Glioblastoma (GBM) is the most malignant brain tumor with profound genomic alterations. Tumor suppressor genes regulate multiple signaling networks that restrict cellular proliferation and present barriers to malignant transformation. While bona fide tumor suppressors such as PTEN and TP53 often undergo inactivation due to mutations, there are several genes for which genomic deletion is the primary route for tumor progression. To functionally identify putative tumor suppressors in GBM, we employed in vivo RNAi screening using patient-derived xenograft models. Here, we identified PIP4K2A, whose functional role and clinical relevance remain unexplored in GBM. We discovered that PIP4K2A negatively regulates phosphoinositide 3-kinase (PI3K) signaling via p85/p110 component degradation in PTEN-deficient GBMs and specifically targets p85 for proteasome-mediated degradation. Overexpression of PIP4K2A suppressed cellular and clonogenic growth in vitro and impeded tumor growth in vivo. Our results unravel a novel tumor-suppressive role of PIP4K2A for the first time and support the feasibility of combining oncogenomics with in vivo RNAi screen.

A common method of three-dimensional (3D) cell cultures is embedding single cells in Matrigel. Separated cells in Matrigel migrate or grow to form spheroids but lack cell-to-cell interaction, which causes difficulty or delay in forming mature spheroids. To address this issue, we proposed a 3D aggregated spheroid model (ASM) to create large single spheroids by aggregating cells in Matrigel attached to the surface of 96-pillar plates. Before gelling the Matrigel, we placed the pillar inserts into blank wells where gravity allowed the cells to gather at the curved end. In a drug screening assay, the ASM with Hepatocellular carcinoma (HCC) cell lines showed higher drug resistance compared to both a conventional spheroid model (CSM) and a two-dimensional (2D) cell culture model. With protein expression, cytokine activation, and penetration analysis, the ASM showed higher expression of cancer markers associated with proliferation (p-AKT, p-Erk), tight junction formation (Fibronectin, ZO-1, Occludin), and epithelial cell identity (E-cadherin) in HCC cells. Furthermore, cytokine factors were increased, which were associated with immune cell recruitment/activation (MIF-3α), extracellular matrix regulation (TIMP-2), cancer interaction (IL-8, TGF-β2), and angiogenesis regulation (VEGF-A). Compared to CSM, the ASM also showed limited drug penetration in doxorubicin, which appears in tissues in vivo. Thus, the proposed ASM better recapitulated the tumor microenvironment and can provide for more instructive data during in vitro drug screening assays of tumor cells and improved prediction of efficacious drugs in HCC patients.

To test the safety and efficacy of drugs via a high does drug heat map, a multi-spheroids array chip was developed by adopting a micropillar and microwell structure. In the chip, patient-derived cells were encapsulated in alginate and grown to maturity for more than 7 days to form cancer multi-spheroids. Multi-spheroids grown in conventional well plates require many cells and are easily damaged as a result of multiple pipetting during maintenance culture or experimental procedures. To address these issues, we applied a micropillar and microwell structure to the multi-spheroids array. Patient-derived cells from patients with Glioblastoma (GBM), the most common and lethal form of central nervous system cancer, were used to validate the array chip performance. After forming multi-spheroids with a diameter greater than 100μm in a 12×36 pillar array chip (25mm × 75mm), we tested 70 drug compounds (6 replicates) using a high-dose to determine safety and efficacy for drug candidates. Comparing the drug response of multi-spheroids derived from normal cells and cancer cells, we found that four compounds (Dacomitinib, Cediranib, LY2835219, BGJ398) did not show toxicity to astrocyte cell and were efficacious to patient-derived GBM cells.

Background: Hepatocellular carcinoma (HCC) is the sixth most common cancer and the third most frequent cause of cancer-related mortality worldwide. The genetic and physiological complexity of HCC is a major barrier to the study of tumorigenesis and identification of therapeutic targets. The three-dimensional (3D) culture of cancer cells within an extracellular matrix (ECM) provides an in vitro tumor model that best recapitulates in vivo tumor pathophysiology.Methods: In the current study, we cultured cells and made spheroids by aggregating cells or diffusing cells in Matrigel attached on the tip of the 96-pillar plate. According to the initial cell position in Matrigel, two 3D-HCC cancer in-vitro models (diffused spheroid model and aggregated spheroid model) were established. These two models were applied to drug sensitivity assays to identify drug sensitivity changes. The protein expression and cytokine activation related to the drug resistance and maintenance of the physiological properties of HCC cells were analyzed in both models. This 3D culture method not only maintained the phenotype of the tumor but allowed easy high-throughput screening (HTS) due to its 96-array plate configuration.Results: The Aggregated Spheroid Model (ASM) showed higher expression of cancer markers associated with proliferation, tight junctions formation and epithelial cell identity in HCC cells, as well as cytokine factors associated with immune cell recruitment/activation, ECM regulation, cancer interaction, and angiogenesis regulation.Conclusions: Overall, the proposed ASM better recapitulates the tumor microenvironment, demonstrating the involvement of the ECM/tight junctions in the cell-to-cell signaling processes. This provides more instructive data for in vitro screening of tumor cells.