The role of mesenchymal stem cells in the tumour microenvironment

The tumour microenvironment consists of a diverse range of cell types, which work together to create favorable conditions to support tumour growth and metastasis. It has been repeatedly reported that bone marrow-derived mesenchymal stem cells (MSCs), also referred to as multipotent stromal cells, infiltrate into the site of developing tumours. These adult multipotent cells show significant therapeutic potential in wound healing, tissue regeneration, immunosuppression and the treatment of cancer. However, there have been numerous contradictory reports on the role and fate of MSCs following engraftment into the tumour microenvironment. Evidence suggests that MSCs may constitute >20% of the stromal component of some solid tumours. In addition, there is evidence that paracrine signalling from cancer cells may cause MSCs to differentiate into cancer-associated myofibroblasts (CAMs), Soluble factors produced by CAMs have been shown to enhance further MSC recruitment and promote tumour progression and metastasis. In light of this information, the aims of this project were: (1) to investigate the changes that occur in MSCs following exposure to cancer cells or CAMs, and conversely, (2) to study the effects that MSCs may have on cancer cells and stromal cells using in vitro co-culture techniques. Exposure to the secretome of the AGS gastric adenocarcinoma cell-line induced a distinct gene expression profile in MSCs that was strongly associated with immune and inflammatory responses. Exposure to primary CAMs did not induce similar changes and exhibited little correlation across MSC and CAM cell-lines. Furthermore, exposure to AGS- or CAM-CM did not promote the acquisition of CAM-like characteristics in MSCs. However, extended exposure to AGS-CM or TNFα prompts the formation of a tumour-promoting MSC (tMSC) phenotype that is supportive of AGS growth via direct cell contact. Nrf2 is a transcription factor that is classically associated with cellular responses to stress that also induces the transcription of various anti-inflammatory genes and can be chemically induced by the synthetic triterpenoid CDDO-Me. Although CDDO-Me could inhibit various pro-inflammatory genes in MSCs that are induced in response to AGS-CM, it also propagates the formation of a tMSC phenotype. Finally, a key trait associated with MSCs is their ability to modulate the immune response. It is widely reported that MSCs have the ability to strongly repress the immune response. We found that MSCs can differentially modulate the maturation of dendritic cells depending on the ratio of cells used, and whether MSCs have been pre-exposed to AGS-CM. These findings suggest that the role and fate of MSCs is heavily dependent on the tissue niche in which they reside. MSCs are a highly responsive set of cells that can be directed towards various functional phenotypes that may inhibit or promote local cellular growth and activity. This highlights the need for high-throughput studies to define MSCs responses to external stimuli that may assist in increasing the efficacy of MSC-based therapies

Molecular signatures of mood stabilisers highlight the role of the transcription factor REST/NRSF

AbstractBackgroundThe purpose of this study was to address the affects of mood modifying drugs on the transcriptome, in a tissue culture model, using qPCR arrays as a cost effective approach to identifying regulatory networks and pathways that might coordinate the cell response to a specific drug.MethodsWe addressed the gene expression profile of 90 plus genes associated with human mood disorders using the StellARray™ qPCR gene expression system in the human derived SH-SY5Y neuroblastoma cell line.ResultsGlobal Pattern Recognition (GPR) analysis identified a total of 9 genes (DRD3⁎, FOS†, JUN⁎, GAD1⁎†, NRG1⁎, PAFAH1B3⁎, PER3⁎, RELN⁎ and RGS4⁎) to be significantly regulated in response to cellular challenge with the mood stabilisers sodium valproate (⁎) and lithium (†). Modulation of FOS and JUN highlights the importance of the activator protein 1 (AP-1) transcription factor pathway in the cell response. Enrichment analysis of transcriptional networks relating to this gene set also identified the transcription factor neuron restrictive silencing factor (NRSF) and the oestrogen receptor as an important regulatory mechanism.LimitationsCell line models offer a window of what might happen in vivo but have the benefit of being human derived and homogenous with regard to cell type.ConclusionsThis data highlights transcription factor pathways, acting synergistically or separately, in the modulation of specific neuronal gene networks in response to mood stabilising drugs. This model can be utilised in the comparison of the action of multiple drug regimes or for initial screening purposes to inform optimal drug design

Whole organism profiling of the Timp gene family

Tissue inhibitor of metalloproteinases (TIMPs/Timps) are an endogenous family of widely expressed matrisome-associated proteins that were initially identified as inhibitors of matrix metalloproteinase activity (Metzincin family proteases). Consequently, TIMPs are often considered simply as protease inhibitors by many investigators. However, an evolving list of new metalloproteinase-independent functions for TIMP family members suggests that this concept is outdated. These novel TIMP functions include direct agonism/antagonism of multiple transmembrane receptors, as well as functional interactions with matrisome targets. While the family was fully identified over two decades ago, there has yet to be an in-depth study describing the expression of TIMPs in normal tissues of adult mammals. An understanding of the tissues and cell-types that express TIMPs 1 through 4, in both normal and disease states are important to contextualize the growing functional capabilities of TIMP proteins, which are often dismissed as non-canonical. Using publicly available single cell RNA sequencing data from the Tabula Muris Consortium, we analyzed approximately 100,000 murine cells across eighteen tissues from non-diseased organs, representing seventy-three annotated cell types, to define the diversity in Timp gene expression across healthy tissues. We describe the unique expression profiles across tissues and organ-specific cell types that all four Timp genes display. Within annotated cell-types, we identify clear and discrete cluster-specific patterns of Timp expression, particularly in cells of stromal and endothelial origins. RNA in-situ hybridization across four organs expands on the scRNA sequencing analysis, revealing novel compartments associated with individual Timp expression. These analyses emphasize a need for specific studies investigating the functional significance of Timp expression in the identified tissues and cell sub-types. This understanding of the tissues, specific cell types and microenvironment conditions in which Timp genes are expressed adds important physiological context to the growing array of novel functions for TIMP proteins

Increased Expression of Chemerin in Squamous Esophageal Cancer Myofibroblasts and Role in Recruitment of Mesenchymal Stromal Cells

Stromal cells such as myofibroblasts influence tumor progression. The mechanisms 45 are unclear but may involve effects on both tumor cells and recruitment of bone 46 marrow-derived mesenchymal stromal cells (MSCs) which then colonize tumors. 47 Using iTRAQ and LC-MS/MS we identified the adipokine, chemerin, as 48 overexpressed in esophageal squamous cancer associated myofibroblasts (CAMs) 49 compared with adjacent tissue myofibroblasts (ATMs). The chemerin receptor, 50 ChemR23, is expressed by MSCs. Conditioned media (CM) from CAMs significantly 51 increased MSC cell migration compared to ATM-CM; the action of CAM-CM was 52 significantly reduced by chemerin-neutralising antibody, pretreatment of CAMs with 53 chemerin siRNA, pretreatment of MSCs with ChemR23 siRNA, and by a ChemR23 54 receptor antagonist, CCX832. Stimulation of MSCs by chemerin increased 55 phosphorylation of p42/44, p38 and JNK-II kinases and inhibitors of these kinases 56 and PKC reversed chemerin-stimulated MSC migration. Chemerin stimulation of 57 MSCs also induced expression and secretion of macrophage inhibitory factor (MIF) 58 that tended to restrict migratory responses to low concentrations of chemerin but not 59 higher concentrations. In a xenograft model consisting of OE21 esophageal cancer 60 cells and CAMs, homing of MSCs administered i.v. was inhibited by CCX832. Thus, 61 chemerin secreted from esophageal cancer myofibroblasts is a potential 62 chemoattractant for MSCs and its inhibition may delay tumor progression

Chemerin stimulates transendothelial migration of MSCs and requires MMP-2.

<p><i>A</i>, Representative fields from MSC transendothelial migration experiments showing migration of PKH67-labelled MSCs (left). CCX832 (1 µM) inhibited chemerin- (center) and CAM-CM stimulated MSC transendothelial migration but CCX826 (1 µM) had no effect (right). <i>B</i>, Chemerin, and IGF-II used as a positive control, promptly (30 min) stimulated proMMP2 abundance in media as detected by Western blot but had no effect on cellular proMMP2 abundance (left); chemerin significantly increased MMP-2 enzyme activity in MSC media detected by the selective substrate MCA-Pro-Leu-Ala-Nva-Dpa-Ala-Arg-NH₂ (right). <i>C</i>, Human recombinant MMP-2 (80 ng/ml) stimulated transendothelial migration and there was dose-dependent inhibition by an MMP-2 selective inhibitor (MMP-2 inhibitor I) (left). The MMP-2 inhibitor (60 µM) significantly inhibited chemerin-stimulated MSC transendothelial migration (centre). Horizontal arrows, p<0.05, t- test (n = 3).</p

Chemerin exhibits increased expression in CAMs and stimulates MSC migration.

<p><i>A</i>. Representative Western analysis of chemerin in media from ESCC CAMs and ATMs (left). Quantitative analysis by densitometry of chemerin abundance in media from ESCC CAMs and ATMs (n = 4 different pairs of myofibroblasts) (right). <i>B</i>. Concentration-dependent stimulation of MSC migration by chemerin in scratch wound migration assays (left) and Boyden chamber migration assays (right)(n = 3). <i>C</i>. Increased migration of MSCs in Boyden chambers in response to conditioned media (CM) from CAMs and their respective ATMs (left) (n = 4 different pairs of myofibroblasts). Stimulation of MSC migration by CAM-CM was inhibited by chemerin neutralizing antibody (Chem.Ab; 10 µg/ml) (center). MSC migration was decreased in response to CM from CAM1 and CAM4 cells transfected with chemerin siRNA#3 (right). Horizontal arrows, p<0.05, t- test (n = 3).</p

Increased MSC homing to xenografts seeded with CAMs and inhibition of homing by the chemR23 receptor antagonist, CCX832.

<p><i>A</i>, Visualisation of PKH67-labelled MSCs in representative fields from xenografts established with OE21 cancer cells alone or co-injected with CAMs followed by treatment with vehicle (top) or CCX832 (bottom) and iv injection of PKH67-labelled MSCs. <i>B</i>, In xenografts with OE21 cancer cells and CAMs there was increased MSC homing expressed as labelled cells per unit area of xenograft compared with xenografts of OE21 cancer cell alone; treatment with CCX832 inhibited homing (OE21/vehicle, n = 3; OE21/CCX832, n = 4; OE21 and CAMs/vehicle, n = 6; OE21 and CAMs/CCX832, n = 6). Horizontal arrows, p<0.05, ANOVA.</p

ChemR23 mediates chemerin stimulation of MSC migration via PKC and MAP kinases.

<p><i>A</i>, Representative images from MSCs stained for vimentin (positive control) and chemR23 revealing knock-down (KD) after ChemR23 siRNA treatment (left). Knockdown of ChemR23, but not GPR1, inhibited MSC migration in response to chemerin (100 ng/ml)(center) and CAM-CM (right). <i>B</i>, Concentration-dependent inhibition of MSC migration in response to chemerin by the ChemR23 antagonist CCX832 (left) but not the control compound CCX826 (1 µM) (center). MSC migration in response to CAM-CM was inhibited similarly by chemerin neutralising antibody, and CCX832, but not CCX826 (1 µM)(right). <i>C</i>, Representative Western blot shows increased phosphorylation of p42/44, p38 and JNK-II kinases in MSCs treated with chemerin (100 ng/ml)(left). In Boyden chamber assays, chemerin-stimulated MSC migration was inhibited by the JNK-II inhibitor, SP600125 (50 µM), the p42/44 inhibitor, UO126 (10 µM), p38 inhibitor SB202190 (3 µM), and the PKC inhibitor Ro320432 (2 µM) but not by PIK3 inhibitor LY294002 (50 µM) (right). Horizontal arrows, p<0.05, ANOVA (n = 3 in each case).</p