THP-1 cell line: an in vitro cell model for immune modulation approach.

The choice of phorbol 12-myristate 13-acetate differentiation protocol influences the response of THP-1 macrophages to a pro-inflammatory stimulus.

MALDI imaging mass spectrometry of human tissue: method challenges and clinical perspectives

Little is known about the polarizing potential of currently used human macrophage cell lines, while a better understanding phenomena can support the prediction of effects in vivo based on in vitro analysis. To test the polarization capability of PMA differentiated-THP-1 macrophages (M0), cells were stimulated with 20 ng ml−1 IFNγ + 1 μg ml−1 LPS and 20 ng ml−1 IL-4, which are known to influence macrophage polarization in vivo and ex vivo into the M1 and M2 state, respectively. Apart from several well-known M1 and M2 markers, also new possible markers for M1 and M2 polarization were analysed in this study. The expression of M1 marker genes was up-regulated in IFNγ + LPS stimulated-M0 THP-1 macrophages. The IL-4 stimulated-M0 THP-1 macrophages expressed M2 cell membrane receptor genes. However, M2 chemokine and their receptor genes were only slightly up-regulated which might be due to the complexity of the secondary cell–cell interaction of the chemokine system. Lipopolysaccharides from E. coli (LPS) and food compounds [lentinan, vitamin D3 (vD3) and the combination of lentinan + vitamin D3 (Len + vD3)] were investigated for their polarizing ability on M0 THP-1 macrophages towards either the M1 or M2 state. LPS (700 ng ml−1) was able to skew M0 THP-1 macrophages towards the M1 direction since all analysed M1 marker genes were strongly expressed. Lentinan, vD3 and Len + vD3 did not induce expression of either M1 or M2 markers, indicating no polarizing ability of these compounds. Based on the expression of M1 and M2 marker genes we concluded that THP-1 macrophages could be successfully polarized into either the M1 or M2 state. Therefore, they can be used as a new macrophage polarizing model to estimate the polarizing/switching ability of test food compounds.

Characterization of polarized THP-1 macrophages and polarizing ability of LPS and food compounds

// Xiaofan Guo 1, 2 , Hao Xue 1, 2 , Qianqian Shao 3 , Jian Wang 1, 2, 4 , Xing Guo 1, 2 , Xi Chen 3 , Jinsen Zhang 1, 2 , Shugang Xu 5 , Tong Li 1, 2 , Ping Zhang 1, 2 , Xiao Gao 1, 2 , Wei Qiu 1, 2 , Qinglin Liu 1, 2, * , Gang Li 1, 2, * 1 Department of Neurosurgery, Qilu Hospital of Shandong University, Jinan, Shandong Province, P.R. China 2 Brain Science Research Institute, Shandong University, Jinan, Shandong Province, P.R. China 3 Institute of Basic Medical Sciences and Key Laboratory of Cardiovascular Proteomics of Shandong Province, P.R. China 4 Department of Biomedicine, University of Bergen, Bergen, Norway 5 Department of Neurosurgery, Dezhou People’s Hospital, Dezhou, Shandong Province, P.R. China * These authors have contributed equally to this work Correspondence to: Gang Li, email: ligangqiluhospital@163.com Qinglin Liu, email: bodystrong@126.com Keywords: glioma, hypoxia, tumor-associated macrophage, M2 macrophage, acriflavine Received: March 09, 2016      Accepted: August 13, 2016      Published: September 02, 2016 ABSTRACT Tumor-associated macrophages (TAMs) are enriched in gliomas and help create a tumor-immunosuppressive microenvironment. A distinct M2-skewed type of macrophages makes up the majority of glioma TAMs, and these cells exhibit pro-tumor functions. Gliomas contain large hypoxic areas, and the presence of a correlation between the density of M2-polarized TAMs and hypoxic areas suggests that hypoxia plays a supportive role during TAM recruitment and induction. Here, we investigated the effects of hypoxia on human macrophage recruitment and M2 polarization. We also investigated the influence of the HIF inhibitor acriflavine (ACF) on M2 TAM infiltration and tumor progression in vivo . We found that hypoxia increased periostin (POSTN) expression in glioma cells and promoted the recruitment of macrophages. Hypoxia-inducible POSTN expression was increased by TGF-α via the RTK/PI3K pathway, and this effect was blocked by treating hypoxic cells with ACF. We also demonstrated that both a hypoxic environment and hypoxia-treated glioma cell supernatants were capable of polarizing macrophages toward a M2 phenotype. ACF partially reversed the M2 polarization of macrophages by inhibiting the upregulation of M-CSFR in macrophages and TGF-β in glioma cells under hypoxic conditions. Administering ACF also ablated tumor progression in vivo . Our findings reveal a mechanism that underlies hypoxia-induced TAM enrichment and M2 polarization and suggest that pharmacologically inhibiting HIFs may reduce M2-polarized TAM infiltration and glioma progression.

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Hypoxia promotes glioma-associated macrophage infiltration via periostin and subsequent M2 polarization by upregulating TGF-beta and M-CSFR.

Aims and background Macrophages are heterogeneous cells with extensive functional plasticity; they can change their functional profiles repeatedly in response to environmental changes anywhere between their extreme phenotypical programs (labeled as M1 and M2 polarization, respectively). In terms of antitumoral immune response, M1 macrophages are considered to be beneficial, while M2 macrophages supposedly promote tumor progression. Tumor-associated macrophages (TAMs) represent a major leukocyte population present in many tumors. Although many studies indicate that TAMs elicit several M2-associated protumoral functions, including promotion of angiogenesis, matrix remodeling and suppression of adaptive immunity, their role regarding tumor progression is still controversial. The aim of the present study was to develop an appropriate in vitro model to study the effect of tumor-secreted soluble factors on the functional phenotype of macrophages. Methods and study design THP-1 human monocytic line cells and peripheral blood mononuclear cells from healthy volunteers were used for macrophage differentiation; primary tumor cell culture supernatants or tumor cell line supernatants were employed along with various cytokines, growth factors and other stimuli to design different model variants and to better mimic the in vivo tumor microenvironment. Results The cytokine secretion patterns of these macrophages suggest that primary tumor cell culture supernatants are able to switch the macrophage phenotype or to induce functional polarization of macrophages toward a mixed M1/M2 phenotype. Conclusions. These data support the hypothesis that TAM behavior is modulated by the tumor microenvironment itself.

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Influence of tumor cell culture supernatants on macrophage functional polarization: in vitro models of macrophage-tumor environment interaction.

Objective Altered immune, inflammatory, and angiogenesis responses have been noticed in head and neck cancer, and many of these responses have been associated with a poor clinical outcome. The objective of this study was to evaluate several immune mediators in the sera of patients with squamous cell carcinoma (SCC) of the larynx undergoing curative surgery in connection with clinicopathologic factors. Methods Multiplex analysis of cytokines (interleukin [IL]-6, IL-8, IL-10, tumour necrosis factor α [TNF-α], interferon-γ [IFN-γ]), chemokines (monocyte chemoattractant protein 1 [MCP-1], macrophage inflammatory protein 1α [MIP-1α], and epithelial neutrophil-activating protein 78 [ENA-78]), and growth factors (vascular endothelial growth factor and basic fibroblast growth factor) in the serum of patients with laryngeal cancer and healthy controls was performed using xMap technology. Results Patients with SCC presented an altered cytokine profile compared to healthy controls, both preoperatively (higher levels of IL-8 and IL-10) and postoperatively (higher values for IL-6, IL-8, IL-10, and TNF-α). Heavy smoking was associated with significantly lower levels of ENA-78 and higher levels of IL-8. Conclusion Differences noticed in patients' immune mediator profiles seem to be attributable to both disease and treatment. Further longitudinal studies are necessary to elucidate the involvement of immune mediators in disease progression and clinical evolution.

Serum perioperative profile of cytokines in patients with squamous cell carcinoma of the larynx.

Cell culture is one of the major tools for oncology research, being an excellent system in which to study the biochemistry and molecular biology associated with individual cancer types and to understand cancer cell physiology Progress in understanding the biology of any type of carcinoma has been impeded by the inability to culture adequately malignant cells from most epithelial tissues The ultimate in vitro tumor model would completely reflect the in vivo tumor microenvironment in function and mechanism Unfortunately, such a model does not currently exist Homogeneous cell lines that can be continuously propagated on plastic surfaces have been extensively used as a surrogate for tumor environment; however they are very different from the in vivo tumor cells Model systems involving primary culture represent the situation most closely related to the original tissue although they have a number of disadvantages over cell lines, such as the limited ability to repeat studies with a well characterized culture system that can be used in multiple laboratories The primary culture may contain many types of stromal and infiltrating cell types potentially complicating the interpretation of data Yet, their properties better reflect the cellular interactions present in intact tissue The present article reviews the critical steps in obtaining, routine maintenance and cryopreservation of primary tumor cell cultures, based on information from literature and personal experience on the subject The article also includes an updated protocol for primary tumor cell isolation and culture

Primary cell culture of human adenocarcinomas--practical considerations.

Immunity plays an important role in the prognosis and the natural development of cancer. Previous studies have shown that the presence of tumor in the body could modify the immune response leading to immunosuppression. The aim of this study was to evaluate the immunological changes of patients with larynx squamous cell carcinoma undergoing potentially curative surgery. We assessed the serum levels of cytokines (IL–1, IL–2, IL–4, IL–6, IL–8, IL–10, IFN–gamma, TNF–alpha, GM–CSF), chemokines (MCP–1 and MIP–1alpha) and growth factors (VEGF and bFGF) in laryngeal cancer patients before, during and after surgery. We have used a novel multianalyte XMap profiling technology that allows simultaneous measurement of multiple parameters in small volumes of samples. To investigate the changes in immune mediators ' profile induced by tumor resection, we assessed the culture supernatants of the peripheral blood mononuclear cells (PBMC) derived from the patients, before and after surgery. The results suggested a predominance of a Th2 type of immune response associated with the presence of the tumor (especially in the case of heavy smokers who smoke more than 40 pack–years). However, shifts towards a Th1 type of immune response as well as an improvement of monocyte functions were noticed after surgery.

Laura Melinceanu

Papers

Influence of tumor cell culture supernatants on macrophage functional polarization: in vitro models of macrophage-tumor environment interaction.

Serum perioperative profile of cytokines in patients with squamous cell carcinoma of the larynx.

Primary cell culture of human adenocarcinomas--practical considerations.

Impact of smoking on the immunological profile of patients with laryngeal carcinoma.