Layer-by-Layer Printing of Alginate for Cancer Cell Migration Assays

Rapid assessment of the invasion potential of various cancers in three-dimensional (3D) cell culture via layer-by-layer printing of cells encapsulated in hydrogels has been studied. Microarray bioprinting technology on microwell chips has been explored to create 3D cancer-like tissue structures and study cancer cell migration. Alginate, a negatively charged biopolymer, forms hydrogels via ionic crosslinking. Oxy-methacrylated alginate (OMA) is polymerized via near-ultraviolet light in the presence of photoinitiators. Our goal is to demonstrate rapid creation of cancer tissuelike structures via microarray 3D-bioprinting and develop a high-throughput, 3D cancer cell migration assay. To achieve this goal, layer-by-layer cell culture conditions were optimized in OMA by varying exposure time, photoinitiator concentration, alginate concentration, and cell seeding density. 3D cancer cell migration was demonstrated by printing two layers of hydrogels into the microwells: the bottom layer with a mixture of alginate and matrigel, and the top layer with Hep3B cells in alginate. Printed cells were cultured for fourteen days to investigate cell migration in 3D. As a result, it was found that migration of liver cancer cells needs to be extended for longer times. Also, bidirectional migration potential and leaching of additives (e.g., Matrigel) over time from alginate matrices will be investigated.https://engagedscholarship.csuohio.edu/u_poster_2016/1045/thumbnail.jp

High-Throughput Assessment of Developmental Stages of NSCs via Promoter-Reporter Assay System Using Recombinant Lentiviruses

Many drugs and chemicals currently available have not been fully evaluated for their toxic effects on the developing brain. Expensive and low-throughput in vivo studies are still being used to evaluate developmental neurotoxicity (DNT). Thus, there is a need to develop an in vitro assay system which is economically feasible and high- throughput. Among various cellular models used for in vitro assay, human neural stem cells (NSCs) are highly desired due to their ability to self-renew and differentiate into neurons, astrocytes and oligodendrocytes. In vitro assessment of developmental stages (proliferation and differentiation) of human NSC is highly important to predict the in vivo effect of various chemicals on developing brain. However, conventional in vitro assay uses immunofluorescence staining to monitor changes in cell morphology and neural cell-specific biomarkers which can either be inaccurate or cumbersome. Therefore, we have developed an in vitro promoter-reporter assay system to monitor the proliferation and differentiation of NSCs using recombinant lentiviruses. Four NSC-specific biomarkers can be monitored by infecting NSCs with recombinant lentiviruses such as synapsin1 for neuron differentiation, glial fibrillary acidic protein (GFAP) for astrocyte differentiation, myelin basic protein (MBP) for oligodendrocyte differentiation, and SOX2 for self-renewal.https://engagedscholarship.csuohio.edu/u_poster_2016/1044/thumbnail.jp

High-Throughput Assessment of Developmental Stages of NSCs via Promoter-Reporter Assay System Using Recombinant Lentiviruses

Many drugs and chemicals currently available have not been fully evaluated for their toxic effects on the developing brain. Expensive and low-throughput in vivo studies are still being used to evaluate developmental neurotoxicity (DNT). Thus, there is a need to develop an in vitro assay system which is economically feasible and high- throughput. Among various cellular models used for in vitro assay, human neural stem cells (NSCs) are highly desired due to their ability to self-renew and differentiate into neurons, astrocytes and oligodendrocytes. In vitro assessment of developmental stages (proliferation and differentiation) of human NSC is highly important to predict the in vivo effect of various chemicals on developing brain. However, conventional in vitro assay uses immunofluorescence staining to monitor changes in cell morphology and neural cell-specific biomarkers which can either be inaccurate or cumbersome. Therefore, we have developed an in vitro promoter-reporter assay system to monitor the proliferation and differentiation of NSCs using recombinant lentiviruses. Four NSC-specific biomarkers can be monitored by infecting NSCs with recombinant lentiviruses such as synapsin1 for neuron differentiation, glial fibrillary acidic protein (GFAP) for astrocyte differentiation, myelin basic protein (MBP) for oligodendrocyte differentiation, and SOX2 for self-renewal.https://engagedscholarship.csuohio.edu/u_poster_2016/1044/thumbnail.jp

Development of a Cx46 Targeting Strategy for Cancer Stem Cells

Gap-junction-mediated cell-cell communication enables tumor cells to synchronize complex processes. We previously found that glioblastoma cancer stem cells (CSCs) express higher levels of the gap junction protein Cx46 compared to non-stem tumor cells (non-CSCs) and that this was necessary and sufficient for CSC maintenance. To understand the mechanism underlying this requirement, we use point mutants to disrupt specific functions of Cx46 and find that Cx46-mediated gap-junction coupling is critical for CSCs. To develop a Cx46 targeting strategy, we screen a clinically relevant small molecule library and identify clofazimine as an inhibitor of Cx46-specific cell-cell communication. Clofazimine attenuates proliferation, self-renewal, and tumor growth and synergizes with temozolomide to induce apoptosis. Although clofazimine does not cross the blood-brain barrier, the combination of clofazimine derivatives optimized for brain penetrance with standard-of-care therapies may target glioblastoma CSCs. Furthermore, these results demonstrate the importance of targeting cell-cell communication as an anti-cancer therapy

Layer-by-Layer Printing of Alginate for Cancer Cell Migration Assays

Rapid assessment of the invasion potential of various cancers in three-dimensional (3D) cell culture via layer-by-layer printing of cells encapsulated in hydrogels has been studied. Microarray bioprinting technology on microwell chips has been explored to create 3D cancer-like tissue structures and study cancer cell migration. Alginate, a negatively charged biopolymer, forms hydrogels via ionic crosslinking. Oxy-methacrylated alginate (OMA) is polymerized via near-ultraviolet light in the presence of photoinitiators. Our goal is to demonstrate rapid creation of cancer tissuelike structures via microarray 3D-bioprinting and develop a high-throughput, 3D cancer cell migration assay. To achieve this goal, layer-by-layer cell culture conditions were optimized in OMA by varying exposure time, photoinitiator concentration, alginate concentration, and cell seeding density. 3D cancer cell migration was demonstrated by printing two layers of hydrogels into the microwells: the bottom layer with a mixture of alginate and matrigel, and the top layer with Hep3B cells in alginate. Printed cells were cultured for fourteen days to investigate cell migration in 3D. As a result, it was found that migration of liver cancer cells needs to be extended for longer times. Also, bidirectional migration potential and leaching of additives (e.g., Matrigel) over time from alginate matrices will be investigated.https://engagedscholarship.csuohio.edu/u_poster_2016/1045/thumbnail.jp

Global immune fingerprinting in glioblastoma patient peripheral blood reveals immune-suppression signatures associated with prognosis.

Glioblastoma (GBM) remains uniformly lethal, and despite a large accumulation of immune cells in the microenvironment, there is limited antitumor immune response. To overcome these challenges, a comprehensive understanding of GBM systemic immune response during disease progression is required. Here, we integrated multiparameter flow cytometry and mass cytometry TOF (CyTOF) analysis of patient blood to determine changes in the immune system among tumor types and over disease progression. Utilizing flow cytometry analysis in a cohort of 259 patients ranging from benign to malignant primary and metastatic brain tumors, we found that GBM patients had a significant elevation in myeloid-derived suppressor cells (MDSCs) in peripheral blood but not immunosuppressive Tregs. In GBM patient tissue, we found that increased MDSC levels in recurrent GBM portended poor prognosis. CyTOF analysis of peripheral blood from newly diagnosed GBM patients revealed that reduced MDSCs over time were accompanied by a concomitant increase in DCs. GBM patients with extended survival also had reduced MDSCs, similar to the levels of low-grade glioma (LGG) patients. Our findings provide a rationale for developing strategies to target MDSCs, which are elevated in GBM patients and predict poor prognosis

Global immune fingerprinting in glioblastoma patient peripheral blood reveals immune-suppression signatures associated with prognosis

Glioblastoma (GBM) remains uniformly lethal, and despite a large accumulation of immune cells in the microenvironment, there is limited antitumor immune response. To overcome these challenges, a comprehensive understanding of GBM systemic immune response during disease progression is required. Here, we integrated multiparameter flow cytometry and mass cytometry TOF (CyTOF) analysis of patient blood to determine changes in the immune system among tumor types and over disease progression. Utilizing flow cytometry analysis in a cohort of 259 patients ranging from benign to malignant primary and metastatic brain tumors, we found that GBM patients had a significant elevation in myeloid-derived suppressor cells (MDSCs) in peripheral blood but not immunosuppressive Tregs. In GBM patient tissue, we found that increased MDSC levels in recurrent GBM portended poor prognosis. CyTOF analysis of peripheral blood from newly diagnosed GBM patients revealed that reduced MDSCs over time were accompanied by a concomitant increase in DCs. GBM patients with extended survival also had reduced MDSCs, similar to the levels of low-grade glioma (LGG) patients. Our findings provide a rationale for developing strategies to target MDSCs, which are elevated in GBM patients and predict poor prognosis