Henry Lopez

OP0326 Modelling the interaction between disease microenvironment and mesenchymal stem cells in scleroderma

Background Mesenchymal stem cells (MSCs) are pleuripotent bone marrow and tissue resident cells implicated in homeostasis and tissue repair. Systemic sclerosis (scleroderma, SSc) is a severe connective tissue disease characterised by progressive fibrotic thickening of the dermis, accompanied by loss of subcutaneous fat and microvasculature. Aberrant activation of MSCs within the disease microenvironment may underly the persistent fibrotic repair process, or account for the failure of adipogenesis and dysregulated vascular repair. Objectives We sought to: 1) determine whether activated MSCs are present within the SSc involved skin lesions, 2) test whether SSc suction blister fluid (BF) derived from involved forearm skin can induce phenotype changes in MSCs, 3) fully profile the altered gene expression in MSCs exposed to SSc BF, 4) investigate the role of key factors present at increased level in SSc BF (IL-31, lactate). Methods Novel post-fixation collagenase tissue dissociation techniques applied to 1 mm tissue sections, combined with Feulgen staining of DNA, were used to identify MSCs undergoing metakaryotic division within the involved skin of SSc patients. Fat derived MSCs from healthy controls were treated in tissue culture with blister fluid derived from the fibrotic skin lesions or from matched sites in healthy individuals, or exposed to key constituent factors, including cytokines (IL-31, 50 ng/ml), metabolites (lactate, 25 mM), and enhanced stiffness matrix (50 kPa gels). The responses of MSCs were studied by analysis of next generation sequencing (NGS) and phenotype changes. Results MSCs undergoing metakaryotic division were identified in SSc skin biopsy material but not in healthy control (HC) tissue (SSc vs HC, superficial dermis 0 vs 0, mid dermis 1.1 vs 0 p<0.0001, deep dermis 1.4 vs 0 p<0.0001 metakaryotic cells per x20 field). SSc BF (diluted 1:125 in media) induced disease-relevant phenotype changes in MSCs, such as αSMA expression (p<0.05), collagen gel contraction (p<0.002) and scratch wound repair (p<0.016), as well as loss of adipogenic potential, more than control BF or media alone, due in part to elevated IL-31 and lactate. NGS indicated that SSc blister fluid induced treatment-specific gene expression in MSCs (figure 1), more differentially than in normal dermal fibroblasts, consistent with activation of fibrosis, wound repair, migration, osteogenesis, connective tissue formation and loss of angiogenesis/vascular repair. Induction of αSMA in MSCs was dependent on the matrix stiffness in model systems. Conclusions Factors present at elevated levels in the disease microenvironment, including cytokines and metabolites, as well as the stiffened ECM, are capable of promoting the migration and differentation of fat derived MSCs, towards tissue reparative cells implicated in the fibrotic process. Conversely, the adipogenic and vascular regenerative potential of these cells may be reduced by exposure to the SSc microenvironment. Disclosure of Interest None declared

Use of Patterned Collagen Coated Slides to Study Normal and Scleroderma Lung Fibroblast Migration

Systemic sclerosis (SSc) is a spreading fibrotic disease affecting the skin and internal organs. We aimed to model pathogenic fibroblast migration in SSc in order to identify enhancing factors, measure the effect of migrating cells on underlying extracellular matrix (ECM) and test possible therapeutic inhibitors. Novel patterned collagen substrates were used to investigate alignment and migration of skin and lung fibroblasts from SSc patients and healthy controls. Normal lung but not skin fibroblasts consistently elongated and aligned with underlying collagen and migrated dependent on PDGF or serum. SSc lung fibroblasts remained growth factor dependent, did not migrate more rapidly and were less restricted to alignment of the collagen. Multiple collagen proline and lysine-modifying enzymes were identified in SSc but not control fibroblast extracellular matrix preparations, indicating differential levels of ECM modification by the diseased cells. Profiling of migrating cells revealed a possible SCF/c-Kit paracrine mechanism contributing to migration via a subpopulation of cells. Heparin, which binds ligands including PDGF and SCF, and imatininib which blocks downstream tyrosine kinase receptors, both inhibited lung fibroblast migration individually but showed synergy in SSc cells. Pathologic lung fibroblasts from SSc patients modify ECM during migration but remain growth factor dependent and sensitive to inhibitors.

Abstract 1640: A novel peptide-suppressing M2 polarized tumor-associated macrophages enhances tumor response to chemotherapy

Introduction: Multiple reports have demonstrated that M2-polarized tumor associated macrophages (TAMs) play a significant role in promoting tumor metastasis, survival and resistance to chemotherapy. Thus, limiting the pro-tumorigenic activity of TAMs may hold great therapeutic promise. Objective: To determine whether targeting the Cluster of Differentiation 206 (CD206) receptor on TAMs with an engineered peptides having both CD206 specificity and apoptotic activity, will limit the protumorigenic effects of TAMs and enhance cancer cells response to chemotherapy. Method: A class of engineered peptides were designed to stimulate phagocytosis via the CD206 receptor, and to initiate apopotic cell death once endocytosed. Primary cells were isolated from C57BL/6J mice from Jackson Laboratory and were polarized to M1 and M2 phenotypes by treating with IL-4 (20ng/ml) and IFN-γ (10ng/ml) respectively from RD gemcitabine @ 40 mg/kg q4D produced a 68% reduction; however, peptide in combination with gemcitabine completely blocked tumor growth. Moreover, immunohistochemistry staining of treated tumors showed a decrease in the expression of CD206 positive M2 macrophages and CD45/CD25 positive Treg cells, and an increase in CD86 positive M1 macrophages. These results were consistent with the in vitro results obtained. Discussion and Conclusion: These results suggest that a targeting CD206 peptide engineered to promote apoptosis, enhances the tumor response to chemotherapy by limiting the pro-tumorigenic activity of TAMs. Citation Format: Anghesom A. Ghebremedhin, Clayton Yates, Jesse Jaynes, George Martin, Henry Lopez, Charles Garvin. A novel peptide-suppressing M2 polarized tumor-associated macrophages enhances tumor response to chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1640. doi:10.1158/1538-7445.AM2017-1640

Abstract PR03: Synthetic peptides suppress M2 macrophages and synergize with chemotherapy in prostate and breast cancer models

Multiple lines of evidence suggest that macrophages, in particular M2-polarized tumor associated macrophages (TAMs), promote tumor aggressiveness and chemoresistance. Thus, limiting the pro-tumorigenic activity of TAMs may hold great therapeutic promise. To assess this potential, we synthesized artificial peptides (10-12mers) with a striapathic arrangement of hydrophobic and hydrophilic amino acids, designed to specifically target TAMs within the tumor microenvironment. These peptides, including drug candidate RP-182, are basic in design and bind to human albumin prior to binding to their eventual target, which enhances peptide half-life to around 2 hours. In vitro experiments demonstrated that exposure to 50uM peptide significantly reduced M2 macrophage viability and increased apoptosis after 48 hours of exposure, but had no effect on M1 macrophages. Interestingly, we did not observe a significant effect of RP-182 in vitro on various cancer cell lines, including MDA-MB-231, C42B or cells isolated from KPC960 mice. To determine whether RP-182 had in vivo efficacy we used multiple animal models, beginning with inflammatory indications where macrophage activity is also implicated, and progressing to solid organ cancer models: (1) In bleomycin animal models of lung and skin fibrosis, mice developed very high levels of inflammatory markers including IL6, which remained at control levels in mice treated with 10 mg/kg RP-182 qD subcu. At increased pulmonary bleomycin levels, all control animals died while 65% of treated animals survived. (2) In xenograft models of triple negative breast cancer (MDA-MB-231) mice treated with peptide (10 mg/kg qD subcu) demonstrated 37% reduction in MD-MB-231 tumor growth; gemcitabine @ 40 mg/kg q4D demonstrated 70% reduction; however, peptide plus gemcitabine completely blocked tumor growth. (3) In a xenograft model of castration resistant prostate cancer (CRPC) (C42B) mice treated with 2.5 mg/kg Docetaxel q7D demonstrated 45% reduction in tumor growth relative to control; mice treated with 10 mg/kg RP-182 qD demonstrated 52% reduction in tumor growth; but Docetaxel plus RP-182 reduced tumor growth 65%. (4) In the KPC960 model of pancreatic cancer, RP-182 treatment caused a 30% reduction in tumor growth and reduced the expression of a number of inflammatory genes in the tumors. (5) Finally, in the KRAS/p53 transgenic model of pancreatic cancer, treatment with peptide for one week reduced tumor growth, and was associated with 5.8 fold reduction in PDL1 and a 5.5 fold reduction in PDL2 expression in tumors. Toxicity testing on animals given much higher doses of RP-182 showed no changes in histology, blood counts, body or tissue weight. These results suggest that these novel designed peptides suppress macrophages in the tumors, enhancing the tumor response to chemotherapy and downregulating certain checkpoint proteins. This abstract is also presented as Poster B23. Citation Format: Henry Lopez, George R. Martin, Jesse M. Jaynes, Clayton Yates. Synthetic peptides suppress M2 macrophages and synergize with chemotherapy in prostate and breast cancer models. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr PR03.

SAT0193 Macrophages Responding To Mechanical Stress in Scleroderma

Background Inflammation, vasculopathy and tissue fibrosis are key features of scleroderma (SSc). While healthy forearm skin which has a Youngs modulus of 4–12kPa, SSc fibrotic skin measures 50–80kPa with its increased mechanical stiffness1. We have shown that mechano-sensing properties of fibroblasts in SSc are mediated by myocardin-related transcription factor A (MRTF-A)3. Monocytes/macrophages are likely to be involved in SSc pathogenesis but the effect of mechanical stress on these cells remain to be elucidated3,4. Objectives To investigate if a mechanically-stressed microenvironment like that in SSc tissue, promotes macrophages towards a pathogenic phenotype, and whether MRTF-A is involved in this process. Methods Control and scleroderma skin sections were immunostained with anti-CD68 and anti-MRTF-A antibodies (n=3). Human PBMC-derived macrophages were cultured in RPMI/M-CSF on 4kPa and 50kPa collagen-fibronectin-coated plates to mimic “soft”/healthy and “stiff”/fibrotic skin, and activated with LPS (10ng/ml) or IL-10 (10ng/ml) for macrophages designated M(LPS) and M(IL-10) (n=4). MRTF-A expression was assessed by qPCR and conditioned media profiled by Luminex array for inflammatory cytokines. Mouse bone marrow-derived macrophages (BMDMs) of wildtype and MRTF-A-null mice were maintained in RPMI/M-CSF on soft and stiff substrates. The data were analysed by two-way ANOVA and Tukey test (p<0.05, CI 95%). Results We observed a greater number of CD68+ macrophages in diffuse SSc skin compared to control skin, mainly around perivascular regions. These macrophages also expressed MRTF-A. Human macrophages expressed MRTF-A mRNA and showed differential cytokine expression when cultured on soft and stiff substrates. M(LPS) on soft matrix expressed IFN-γ, which was undetectable with M(LPS) on stiff substrate (mean difference 0.2075±0.1576pg/ml, p<0.01). LPS- and IL-10 activation on soft substrate increased MCP-3 expression compared to controls (mean difference 68.51±49.19pg/ml, p<0.01, 92.88±49.22pg/ml, p<0.0001, respectively). M(LPS) on stiff compared to soft substrate showed lower MCP-3 expression (mean difference 57.01±49.22pg/ml, p<0.05). M(IL-10) on soft substrate showed higher MCP-1 expression compared to controls (mean difference 2448±2232pg/ml, p<0.05). M(IL-10) on stiff substrate decreased expression of MCP-1 (mean difference 2590±2233pg/ml, p<0.05) and increased fractalkine levels compared to soft substrate (mean difference 51.22±36.28pg/ml, p<0.01). Wildtype BMDMs on stiff compared to soft substrate displayed a more elongated morphology. MRTF-A-null BMDMs remained rounded on stiff substrate. Conclusions MRTF-A is a mechanical stress-responsive factor which co-activates transcription of cytoskeletal and extracellular matrix-modifying genes. MRTF-A may couple mechanical stress to macrophage activation in SSc, where stiff matrix promotes macrophage secretion of cytokines and growth factors that exacerbate fibrosis. References Sacksen et al., Arthritis Rheum 2013. Shiwen et al., PLoS One 2015. Stifano et al., Curr Rheumatol Rep 2015. Chia et al., Curr Opin Rheumatol 2015. Disclosure of Interest None declared