Rafał Szatanek

Tumour-derived microvesicles carry several surface determinants and mRNA of tumour cells and transfer some of these determinants to monocytes

This study was designed to determine the characteristics of tumour cell-derived microvesicles (TMV) and their interactions with human monocytes. TMV were shed spontaneously by three different human cancer cell lines but their release was significantly increased upon activation of the cells with phorbol 12-myristate 13-acetate (PMA). TMV showed the presence of several surface determinants of tumour cells, e.g. HLA class I, CD29, CD44v7/8, CD51, chemokine receptors (CCR6, CX3CR1), extracellular matrix metalloproteinase inducer (EMMPRIN), epithelial cell adhesion molecule (EpCAM), but their level of expression differed from that on cells they originated from. TMV also carried mRNA for growth factors: vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), interleukin-8 (IL-8) and surface determinants (CD44H). TMV were localized at the monocytes surface following their short exposure to TMV, while at later times intracellularly. TMV transferred CCR6 and CD44v7/8 to monocytes, exerted antiapoptotic effect on monocytes and activated AKT kinase (Protein Kinase B). Thus, TMV interact with monocytes, alter their immunophenotype and biological activity. This implicates the novel mechanism by which tumour infiltrating macrophages may be affected by tumour cells not only by a direct cell to cell contact, soluble factors but also by TMV.

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 α, 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.

Isolation of extracellular vesicles: Determining the correct approach (Review)

The discovery of extracellular vesicles (EVs) has revised the interpretation of intercellular communication. It is now well established that EVs play a significant role in coagulation, inflammation, cancer and stem cell renewal and expansion. Their release presents an intriguing, transporting/trafficking network of biologically active molecules, which are able to reach and modulate the function/behavior of the target cells in a variety of ways. Moreover, the presence of EVs in various body fluids points to their potential for use as biomarkers and prognostic indicators in the surveillance/monitoring of a variety of diseases. Although vast knowledge on the subject of EVs has accumulated over the years, there are still fundamental issues associated with the correct approach for their isolation. This review comprises the knowledge on EV isolation techniques that are currently available. The aim of this review was to make both experienced researchers and newcomers to the field aware that different types of EVs require unique isolation approaches. The realization of this ‘uniqueness’ is the first step in the right direction for the complete assessment of EVs.

The Methods of Choice for Extracellular Vesicles (EVs) Characterization

In recent years, extracellular vesicles (EVs) have become a subject of intense study. These membrane-enclosed spherical structures are secreted by almost every cell type and are engaged in the transport of cellular content (cargo) from parental to target cells. The impact of EVs transfer has been observed in many vital cellular processes including cell-to-cell communication and immune response modulation; thus, a fast and precise characterization of EVs may be relevant for both scientific and diagnostic purposes. In this review, the most popular analytical techniques used in EVs studies are presented with the emphasis on exosomes and microvesicles characterization.

Induction of intracellular cytokine production in human monocytes/macrophages stimulated with ligands of pattern recognition receptors.

AbstractObjective: This study addressed the role of the pattern recognition receptors (PRR), which recognize different molecular structures present on microorganisms, apoptotic, senescent and tumor cells, in the stimulation of human monocyte and monocyte-derived macrophages (MDM) for the production of intracellular cytokines. Materials and methods: Monocytes and MDM were stimulated with different ligands of scavenger receptors (SR) and mannose receptor (MR). Production of intracellular cytokines: tumor necrosis factor alpha (TNFα), interleukin 10 and 12 (IL-10, IL-12) was determined by flow cytometry following staining with anti-cytokine monoclonal antibodies (mAbs). Results: The ligands of SR type A: fucoidan, polyguanylic acid (polyG), chemically modified low density lipoproteins (LDL), ligands of SR-B: native and chemically modified LDL, and ligand of mannose receptor (MR) – mannan induced strong expression of intracellular TNFα and weaker IL-10 in monocytes, while phosphatidylserine (PdS) was without effect. IL-12 was stimulated only by fucoidan and polyG. The induction of cytokine m-RNA generally followed the pattern and the magnitude of intracellular cytokine production. In MDM, intracellular TNFα and IL-12 expression was induced by mannan, native and modified LDL, but not other ligands. Expression of IL-10 was less pronounced and occurred following stimulation with fucoidan, polyG and modified, but not native, LDL. Conclusions: These results suggest that some PRR ligands may be involved in activation of monocytes/MDM for the production of mainly proinflammatory cytokines (TNFα, IL-12) implicating their role in the response to microbial and tumor invasion.

Depressed tumor necrosis factor alpha and interleukin-12p40 production by peripheral blood mononuclear cells of gastric cancer patients: Association with IL-1R-associated kinase-1 protein expression and disease stage

Our study investigated the ability of peripheral blood mononuclear cells (PBMCs) isolated from patients with different clinical stages of gastric cancer to produce proinflammatory (tumor necrosis factor alpha [TNFα], interleukin 12p40 [IL‐12p40] and interleukin 6 [IL‐6]) and antiinflammatory (interleukin‐10 [IL‐10]) cytokines after stimulation with lipopolysaccharide (LPS) or tumor cells, and its correlation with IL‐1R‐associated kinase‐1 (IRAK‐1) protein expression. The data showed that TNF production by tumor cell–stimulated PBMCs obtained from patients with advanced gastric cancer was significantly depressed in comparison to the control group. The response to LPS was less affected. IL‐12p40 production was depressed in all stages of disease, while the release of IL‐10 and IL‐6 remained unchanged. Depressed tumor cell–induced TNF and IL‐12p40 production was associated with diminished IRAK‐1 protein expression in PBMC. These findings may suggest that in advanced gastric cancer (at least in some cancer patients) diminished IRAK‐1 protein expression may be a novel mechanism responsible for or facilitating downregulation of innate immune response to tumor cells.

Hyaluronan carried by tumor-derived microvesicles induces IL-10 production in classical (CD14(++)CD16(-)) monocytes via PI3K/Akt/mTOR-dependent signalling pathway.

Tumor-derived microvesicles (TMV) can mimic effects of tumor cells leading to an increased anti-inflammatory cytokine production, such as interleukin 10 (IL-10), by tumor-infiltrating monocytes and macrophages. Yet, the mechanism of IL-10 induction by TMV in monocytes remains unclear. The co-incubation of TMV derived from the human pancreas carcinoma cell line (HPC-4) with human monocytes resulted in a nearly 30-fold increase in IL-10 protein production. This effect operates at the level of transcription since monocytes transduced with an adenovirus containing IL-10-promoter luciferase reporter gene showed a 5-fold induction of luciferase activity after treatment with TMV. Since tumor cells can express hyaluronan (HA), which participates in tumor invasion and metastases, we have tested its effect on IL-10 expression. We showed that HA at the concentration of 100μg/ml induces IL-10 protein expression and the IL-10 promoter activation in monocytes. Moreover, hyaluronidase treatment of TMV reduced IL-10 protein production by 50% and promoter activity by 40%. Inhibitors of the PI3K/Akt/mTOR pathway reduced both, TMV-induced IL-10 promoter activity and protein production, and the same was observed in monocytes when stimulated by HPC-4 cells or HA. Inhibition of PI3K activity down-regulated phosphorylation of the Akt and (to a lesser extent) mTOR proteins in monocytes following TMV or HA stimulation. When comparing monocyte subsets, TMV induced IL-10 protein and mRNA synthesis only in classical CD14++CD16- but not in CD16-positive monocytes. Our data show that TMV induce IL-10 synthesis in human classical monocytes via HA, which, in turn, activates the PI3K/Akt/mTOR pathway.

Application of Flow Cytometry in the Studies of Microparticles

Many cell types including leukocytes, platelets and endothelial cells release small membrane fragments called microparticles (MP). MP are shed during cell growth, activation, proliferation, senescence and apoptosis. MP contain proteins (intracellular as well as surface markers), mRNA and miRNA of the cells they have originated from. Based on the release mechanism, size and phenotype, MP are frequently divided into two categories: exosomes and ectosomes called also microvesicles. There is no doubt that the biological significance of MP has been largely overlooked for many years, regarding them as merely cellular fragments or debris. Nowadays, MP are being recognized as an important regulator of cellular interactions under physiological and pathological conditions. MP are present in all body fluids and physiologically serve various functions like blood clotting, enhance cell adhesiveness, increase cell aggregation, etc. They mediate cell-to-cell communication by transferring cell surface receptors, mRNA, and miRNA from the cell of origin to target cells. The growing body of literature regarding the role of MP in many pathologies has recently progressed from describing the association of elevated MP number with disease stage (e.g. cancer, sepsis) through understanding how MP may contribute to thrombosis, preeclampsia and tumor progression, and finally, to using MP as a source of antigens in new forms of vaccines against infectious or malignant diseases.

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.