Correction: High-dose drug heat map analysis for drug safety and efficacy in multi-spheroid brain normal cells and GBM patient-derived cells.

[This corrects the article DOI: 10.1371/journal.pone.0251998.].

High-dose drug heat map analysis for drug safety and efficacy in multi-spheroid brain normal cells and GBM patient-derived cells.

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.

Three-Dimensional Aggregated Spheroid Model of Hepatocellular Carcinoma Using a 96-Pillar/Well Plate.

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-ß2), 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.

PIP4K2A as a negative regulator of PI3K in PTEN-deficient glioblastoma.

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.

Transgenerational effects of proton beam irradiation on Caenorhabditis elegans germline apoptosis.

When treating cancer using radiation therapy, it is critical to increase patient survival rates and to reduce side effects. In this respect, proton beam radiation treatment performs better than other radiation treatments because of its high target specificity. However, complications still remain after proton beam radiation treatment. Among them, the risk to progeny after irradiation of their parents is a major concern. In this study, we analyzed the transgenerational effects of proton beam irradiation using the model organism Caenorhabditis. elegans. We found that germline apoptosis increased after proton beam irradiation and its effects were sustained transgenerationally. Moreover, we identified that a germline-specific histone methyltransferase component, SET-2, has a critical role in transmitting the transgenerational effect on germline apoptosis to the next generation after proton beam irradiation.

LIN-23, an E3 Ubiquitin Ligase Component, Is Required for the Repression of CDC-25.2 Activity during Intestinal Development in Caenorhabditis elegans.

Caenorhabditis elegans (C. elegans) utilizes two different cell-cycle modes, binucleations during the L1 larval stage and endoreduplications at four larval moltings, for its postembryonic intestinal development. Previous genetic studies indicated that CDC-25.2 is specifically required for binucleations at the L1 larval stage and is repressed before endoreduplications. Furthermore, LIN-23, the C. elegans ß-TrCP ortholog, appears to function as a repressor of CDC-25.2 to prevent excess intestinal divisions. We previously reported that intestinal hyperplasia in lin-23(e1883) mutants was effectively suppressed by the RNAi depletion of cdc-25.2. Nevertheless, LIN-23 targeting CDC-25.2 for ubiquitination as a component of E3 ubiquitin ligase has not yet been tested. In this study, LIN-23 is shown to be the major E3 ubiquitin ligase component, recognizing CDC-25.2 to repress their activities for proper transition of cell-cycle modes during the C. elegans postembryonic intestinal development. In addition, for the first time that LIN-23 physically interacts with both CDC-25.1 and CDC-25.2 and facilitates ubiquitination for timely regulation of their activities during the intestinal development.

CDC-25.2, a C. elegans ortholog of cdc25, is essential for the progression of intestinal divisions.

Intestinal divisions in Caenorhabditis elegans take place in 3 stages: (1) cell divisions during embryogenesis, (2) binucleations at the L1 stage, and (3) endoreduplications at the end of each larval stage. Here, we report that CDC-25.2, a C. elegans ortholog of Cdc25, is required for these specialized division cycles between the 16E cell stage and the onset of endoreduplication. Results of our genetic analyses suggest that CDC-25.2 regulates intestinal cell divisions and binucleations by counteracting WEE-1.3 and by activating the CDK-1/CYB-1 complex. CDC-25.2 activity is then repressed by LIN-23 E3 ubiquitin ligase before the onset of intestinal endoreduplication, and this repression is maintained by LIN-35, the C. elegans ortholog of Retinoblastoma (Rb). These findings indicate that timely regulation of CDC-25.2 activity is essential for the progression of specialized division cycles and development of the C. elegans intestine.