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Various methods of generating 2D and 3D in vitro blood-brain barrier (BBB) models have previously been published with the objective of developing therapeutics for brain diseases. In general, published methods including our published method demonstrate that in vivo-like semi-permeable barrier can be generated. To further verify that an in vitro BBB model closely represents BBB, functional validation is required. Here, we functionally validate our in vitro 3D BBB model using rituximab as a representative therapeutic antibody and previously published anti-TfR (transferrin receptor) antibodies as representative BBB-penetrating antibodies. We demonstrate that our BBB model can efficiently block rituximab while allowing receptor-mediated transcytosis (RMT) of anti-TfR antibodies. In addition, we showed that RMT efficacy of anti-TfR antibodies with different binding affinity can be displayed using our BBB model. In conclusion, this demonstrates that our BBB model functionally mimics the BBB as well as having BBB-like physical properties, further establishing our BBB model as a screening tool for discovery and development of therapeutics for brain diseases.

PDF file - 340K, Effects of BUB1B knockdown on in human astrocytes, RasV12 transformed astrocytes, Hela cells and RPE-1 cells

Due to the innate high metastatic ability of renal cell carcinoma (RCC), many patients with RCC experience local or systemic relapses after surgical resection. A deeper understanding of the molecular pathogenesis underlying advanced RCC is essential for novel innovative therapeutics. Tumor progression locus 2 (Tpl2), upregulated in various tumor types, has been reported to be associated with oncogenesis and metastatic progression via activation of the MAPK signaling pathway. Herein, the relevance of Tpl2 in tumor growth and metastasis of RCC is explored. Inspection of The Cancer Genome Atlas (TCGA) indicated that Tpl2 overexpression was significantly related to the presence of metastases and poor outcome in clear cell RCC (ccRCC), which is the most aggressive subtype of RCC. Moreover, expression of Tpl2 and CXCR4 showed a positive correlation in ccRCC patients. Depletion of Tpl2 by RNAi or activity by a Tpl2 kinase inhibitor in human ccRCC cells remarkably suppressed MAPK pathways and impaired in vitro cell proliferation, clonogenicity, anoikis resistance, migration, and invasion capabilities. Similarly, orthotopic xenograft growth and lung metastasis were significantly inhibited by Tpl2 silencing. Furthermore, Tpl2 knockdown reduced CXCL12-directed chemotaxis and chemoinvasion accompanied with impaired downstream signaling, indicating potential involvement of Tpl2 in CXCR4-mediated metastasis. Taken together, these data indicate that Tpl2 kinase is associated with and contributes to disease progression of ccRCC.Implications:Tpl2 kinase activity has prognostic and therapeutic targeting potential in aggressive clear cell renal cell carcinoma. Mol Cancer Res; 11(11); 1375–86. ©2013 AACR.

PDF file - 133K, BTIC and NSC growth phenotypes resulting from knockdown of PLK1 and pharmacological inhibition of Aurora B kinase

PDF file - 134K, Ras-Mapk signaling is inappropriately high BTICs and NHA-Ras cells

PDF file - 117K

Supplementary Figure 2. The EGFR-GC1118 interaction is not disrupted by excess amounts of high- or low-affinity EGFR ligands. The indicated colorectal cancer cell lines were pre-incubated with 0.1 ug/ml of cetuximab or GC1118 for 2 h prior to the addition of EGFR ligands for 10 min (EGF, HB-EGF, BTC, and TGF-�, 20~500 ng/ml; AREG and EREG, 40~1000 ng/ml). The blue and red lines indicate the relative % of cetuximab and GC1118, respectively, bound to EGFR in the presence of competing ligands compared with bound antibodies in the absence of blocking ligands. Antibody binding to EGFR was assessed by measuring the fluorescence intensity using flow cytometry. The results are expressed as the average{plus minus}SE of % maximal binding of the antibody of triplicate experiments.

PDF file - 39K, BUB1B knockdown prevents longer-term expansion BTICs and SSEA1+ BTIC subpopulations

Supplementary Figure 1. Kinetic binding interactions between GC1118 and EGFR as analyzed by SPR. SPR sensorgrams were obtained from injections of (A) GC1118 or (B) cetuximab over an EGFR-immobilized surface at a flow rate of 30 µl/min. The results of a quantitative evaluation of the experimental data are shown in Supplementary Table 2.

PDF file - 53K, Kaplan-Meier graphs representing the probability of cumulative overall survival (OS) in patients with papRCCs stratified according to Tpl2 level in their primary tumors. For TCGA data, high, moderate and low expression were designated as >mean+SD, mean-SD~mean+SD and

PDF file - 120K, Supplemental data for Figure 5

XLS file - 42K, Candidate genes differentially required for expansion of G166-BTICs

PDF file - 27K, Measurement of mitotic transit times BTIC-G166 cells using live microscopy for mBUB1B allelic complementation experiments (from Figure 5)

PDF file - 34K, Tpl2 silencing delayed cell cycle progression in human ccRCC Caki-1 cells. *P

PDF file - 74K, BUB1B and other mitotic checkpoint genes are up-regulated in BTIC isolates and Ras- transformed astrocytes

PDF file - 1.3MB, Examination of the effects of BUB1B knockdown on mitotic checkpoint activity

PDF file - 1MB, Additional information showing cancer cells have added requirement on BUB1B for chromosome alignment and KT-MT attachment

Supplementary Table 1. X-ray data collection and refinement statistics.

Surface plasmon resonance (SPR)

PurposeThe KNOG-1101 study showed improved 2-year PFS with temozolomide during and after radiotherapy compared to radiotherapy alone for patients with anaplastic gliomas. This trial investigates the effect of concurrent and adjuvant temozolomide on health-related quality of life (HRQoL).Materials and MethodsIn this randomized, open-label, phase II trial, 90 patients with World Health Organization grade III glioma were enrolled across multiple centers in South Korea between March 2012 to February 2015 and followed up through 2017. The European Organization for Research and Treatment of Cancer Quality of Life Questionnaire 30 (EORTC QLQ-C30) and 20-item EORTC QLQ-Brain Neoplasm (QLQ-BN20) were used to compare HRQoL between patients assigned to concurrent chemoradiotherapy with temozolomide followed by 6 cycles of adjuvant temozolomide (arm A) and radiotherapy (RT) alone (arm B).ResultsOf the 90 patients in the study, 84 patients (93.3%) completed the baseline HRQoL questionnaire. Emotional functioning, fatigue, nausea and vomiting, dyspnea, constipation, appetite loss, diarrhea, seizures, itchy skin, drowsiness, hair loss, and bladder control were not affected by the addition of temozolomide. All other items did not differ significantly between arm A and arm B throughout treatment. Global health status particularly stayed consistent at the end of adjuvant temozolomide (p=0.47) and at the end of RT (p=0.33).ConclusionThe addition of concurrent and adjuvant temozolomide did not show negative influence on HRQoL with improvement of progression-free survival for patients with anaplastic gliomas. The absence of systematic and clinically relevant changes in HRQoL suggests that an overall long-term net clinical benefit exists for concurrent and adjuvant temozolomide.

Objective To analyze radiological characteristics, treatment strategy, and treatment outcomes of posterior fossa giant solid hemangioblastomas (GSHBs; ≥ 4 cm in maximum diameter). Methods We performed a retrospective study on 13 consecutive patients (9 male and 4 female patients [mean age 57.5 ± 13.3 years; range 24-76 years]) with GSHB who underwent surgical resection at a single institution between 2002 and 2018. The median follow-up was 33 months (range 12-120 months). For each patient, neuroimaging findings, operative record, and treatment outcome were reviewed. Results Seven tumors (53.8%) were located within cerebellar hemispheres, 4 (30.8%) in proximity to the brainstem, 1 (7.7%) within the cerebellar vermis, and 1 (7.7%) in the cerebellopontine angle. The mean maximum diameter was 4.8 ± 0.8 cm (range 4.0-6.7 cm). Gross total resection was achieved in 11 patients (84.6%), and near-total resection (NTR) in 2 patients (15.4%). Surgical complications occurred in 5 patients (38.5%); persistent neurological deficits occurred in 2 patients (15.4%). Estimated progression-free survival rates after surgery were 92.3%, 80.8%, and 80.8% at 1, 5, and 10 years, respectively, while the estimated 1-, 5- and 10-year overall survival rates were 100%, 90% and 90%, respectively. Conclusions GSHBs are surgically challenging. The current study shows that favorable outcome can be achieved for GSHBs in the cerebellar hemispheres and vermis. For those involving the brainstem, planned NTR or STR in a piecemeal fashion can be attempted if en bloc resection is judged to be infeasible, and further intervention can be considered as needed.

OBJECTIVE The petrous apex (PA) is one of the most challenging areas in skull base surgery because it is surrounded by numerous critical neurovascular structures. The authors analyzed the clinical outcomes of patients who underwent endoscopic endonasal approach (EEA) and transorbital approach (TOA) procedures for lesions involving PA to determine the perspectives and proper applications of these two approaches. METHODS The authors included patients younger than 80 years with lesions involving PA who were treated between May 2015 and December 2019 and had regular follow-up MR images available for analysis. Patients with meningioma involving petroclival regions were excluded. The authors classified PA into three regions: superior to the petrous segment of the internal carotid artery (p-ICA) (zone 1); posterior to p-ICA (zone 2); and inferior to p-ICA (zone 3). Demographic data, preoperative clinical and radiological findings, surgical outcomes, and morbidities were reviewed. RESULTS A total of 19 patients with lesions involving PA were included. Ten patients had malignant tumor (chondrosarcoma, chordoma, and osteosarcoma), and 6 had benign tumor (schwannoma, Cushing’s disease, teratoma, etc.). Three patients had PA cephalocele (PAC). Thirteen patients underwent EEA, and 5 underwent TOA. Simultaneous combined EEA and TOA was performed on 1 patient. Thirteen of 16 patients (81.3%) had gross- or near-total resection. Tumors within PA were completely resected from 13 of 16 patients using a view limited to only the PA. Complete obliteration of PAC was achieved in all patients. Postoperative complications included 2 cases of CSF leak, 1 case of injury to ICA, 1 fatality due to sudden herniation of the brainstem, and 1 case of postoperative diplopia. CONCLUSIONS EEA is a versatile surgical approach for lesions involving all three zones of PA. Clival tumor spreading to PA in a medial-to-lateral direction is a good indication for EEA. TOA provided a direct surgical corridor to the superior portion of PA (zone 1). Patients with disease with cystic nature are good candidates for TOA. TOA may be a reasonable alternative surgical treatment for select pathologies involving PA.

Supplementary Figure S1. Histopathology of NSCLC brain metastases and their corresponding PDXs. Histological subtype and differentiation status was indicated for each case. ADC = adenocarcinoma, LCC = large cell carcinoma, SQCC = squamous cell carcinoma.

Supplementary Figure S1 - PDF file 84K, Supplementary Figure S1. Verification of specific detection of ACTA2 in lung ADC primary tumors Immunohistochmical staining patterns by two different antibodies against ACTA2 purchased from Dako and Thermo Scientific Pierce were compared in primary lung tumors from ACTA2-High and Low group

PDF file - 61K, Primers for RT-qPCR.

Necrosis is a hallmark of glioblastoma (GBM) and is responsible for poor prognosis and resistance to conventional therapies. However, the molecular mechanisms underlying necrotic microenvironment-induced malignancy of GBM have not been elucidated. Here, we report that transglutaminase 2 (TGM2) is upregulated in the perinecrotic region of GBM and triggered mesenchymal (MES) transdifferentiation of glioma stem cells (GSC) by regulating master transcription factors (TF), such as C/EBPβ, TAZ, and STAT3. TGM2 expression was induced by macrophages/microglia-derived cytokines via NF-κB activation and further degraded DNA damage–inducible transcript 3 (GADD153) to induce C/EBPβ expression, resulting in expression of the MES transcriptome. Downregulation of TGM2 decreased sphere-forming ability, tumor size, and radioresistance and survival in a xenograft mouse model through a loss of the MES signature. A TGM2-specific inhibitor GK921 blocked MES transdifferentiation and showed significant therapeutic efficacy in mouse models of GSC. Moreover, TGM2 expression was significantly increased in recurrent MES patients and inversely correlated with patient prognosis. Collectively, our results indicate that TGM2 is a key molecular switch of necrosis-induced MES transdifferentiation and an important therapeutic target for MES GBM. Cancer Res; 77(18); 4973–84. ©2017 AACR.

Supplementary table S3. Clinicopathological characteristics and in vivo tumorigenicity of primary NSCLC cases.

Purpose: The increasing prevalence of distant metastases from non–small cell lung cancer (NSCLC) indicates an urgent need for novel therapeutic modalities. Brain metastasis is particularly common in NSCLC, with severe adverse effects on clinical prognosis. Although the molecular heterogeneity of NSCLC and availability of various targeted agents suggest personalized therapeutic approaches for such brain metastases, further development of appropriate preclinical models is needed to validate the strategies.Experimental Design: We established patient-derived xenografts (PDX) using NSCLC brain metastasis surgical samples and elucidated their possible preclinical and clinical implications for personalized treatment.Results: NSCLC brain metastases (n = 34) showed a significantly higher successful PDX establishment rate than primary specimens (n = 64; 74% vs. 23%). PDXs derived from NSCLC brain metastases recapitulated the pathologic, genetic, and functional properties of corresponding parental tumors. Furthermore, tumor spheres established in vitro from the xenografts under serum-free conditions maintained their in vivo brain metastatic potential. Differential phenotypic and molecular responses to 20 targeted agents could subsequently be screened in vitro using these NSCLC PDXs derived from brain metastases. Although PDX establishment from primary NSCLCs was significantly influenced by histologic subtype, clinical aggressiveness, and genetic alteration status, the brain metastases exhibited consistently adequate in vivo tumor take rate and in vitro tumor sphere formation capacity, regardless of clinical and molecular conditions.Conclusions: Therefore, PDXs from NSCLC brain metastases may better represent the heterogeneous advanced NSCLC population and could be utilized as preclinical models to meet unmet clinical needs such as drug screening for personalized treatments. Clin Cancer Res; 21(5); 1172–82. ©2014 AACR.

Supplementary Figure Legends - PDF file 75K, Supplementary figure legends

Purpose: The potential of metastasis can be predicted from clinical features like tumor size, histologic grade, and gene expression patterns. We examined the whole-genome transcriptomic profile of a xenograft model of breast cancer to understand the characteristics of brain metastasis.Experimental Design: Variants of the MDA-MB-435 cell were established from experimental brain metastases. The LvBr2 variant was isolated from lesions in a mouse injected in the left ventricle of the heart, and these cells were used for two cycles of injection into the internal carotid artery and selection of brain lesions, resulting in the Br4 variant. To characterize the different metastatic variants, we examined the gene expression profile of MDA-MB-435, LvBr2, and Br4 cells using microarrays.Results: We could identify 2,016 differentially expressed genes in Br4 by using the F test. Various metastasis-related genes and a number of genes related to angiogenesis, migration, tumorigenesis, and cell cycle were differentially expressed by the Br4 cells. Notably, the Notch signaling pathway was activated in Br4, with increased Jag2 mRNA, activated Notch intracellular domain, and Notch intracellular domain/CLS promoter-luciferase activity. Br4 cells were more migratory and invasive than MDA-MB-435 cells in collagen and Matrigel Transwell assays, and the migration and invasion of Br4 cells were significantly inhibited by inactivation of Notch signaling using DAPT, a γ-secretase inhibitor, and RNA interference–mediated knockdown of Jagged 2 and Notch1.Conclusions: Taken together, these results suggest that we have isolated variants of a human cancer cell line with enhanced brain metastatic properties, and the activation of Notch signaling might play a crucial role in brain metastasis.

Supplementary Data from Activation of Notch Signaling in a Xenograft Model of Brain Metastasis

Purpose: Metastatic relapse of primary lung cancer leads to therapeutic resistance and unfavorable clinical prognosis; therefore, identification of key molecules associated with metastatic conversion has significant clinical implications. We previously identified a link between early brain metastasis of lung adenocarcinoma and amplification of the α-smooth muscle actin (ACTA2) gene. The aim of present study was to investigate the prognostic and functional significance of ACTA2 expression in cancer cells for the metastatic potential of lung adenocarcinomas.Experimental Design:ACTA2 expression was analyzed in tumor cells from 263 patients with primary lung adenocarcinomas by immunohistochemistry, and was correlated with clinicopathologic parameters. The expression of ACTA2 in human lung adenocarcinoma cells was modulated with short hairpin RNAs (shRNA) and siRNAs specifically targeting ACTA2.Results: The patients with lung adenocarcinomas with high ACTA2 expression in tumor cells showed significantly enhanced distant metastasis and unfavorable prognosis. ACTA2 downregulation remarkably impaired in vitro migration, invasion, clonogenicity, and transendothelial penetration of lung adenocarcinoma cells without affecting proliferation. Consistent with the in vitro results, depletion of ACTA2 in human lung adenocarcinoma PC14PE6 cells significantly reduced their metastatic potential without altering their tumorigenic potential. Expression of c-MET and FAK in lung adenocarcinoma cells was also reduced by ACTA2-targeting siRNAs and shRNAs, and was accompanied by a loss of mesenchymal characteristics.Conclusions: These findings indicate that ACTA2 regulates c-MET and FAK expression in lung adenocarcinoma cells, which positively and selectively influence metastatic potential. Therefore, ACTA2 could be a promising prognostic biomarker and/or therapeutic target for metastatic lung adenocarcinoma. Clin Cancer Res; 19(21); 5879–89. ©2013 AACR.

Supplementary Figure 1 related to Figure 1. TGM2 is highly expressed in the necrotic region of MES GBM. Supplementary Figure 2 related to Figure 2. TGM2 regulates stemness of MES, PN, and CL GSCs. Supplementary Figure 3 related to Figure 4. A negative correlation between C/EBPβ and GADD153.

Supplementary Figure 5 from Negative Regulation of p53 by the Long Isoform of ErbB3 Binding Protein Ebp1 in Brain Tumors