INTRODUCTION: Administration of ineffective anticancer therapy is associated with unnecessary toxicity and development of resistant clones. Cancer stem cells (CSCs) resist chemotherapy, thereby causing relapse of the disease. Thus, development of a test that identifies the most effective chemotherapy management offers great promise for individualized anticancer treatments. We have developed an ex vivo chemotherapy drug response assay (ChemoID ^® ), which measures the sensitivity of CSCs as well as the bulk of tumor cells to a variety of chemotherapy agents to assist an oncologist in making treatment decisions. METHODS: Three patients affected by oral cancer were referred. RESULTS: Biopsy showed a well-differentiated squamous cell carcinoma (G1) in case 1, a G2 adenocarcinoma in case 2 and a G3 squamous cell carcinoma in case 3. In all of the three cases, after clinical inspection and suspicion of a diagnosis of cancer, a double biopsy was performed. One specimen was sent to the ChemoID laboratory for chemosensitivity assay and the other for histological analysis. Chemotherapy dose response curves were generated, and grouped in 3 categories: 1. No response (less than 30% cell kill), Intermediate (30–60% cell kill), and 3. Sensitive (60% cell kill or above). CONCLUSIONS: This procedure may be useful in helping physicians choose an effective chemotherapy regimen for head and neck cancer patients and lower treatment costs by eliminating ineffective chemotherapies and unnecessary toxicity particularly in elderly patients. Highlights:

Lack of standardization of clinically compliant culture protocols of mesenchymal stem cells for re-implantation in humans have hindered clinical progress in the field of tissue regeneration to repair maxillofacial and orthopedic defects. The goal of this study was to establish a clinically relevant osteogenic protocol for collection and expansion of autologous stem cells to be used at Marshall University for re-implantation and repair of maxillofacial and orthopedic conditions. Human bone marrow (hBM) samples were collected from patients undergoing intramedullary nail fixation for closed femoral fractures. hBM mesenchymal cells were expanded by growing them first in Petri dishes for two weeks, followed by a week of culture using Perfecta 3D Hanging Drop Plates^®. Various scaffold materials were tested and analyzed for cellular integration, vitality, and differentiation capacity of harvested hBM-MSCs including: 60/40 blend of hydroxyapatite biomatrix; Acellular bone composite discs; Allowash^®, cancellous bone cubes; PLGA (poly lactic-co-glycolic acid); and Woven chitin derived fiber. We found that the 3D spheroid culture allowed production of hBM mesenchymal cells that retained osteoblast differentiation capacity over a monolayer culture of hBM-MSCs without the need to use chemical or hormonal modulation. We also observed that hydroxyapatite and Allowash cancellous bone scaffolds allowed better cell integration and viability properties as compared to other materials tested in this study. In conclusion, the multimodal culture methodology we developed creates actively differentiating stem-cell spheroids that can then be readily utilized in clinical practices to improve the regeneration of tissues of the head and the body.

The mechanistic target of rapamycin (mTOR) kinase is a component of two signaling complexes that are known as mTOR complex 1 (mTORC1) and mTORC2. We sought to identify mTOR-phosphorylated proteins that are differently expressed in clinically resected clear cell renal cell carcinoma (ccRCC) relative to pair-matched normal renal tissue. Using a proteomic array, we found N-Myc Downstream Regulated 1 (NDRG1) showed the greatest increase (3.3-fold) in phosphorylation (on Thr346) in ccRCC. This was associated with an increase in total NDRG1. RICTOR is a required subunit in mTORC2, and its knockdown decreased total and phospho-NDRG1 (Thr346) but not NDRG1 mRNA. The dual mTORC1/2 inhibitor, Torin 2, significantly reduced (by ~100%) phospho-NDRG1 (Thr346). Rapamycin is a selective mTORC1 inhibitor that had no effect on the levels of total NDRG1 or phospho-NDRG1 (Thr346). The reduction in phospho-NDRG1 (Thr346) due to the inhibition of mTORC2 corresponded with a decrease in the percentage of live cells, which was correlated with an increase in apoptosis. Rapamycin had no effect on ccRCC cell viability. Collectively, these data show that mTORC2 mediates the phosphorylation of NDRG1 (Thr346) in ccRCC. We hypothesize that RICTOR and mTORC2-mediated phosphorylation of NDRG1 (Thr346) promotes the viability of ccRCC cells.

E13012 Background: Cancer stem-like cells (CSLCs) in primary brain tumors can resist certain chemotherapies, thereby causing relapse of the disease. Thus, development of a test that identifies the most effective chemotherapy management offers great promise for personalized anticancer treatments. Methods: We have developed an ex vivo chemosensitivity assay (ChemoID) designed to predict the sensitivity and resistance of CSLCs and bulk of tumor cells of a given patient's solid tumor to a variety of chemotherapy agents by measuring cellular viability following chemotherapy treatments at clinically relevant doses. In a retrospective study of 7 patients with malignancies of the central nervous system (2 Ependymoma Grade III, 4 Glioblastoma Grade IV, and 1 Medulloblastoma), we assessed the accuracy of the ChemoID assay by comparing the results to the actual clinical response. Tumors were classified as responsive (60-100% cell kill), intermediately responsive (30-60% cell kill), and non-responsive (0-30% cell kill) to chemotherapy. Treatment selection was blinded to assay results. MRI and CT scan determined response to therapy. Additionally, the effectiveness of the ex vivo ChemoID predicted drugs and clinical response was compared to the response of patients’ derived xenografts treated with the most effective and least effective ChemoID predicted drugs. Results: The ChemoID assay performed on the CSLCs and tumor bulk produced a correct prediction in 7 out of 7 cases (p=0.02857, Fisher's Exact Test; PPV=100% (3/3), NPV=100% (4/4) when compared to the drugs received. Results from animal studies conducted in 3 patients’ derived xenografts were also found in agreement with the clinical outcomes. Conclusions: An assay such as ChemoID that measures cell death of CSLCs and bulk of tumor cells appears to be beneficial in selecting specific standard chemotherapy agents ex vivo for patients with malignancies of the central nervous system.

Glioblastomas (GBMs) are a form of malignant gliomas characterized by a dismal prognosis. Standard treatment for glioblastoma patients is combined maximal surgical removal of the tumor with postoperative radiotherapy and concomitant chemotherapy with Temozolomide (TMZ). Among the histological characteristics that contribute to GBM progression are the rapid proliferation and neo-angiogenetic processes. The Na⁺/K⁺-ATPase is a transporter that promotes the migration of cancer cells, and its aberrant expression and activity have been associated with several cancers, including GBM. Using cardiac glycosides, we examined the effects of direct inhibition of the Na⁺/K⁺-ATPase in glioblastoma cells in vitro. We found that cardiac glycoside Digoxin is an effective anticancer agent on several glioma cell lines via Na⁺/K⁺-ATPase inhibition. Drug cytotoxicity assays showed that Digoxin as monotherapy significantly increased cell death and increased the efficacy of Temozolomide (TMZ) in the glioma cell lines T98G, U-97 MG, and primary GBM cells BNC-6. Additionally, Digoxin exhibited important anti-migratory effects on the highly aggressive and chemotherapy-resistant T98G glioma cell-line, demonstrating a potential therapeutic role for cardiac glycosides.

The overarching goal of this research work is to fabricate biocompatible, porous bone tissue scaffolds, incorporating patient-derived bone marrow mesenchymal stem cells (hBMSCs), for the treatment of osseous fractures, defects, and diseases. In pursuit of this goal, the objective of the work is to investigate the influence of consequential scaffold design factors as well as