Preoperative Neutrophil-Lymphocyte and Lymphocyte-Monocyte Ratios Reflect Immune Cell Population Rearrangement in Resectable Pancreatic Cancer.

BACKGROUND: Neutrophil-lymphocyte ratio (NLR), platelet-lymphocyte ratio (PLR), and lymphocyte-monocyte ratio (LMR) may serve as a simple index of the immune function. The aim of this study was to investigate the prognostic significance of NLR, PLR, and LMR in patients with resectable pancreatic ductal adenocarcinoma (PDAC) and to verify whether such biomarkers are associated with changes in populations of lymphoid cells. METHODS: The prognostic implications of blood count parameters were evaluated in a retrospective cohort of 442 subjects undergoing pancreatic resections for PDAC. Subpopulations of lymphocytes and monocytes in peripheral blood were identified by FACS in a prospective cohort of 54 patients. RESULTS: In the univariate analysis, NLR < 5 and LMR ≥ 3 were associated with significantly longer median survival of 25.7 vs 12.6 months and 29.2 vs 13.1 months, respectively. PLR did not influence survival. The Cox proportional hazards model showed that high NLR (HR 1.66, 95 % CI 1.12 to 2.46, P = 0.012) and low LMR (HR 1.65, 95 % CI 1.06 to 2.58, P = 0.026) were independent predictors of poor prognosis. NLR ≥ 5 and LMR < 3 correlated with an approximately twofold decrease in counts of helper and cytotoxic T cells, B cells, and NK cells. High NLR was also accompanied with increased neutrophil counts, while low LMR showed increased numbers of monocytes, mostly classical. CONCLUSIONS: NLR and LMR may carry important prognostic information for patients with resected PDAC. The unfavorable prognosis likely correlates with reduced numbers of immune cells effective against the tumor and increased populations of cells involved in immune suppression.

Colorectal cancer-derived microvesicles modulate differentiation of human monocytes to macrophages.

BACKGROUND: Tumour-derived microvesicles (TMVs) are important players in tumour progression, modulating biological activity of immune cells e.g. lymphocytes, monocytes and macrophages. This phenomenon is particularly interesting in the progression of colon cancer, as macrophages in this type of tumour are relevant for the recovery processes. In the present study, the role of colon cancer cell-derived microvesicles in monocyte differentiation and activity profile (polarization) was investigated. METHODS: Monocyte-derived macrophages (MDM) were differentiated in vitro in the presence of TMVs obtained from colon cancer: Caco-2, SW620, LoVo or SW480 cell lines and analysed according to their morphology and biological functions, as defined by cytokine secretion, reactive oxygen intermediate (ROI) production and cytotoxic activity against respective colon cancer cells. RESULTS: Monocytes differentiated with TMVs exhibited morphological and phenotypical characteristics of macrophages. An early contact (beginning with the first day of the in vitro culture) of monocytes with TMVs resulted in increased IL-10 secretion and only slightly elevated TNF release. Early, or prolonged contact resulted in low ROI production and low cytotoxicity against tumour cells. On the other hand, late contact of MDM with TMVs, stimulated MDM to significant TNF and IL-12 secretion, ROI production and enhanced cytotoxicity against tumour cells in vitro. In addition, differences in MDM response to TMVs from different cell lines were observed (according to cytokine secretion, ROI production and cytotoxicity against tumour cells in vitro). Biological activity, STATs phosphorylation and microRNA profiling of MDMs indicated differences in their polarization/activation status which may suggest mixed polarization type M1/M2 with the predominance of proinflammatory cells after late contact with TMVs. CONCLUSIONS: Macrophage activity (polarization status) may be regulated by contact with not only tumour cells but also with TMVs. Their final polarization status depends on the contact time, and probably on the vesicle "cargo", as signified by the distinct impact of TMVs which enabled the switching of MDM maturation to regulatory macrophages.

Interactions of tumour-derived micro(nano)vesicles with human gastric cancer cells.

BACKGROUND: Tumour cells release membrane micro(nano)fragments called tumour-derived microvesicles (TMV) that are believed to play an important role in cancer progression. TMV suppress/modify antitumour response of the host, but there is also some evidence for their direct interaction with cancer cells. In cancer patients TMV are present in body fluid and tumour microenvironment. The present study aimed at characterization of whole types/subpopulations, but not only exosomes, of TMV from newly established gastric cancer cell line (called GC1415) and to define their interactions with autologous cells. METHODS: TMV were isolated from cell cultures supernatants by centrifugation at 50,000×g and their phenotype was determined by flow cytometry. The size of TMV was analysed by dynamic light scattering and nanoparticle tracking analysis, while morphology by transmission electron microscopy and atomic force microscopy. Interactions of TMV with cancer cells were visualized using fluorescence-activated cell sorter, confocal and atomic force microscopy, biological effects by xenografts in NOD SCID mice. RESULTS: Isolated TMV showed expression of CD44H, CD44v6 (hyaluronian receptors), CCR6 (chemokine receptor) and HER-2/neu molecules, exhibited different shapes and sizes (range 60-900 nm, highest frequency of particles with size range of 80-120 nm). TMV attached to autologous cancer cells within 2 h and then were internalized by them at 24 h. CD44H, CD44v6 and CCR6 molecules may play a role in attachment of TMV to cancer cells, while HER-2 associated with CD24 be involved in promoting cancer cells growth. Pre-exposure of cancer cells to TMV resulted in enhancement of tumour growth and cancer cell-induced angiogenesis in NOD SCID mice model. CONCLUSIONS: TMV interact directly with cancer cells serving as macro-messengers and molecular cargo transfer between gastric cancer cells resulting in enhancement of tumour growth. TMV should be considered in future as target of anticancer therapy.

Properties of monocytes generated from haematopoietic CD34(+) stem cells from bone marrow of colon cancer patients.

Monocytes exhibit direct and indirect antitumour activities and may be potentially useful for various forms of adoptive cellular immunotherapy of cancer. However, blood is a limited source of them. This study explored whether monocytes can be obtained from bone marrow haematopoietic CD34(+) stem cells of colon cancer patients, using previously described protocol of expansion and differentiation to monocytes of cord blood-derived CD34(+) haematopoietic progenitors. Data show that in two-step cultures, the yield of cells was increased approximately 200-fold, and among these cells, up to 60 % of CD14(+) monocytes were found. They consisted of two subpopulations: CD14(++)CD16(+) and CD14(+)CD16(-), at approximately 1:1 ratio, that differed in HLA-DR expression, being higher on the former. No differences in expression of costimulatory molecules were observed, as CD80 was not detected, while CD86 expression was comparable. These CD14(+) monocytes showed the ability to present recall antigens (PPD, Candida albicans) and neoantigens expressed on tumour cells and tumour-derived microvesicles (TMV) to autologous CD3(+) T cells isolated from the peripheral blood. Monocytes also efficiently presented the immunodominant HER-2/neu369-377 peptide (KIFGSLAFL), resulting in the generation of specific cytotoxic CD8(+) T lymphocytes (CTL). The CD14(++)CD16(+) subset exhibited enhanced cytotoxicity, though nonsignificant, towards tumour cells in vitro. These observations indicate that generation of monocytes from CD34(+) stem cells of cancer patients is feasible. To our knowledge, it is the first demonstration of such approach that may open a way to obtain autologous monocytes for alternative forms of adaptive and adoptive cellular immunotherapy of cancer.

Interactions of human monocytes with TMVs (tumour-derived microvesicles).

The tumour microenvironment represents a dynamic complex milieu, which includes tumour cells, cells of the immune system and other (cellular and non-cellular) components. The role of these particular 'puzzle pieces' may change substantially due to their mutual interactions. The present review concerns different opinions on interactions that occur between monocytes, tumour cells and TMVs (tumour-derived microvesicles).

The role of CD44H molecule in the interactions between human monocytes and pancreatic adenocarcinoma-derived microvesicles.

INTRODUCTION: CD44H is a transmembrane molecule important for cell-cell and cell-extracellular matrix interactions. In monocytes, CD44H is implicated in phagocytosis of particles coated by hyaluronan (HA). HA fragments were shown to induce chemokine secretion by monocytes. Tumour derived microvesicles (TMVs), which are small membrane fragments derived from tumour cells can carry fragments of HA. The aim of the study was to examine whether monocyte's CD44H is involved in the engulfment of pancreatic adenocarcinoma-derived microvesicles and in the production of chemokines induced by TMVs. MATERIALS AND METHODS: TMVs engulfment and chemokines' secretion stimulated with TMVs were determined in control human monocytes and cells incubated with anti-CD44H monoclonal antibody (mAb) by flow cytometry and ELISA, respectively. Phosphorylation of STAT3, transcription factor essential for chemokines' production and CD44 signal transduction, was determined by Western blotting. RESULTS: Blocking of CD44H by anti-CD44H mAb on monocytes decreased the engulfment of TMVs and the secretion of CCL4 and CCL5, but had no effect on CCL2, CCL3 and CXCL8. STAT-3 phosphorylation in monocytes incubated with TMVs after CD44 blocking was also reduced. CONCLUSION: The results suggest that tumour-derived microvesicles (TMVs) may carry bioactive cargo(s) which induces STAT3 dependent signalling pathway in human monocytes via CD44 molecules.

Human monocytes both enhance and inhibit the growth of human pancreatic cancer in SCID mice.

BACKGROUND: Monocytes/macrophages exhibit antitumour potential, but clinicopathological evidence suggests that they may both inhibit and enhance tumour growth. The purpose of this study was to determine the effect of monocytes on the growth of human pancreatic cancer (HPC-4) in severe combined immunodeficient (SCID) mice. MATERIALS AND METHODS: Freshly isolated human monocytes or CD14+ cells from cocultures with tumour cells were coengrafted, at various ratios, with HPC-4 cells into SCID mice. The tumour size and angiogenesis were determined. RESULTS: At a high ratio of monocytes to cancer cells the enhancement and, at a low ratio, the inhibition of tumour growth was observed. Multiple intratumoral applications of monocytes in large numbers also enhanced tumour growth. Deactivation of monocytes by a short pre-exposure to tumour cells in vitro before engraftment led to increased tumour growth. Monocytes used in large numbers and deactivated monocytes in low doses enhanced tumour-induced angiogenesis. CONCLUSION: Monocytes may both facilitate and suppress the growth of human tumours in SCID mice and both the number of monocytes, as well as the state of monocyte deactivation are critical for the final outcome of monocyte-tumour interactions.

Expansion and differentiation of CD14+CD16(-) and CD14+ +CD16+ human monocyte subsets from cord blood CD34+ hematopoietic progenitors.

To determine whether monocytes can be generated from CD34+ hematopoietic progenitors in large numbers, cord blood CD34+ cells were first expanded for 3-10 days in X-VIVO 10 medium supplemented with FCS, stem cell factor (SCF), thrombopoietin (TPO), and Flt-3 Ligand (Flt-3L), and then differentiated in IMDM medium supplemented with FCS, SCF, Flt-3L, IL-3 and M-CSF for 7-14 days. These two step cultures resulted in up to a 600-fold mean increase of total CD14+ cells. Using this approach, two subpopulations of monocytes were obtained: CD14+CD16(-) and CD14++CD16+ occurring at 2:1 ratio. 1.25(OH)2 Vitamin D3 added to the differentiation medium altered this ratio by decreasing proportion of CD14++CD16+ monocytes. In comparison to CD14+CD16(-), the CD14++CD16+ cells showed different morphology and an enhanced expression of CD11b, CD33, CD40, CD64, CD86, CD163, HLA-DR, and CCR5. Both subpopulations secreted TNF and IL-12p40 but little or no IL-10. CD14++CD16+ monocytes released significantly more IL-12p40, were better stimulators of MLR but showed less S. aureus phagocytosis. These subpopulations are clearly different from those present in the blood and may be novel monocyte subsets that represent different stages in monocyte differentiation with distinct biological function.

Characterization of human gastric adenocarcinoma cell lines established from peritoneal ascites.

The three cell lines, designated as gastric cancer (GC)1401, GC1415 and GC1436 were derived from peritoneal effusions from patients with gastric adenocarcinoma. Cell lines were established in tissue culture and in immunodeficient, non-obese diabetic/severe combined immunodeficiency (NOD/SCID) mice. All cell lines were cultured in Dulbecco's modified Eagle's medium supplemented with 5% fetal bovine serum. These cell lines were grown as an adherent monolayer with doubling time ranging between 25 h (GC1436 cell line) and 30-34 h (GC1401 and GC1415, respectively). All cells showed morphological features of epithelial-like cells, forming sheets of polygonal cells. Chromosomal analysis showed that the modal numbers ranged from 52 (GC1401), 51-56 (GC1415) and 106 (GC1436). High heterogeneity, resulting from several structural and numerical chromosomal abnormalities were evident in all cell lines. The surface marker expression suggested a tumor origin of the cells, and indicated the intestinal phenotype of a GC (CD10+, MUC1). All three cell lines were tumorigenic but not metastatic, in vivo, in NOD/SCID mice. The lack of metastatic potential was suggested by the lack of aldehyde dehydrogenase 1A1 activity. In conclusion, these newly established GC cell lines widen the feasibility of the functional studies on biology of GC as well as drug testing for potential therapeutic purposes.

Tumor cell-induced deactivation of human monocytes.

Although blood monocytes exhibit significant cytotoxic activity against tumor cells, the function of tumor infiltrating macrophages (TIM) is depressed in cancer patients. This study addresses the question of how the antitumor response of human monocytes, assessed by production of cytokines (tumor necrosis factor alpha, TNF; IL-10; IL-12p40) and cytotoxicity, is altered by exposure to cancer cells. Tumor cell--pre-exposed monocytes restimulated with tumor cells showed significantly decreased production of TNF, IL-12, increased IL-10 (mRNA and release) and inhibition of IL-1 receptor-associated kinase-1 (IRAK-1) expression. This down-regulation of cytokine production was selective, as the response of pre-exposed monocytes to lipopolysaccharide (LPS) was unaffected. Treatment of tumor cell--pre-exposed monocytes with hyaluronidase (HAase) improved their depressed production of TNF, while HAase-treated cancer cells did not cause monocyte dysfunction. The response of hyaluronan (HA)--pre-exposed monocytes to stimulation with tumor cells was also inhibited. Cytotoxic activity of monocytes pretreated with cancer cells was also decreased. This study shows that tumor cells selectively deactivate monocytes and suggests that tumor cell-derived HA by blocking CD44 on monocytes inhibits their antitumor response. These observations may provide some explanation for the depressed function of TIM in human malignancy.

Immunophenotypic changes and induction of apoptosis of monocytes and tumour cells during their interactions in vitro.

The in vitro model of tumour infiltrating macrophages (TIM)-tumour interactions in which monocytes and monocyte-derived macrophages (MDM) are cultured with cancer cells was used to assess immunophenotypic changes of interacting cells. Following short cocultures, monocytes, MDM and tumour cells were sorted out by FACS and the expression of several determinants was evaluated. Monocytes showed the induction of CD44v6 and v7/8, and up-regulation of CD16 (Fc gamma RIII), CD54 (ICAM-1), CD68 (macrophage maturation marker) and CD86 (costimulatory molecule B7.2). The increased expression of CD11a (LFA-1) and CD58 (LFA-3) was noted on some cancer cells. Up-regulation of TNFRII and HLA-DR was observed on both types of cells. MDM shared similar changes. Contact of monocytes, but not of MDM, with tumour cells led to Fas-FasL-dependent apoptosis of both types of cells. This study suggests that the immunophenotype of monocytes/macrophages and cancer cells may be modified during their bidirectional interactions in the absence of other microenvironmental elements that are present in the tumour stroma.

Involvement of pattern recognition receptors in the induction of cytokines and reactive oxygen intermediates production by human monocytes/macrophages stimulated with tumour cells.

BACKGROUND: Some ligands of pattern recognition receptors (PRR) are present on tumour cells. The role of PRR in signalling for cytokine and reactive oxygen intermediates (ROI) production by monocytes and monocyte-derived macrophages (MDM) stimulated with tumour cells was studied. MATERIALS AND METHODS: Monocytes/MDM were pretreated with PRR ligands or anti-PRR monoclonal antibodies (mAbs) and stimulated with tumour cells. Cytokine secretion was measured by enzyme-linked immunoassay (ELISA) and ROI production by luminol-dependent chemiluminescence (CL). RESULTS: The ligands of scavenger receptor A (SR-A): (fucoidan, polyguanylic acid (polyG) and modified low density lipoproteins (LDL)) and B (SR-B) (native and modified LDL, phosphatidylserine (PdS)) and of mannose receptor (MR) (mannan), induced tumour necrosis factor alpha (TNF) and ROI (except LDL) release by monocytes. Production of TNF and interleukin-10 (IL-10) by MDM was stimulated by SR-A ligands and mannan. Tumour cell-induced TNF and IL-10 production by monocytes, but not MDM, was diminished by fucoidan and polyG, while ROI release was reduced by MR and SR-A ligands. Supplementation of tumour cells with modified LDL and PdS enhanced their stimulatory capacity. TNF and ROI release by tumour cells-stimulated monocytes was inhibited by anti-CD36 and anti-MR (clone PAM-1) mAbs. CONCLUSION: SR and MR may be involved to different extents in the induction of cytokines and ROI production by monocytes, but not MDM, stimulated with tumour cells.

Interactions of monocyte subpopulations generated from cord blood CD34(+) hematopoietic progenitors with tumor cells: assessment of antitumor potential.

Monocytes and their subsets (CD14(++)CD16(+) and CD14(+)CD16(-)) generated from cord blood CD34(+) progenitor cells were used for determination of their capacity to interact with tumor cells in vitro and in vivo. The studies in vitro included adhesion to human umbilical vein endothelial cells, cytotoxicity, production of toxic mediators: reactive oxygen and nitrogen intermediates (ROI and RNI, respectively), and finally their effect on transplantable human tumor growth in nonobese diabetic severe combined immunodeficient mice. The CD14(++)CD16(+) subset exhibited an increased adherence to human umbilical vein endothelial cells and cytotoxicity toward tumor cells in vitro. CD14(+)CD16(-) monocytes showed a higher production of reactive oxygen and nitrogen intermediates after stimulation with tumor cells, and more pronounced inhibition of tumor growth in vivo. The results revealed significant differences in the behavior of CD14(++)CD16(+) and CD14(+)CD16(-) monocyte subsets toward tumor cells, thus providing further evidence that CD34(+) cell-derived monocytes differ in this respect from blood monocytes. The protocol for generation of monocytes with antitumor reactivity described here may be useful to obtain monocytes from CD34(+) progenitor cells of cancer patients. This might offer a basis for a novel approach for various forms of cellular immunotherapy of cancer.

Induction of monocyte antitumor response by human cancer cells transduced with TNF-GFP fusion gene: possible implications for immunotherapy of cancer.

This study was undertaken to determine how human pancreatic cancer (HPC-4) cells transduced with the TNF-GFP fusion gene (TLG) alter the antitumor response of human monocytes in vitro and whether they could act as an antitumor vaccine. In our model, HPC-4 cells were transduced with retroviral vector harboring TLG gene and designated as HPC-4(TLG). The TLG protein expression was confirmed by Western blot and flow cytometry analysis. Monocytes were co-cultured with transduced and control HPC-4 cells. The secretion of TNF, IL-10 and IL-12 was measured by ELISA. The cytotoxicity of monocytes against HPC-4 cells was determined by MTT test. The results show that the HPC-4(TLG) cells expressed membrane-bound, intracellular and secretory TLG protein. When cultured with HPC-4(TLG) cells, monocytes released a higher amount of TNF, but IL-10 and IL-12 secretion was inhibited. The pre-exposure of monocytes to HPC-4(TLG), but not to HPC-4, cells did not decrease TNF nor increase IL-10 production, thus not leading to monocyte deactivation. Also, the antitumor cytotoxicity of monocytes stimulated with HPC-4(TLG) was not downregulated, which occurred when non-transduced HPC-4 cells were used. In conclusion, compared to parental HPC-4 cells, TLG gene transduced HPC-4 cells induced stronger antitumor response of monocytes in vitro and prevented deactivation of monocytes.

Tumour-derived microvesicles contain interleukin-8 and modulate production of chemokines by human monocytes.

BACKGROUND: Tumour-derived microvesicles (TMVs) may interact with cells of the immune system. Our previous observations indicated that TMVs modulate production of cytokines and reactive oxygen species (ROS) by monocytes. This study was designed to determine the role of TMVs in stimulation of chemokine production by human monocytes. MATERIALS AND METHODS: Chemokines at the mRNA and protein level were detected by real-time PCR and by Western blot, respecively. Chemokine release and chemotaxis of blood leukocytes were analysed by flow cytometry. Matrigel assay was used to determine angiogenesis in a NOD-SCID mice model. RESULTS: TMVs induced secretion of interleukin-8 (CXCL8), monocyte chemoattractant protein-1 (CCL2), macrophage inflammatory protein-1α (CCL3) and MIP-1ß (CCL4), and regulated on activation normal T-cells expressed and secreted (CCL5) chemokines and accumulation of their mRNA in monocytes. Moreover, TMVs enhanced angiogenesis in NOD-SCID mice by delivering chemokines and via stimulation of monocytes. In addition, TMVs may be storage for chemokines thus inducing chemotaxis of blood leukocytes. CONCLUSION: These results further support the role of TMVs in modulation of monocyte biological activity.

Biological activity of dendritic cells generated from cord blood CD34+ hematopoietic progenitors in IL-7- and IL-13-conditioned cultures.

INTRODUCTION: Dendritic cells (DCs) are required for initiation of the immune response and may therefore be used for the production of cancer vaccines. As mature DCs (mDCs) are the most potent antigen-presenting cells, there is increasing interest in generating them ex vivo. The present study was designed to obtain mDCs from CD34+ hematopoietic progenitors by culturing them in different media. MATERIALS AND METHODS: Cord blood CD34+ hematopoietic progenitors were expanded for 7 days in FST medium containing fms-related tyrosine kinase 3 ligand (Flt3-L), stem cell factor (SCF), and thrombopoietin (TPO). Then the cells were divided into three parts and cultured for 21 days in different media: FST medium or FST enriched in interleukin (IL)-3 (FST3 medium) or supplemented with IL-7 and IL-13 (FST713 medium). At the end of culture part of the cells was harvested, counted, and analyzed while the other part was matured with proinflammatory cytokines for 2 days. The cells' phenotypes, ability to induce proliferation of allogeneic lymphocytes in the mixed lymphocyte reaction (allo-MLR), chemotaxis, phagocytosis, and O2(-) production were determined. RESULTS: The average fold increase of DCs at the end of culture in FST medium was 127, in FST3 1043, and in FST713 71. In comparison with the other media, FST713 medium supported the generation of mDCs that were characterized by higher expression of CD83, costimulatory molecules, and HLA-DR, enhanced ability to induce allo-MLR and migration to -macrophage inflammatory protein (MIP) 3beta poor phagocytosis, and O2(-) production. CONCLUSIONS: This study indicates that FST713 medium allows the generation of limited numbers of more mature DCs, while FST3 medium leads to the production of immature DCs in high numbers.

Blood monocytes stimulate migration of human pancreatic carcinoma cells in vitro: the role of tumour necrosis factor - alpha.

In some types of cancers, tumour-infiltrating monocytes/macrophages (TIM) may be responsible for the formation of an invasive microenvironment in a manner dependent on the secretion of soluble mediators such as tumour necrosis factor-alpha (TNF). Human pancreatic carcinoma (HPC-4) cells are able to induce TNF production by monocytes. Here, the effect of human peripheral blood monocytes, precursors of TIM, on the motility of co-cultured HPC-4 cells, was directly analysed in vitro. A phenotypic transition, i.e., the appearance of rear-front polarised HPC-4 cells paralleled by their increased motility, and increased motility of monocytes, were observed. This effect was attenuated when HPC-4 cells and monocytes were co-cultured in the presence of inhibitors of TNF production and anti-TNF monoclonal antibodies, indicating the specific role of this cytokine in determining paracrine loops between monocytes and cancer cells. Moreover, exogenous TNF induced HPC-4 cell motility concomitantly to the appearance of cellular features characteristic for epithelial-mesenchymal transition (EMT) such as rear-front polarisation, rearrangements of the actin cytoskeleton characteristic for motile cells and the induction of Snail-1 expression. Since cell movement is crucial for cancer invasion and the formation of metastases, these findings demonstrate an EMT-dependent mechanism of cancer progression which acts through the phenotypic transition of pancreatic cancer cells dependent on monocyte-derived TNF.