Differentiated glioblastoma cells accelerate tumor progression by shaping the tumor microenvironment via CCN1-mediated macrophage infiltration

AbstractGlioblastoma (GBM) is the most lethal primary brain tumor characterized by significant cellular heterogeneity, namely tumor cells, including GBM stem-like cells (GSCs) and differentiated GBM cells (DGCs), and non-tumor cells such as endothelial cells, vascular pericytes, macrophages, and other types of immune cells. GSCs are essential to drive tumor progression, whereas the biological roles of DGCs are largely unknown. In this study, we focused on the roles of DGCs in the tumor microenvironment. To this end, we extracted DGC-specific signature genes from transcriptomic profiles of matched pairs of in vitro GSC and DGC models. By evaluating the DGC signature using single cell data, we confirmed the presence of cell subpopulations emulated by in vitro culture models within a primary tumor. The DGC signature was correlated with the mesenchymal subtype and a poor prognosis in large GBM cohorts such as The Cancer Genome Atlas and Ivy Glioblastoma Atlas Project. In silico signaling pathway analysis suggested a role of DGCs in macrophage infiltration. Consistent with in silico findings, in vitro DGC models promoted macrophage migration. In vivo, coimplantation of DGCs and GSCs reduced the survival of tumor xenograft-bearing mice and increased macrophage infiltration into tumor tissue compared with transplantation of GSCs alone. DGCs exhibited a significant increase in YAP/TAZ/TEAD activity compared with GSCs. CCN1, a transcriptional target of YAP/TAZ, was selected from the DGC signature as a candidate secreted protein involved in macrophage recruitment. In fact, CCN1 was secreted abundantly from DGCs, but not GSCs. DGCs promoted macrophage migration in vitro and macrophage infiltration into tumor tissue in vivo through secretion of CCN1. Collectively, these results demonstrate that DGCs contribute to GSC-dependent tumor progression by shaping a mesenchymal microenvironment via CCN1-mediated macrophage infiltration. This study provides new insight into the complex GBM microenvironment consisting of heterogeneous cells.

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Integrated in silico and 3D in vitro model of macrophage migration in response to physical and chemical factors in the tumor microenvironment

Integrative Biology ◽

10.1093/intbio/zyaa007 ◽

2020 ◽

Vol 12 (4) ◽

pp. 90-108

Author(s):

Sharon Wei Ling Lee ◽

R J Seager ◽

Felix Litvak ◽

Fabian Spill ◽

Je Lin Sieow ◽

...

Keyword(s):

Tumor Microenvironment ◽

Tumor Cells ◽

Rho Gtpases ◽

In Silico ◽

Three Dimensional ◽

Potential Contribution ◽

Macrophage Migration ◽

Interstitial Flow ◽

Co Addition

Abstract Macrophages are abundant in the tumor microenvironment (TME), serving as accomplices to cancer cells for their invasion. Studies have explored the biochemical mechanisms that drive pro-tumor macrophage functions; however the role of TME interstitial flow (IF) is often disregarded. Therefore, we developed a three-dimensional microfluidic-based model with tumor cells and macrophages to study how IF affects macrophage migration and its potential contribution to cancer invasion. The presence of either tumor cells or IF individually increased macrophage migration directedness and speed. Interestingly, there was no additive effect on macrophage migration directedness and speed under the simultaneous presence of tumor cells and IF. Further, we present an in silico model that couples chemokine-mediated signaling with mechanosensing networks to explain our in vitro observations. In our model design, we propose IL-8, CCL2, and β-integrin as key pathways that commonly regulate various Rho GTPases. In agreement, in vitro macrophage migration remained elevated when exposed to a saturating concentration of recombinant IL-8 or CCL2 or to the co-addition of a sub-saturating concentration of both cytokines. Moreover, antibody blockade against IL-8 and/or CCL2 inhibited migration that could be restored by IF, indicating cytokine-independent mechanisms of migration induction. Importantly, we demonstrate the utility of an integrated in silico and 3D in vitro approach to aid the design of tumor-associated macrophage-based immunotherapeutic strategies.

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Integrated in silico and 3D in vitro model of macrophage migration in response to physical and chemical factors in the tumor microenvironment

10.1101/2020.02.17.953208 ◽

2020 ◽

Author(s):

Sharon Wei Ling Lee ◽

R.J. Seager ◽

Felix Litvak ◽

Fabian Spill ◽

Je Lin Sieow ◽

...

Keyword(s):

Tumor Microenvironment ◽

Tumor Cells ◽

Rho Gtpases ◽

In Silico ◽

Three Dimensional ◽

Potential Contribution ◽

Macrophage Migration ◽

Interstitial Flow ◽

Co Addition

AbstractMacrophages are abundant in the tumor microenvironment (TME), serving as accomplices to cancer cells for their invasion. Studies have explored the biochemical mechanisms that drive pro-tumor macrophage functions, however the role of TME interstitial flow (IF) is often disregarded. Therefore, we developed a three-dimensional microfluidic-based model with tumor cells and macrophages to study how IF affects macrophage migration and its potential contribution to cancer invasion. The presence of either tumor cells or IF individually increased macrophage migration directedness and speed. Interestingly, there was no additive effect on macrophage migration directedness and speed under the simultaneous presence of tumor cells and IF. Further, we present an in silico model that couples chemokine-mediated signaling with mechanosensing networks to explain our in vitro observations. The model proposes IL-8, CCL2 and β-integrin as key pathways that commonly regulate various Rho GTPases. In agreement, in vitro macrophage migration remained elevated when exposed to a saturating concentration of recombinant IL-8 or CCL2, or to the co-addition of a sub-optimal concentration of both cytokines. Moreover, antibody blockade against IL-8 and/or CCL2 inhibited migration that could be restored by IF, indicating cytokine-independent mechanisms of migration induction. Importantly, we demonstrate the utility of an integrated in silico and 3D in vitro approach to aid the design of tumor-associated macrophage-based immunotherapeutic strategies.

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Hypoxic stress suppresses lung tumor-secreted exosomal miR101 to activate macrophages and induce inflammation

Cell Death and Disease ◽

10.1038/s41419-021-04030-x ◽

2021 ◽

Vol 12 (8) ◽

Author(s):

Jie Li ◽

Peng Xu ◽

Di Wu ◽

Minjie Guan ◽

Xuanwen Weng ◽

...

Keyword(s):

Tumor Microenvironment ◽

Tumor Cells ◽

Lung Tumor ◽

Hypoxia Inducible Factor ◽

Lung Squamous Cell Carcinoma ◽

In Vitro Study ◽

The Cancer Genome Atlas ◽

Hypoxic Stress

AbstractHypoxia promotes inflammation in the tumor microenvironment. Although hypoxia-inducible factor 1α (HIF1α) is a master modulator of the response to hypoxia, the exact mechanisms through which HIF1α regulates the induction of inflammation remain largely unclear. Using The Cancer Genome Atlas Lung Squamous Cell Carcinoma (TCGA-LUSC) database, we divided patients with LUSC into two groups based on low or high HIF1α expression. After analyzing the differentially expressed genes in these two groups, we found that HIF1α was positively correlated with interleukin 1A (IL1A) and IL6 expression. Our in vitro study showed that hypoxic stress did not induce IL1A or IL6 expression in tumor cells or macrophages but dramatically enhanced their expression when co-cultured with tumor cells. We then investigated the effect of tumor-derived exosomes on macrophages. Our data suggested that the changes in miR101 in the tumor-derived exosomes played an important role in IL1A and IL6 expression in macrophages, although the hypoxic stress did not change the total amount of exosome secretion. The expression of miR101 in exosomes was suppressed by hypoxic stress, since depletion of HIF1α in tumor cells recovered the miR101 expression in both tumor cells and exosomes. In vitro, miRNA101 overexpression or uptake enriched exosomes by macrophages suppressed their reprogramming into a pro-inflammatory state by targeting CDK8. Injection of miR101 into xenografted tumors resulted in the suppression of tumor growth and macrophage tumor infiltration in vivo. Collectively, this study suggests that the HIF1α-dependent suppression of exosome miR101 from hypoxic tumor cells activates macrophages to induce inflammation in the tumor microenvironment.

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EphA2 super-enhancer promotes tumor progression by recruiting FOSL2 and TCF7L2 to activate the target gene EphA2

Cell Death and Disease ◽

10.1038/s41419-021-03538-6 ◽

2021 ◽

Vol 12 (3) ◽

Author(s):

Shuang Cui ◽

Qiong Wu ◽

Ming Liu ◽

Mu Su ◽

ShiYou Liu ◽

...

Keyword(s):

Tumor Progression ◽

Tumor Cells ◽

Target Gene ◽

Super Enhancer ◽

Proliferation And Metastasis ◽

Active Transcription ◽

Hct 116 ◽

Large Clusters

AbstractSuper-enhancers or stretch enhancers (SEs) consist of large clusters of active transcription enhancers which promote the expression of critical genes that define cell identity during development and disease. However, the role of many super-enhancers in tumor cells remains unclear. This study aims to explore the function and mechanism of a new super-enhancer in various tumor cells. A new super-enhancer that exists in a variety of tumors named EphA2-Super-enhancer (EphA2-SE) was found using multiple databases and further identified. CRISPR/Cas9-mediated deletion of EphA2-SE results in the significant downregulation of its target gene EphA2. Mechanistically, we revealed that the core active region of EphA2-SE comprises E1 component enhancer, which recruits TCF7L2 and FOSL2 transcription factors to drive the expression of EphA2, induce cell proliferation and metastasis. Bioinformatics analysis of RNA-seq data and functional experiments in vitro illustrated that EphA2-SE deletion inhibited cell growth and metastasis by blocking PI3K/AKT and Wnt/β-catenin pathway in HeLa, HCT-116 and MCF-7 cells. Overexpression of EphA2 in EphA2-SE−/− clones rescued the effect of EphA2-SE deletion on proliferation and metastasis. Subsequent xenograft animal model revealed that EphA2-SE deletion suppressed tumor proliferation and survival in vivo. Taken together, these findings demonstrate that EphA2-SE plays an oncogenic role and promotes tumor progression in various tumors by recruiting FOSL2 and TCF7L2 to drive the expression of oncogene EphA2.

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Assessment of phagocytic activity in live macrophages-tumor cells co-cultures by Confocal and Nomarski Microscopy

Biology Methods and Protocols ◽

10.1093/biomethods/bpx002 ◽

2017 ◽

Vol 2 (1) ◽

Cited By ~ 5

Author(s):

Dalia Martinez-Marin ◽

Courtney Jarvis ◽

Thomas Nelius ◽

Stéphanie Filleur

Keyword(s):

Tumor Microenvironment ◽

Tumor Cells ◽

Phagocytic Activity ◽

Molecular Mechanisms ◽

Cell Types ◽

Functional Assay ◽

Loss Of Function ◽

Multiple Cancer

Abstract Macrophages have been recognized as the main inflammatory component of the tumor microenvironment. Although often considered as beneficial for tumor growth and disease progression, tumor-associated macrophages have also been shown to be detrimental to the tumor depending on the tumor microenvironment. Therefore, understanding the molecular interactions between macrophages and tumor cells in relation to macrophages functional activities such as phagocytosis is critical for a better comprehension of their tumor-modulating action. Still, the characterization of these molecular mechanisms in vivo remains complicated due to the extraordinary complexity of the tumor microenvironment and the broad range of tumor-associated macrophage functions. Thus, there is an increasing demand for in vitro methodologies to study the role of cell–cell interactions in the tumor microenvironment. In the present study, we have developed live co-cultures of macrophages and human prostate tumor cells to assess the phagocytic activity of macrophages using a combination of Confocal and Nomarski Microscopy. Using this model, we have emphasized that this is a sensitive, measurable, and highly reproducible functional assay. We have also highlighted that this assay can be applied to multiple cancer cell types and used as a selection tool for a variety of different types of phagocytosis agonists. Finally, combining with other studies such as gain/loss of function or signaling studies remains possible. A better understanding of the interactions between tumor cells and macrophages may lead to the identification of new therapeutic targets against cancer.

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The MEMIC: An ex vivo system to model the complexity of the tumor microenvironment

Disease Models & Mechanisms ◽

10.1242/dmm.048942 ◽

2021 ◽

Author(s):

Libuše Janská ◽

Libi Anandi ◽

Nell C. Kirchberger ◽

Zoran S. Marinkovic ◽

Logan T. Schachtner ◽

...

Keyword(s):

Tumor Microenvironment ◽

Tumor Cells ◽

Ex Vivo ◽

Tumor Stroma ◽

Stem Cell Differentiation ◽

Blood Stream ◽

Direct Access ◽

Ex Vivo Model

There is an urgent need for accurate, scalable, and cost-efficient experimental systems to model the complexity of the tumor microenvironment. Here, we detail how to fabricate and use the Metabolic Microenvironment Chamber (MEMIC) – a 3D-printed ex vivo model of intratumoral heterogeneity. A major driver of the cellular and molecular diversity in tumors is the accessibility to the blood stream that provides key resources such as oxygen and nutrients. While some tumor cells have direct access to these resources, many others must survive under progressively more ischemic environments as they reside further from the vasculature. The MEMIC is designed to simulate the differential access to nutrients and allows co-culturing different cell types, such as tumor and immune cells. This system is optimized for live imaging and other microscopy-based approaches, and it is a powerful tool to study tumor features such as the effect of nutrient scarcity on tumor-stroma interactions. Due to its adaptable design and full experimental control, the MEMIC provide insights into the tumor microenvironment that would be difficult to obtain via other methods. As a proof of principle, we show that cells sense gradual changes in metabolite concentration resulting in multicellular spatial patterns of signal activation and cell proliferation. To illustrate the ease of studying cell-cell interactions in the MEMIC, we show that ischemic macrophages reduce epithelial features in neighboring tumor cells. We propose the MEMIC as a complement to standard in vitro and in vivo experiments, diversifying the tools available to accurately model, perturb, and monitor the tumor microenvironment, as well as to understand how extracellular metabolites affect other processes such as wound healing and stem cell differentiation.

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Thioholgamide A, a New Anti-Proliferative Anti-Tumor Agent, Modulates Macrophage Polarization and Metabolism

Cancers ◽

10.3390/cancers12051288 ◽

2020 ◽

Vol 12 (5) ◽

pp. 1288 ◽

Cited By ~ 2

Author(s):

Charlotte Dahlem ◽

Wei Xiong Siow ◽

Maria Lopatniuk ◽

William K. F. Tse ◽

Sonja M. Kessler ◽

...

Keyword(s):

Tumor Microenvironment ◽

Tumor Cells ◽

Cell Metabolism ◽

Macrophage Polarization ◽

Human Monocyte ◽

Live Cell ◽

Hallmarks Of Cancer ◽

Culture Models

Natural products represent powerful tools searching for novel anticancer drugs. Thioholgamide A (thioA) is a ribosomally synthesized and post-translationally modified peptide, which has been identified as a product of Streptomyces sp. MUSC 136T. In this study, we provide a comprehensive biological profile of thioA, elucidating its effects on different hallmarks of cancer in tumor cells as well as in macrophages as crucial players of the tumor microenvironment. In 2D and 3D in vitro cell culture models thioA showed potent anti-proliferative activities in cancer cells at nanomolar concentrations. Anti-proliferative actions were confirmed in vivo in zebrafish embryos. Cytotoxicity was only induced at several-fold higher concentrations, as assessed by live-cell microscopy and biochemical analyses. ThioA exhibited a potent modulation of cell metabolism by inhibiting oxidative phosphorylation, as determined in a live-cell metabolic assay platform. The metabolic modulation caused a repolarization of in vitro differentiated and polarized tumor-promoting human monocyte-derived macrophages: ThioA-treated macrophages showed an altered morphology and a modulated expression of genes and surface markers. Taken together, the metabolic regulator thioA revealed low activities in non-tumorigenic cells and an interesting anti-cancer profile by orchestrating different hallmarks of cancer, both in tumor cells as well as in macrophages as part of the tumor microenvironment.

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RANKL Is Involved in Runx2-Triggered Hepatic Infiltration of Macrophages in Mice with NAFLD Induced by a High-Fat Diet

BioMed Research International ◽

10.1155/2020/6953421 ◽

2020 ◽

Vol 2020 ◽

pp. 1-8 ◽

Cited By ~ 1

Author(s):

Li Zhong ◽

Jianghan Yuan ◽

Lu Huang ◽

Shan Li ◽

Liang Deng

Keyword(s):

High Fat Diet ◽

Macrophage Infiltration ◽

Macrophage Migration ◽

High Fat ◽

Transwell Assay ◽

Nonalcoholic Fatty Liver ◽

Related Transcription Factor

Background. Receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL) is significant in the activation of inflammation. Runt-related transcription factor 2 (Runx2) promotes the hepatic infiltration of macrophages in nonalcoholic fatty liver disease (NAFLD). We studied how RANKL affects Runx2-triggered macrophage infiltration in NAFLD. Method. 30 male C57BL/6J mice at 4 weeks of age were utilized in this study, 20 mice received a high-fat diet (HFD), and 10 mice received standard rodent chow over 8 months. The histopathologic features of the liver were identified by H&E, Oil red O, and Masson staining. Runx2, RANKL, and F4/80 were analyzed by western blot, real-time PCR, and immunohistochemistry in vivo, respectively. Lentivirus or siRNA was utilized for transwell assay to investigate the role of RANKL in Runx2-induced macrophage migration in vitro. Results. Compared to controls, during NAFLD development, HFD increased Runx2 and RANKL in vivo in NASH (P<0.01). Meanwhile, a correlation between the expression of Runx2 and RANKL (P<0.05) was evident. In addition, the hepatic infiltration of macrophages was increased upon HFD feeding, and analysis showed that the macrophage infiltration was correlated with the expression of Runx2 or RANKL (P<0.05). In vitro, we found that overexpression or deficiency of Runx2 increased or decreased the production of RANKL in mHSCs. Then, transwell assay revealed that RANKL was involved in Runx2-induced macrophage migration. Conclusions. Overall, RANKL is involved in Runx2-triggered macrophage migration during NAFLD pathogenesis, which may provide an underlying therapeutic target for NAFLD.

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Melanoma-specific bcl-2 promotes a protumoral M2-like phenotype by tumor-associated macrophages

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2019-000489 ◽

2020 ◽

Vol 8 (1) ◽

pp. e000489 ◽

Cited By ~ 3

Author(s):

Marta Di Martile ◽

Valentina Farini ◽

Francesca Maria Consonni ◽

Daniela Trisciuoglio ◽

Marianna Desideri ◽

...

Keyword(s):

Tumor Microenvironment ◽

Tumor Progression ◽

Tumor Cells ◽

Macrophage Polarization ◽

Melanoma Cells ◽

Response To Therapy ◽

Major Constituent ◽

M2 Macrophage ◽

Tumor Associated Macrophages

BackgroundA bidirectional crosstalk between tumor cells and the surrounding microenvironment contributes to tumor progression and response to therapy. Our previous studies have demonstrated that bcl-2 affects melanoma progression and regulates the tumor microenvironment. The aim of this study was to evaluate whether bcl-2 expression in melanoma cells could influence tumor-promoting functions of tumor-associated macrophages, a major constituent of the tumor microenvironment that affects anticancer immunity favoring tumor progression.MethodsTHP-1 monocytic cells, monocyte-derived macrophages and melanoma cells expressing different levels of bcl-2 protein were used. ELISA, qRT-PCR and Western blot analyses were used to evaluate macrophage polarization markers and protein expression levels. Chromatin immunoprecipitation assay was performed to evaluate transcription factor recruitment at specific promoters. Boyden chamber was used for migration experiments. Cytofluorimetric and immunohistochemical analyses were carried out to evaluate infiltrating macrophages and T cells in melanoma specimens from patients or mice.ResultsHigher production of tumor-promoting and chemotactic factors, and M2-polarized activation was observed when macrophages were exposed to culture media from melanoma cells overexpressing bcl-2, while bcl-2 silencing in melanoma cells inhibited the M2 macrophage polarization. In agreement, the number of melanoma-infiltrating macrophages in vivo was increased, in parallel with a greater expression of bcl-2 in tumor cells. Tumor-derived interleukin-1β has been identified as the effector cytokine of bcl-2-dependent macrophage reprogramming, according to reduced tumor growth, decreased number of M2-polarized tumor-associated macrophages and increased number of infiltrating CD4+IFNγ+and CD8+IFNγ+effector T lymphocytes, which we observed in response to in vivo treatment with the IL-1 receptor antagonist kineret. Finally, in tumor specimens from patients with melanoma, high bcl-2 expression correlated with increased infiltration of M2-polarized CD163+macrophages, hence supporting the clinical relevance of the crosstalk between tumor cells and microenvironment.ConclusionsTaken together, our results show that melanoma-specific bcl-2 controls an IL-1β-driven axis of macrophage diversion that establishes tumor microenvironmental conditions favoring melanoma development. Interfering with this pathway might provide novel therapeutic strategies.

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The Brain Penetrating and Dual TORC1/TORC2 Inhibitor, RES529, Elicits Anti-Glioma Activity and Enhances the Therapeutic Effects of Anti-Angiogenetic Compounds in Preclinical Murine Models

Cancers ◽

10.3390/cancers11101604 ◽

2019 ◽

Vol 11 (10) ◽

pp. 1604 ◽

Cited By ~ 1

Author(s):

Giovanni Luca Gravina ◽

Andrea Mancini ◽

Alessandro Colapietro ◽

Simona Delle Monache ◽

Roberta Sferra ◽

...

Keyword(s):

Tumor Progression ◽

Tumor Cells ◽

Kinase Inhibitor ◽

Vasculogenic Mimicry ◽

Disease Free Survival ◽

Therapeutic Effects ◽

Murine Models ◽

Tubule Formation

Background. Glioblastoma multiforme (GBM) is a devastating disease showing a very poor prognosis. New therapeutic approaches are needed to improve survival and quality of life. GBM is a highly vascularized tumor and as such, chemotherapy and anti-angiogenic drugs have been combined for treatment. However, as treatment-induced resistance often develops, our goal was to identify and treat pathways involved in resistance to treatment to optimize the treatment strategies. Anti-angiogenetic compounds tested in preclinical and clinical settings demonstrated recurrence associated to secondary activation of the phosphatidylinositol 3-kinase (PI3K)/AKT/mTOR pathway. Aims. Here, we determined the sensitizing effects of the small molecule and oral available dual TORC1/TORC2 dissociative inhibitor, RES529, alone or in combination with the anti-VEGF blocking antibody, bevacizumab, or the tyrosine kinase inhibitor, sunitinib, in human GBM models. Results. We observed that RES529 effectively inhibited dose-dependently the growth of GBM cells in vitro counteracting the insurgence of recurrence after bevacizumab or sunitinib administration in vivo. Combination strategies were associated with reduced tumor progression as indicated by the analysis of Time to Tumor Progression (TTP) and disease-free survival (DSF) as well as increased overall survival (OS) of tumor bearing mice. RES529 was able to reduce the in vitro migration of tumor cells and tubule formation from both brain-derived endothelial cells (angiogenesis) and tumor cells (vasculogenic mimicry). Conclusions. In summary, RES529, the first dual TORC1/TORC2 dissociative inhibitor, lacking affinity for ABCB1/ABCG2 and having good brain penetration, was active in GBM preclinical/murine models giving credence to its use in clinical trial for patients with GBM treated in association with anti-angiogenetic compounds.

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