Development of a New Enzyme-Linked Immunosorbent Assay (ELISA) for Measuring the Content of PACAP in Mammalian Tissue and Plasma.

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a naturally occurring neuropeptide found in both the central and peripheral nervous systems of vertebrates. Recent studies have revealed the presence of PACAP and its corresponding receptors, namely, the pituitary adenylate cyclase-activating polypeptide type I receptor (PAC1R), vasoactive intestinal peptide receptor 1 (VIPR1), and vasoactive intestinal peptide receptor 2 (VIPR2), in various structures implicated in migraine pathophysiology, including sensory trigeminal neurons. Human studies have demonstrated that when infused, PACAP can cause dilation of cranial vessels and result in delayed migraine-like attacks. In light of this, we present a novel ELISA assay that has been validated for quantifying PACAP in tissue extracts and human plasma. Using two well characterized antibodies specifically targeting PACAP, we successfully developed a sandwich ELISA assay, capable of detecting and accurately quantifying PACAP without any cross-reactivity to closely related peptides. The quantification range was between 5.2 pmol/L and 400 pmol/L. The recovery in plasma ranged from 98.2% to 100%. The increasing evidence pointing to the crucial role of PACAP in migraine pathophysiology necessitates the availability of tools capable of detecting changes in the circulatory levels of PACAP and its potential application as a reliable biomarker.

Perspective: targeting VEGF-A and YKL-40 in glioblastoma - matter matters.

Glioblastomas (GBM) are heterogeneous highly vascular brain tumors exploiting the unique microenvironment in the brain to resist treatment and anti-tumor responses. Anti-angiogenic agents, immunotherapy, and targeted therapy have been studied extensively in GBM patients over a number of decades with minimal success. Despite maximal efforts, prognosis remains dismal with an overall survival of approximately 15 months.Bevacizumab, a humanized anti-vascular endothelial growth factor (VEGF) antibody, underwent accelerated approval by the U.S. Food and Drug Administration in 2009 for the treatment of recurrent GBM based on promising preclinical and early clinical studies. Unfortunately, subsequent clinical trials did not find overall survival benefit. Pursuing pleiotropic targets and leaning toward multitarget strategies may be a key to more effective therapeutic intervention in GBM, but preclinical evaluation requires careful consideration of model choices. In this study, we discuss bevacizumab resistance, dual targeting of pro-angiogenic modulators VEGF and YKL-40 in the context of brain tumor microenvironment, and how model choice impacts study conclusions and its translational significance.

SPT6-driven error-free DNA repair safeguards genomic stability of glioblastoma cancer stem-like cells.

Glioblastoma cancer-stem like cells (GSCs) display marked resistance to ionizing radiation (IR), a standard of care for glioblastoma patients. Mechanisms underpinning radio-resistance of GSCs remain largely unknown. Chromatin state and the accessibility of DNA lesions to DNA repair machineries are crucial for the maintenance of genomic stability. Understanding the functional impact of chromatin remodeling on DNA repair in GSCs may lay the foundation for advancing the efficacy of radio-sensitizing therapies. Here, we present the results of a high-content siRNA microscopy screen, revealing the transcriptional elongation factor SPT6 to be critical for the genomic stability and self-renewal of GSCs. Mechanistically, SPT6 transcriptionally up-regulates BRCA1 and thereby drives an error-free DNA repair in GSCs. SPT6 loss impairs the self-renewal, genomic stability and tumor initiating capacity of GSCs. Collectively, our results provide mechanistic insights into how SPT6 regulates DNA repair and identify SPT6 as a putative therapeutic target in glioblastoma.

Reciprocal Signaling between Glioblastoma Stem Cells and Differentiated Tumor Cells Promotes Malignant Progression.

Glioblastoma is the most lethal primary brain tumor; however, the crosstalk between glioblastoma stem cells (GSCs) and their supportive niche is not well understood. Here, we interrogated reciprocal signaling between GSCs and their differentiated glioblastoma cell (DGC) progeny. We found that DGCs accelerated GSC tumor growth. DGCs preferentially expressed brain-derived neurotrophic factor (BDNF), whereas GSCs expressed the BDNF receptor NTRK2. Forced BDNF expression in DGCs augmented GSC tumor growth. To determine molecular mediators of BDNF-NTRK2 paracrine signaling, we leveraged transcriptional and epigenetic profiles of matched GSCs and DGCs, revealing preferential VGF expression by GSCs, which patient-derived tumor models confirmed. VGF serves a dual role in the glioblastoma hierarchy by promoting GSC survival and stemness in vitro and in vivo while also supporting DGC survival and inducing DGC secretion of BDNF. Collectively, these data demonstrate that differentiated glioblastoma cells cooperate with stem-like tumor cells through BDNF-NTRK2-VGF paracrine signaling to promote tumor growth.