Curcumin Suppresses Crosstalk between Colon Cancer Stem Cells and Stromal Fibroblasts in the Tumor Microenvironment: Potential Role of EMT

Objective

Interaction of stromal and tumor cells plays a dynamic role in initiating and enhancing carcinogenesis. In this study, we investigated the crosstalk between colorectal cancer (CRC) cells with stromal fibroblasts and the anti-cancer effects of curcumin and 5-Fluorouracil (5-FU), especially on cancer stem cell (CSC) survival in a 3D-co-culture model that mimics in vivo tumor microenvironment.

Methods

Colon carcinoma cells HCT116 and MRC-5 fibroblasts were co-cultured in a monolayer or high density tumor microenvironment model in vitro with/without curcumin and/or 5-FU.

Results

Monolayer tumor microenvironment co-cultures supported intensive crosstalk between cancer cells and fibroblasts and enhanced up-regulation of metastatic active adhesion molecules (β1-integrin, ICAM-1), transforming growth factor-β signaling molecules (TGF-β3, p-Smad2), proliferation associated proteins (cyclin D1, Ki-67) and epithelial-to-mesenchymal transition (EMT) factor (vimentin) in HCT116 compared with tumor mono-cultures. High density tumor microenvironment co-cultures synergistically increased tumor-promoting factors (NF-κB, MMP-13), TGF-β3, favored CSC survival (characterized by up-regulation of CD133, CD44, ALDH1) and EMT-factors (increased vimentin and Slug, decreased E-cadherin) in HCT116 compared with high density HCT116 mono-cultures. Interestingly, this synergistic crosstalk was even more pronounced in the presence of 5-FU, but dramatically decreased in the presence of curcumin, inducing biochemical changes to mesenchymal-epithelial transition (MET), thereby sensitizing CSCs to 5-FU treatment.

Conclusion

Enrichment of CSCs, remarkable activation of tumor-promoting factors and EMT in high density co-culture highlights that the crosstalk in the tumor microenvironment plays an essential role in tumor development and progression, and this interaction appears to be mediated at least in part by TGF-β and EMT. Modulation of this synergistic crosstalk by curcumin might be a potential therapy for CRC and suppress metastasis.     

Stromal Fibroblasts Mediate Extracellular Matrix Remodeling and Invasion of Scirrhous Gastric Carcinoma Cells

Scirrhous gastric carcinoma (SGC) has the worst prognosis of all gastric cancers, owing to its rapid expansion by invasion and frequent peritoneal dissemination. Due to the increased proliferation of stromal fibroblasts (SFs) that occurs within SGC lesions and the peritoneal metastatic sites, SFs have been proposed to support the progression of this disease. However, the biological and molecular basis and the pathological role of the intercellular interaction between SGC cells and SFs remain largely unknown. In this study, we investigated the role of SFs in the invasion of the extracellular matrix (ECM) by SGC cells. When SGC cells were cocultured with SFs derived from SGC tissue on three-dimensional (3D) Matrigel, they were attracted together to form large cellular aggregates that invaded within the Matrigel. Time-lapse imaging revealed that this process was associated with extensive contraction and remodeling of the ECM. Immunofluorescence and biochemical analysis showed that SGC cells stimulate phosphorylation of myosin light chain and actomyosin-mediated mechanical remodeling of the ECM by SFs. By utilizing this assay system for inhibitor library screening, we have identified several inhibitors that potently suppress the cooperation between SGC cells and SFs to form the invasive structures. Among them, a Src inhibitor dasatinib impaired the interaction between SGC cells and SFs both in vitro and in vivo and effectively blocked peritoneal dissemination of SGC cells. These results indicate that SFs mediate mechanical remodeling of the ECM by SGC cells, thereby promoting invasion and peritoneal dissemination of SGC.     

Chromogranin A and the Tumor Microenvironment

Chromogranin A (CgA) is an acidic glycoprotein belonging to a family of regulated secretory proteins stored in the dense core granules of the adrenal medulla and of many other neuroendocrine cells and neurons. This protein is frequently used as a diagnostic and prognostic serum marker for a range of neuroendocrine tumors. Circulating CgA is also increased in patients with other diseases, including subpopulations of patients with non-neuroendocrine tumors, with important prognostic implications. A growing body of evidence suggests that CgA is more than a diagnostic/prognostic marker for cancer patients. Indeed, results of in vitro experiments and in vivo studies in animal models suggest that this protein and its fragments can affect several elements of the tumor microenvironment, including fibroblasts and endothelial cells. In this article, recent findings implicating CgA as a modulator of the tumor microenvironment and suggesting that abnormal secretion of CgA could play important roles in tumor progression and response to therapy in cancer patients are reviewed and discussed.     

Numerical Simulation of the Inhibitory Effect of Angiostatin on Metastatic Tumor Angiogenesis and Microenvironment

The present work formulates and analyzes the inhibitory effect of anti-angiogenic factor angiostatin excreted by the primary tumor on metastatic tumor angiogenesis, blood perfusion, and interstitial fluid flow in the tumor microenvironment by means of a numerical experiment. The simulation results demonstrate that angiostatin has an obvious impact on the morphology, growth rate, and the number of branches of microvascular network inside and outside the metastatic tumor, and angiostatin has the capacity to regulate and inhibit the formation of new blood vessels. Heterogeneous blood perfusion, widespread interstitial hypertension, and low convection within the metastatic tumor have obviously improved under the inhibitory effect of angiostatin, which are consistent with physiological observed facts. The simulation results may provide beneficial information and theoretical models for clinical research of anti-angiogenic therapy strategies.     

Stromal Cells Induce Th17 during Helicobacter pylori Infection and in the Gastric Tumor Microenvironment

Gastric cancer is associated with chronic inflammation and Helicobacter pylori infection. Th17 cells are CD4⁺ T cells associated with infections and inflammation; but their role and mechanism of induction during carcinogenesis is not understood. Gastric myofibroblasts/fibroblasts (GMF) are abundant class II MHC expressing cells that act as novel antigen presenting cells. Here we have demonstrated the accumulation of Th17 in H. pylori-infected human tissues and in the gastric tumor microenvironment. GMF isolated from human gastric cancer and H. pylori infected tissues co-cultured with CD4⁺ T cells induced substantially higher levels of Th17 than GMF from normal tissues in an IL-6, TGF-β, and IL-21 dependent manner. Th17 required interaction with class II MHC on GMF for activation and proliferation. These studies suggest that Th17 are induced during both H. pylori infection and gastric cancer in the inflammatory milieu of gastric stroma and may be an important link between inflammation and carcinogenesis.     

Loss of S6K1 But Not S6K2 in the Tumor Microenvironment Suppresses Tumor Growth by Attenuating Tumor Angiogenesis

Two isoforms of the 70-kDa ribosomal protein S6 kinase, S6K1 and S6K2, have been identified and are considered key downstream effectors of the mTOR signaling pathway, which is involved in tumor growth and progression. However, their biological roles in the tumor microenvironment are poorly understood. In this study, utilizing tumor xenograft models in S6k1^−/− and S6k2^−/− mice, we show that loss of S6K1 but not S6K2 in the tumor stroma suppresses tumor growth, accompanied by attenuated tumor angiogenesis. We found that while S6K1 depletion had no effect on the proangiogenic phenotype of endothelial cells, the growth and angiogenesis of tumor xenografts were significantly reduced in wild-type mice upon reconstitution with S6K1-deficient bone marrow cells. Furthermore, upon S6K1 loss, induction of both mRNA and protein levels of Hif-1α and those of the downstream target, Vegf, was compromised in bone marrow–derived macrophages stimulated with lactate. These findings indicate that S6K1 but not S6K2 contributes to establishing a microenvironment that favors tumor growth through mediating angiogenesis, and suggest that attenuated tumor angiogenesis upon loss of S6K1 in the tumor stroma is, at least in part, attributable to impaired upregulation of Vegf in tumor-associated macrophages.     

Secretome Analysis of an Osteogenic Prostate Tumor Identifies Complex Signaling Networks Mediating Cross-talk of Cancer and Stromal Cells Within the Tumor Microenvironment

A distinct feature of human prostate cancer (PCa) is the development of osteoblastic (bone-forming) bone metastases. Metastatic growth in the bone is supported by factors secreted by PCa cells that activate signaling networks in the tumor microenvironment that augment tumor growth. To better understand these signaling networks and identify potential targets for therapy of bone metastases, we characterized the secretome of a patient-derived xenograft, MDA-PCa-118b (PCa-118b), generated from osteoblastic bone lesion. PCa-118b induces osteoblastic tumors when implanted either in mouse femurs or subcutaneously. To study signaling molecules critical to these unique tumor/microenvironment-mediated events, we performed mass spectrometry on conditioned media of isolated PCa-118b tumor cells, and identified 26 secretory proteins, such as TGF-β2, GDF15, FGF3, FGF19, CXCL1, galectins, and β2-microglobulin, which represent both novel and previously published secreted proteins. RT-PCR using human versus mouse-specific primers showed that TGFβ2, GDF15, FGF3, FGF19, and CXCL1 were secreted from PCa-118b cells. TGFβ2, GDF15, FGF3, and FGF19 function as both autocrine and paracrine factors on tumor cells and stromal cells, that is, endothelial cells and osteoblasts. In contrast, CXCL1 functions as a paracrine factor through the CXCR2 receptor expressed on endothelial cells and osteoblasts. Thus, our study reveals a complex PCa bone metastasis secretome with paracrine and autocrine signaling functions that mediate cross-talk among multiple cell types within the tumor microenvironment.A distinct feature of human prostate cancer (PCa)¹ with lethal potential is the development of metastases in bone with a bone-forming phenotype (1). This property of PCa bone metastasis suggests that PCa cells have unique interactions with cells in the bone microenvironment. Cells that are known to be present in the bone microenvironment include osteoblasts, osteoclasts, adipocytes, fibroblasts, and endothelial cells. Communication between PCa cells and each of these cells in the microenvironment is known to promote metastatic growth. This communication involves metastatic PCa cells that secrete factors to affect stromal cells in the bone microenvironment. The tumor-modified stromal cells may further alter the properties of the PCa cells to allow them to progress in the bone environment (1). Determining how secretory proteins from the metastatic PCa cells affect the PCa/stromal communication network will lead to the development of strategies to treat bone metastases.Although men with PCa and bone metastasis most frequently present with osteoblastic bone lesions, the commonly-used PCa cell lines to study metastatic properties, for example, PC3 and C4–2B, induce osteolytic or mixed osteoblastic/osteolytic lesions, respectively, when the cells are implanted into mouse femurs or tibia (2). In contrast, the PCa-118b patient-derived xenograft (PDX), generated from an osteoblastic bone lesion of a patient with PCa and bone metastasis, shows phenotypic characteristics similar to the tumor from which it was derived, including induction of a strong osteoblastic response when implanted into femurs (3). Interestingly, PCa-118b cells are also able to induce ectopic bone formation when implanted subcutaneously (3, 4). The capacity of PCa-118b cells to induce bone formation, in which human tumor cells interact with the murine stromal microenvironment, makes this PDX an ideal model system to study tumor-microenvironment signaling pathways that create a bone-like tumor microenvironment conducive to metastatic PCa growth.In this study, we identified secreted factors from the conditioned medium of isolated PCa-118b cells by mass spectrometry. A total of 26 secretory proteins, including cytokines and growth factors, were identified. Human- and mouse-specific PCR probes were used to identify the cells that expressed these factors. Analysis of the receptor for the corresponding secreted factor determined whether the factor exerted activities in a paracrine and/or autocrine manner. The effects of selected factors on PCa cells or stromal cells, including osteoblasts and endothelial cells, were also examined. Our studies showed that PCa-118b cells secreted multiple factors that establish an autocrine or paracrine signaling network that can mediate cross-talk among multiple cell types within the bone microenvironment.     

Cancer Associated Fibroblasts Promote Tumor Growth and Metastasis by Modulating the Tumor Immune Microenvironment in a 4T1 Murine Breast Cancer Model

Background

Local inflammation associated with solid tumors commonly results from factors released by tumor cells and the tumor stroma, and promotes tumor progression. Cancer associated fibroblasts comprise a majority of the cells found in tumor stroma and are appealing targets for cancer therapy. Here, our aim was to determine the efficacy of targeting cancer associated fibroblasts for the treatment of metastatic breast cancer.

Methodology/Principal Findings

We demonstrate that cancer associated fibroblasts are key modulators of immune polarization in the tumor microenvironment of a 4T1 murine model of metastatic breast cancer. Elimination of cancer associated fibroblasts in vivo by a DNA vaccine targeted to fibroblast activation protein results in a shift of the immune microenvironment from a Th2 to Th1 polarization. This shift is characterized by increased protein expression of IL-2 and IL-7, suppressed recruitment of tumor-associated macrophages, myeloid derived suppressor cells, T regulatory cells, and decreased tumor angiogenesis and lymphangiogenesis. Additionally, the vaccine improved anti-metastatic effects of doxorubicin chemotherapy and enhanced suppression of IL-6 and IL-4 protein expression while increasing recruitment of dendritic cells and CD8⁺ T cells. Treatment with the combination therapy also reduced tumor-associated Vegf, Pdgfc, and GM-CSF mRNA and protein expression.

Conclusions/Significance

Our findings demonstrate that cancer associated fibroblasts promote tumor growth and metastasis through their role as key modulators of immune polarization in the tumor microenvironment and are valid targets for therapy of metastatic breast cancer.     

Proteomic Profiling of Human Prostate Cancer-associated Fibroblasts (CAF) Reveals LOXL2-dependent Regulation of the Tumor Microenvironment

1. Download : Download high-res image (105KB)
2. Download : Download full-size image

Highlights

• •Quantitative (phoshpo)proteome of primary cell cultures of patient-matched prostate CAF and NPF.
• •Key CAF-associated proteins validated using orthogonal methodologies.
• •LOXL2 inhibitors D-penicillamine and PXS-S2A impaired CAF migration and ECM alignment.
• •Pre-treatment with LOXL2 inhibitors impaired migratory capacity of RWPE-2 cells in co-culture.

     

Changes in the Secretory Profile of NSCLC-Associated Fibroblasts after Ablative Radiotherapy: Potential Impact on Angiogenesis and Tumor Growth

In the context of radiotherapy, collateral effects of ablative doses of ionizing radiation (AIR) on stromal components of tumors remains understudied. In this work, cancer-associated fibroblasts (CAFs) isolated from freshly resected human lung tumors were exposed to AIR (1x 18 Gy) and analyzed for their release of paracrine factors. Inflammatory mediators and regulators of angiogenesis and tumor growth were analyzed by multiplex protein assays in conditioned medium (CM) from irradiated and non-irradiated CAFs. Additionally, the profile of secreted proteins was examined by proteomics. In functional assays, effects of CAF-CM on proliferative and migratory capacity of lung tumor cells (H-520/H-522) and human umbilical vein endothelial cells (HUVECs) and their tube-forming capacity were assessed. Our data show that exposure of CAFs to AIR results in 1) downregulated release of angiogenic molecules such as stromal cell-derived factor-1, angiopoietin, and thrombospondin-2 (TSP-2); 2) upregulated release of basic fibroblast growth factor from most donors; and 3) unaffected expression levels of hepatocyte growth factor, interleukin-6 (IL-6), IL-8, IL-1β, and tumor necrosis factor-α. CM from irradiated and control CAFs did not affect differently the proliferative or migratory capacity of tumor cells (H-520/H-522), whereas migratory capacity of HUVECs was partially reduced in the presence of irradiated CAF-CM. Overall, we conclude that AIR mediates a transformation on the secretory profile of CAFs that could influence the behavior of other cells in the tumor tissue and hence guide therapeutic outcomes. Downstream consequences of the changes observed in this study merits further investigations.     

Collagenase-3 Induction in Rat Lung Fibroblasts Requires the Combined Effects of Tumor Necrosis Factor-α and 12-Lipoxygenase Metabolites: A Model of Macrophage-induced, Fibroblast-driven Extracellular Matrix Remodeling during Inflammatory Lung Injury

The mechanisms responsible for the induction of matrix-degrading proteases during lung injury are ill defined. Macrophage-derived mediators are believed to play a role in regulating synthesis and turnover of extracellular matrix at sites of inflammation. We find a localized increase in the expression of the rat interstitial collagenase (MMP-13; collagenase-3) gene from fibroblastic cells directly adjacent to macrophages within silicotic rat lung granulomas. Conditioned medium from macrophages isolated from silicotic rat lungs was found to induce rat lung fibroblast interstitial collagenase gene expression. Conditioned medium from primary rat lung macrophages or J774 monocytic cells activated by particulates in vitro also induced interstitial collagenase gene expression. Tumor necrosis factor-α (TNF-α) alone did not induce interstitial collagenase expression in rat lung fibroblasts but did in rat skin fibroblasts, revealing tissue specificity in the regulation of this gene. The activity of the conditioned medium was found to be dependent on the combined effects of TNF-α and 12-lipoxygenase-derived arachidonic acid metabolites. The fibroblast response to this conditioned medium was dependent on de novo protein synthesis and involved the induction of nuclear activator protein-1 activity. These data reveal a novel requirement for macrophage-derived 12-lipoxygenase metabolites in lung fibroblast MMP induction and provide a mechanism for the induction of resident cell MMP gene expression during inflammatory lung processes.     

Inhibition of P38 MAPK Reduces Tumor Conditioned Medium-Induced Angiogenesis in Co-Cultured Human Umbilical Vein Endothelial Cells and Fibroblasts

Tumor conditioned medium (CM) has been widely used to stimulate endothelial cells to form capillary-like structures in in vitro angiogenesis models. We report herein the effect of HT1080 and A549 CM after they were mixed with microvascular endothelial cells medium-2 (EGM-2) on angiogenesis in human umbilical vein endothelial cells (HUVECs). Both HT1080 and A549 CM decreased HUVEC proliferation, to different extents. While A549 CM significantly increased capillary-like structure formation in a co-culture system, no effect of HT1080 was apparent. Inhibition of p38 mitogen-activated protein kinase (MAPK) blocked both basal and A549 CM induced capillary-like structure formation, but inhibition of extracellular signal-regulated kinases (ERK) and that of c-Jun N-terminal protein kinases (JNK) MAPK had no such effect. Activation of ERK MAPK was inhibited by both CMs, whereas p38 MAPK was inactivated by HT1080 and activated by A549 CM and a control. Neither CM had an effect on JNK MAPK. The results suggest that p38 MAPK played a critical role in capillary-like structure formation in the co-culture, partly via promotion of apoptosis in HUVECs.     

Direct Melanoma Cell Contact Induces Stromal Cell Autocrine Prostaglandin E2-EP4 Receptor Signaling That Drives Tumor Growth,Angiogenesis, and Metastasis

The stromal cells associated with tumors such as melanoma are significant determinants of tumor growth and metastasis. Using membrane-bound prostaglandin E synthase 1 (mPges1^−/−) mice, we show that prostaglandin E₂ (PGE₂) production by host tissues is critical for B16 melanoma growth, angiogenesis, and metastasis to both bone and soft tissues. Concomitant studies in vitro showed that PGE₂ production by fibroblasts is regulated by direct interaction with B16 cells. Autocrine activity of PGE₂ further regulates the production of angiogenic factors by fibroblasts, which are key to the vascularization of both primary and metastatic tumor growth. Similarly, cell-cell interactions between B16 cells and host osteoblasts modulate mPGES-1 activity and PGE₂ production by the osteoblasts. PGE₂, in turn, acts to stimulate receptor activator of NF-κB ligand expression, leading to osteoclast differentiation and bone erosion. Using eicosanoid receptor antagonists, we show that PGE₂ acts on osteoblasts and fibroblasts in the tumor microenvironment through the EP4 receptor. Metastatic tumor growth and vascularization in soft tissues was abrogated by an EP4 receptor antagonist. EP4-null Ptger4^−/− mice do not support B16 melanoma growth. In vitro, an EP4 receptor antagonist modulated PGE₂ effects on fibroblast production of angiogenic factors. Our data show that B16 melanoma cells directly influence host stromal cells to generate PGE₂ signals governing neoangiogenesis and metastatic growth in bone via osteoclast erosive activity as well as angiogenesis in soft tissue tumors.     

Lycosin-II Suppresses the Growth of Tumor Cells and Kills them Through Membrane Disruption and Apoptosis Induction

International Journal of Peptide Research and Therapeutics - Recently, there has been a growing interest in the discovery of novel chemotherapeutic agents that have fewer side effects and are less...     

Regional Activation of Myosin II in Cancer Cells Drives Tumor Progression via a Secretory Cross-Talk with the Immune Microenvironment

1. Download high-res image (352KB)
2. Download full-size image

     

乏氧微环境、“瓦伯格”效应及上皮间质转化相关性

肿瘤细胞在氧气充足的情况下以糖酵解的方式供能,这一现象称为“瓦伯格”效应,被认为是肿瘤的第七大特征。上皮间质转化（epithelial mesenchymal transition,EMT）是一种重要的细胞过程,参与胚胎发育、伤口愈合及肿瘤的发生等过程中,被认为是恶性肿瘤的重要特征。近年研究表明,“瓦伯格”效应和上皮间质转化的发生均与肿瘤处于乏氧微环境密切相关。乏氧微环境除可直接诱导上皮间质转化发生外,还可诱导肿瘤细胞产生“瓦伯格”效应,进一步促进上皮间质转化的发生。本文就乏氧微环境、“瓦伯格”效应、以及上皮间质转化的相关性的研究进展做一综述,有助于揭示乏氧微环境、肿瘤能量代谢改变以及肿瘤迁移侵袭之间的因果关联。