The quadruplex forming G-rich sequences are unevenly distributed throughout the human genome. Their enrichment in oncogenic promoters and telomeres has generated interest in targeting G-quadruplex (GQ) for an anticancer therapy. Here, we present a quantitative analysis on the conformations and dynamics of GQ forming sequences measured by single molecule fluorescence. Additionally, we relate these properties to GQ targeting ligands and G4 resolvase 1 (G4R1) protein binding. Our result shows that both the loop (non-G components) length and sequence contribute to the conformation of the GQ. Real time single molecule traces reveal that the folding dynamics also depend on the loop composition. We demonstrate that GQ-stabilizing small molecules, N-methyl mesoporphyrin IX (NMM), its analog, NMP and the G4R1 protein bind selectively to the parallel GQ conformation. Our findings point to the complexity of GQ folding governed by the loop length and sequence and how the GQ conformation determines the small molecule and protein binding propensity.

Coherent diffractive imaging (CDI) has been widely applied in the physical and biological sciences using synchrotron radiation, X-ray free-electron laser, high harmonic generation, electrons, and optical lasers. One of CDI’s important applications is to probe dynamic phenomena with high spatiotemporal resolution. Here, we report the development of a general in situ CDI method for real-time imaging of dynamic processes in solution. By introducing a time-invariant overlapping region as real-space constraint, we simultaneously reconstructed a time series of complex exit wave of dynamic processes with robust and fast convergence. We validated this method using optical laser experiments and numerical simulations with coherent X-rays. Our numerical simulations further indicated that in situ CDI can potentially reduce radiation dose by more than an order of magnitude relative to conventional CDI. With further development, we envision in situ CDI could be applied to probe a range of dynamic phenomena in the future.

Glioblastoma (GBM) tumors exhibit potentially actionable genetic alterations against which targeted therapies have been effective in treatment of other cancers. However, these therapies have largely failed in GBM patients. A notable example is kinase inhibitors of EGFR, which display poor clinical efficacy despite overexpression and/or mutation of EGFR in >50% of GBM. In addressing this issue, preclinical models may be limited by the inability to accurately replicate pathophysiologic interactions of GBM cells with unique aspects of the brain extracellular matrix (ECM), which is relatively enriched in hyaluronic acid (HA) and flexible. In this study, we present a brain-mimetic biomaterial ECM platform for 3D culturing of patient-derived GBM cells, with improved pathophysiologic properties as an experimental model. Compared with orthotopic xenograft assays, the novel biomaterial cultures we developed better preserved the physiology and kinetics of acquired resistance to the EGFR inhibition than gliomasphere cultures. Orthogonal modulation of both HA content and mechanical properties of biomaterial scaffolds was required to achieve this result. Overall, our findings show how specific interactions between GBM cell receptors and scaffold components contribute significantly to resistance to the cytotoxic effects of EGFR inhibition. Significance: Three-dimensional culture scaffolds of glioblastoma provide a better physiological representation over current methods of patient-derived cell culture and xenograft models. Cancer Res; 78(5); 1358–70. ©2017 AACR.

Glioblastoma (GBM) is the most lethal cancer originating in the brain. Its high mortality rate has been attributed to therapeutic resistance and rapid, diffuse invasion – both of which are strongly influenced by the unique microenvironment. Thus, there is a need to develop new models that mimic individual microenvironmental features and are able to provide clinically relevant data. Current understanding of the effects of the microenvironment on GBM progression, established experimental models of GBM and recent developments using bioengineered microenvironments as ex vivo experimental platforms that mimic the biochemical and physical properties of GBM tumors are discussed. Lay abstract Glioblastoma (GBM), the most common form of brain cancer, is extremely lethal due to its rapid progression, resistance to treatment and high rates of recurrence. As reviewed here, the microenvironment that surrounds GBM tumors drives their progression. Development of effective treatments for GBM will require better experimental models that account for the tumor microenvironment and thus, can provide clinically translatable results. This review focuses on using biomaterials to create tumor-mimetic microenvironments as experimental platforms for cell culture.

Pancreatic ductal adenocarcinoma (PDAC) is currently the third leading cause of cancer-related death in the United States. Even though the poor prognosis of PDAC is often attributed to late diagnosis, patients with an early diagnosis who undergo tumor resection and adjuvant chemotherapy still show tumor recurrence, highlighting a need to develop therapies which can overcome chemoresistance. Chemoresistance has been linked to the high expression of microRNAs (miRs), such as miR-21, within tumor cells. Tumor cells can collect miRs through the uptake of miR-containing lipid extracellular vesicles called exosomes. These exosomes are secreted in high numbers from cancer-associated fibroblasts (CAFs) within the tumor microenvironment during gemcitabine treatment and can contribute to cell proliferation and chemoresistance. Here, we show a novel mechanism in which CAF-derived exosomes may promote proliferation and chemoresistance, in part, through suppression of the tumor suppressor PTEN. We identified five microRNAs: miR-21, miR-181a, miR-221, miR-222, and miR-92a, that significantly increased in number within the CAF exosomes secreted during gemcitabine treatment which target PTEN. Furthermore, we found that CAF exosomes suppressed PTEN expression in vitro and that treatment with the exosome inhibitor GW4869 blocked PTEN suppression in vivo. Collectively, these findings highlight a mechanism through which the PTEN expression loss, often seen in PDAC, may be attained and lend support to investigations into the use of exosome inhibitors as potential therapeutics to improve the effectiveness of chemotherapy.