Characterizing Epithelial-Mesenchymal-Transition (EMT)-Like Behavior in Breast Cancer Cells in a Tissue Engineering Co-Culture Model

Tissue engineered 3D cell culture models have shown promise investigating the influence of the tumor microenvironment on cancer cell behavior. Synthetic hydrogels, such as those made from poly(ethylene glycol) (PEG), can be customized with bio-instructive moieties that mimic different parts of the extracellular matrix (ECM) that cells can adhere to through integrins and regions of cadherins that cells can interact with through their own cadherins. The question remains of how the composition of the ECM and neighboring cell interactions impact the transition from an epithelial phenotype to a mesenchymal-like phenotype, termed EMT, which is considered crucial in tumor progression to metastatic cancer. Therefore, in this project, a cell-adhesive peptide that mimics ECM and an cadherin mimetic peptide that mimics mesenchymal N-cadherin were utilized to investigate how cell-ECM interactions through integrin binding and how cell-cell interactions through cadherin binding can direct the emergence of EMT markers in breast cancer. Cytotoxicity studies demonstrated that the peptides allowed high SKBR3 breast cancer cell cytocompatibility but significantly lowered 3T3 fibroblast viability in a 3D environment, indicating that the peptide interactions were changing the behavior of the 3T3’s in a way that did not affect the SKBR3’s. Also, encapsulated as single cells in a gel with a cell-ECM peptide, the 3T3’s began to obtain a spindle-like phenotype, but not the SKBR3’s, showing that the 3T3’s were starting to form focal adhesions and take on a more mesenchymal-like phenotype not taken on by SKBR3’s in the same conditions. When putting the 3T3’s and SKBR3’s in co-culture, the SKBR3’s obtained a spindle-like phenotype when the 3T3’s were in a gel with a cadherin peptide, showing the beginning of an epithelial-mesenchymal transition-like morphology. After running quantitative PCR on said SKBR3’s, it was found that SKBR3’s with 3T3’s in contact with both cell adhesive and cadherin peptide and without either peptides were making significantly more mRNA for Collagen III and vimentin than SKBR3’s in contact with 3T3’s in only a cell-ECM peptide gel, both proteins made as a sign of becoming more mesenchymal-like. Collectively, these results demonstrate that the ability to incorporate mimetic peptides of cell-cell and cell-ECM interaction into PEG hydrogels makes them a powerful platform for studying how the composition of the ECM impacts EMT

Identification of a humanized mouse model for functional testing of immune-mediated biomaterial foreign body response.

Biomedical devices comprise a major component of modern medicine, however immune-mediated fibrosis and rejection can limit their function over time. Here, we describe a humanized mouse model that recapitulates fibrosis following biomaterial implantation. Cellular and cytokine responses to multiple biomaterials were evaluated across different implant sites. Human innate immune macrophages were verified as essential to biomaterial rejection in this model and were capable of cross-talk with mouse fibroblasts for collagen matrix deposition. Cytokine and cytokine receptor array analysis confirmed core signaling in the fibrotic cascade. Foreign body giant cell formation, often unobserved in mice, was also prominent. Last, high-resolution microscopy coupled with multiplexed antibody capture digital profiling analysis supplied spatial resolution of rejection responses. This model enables the study of human immune cell-mediated fibrosis and interactions with implanted biomaterials and devices

Interleukin-17-induced neutrophil extracellular traps mediate resistance to checkpoint blockade in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) remains a lethal malignancy with an immunosuppressive microenvironment that is resistant to most therapies. IL17 is involved in pancreatic tumorigenesis, but its role in invasive PDAC is undetermined. We hypothesized that IL17 triggers and sustains PDAC immunosuppression. We inhibited IL17/IL17RA signaling using pharmacological and genetic strategies alongside mass cytometry and multiplex immunofluorescence techniques. We uncovered that IL17 recruits neutrophils, triggers neutrophil extracellular traps (NETs), and excludes cytotoxic CD8 T cells from tumors. Additionally, IL17 blockade increases immune checkpoint blockade (PD-1, CTLA4) sensitivity. Inhibition of neutrophils or Padi4-dependent NETosis phenocopies IL17 neutralization. NMR spectroscopy revealed changes in tumor lactate as a potential early biomarker for IL17/PD-1 combination efficacy. Higher expression of IL17 and PADI4 in human PDAC corresponds with poorer prognosis, and the serum of patients with PDAC has higher potential for NETosis. Clinical studies with IL17 and checkpoint blockade represent a novel combinatorial therapy with potential efficacy for this lethal disease