. This study aimed to investigate the expression of such ligands in CRC in relation to the phenotype of circulating NK- and NKT cells, and clinical outcome. METHODS: Primary tumor tissues were analyzed for protein expression of NK cell ligands using immunohistochemistry with automated image analysis in a cohort of 78 CRC patients. For 24 of the 78 patients, RNA expression of NK cell ligands was analyzed in primary tumor tissue using RNA sequencing. Receptor expression on circulating NK- and NKT cells was previously measured by us in 71 of the 78 patients using flow cytometry. RESULTS: High Proliferating Cell Nuclear Antigen (PCNA) protein expression in the primary tumor associated with shorter disease-free survival (DFS) of CRC patients (P = 0.026). A trend was observed towards shorter DFS in CRC patients with above-median galectin-3 protein expression in the primary tumor (P = 0.055). High protein expression of galectin-3, CD1d, and human leukocyte antigen (HLA) class I, and high RNA expression of UL16-binding protein (ULBP)-1, -2, and -5, and HLA-E in the tumor tissue correlated with low expression of the corresponding receptors on circulating NK- or NKT cells (P < 0.05). CONCLUSIONS: Galectin-3 and PCNA expression in the primary tumor may be prognostic biomarkers in CRC patients. Furthermore, our results suggest that NK cell receptor ligands expressed by tumor cells may modulate the phenotype of circulating NK- and NKT cells, and facilitate immune escape of metastasizing cells.

CD163 as a Biomarker in Colorectal Cancer: The Expression on Circulating Monocytes and Tumor-Associated Macrophages, and the Soluble Form in the Blood.

The macrophage-associated molecule CD163 has been reported as a prognostic biomarker in different cancer types, but its role in colorectal cancer (CRC) is unclear. We studied CD163 in the tumor microenvironment and circulation of patients with CRC in relation to clinicopathological parameters. An enzyme-linked immunosorbent assay (ELISA) was used to measure the serum sCD163 levels and multiparameter flow cytometry was used to study the peripheral blood monocytes and their CD163 expression in CRC patients (N = 78) and healthy donors (N = 50). The distribution of tumor-associated macrophages (TAMs) was studied in primary colorectal tumors with multiplex immunofluorescence. We showed that CRC patients with above-median sCD163 level had a shorter overall survival (OS, p = 0.035) as well as disease-free survival (DFS, p = 0.005). The above-median sCD163 remained significantly associated with a shorter DFS in the multivariate analysis (p = 0.049). Moreover, a shorter OS was observed in CRC patients with an above-median total monocyte percentage (p = 0.007). The number and phenotype of the stromal and intraepithelial TAMs in colorectal tumors were not associated with clinical outcome. In conclusion, sCD163 and monocytes in the circulation may be potential prognostic biomarkers in CRC patients, whereas TAMs in the tumor showed no association with clinical outcome. Thus, our results emphasize the importance of the innate systemic immune response in CRC disease progression.

The effects of tumor resection and adjuvant therapy on the peripheral blood immune cell profile in patients with colon carcinoma.

OBJECTIVE: The subset distribution and immunophenotype of circulating immune cells ("peripheral blood immune cell profile") may reflect tumor development and response to cancer treatment. In order to use the peripheral blood immune cell profile as biomarker to monitor patients over time, it is crucial to know how immune cell subsets respond to therapeutic interventions. In this study, we investigated the effects of tumor resection and adjuvant therapy on the peripheral blood immune cell profile in patients with colon carcinoma (CC). METHODS: The subset distribution and immunophenotype of T cells (CD3+CD56-), CD56dim NK cells (CD3-CD56dim), CD56bright NK cells (CD3-CD56bright) and NKT-like cells (CD3+CD56+) were studied in preoperative and postoperative peripheral blood mononuclear cell (PBMC) samples of 24 patients with CC by multiparameter flow cytometry. Changes in immunophenotype of circulating immune cells after tumor resection were studied in patients treated with and without (capecitabine-based) adjuvant therapy. RESULTS: The NKT-like cell (% of total PBMCs) and CD8+ T cell (% of total T cells) populations expanded in the peripheral blood of non-adjuvant-treated CC patients after surgery. NK- and NKT-like cells showed upregulation of activating receptors and downregulation of inhibitory receptors in non-adjuvant-treated CC patients after surgery. These changes were not observed in the peripheral blood of adjuvant-treated CC patients. CONCLUSIONS: Our results suggest tumor-induced suppression of NK- and NKT-like cells in CC patients, an effect that could not be detected after tumor resection. In contrast, adjuvant therapy maintained tumor-induced immunosuppression of NK- and NKT-like cells in CC patients.

Application Settings versus Fixed Photomultiplier Tube Voltages for Optimal Cytometer Stability over Time, and Effect on Reusing a Compensation Matrix.

In flow cytometry, a compensation matrix is commonly reused over time, especially in clinical laboratories, to save time and reagents. However, generating a same-day compensation matrix is considered the best practice by many experts. BD Biosciences developed Cytometer Setup and Tracking software to deliver proper instrument characterization, performance tracking, and stability. BD's "Application Settings" enable daily cytometer adjustments of photomultiplier tube (PMT) settings to correct for day-to-day variations in instrument performance. Here, we investigated if using Application Settings would improve data stability over time, including the impact on data stability when reusing a compensation matrix. We consecutively analyzed peripheral blood mononuclear cell (PBMC) aliquots from a single healthy donor together with 8-peak Rainbow beads and daily compensation controls (22 runs in total over 6.5 months). We found larger variation within both PBMC subset quantifications and median fluorescence intensity (MFI) levels when using Application Settings (i.e., daily adjusted PMTs) compared to fixed PMT voltages (both with the same Day 0 compensation matrix applied). This larger variation was partly due to errors in compensation, but was also seen for Rainbow beads MFI data (not impacted by compensation), and thus likely produced by imprecise adjustments of PMTs by Applications Settings. Notably, the larger variation observed with Application Settings was most pronounced on a few days of the experiment with very large deviations, whereas on most days Application Settings and Fixed PMTs performed similar. The present results call for caution in using Application Settings in longitudinal studies, especially if also reusing a compensation matrix. In contrast, reusing a compensation matrix over time with fixed PMT voltages yielded stable results comparable with running same-day compensation controls. © 2020 International Society for Advancement of Cytometry.

Circulating extracellular vesicle-associated CD163 and CD206 in multiple myeloma.

OBJECTIVES: Extracellular vesicles (EVs) are important for intercellular signalling in cancer. Tumour-associated macrophages, expressing the haemoglobin-haptoglobin and mannose receptors CD163 and CD206, are crucial for cancer progression. We recently identified CD163 on EVs in the circulation as a fraction of total soluble CD163 (sCD163). Here, we investigated the presence of CD163 and CD206-positive EVs (EV-CD163, EV-CD206) in patients with multiple myeloma (MM). METHODS: We enrolled patients with MM (n = 32), monoclonal gammopathy of undetermined significance (MGUS) (n = 8) and healthy donors (n = 16). Plasma protein levels were determined by ELISA before and after vesicle precipitation. Monocytes were examined by flow cytometry, and leucocyte CD163 mRNA by qPCR. RESULTS: Fractions of EV-CD163 and EV-CD206 were significantly elevated in patients with newly diagnosed MM (median = 39.8%, 76.5%, respectively) compared to patients with relapse (15.6%, P = .02, 42.5%, P = .003), remission (16.9%, P < .0001, 25.2%, P < .0001), MGUS (17.8%, P < .01, 33.1%, P = .0005) and healthy donors (14.8%, P < .0001, 35.5%, P < .0001). Whole blood CD163 mRNA did not vary between the groups. The intermediate monocyte subset showed a higher CD163 expression in newly diagnosed patients. CONCLUSIONS: Our results indicate that macrophage-derived EVs may play a role in the late phase of malignant progression of MM, and encourage further EV investigations in functional experiments.

Characterization of circulating T-, NK-, and NKT cell subsets in patients with colorectal cancer: the peripheral blood immune cell profile.

OBJECTIVE: As the development and progression of colorectal cancer (CRC) are known to be affected by the immune system, cell subsets such as T cells, natural killer (NK) cells, and natural killer T (NKT) cells are considered interesting targets for immunotherapy and clinical biomarker research. Until now, the role of systemic immune profiles in tumor progression remains unclear. In this study, we aimed to characterize the immunophenotype of circulating T cells, NK cells, and NKT-like cells in patients with CRC, and to subsequently correlate these immunophenotypes to clinical follow-up data. METHODS: Using multiparameter flow cytometry, the subset distribution and immunophenotype of T cells (CD3+CD56-), CD56dim NK cells (CD3-CD56dim), CD56bright NK cells (CD3-CD56bright), and NKT-like (CD3+CD56+) cells were investigated in peripheral blood mononuclear cell (PBMC) samples from 71 CRC patients and 19 healthy donors. RESULTS: CRC patients showed profound differences in immune cell subset distribution and their immunophenotype compared to healthy donors, as characterized by increased percentage of regulatory T cells, and reduced expression level of the natural cytotoxicity receptors NKp44 and NKp46 on both CD56dim NK cells and NKT-like cells. Finally, we showed in a multivariate analysis that above-median percentage of CD16+ NKT-like cells was independently associated with shorter disease-free survival in CRC patients. CONCLUSION: The altered phenotype of circulating immune cell subsets in CRC and its association with clinical outcome highlight the potential use of PBMC subsets as prognostic biomarkers in CRC, thereby contributing to better insight into the role of systemic immune profiles in tumor progression.

STAT3 inhibition specifically in human monocytes and macrophages by CD163-targeted corosolic acid-containing liposomes.

Tumor-associated macrophages (TAMs) are of major importance in cancer-related immune suppression, and tumor infiltration by CD163pos TAMs is associated with poor outcome in most human cancers. Therefore, therapeutic strategies for reprogramming TAMs from a tumor-supporting (M2-like) phenotype towards a tumoricidal (M1-like) phenotype are of great interest. Activation of the transcription factor STAT3 within the tumor microenvironment is associated with worse prognosis, and STAT3 activation promotes the immunosuppressive phenotype of TAMs. Therefore, we aimed to develop a drug for inhibition of STAT3 specifically within human TAMs by targeting the endocytic CD163 scavenger receptor, which is highly expressed on TAMs. Here, we report the first data on a CD163-targeted STAT3-inhibitory drug consisting of corosolic acid (CA) packaged within long-circulating liposomes (LCLs), which are CD163-targeted by modification with monoclonal anti-CD163 antibodies (αCD163)-CA-LCL-αCD163. We show, that activation of STAT3 (by phosphorylation) was inhibited by CA-LCL-αCD163 specifically within CD163pos cells, with minor effect on CD163neg cells. Furthermore, CA-LCL-αCD163 inhibited STAT3-regulated gene expression of IL-10, and increased expression of TNFα, thus indicating a pro-inflammatory effect of the drug on human macrophages. This M1-like reprogramming at the mRNA level was confirmed by significantly elevated levels of pro-inflammatory cytokines (IFNγ, IL-12, TNFα, IL-2) in the culture medium. Since liposomes are attractive vehicles for novel anti-cancer drugs, and since direct TAM-targeting may decrease adverse effects of systemic inhibition of STAT3, the present results encourage future investigation of CA-LCL-αCD163 in the in vivo setting.

Quantification of NK cell activity using whole blood: Methodological aspects of a new test.

Natural Killer (NK) cells are essential in the biological fight against cancer and intracellular pathogens, and their level of activity has in many settings been used as a biomarker for a functional immune response. Currently, NK cell activity is measured using either 51Cr-release assays or flow cytometry based assays revealing the cells´ cytotoxic capacity or by stimulating them to produce cytokines. Although very effective, these are cumbersome techniques not suitable for high volume clinical laboratories. Recently, an assay has been introduced to measure NK cell activity in a simple and standardized manner. Following stimulation of NK cells in whole blood with a recombinant protein, it utilizes the concentration of IFNγ released to the plasma as a surrogate marker for NK cell activity. However, whole blood holds several sources of IFNγ which may blur the results and hamper the interpretation of the test. Therefore, the present study aimed at analyzing how specifically the test is measuring the activity of NK cells. Intracellular flow cytometry showed that NK cells, T cells, and Natural Killer T (NKT) cells were producing IFNγ in the assay, however when analyzing the distribution of lymphocytes in the IFNγ-expressing subset, the proportion of NK cells far exceeded the percentage of T-, and NKT cells (p < .0001). Hence, our data indicate that the readout of the test was indicative of the NK cells´ ability to mount a response and thus the results may pave the way for the assay to become applicable in the clinical setting as an estimate of NK cell activity for both diagnostic and prognostic purposes.

The Role of Natural Killer T Cells in Cancer-A Phenotypical and Functional Approach.

Natural killer T (NKT) cells are a subset of CD1d-restricted T cells at the interface between the innate and adaptive immune system. NKT cells can be subdivided into functional subsets that respond rapidly to a wide variety of glycolipids and stress-related proteins using T- or natural killer (NK) cell-like effector mechanisms. Because of their major modulating effects on immune responses via secretion of cytokines, NKT cells are also considered important players in tumor immunosurveillance. During early tumor development, T helper (TH)1-like NKT cell subsets have the potential to rapidly stimulate tumor-specific T cells and effector NK cells that can eliminate tumor cells. In case of tumor progression, NKT cells may become overstimulated and anergic leading to deletion of a part of the NKT cell population in patients via activation-induced cell death. In addition, the remaining NKT cells become hyporesponsive, or switch to immunosuppressive TH2-/T regulatory-like NKT cell subsets, thereby facilitating tumor progression and immune escape. In this review, we discuss this important role of NKT cells in tumor development and we conclude that there should be three important focuses of future research in cancer patients in relation with NKT cells: (1) expansion of the NKT cell population, (2) prevention and breaking of NKT cell anergy, and (3) skewing of NKT cells toward TH1-like subsets with antitumor activity.

The microbial metabolite indole-3-propionic acid improves glucose metabolism in rats, but does not affect behaviour.

CONTEXT: We have previously shown that an antidepressant-like effect of probiotics in rats was associated with a higher plasma level of the microbial tryptophan metabolite indole-3-propionic acid (IPA). OBJECTIVE: We therefore wanted to study the isolated effect of IPA on behaviour and glucose metabolism in rats. METHODS: Male Sprague-Dawley rats were fed control or IPA-enriched diet for six weeks (n = 12 per group) and assessed in the elevated plus maze, open field and forced swim test. Blood glucose, metabolic hormones and the white blood cell (WBC) composition were analysed. RESULTS: IPA (mean intake 27.3 mg/kg/day) significantly lowered fasting blood glucose level by 0.42 mM (95% CI 0.11-0.73). Similarly, fasting plasma insulin levels and the homeostatic model assessment (HOMA) index of insulin resistance were reduced, whereas plasma metabolic hormones, behaviour and WBC composition remained unaffected by IPA. CONCLUSIONS: Our findings highlight IPA as a promising candidate for treatment of metabolic disorders associated with insulin resistance.

Probiotic treatment protects against the pro-depressant-like effect of high-fat diet in Flinders Sensitive Line rats.

Major depressive disorder (MDD) is highly associated with dysmetabolic conditions, such as obesity and diabetes mellitus type 2, and the gut microbiota may interact with both disease entities. We have previously shown that a high-fat diet (HFD) exacerbated depressive-like behaviour uniquely in Flinders Sensitive Line (FSL) rats that inherently present with an increased level of depressive-like behaviour compared with Flinders Resistant Line (FRL) rats. We therefore investigated whether multispecies probiotics possessed anti-depressant-like effect in FSL rats or protected against the pro-depressant-like effect of HFD. We also examined blood and cerebral T cell subsets as well as plasma cytokines. Lastly, we investigated the effect of HFD in outbred Sprague-Dawley (SD) rats to substantiate the association between depressive-like behaviour and any immunological measures affected by HFD. HFD exacerbated the depressive-like behaviour in FSL rats in the forced swim test, whereas SD rats remained unaffected. Probiotic treatment completely precluded the pro-depressant-like effect of HFD, but it did not affect FSL rats on control diet. Cerebral T lymphocyte CD4/8 ratios closely mirrored the behavioural changes, whereas the proportions of Treg and Th17 subsets were unaltered. No association between blood and brain CD4/8 ratios were evident; nor did plasma cytokine levels change as a consequence of HFD of probiotic treatment. Our findings suggest that MDD may hold a dysmetabolic component that responds to probiotic treatment. This finding has wide implications owing to the high metabolic comorbidity in MDD. Furthermore, the close association between depressive-like behaviour and cerebral T cell populations demonstrate lymphocyte-brain interactions as a promising future research area in the field of psychoneuroimmunology.

Probiotic treatment reduces depressive-like behaviour in rats independently of diet.

The gut microbiota has recently emerged as an important regulator of brain physiology and behaviour in animals, and ingestion of certain bacteria (probiotics) therefore appear to be a potential treatment for major depressive disorder (MDD). However, some conceptual and mechanistical aspects need further elucidation. We therefore aimed at investigating whether the habitual diet may interact with the effect of probiotics on depression-related behaviour and further examined some potentially involved mechanisms underlying the microbe-mediated behavioural effects. Forty male Sprague-Dawley rats were fed a control (CON) or high-fat diet (HFD) for ten weeks and treated with either a multi-species probiotic formulation or vehicle for the last five weeks. Independently of diet, probiotic treatment markedly reduced depressive-like behaviour in the forced swim test by 34% (95% CI: 22-44%). Furthermore, probiotic treatment skewed the cytokine production by stimulated blood mononuclear cells towards IFNγ, IL2 and IL4 at the expense of TNFα and IL6. In addition, probiotics lowered hippocampal transcript levels of factors involved in HPA axis regulation (Crh-r1, Crh-r2 and Mr), whereas HFD increased these levels. A non-targeted plasma metabolomics analysis revealed that probiotics raised the level of indole-3-propionic acid, a potential neuroprotective agent. Our findings clearly support probiotics as a potential treatment strategy in MDD. Importantly, the efficacy was not attenuated by intake of a "Western pattern" diet associated with MDD. Mechanistically, the HPA axis, immune system and microbial tryptophan metabolism could be important in this context. Importantly, our study lend inspiration to clinical trials on probiotics in depressed patients.

Elimination of erroneous results in flow cytometry caused by antibody binding to Fc receptors on human monocytes and macrophages.

Nonspecific binding of monoclonal antibodies (mAbs) to Fc-receptors on leukocytes is an important cause of background fluorescence in flow cytometry, and failing to block such nonspecific binding can lead to erroneous results. A major part of previous studies on blocking reagents for flow cytometry have been done in mice, and published results are not completely in agreement. In humans, Fc-receptors are found on most leukocytes, with highest abundance on monocytes/macrophages. Therefore, in the present study our aim was to thoroughly investigate the efficiency of different commonly used blocking reagents regarding inhibition of nonspecific binding of mouse mAbs to human peripheral blood mononuclear cells (MNCs) and monocyte-derived macrophages (MDMs). Monocytes and MDMs showed strong nonspecific binding of IgG1 and IgG2a isotypes, but not IgG2b. In contrast, B-cells, T-cells, and NK-cells did not substantially bind any of the mouse isotype control antibodies evaluated (IgG1, IgG2a, and IgG2b). Importantly, we show that binding of IgG1 and IgG2a to monocytes and MDMs can be eliminated by blocking, either with a commercial Fc-blocking reagent, with mouse or human serum, or with mouse or human IgG in high concentration. Previously, isotype controls have been widely used in flow cytometry assays. However, we show that such controls may be highly unreliable, and we believe they should not be used as gating controls, or to determine background signal. Based on these results, as well as considerations of price and applicability, our recommendation is not to use isotypes as gating controls in flow cytometry, but instead to use 100 µg/mL of purified human IgG as blocking reagent for elimination of nonspecific binding of mouse mAbs to human MNCs and MDMs. © 2016 International Society for Advancement of Cytometry.

The Immunogenicity of Colorectal Cancer in Relation to Tumor Development and Treatment.

Although most cancer types have been viewed as immunologically silent until recently, it has become increasingly clear that the immune system plays key roles in the course of tumor development. Remarkable progress towards understanding cancer immunogenicity and tumor-immune system interactions has revealed important implications for the design of novel immune-based therapies. Natural immune responses, but also therapeutic interventions, can modulate the tumor phenotype due to selective outgrowth of resistant subtypes. This is the result of heterogeneity of tumors, with genetic instability as a driving force, and obviously changes the immunogenicity of tumors. In this review, we discuss the immunogenicity of colorectal cancer (CRC) in relation to tumor development and treatment. As most tumors, CRC activates the immune system in various ways, and is also capable of escaping recognition and elimination by the immune system. Tumor-immune system interactions underlie the balance between immune control and immune escape, and may differ in primary tumors, in the circulation, and in liver metastases of CRC. Since CRC immunogenicity varies between tumors and individuals, novel immune-based therapeutic strategies should not only anticipate the molecular profile, but also the immunological profile of a specific tumor.