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Significance Delivery remains a significant challenge for robust implementation of CRISPR/Cas9. We report an efficient CRISPR/Cas9 delivery system comprising PEGylated nanoparticles based on the α-helical polypeptide PPABLG. Assisted by the high membrane-penetrating ability of the polypeptide, P-HNPs achieved efficient cellular internalization and endosomal escape. The CRISPR/Cas9 delivery system could reach 47.3% gene editing in cells, 35% gene deletion in vivo, and HeLa tumor growth suppression >71%, demonstrating an advantage over the existing conventional polycationic transfection reagents. Efficient also in knock-in and gene activation, the reported CRISPR/Cas9 delivery system serves to advance gene editing in vitro and in vivo., Effective and safe delivery of the CRISPR/Cas9 gene-editing elements remains a challenge. Here we report the development of PEGylated nanoparticles (named P-HNPs) based on the cationic α-helical polypeptide poly(γ-4-((2-(piperidin-1-yl)ethyl)aminomethyl)benzyl-l-glutamate) for the delivery of Cas9 expression plasmid and sgRNA to various cell types and gene-editing scenarios. The cell-penetrating α-helical polypeptide enhanced cellular uptake and promoted escape of pCas9 and/or sgRNA from the endosome and transport into the nucleus. The colloidally stable P-HNPs achieved a Cas9 transfection efficiency up to 60% and sgRNA uptake efficiency of 67.4%, representing an improvement over existing polycation-based gene delivery systems. After performing single or multiplex gene editing with an efficiency up to 47.3% in vitro, we demonstrated that P-HNPs delivering Cas9 plasmid/sgRNA targeting the polo-like kinase 1 (Plk1) gene achieved 35% gene deletion in HeLa tumor tissue to reduce the Plk1 protein level by 66.7%, thereby suppressing the tumor growth by >71% and prolonging the animal survival rate to 60% within 60 days. Capable of delivering Cas9 plasmids to various cell types to achieve multiplex gene knock-out, gene knock-in, and gene activation in vitro and in vivo, the P-HNP system offers a versatile gene-editing platform for biological research and therapeutic applications.

Emergence of tumor resistance to an anti-cancer therapy directed against a putative target raises several questions including: (1) do mutations in the target/pathway confer resistance? (2) Are these mutations pre-existing? (3) What is the relative fitness of cells with/without the mutation? We addressed these questions in patients with metastatic colorectal cancer (mCRC). We conducted an exhaustive review of published data to establish a median doubling time for CRCs and stained a cohort of CRCs to document mitotic indices. We analyzed published data and our own data to calculate rates of growth (g) and regression (d, decay) of tumors in patients with CRC correlating these results with the detection of circulating MT-KRAS DNA. Additionally we estimated mathematically the caloric burden of such tumors using data on mitotic and apoptotic indices. We conclude outgrowth of cells harboring intrinsic or acquired MT-KRAS cannot explain resistance to anti-EGFR (epidermal growth factor receptor) antibodies. Rates of tumor growth with panitumumab are unaffected by presence/absence of MT-KRAS. While MT-KRAS cells may be resistant to anti-EGFR antibodies, WT-KRAS cells also rapidly bypass this blockade suggesting inherent resistance mechanisms are responsible and a neutral evolution model is most appropriate. Using the above clinical data on tumor doubling times and mitotic and apoptotic indices we estimated the caloric intake required to support tumor growth and suggest it may explain in part cancer-associated cachexia.

In the present study, we investigated the effect of voluntary exercise on the formation and growth of the human pancreas Panc-1 and prostate PC-3 tumors in immunodeficient mice. Female severe combined immunodeficient (SCID) mice were injected subcutaneously with human pancreatic cancer Panc-1 cells, and male SCID mice were injected subcutaneously with human prostate cancer PC-3 cells. Voluntary running wheel exercise for 63 days, starting one week before the subcutaneous injection of Panc-1 or PC-3 tumor cells into SCID mice, suppressed the growth of Panc-1 and PC-3 tumors. The exercise regimen increased the food and fluid consumption in the female and male mice. Exercise also decreased the size of the parametrial fat pads in the female mice and the paradidymis fat pads in the male mice, but there was no effect on the body weight. Mechanistic studies showed that voluntary running wheel exercise inhibited proliferation as reflected by a decreased mitosis, and the exercise regimen also stimulated apoptosis as reflected by the increased caspase-3 (active form) expression in the Panc-1 and PC-3 tumors. Voluntary running wheel exercise decreased the ratio of the percent mitotic cells/apoptotic cells in Panc-1 and PC-3 tumors by 38 and 32%, respectively. The present study demonstrated an inhibitory effect of voluntary exercise on the growth of pancreas and prostate tumors in a SCID mouse xenograft model.

Vitamin E succinate selenium-conjugated molecules were synthesized and their apoptogenic properties were evaluated. 4-Methyl-2-phenylselenyl succinate (4) was prepared by the reaction of sodium benzeneselenolate with 2-bromosuccinic anhydrite in methanol solution. The methyl ester was converted to the acid (5) by hydrolysis with aqueous hydrochloric acid. Reaction of the 2-phenylselenyl succinic anhydrite (6) with α-tocopherol (1a), γ-tocopherol (1c), and γ-tocotrienol (2c) in acidic conditions gave the respective esters. The free radical scavenging properties of α-tocopheryl-2-phenylselenyl succinate (7), γ-tocopheryl-2- phenylselenyl succinate (8), and γ-tocotrienyl-2-phenylselenyl succinate (9) were evaluated in comparison with those of α-tocopheryl succinate (10), γ-tocopheryl succinate (11), and γ-tocotrienyl succinate (12), respectively, and the free tocopherols and γ-tocotrienol. Compounds 7-9 induced a statistically significant decrease in prostate cancer cell viability compared to 10-12, respectively, or 5, exhibiting features of apoptotic cell death and associated with caspase-3 activation. These data show that structural modifications of vitamin E components by 5 enhance their apoptogenic properties in cancer cells. © 2005 Elsevier Ltd. All rights reserved. 14 8 2684 2696 Cited By :23

3D cell tumour models are generated mainly in non-scalable culture systems, using bioactive scaffolds. Many of these models fail to reflect the complex tumour microenvironment and do not allow long-term monitoring of tumour progression. To overcome these limitations, we have combined alginate microencapsulation with agitation-based culture systems, to recapitulate and monitor key aspects of the tumour microenvironment and disease progression. Aggregates of MCF-7 breast cancer cells were microencapsulated in alginate, either alone or in combination with human fibroblasts, then cultured for 15 days. In co-cultures, the fibroblasts arranged themselves around the tumour aggregates creating distinct epithelial and stromal compartments. The presence of fibroblasts resulted in secretion of proinflammatory cytokines and deposition of collagen in the stromal compartment. Tumour cells established cellecell contacts and polarised around small lumina in the interior of the aggregates. Over the culture period, there was a reduction in oestrogen receptor and membranous E-cadherin alongside loss of cell polarity, increased collective cell migration and enhanced angiogenic potential in co-cultures. These phenotypic alterations, typical of advanced stages of cancer, were not observed in the monocultures of MCF-7 cells. The proposed model system constitutes a new tool to study tumour-stroma crosstalk, disease progression and drug resistance mechanisms. We gratefully acknowledge Dr Cathrin Brisken for the supply of the MCF-7-dsRED cell line within the scope of the IMI-funded project PREDECT and for valuable advice. We are grateful to the PREDECT consortium partners, especially to Dr John Hickman, Dr Juha Klefstrom and Dr Georgios A. S € flomos for fruitful discussions. We also thank Dr Joana Paredes for critical advice. We acknowledge support from the Innovative Medicines Initiative Joint Undertaking Fig. 6. Characterization of the inflammatory environment and angiogenic potential of tumour cells microencapsulated in co-cultures. (A) Cytokine arrays of mono and cocultures from days 5 and 15, were performed for both culture and capsule supernatant. Data are mean ± SD from two independent experiments. * or þ indicate significant difference with p < 0.01 by a non-parametric Man-Whitney U statistics Test (two-tailed p-value). Microencapsulated mono-cultures of fibroblasts were used as controls. (B) Quantification and representative images of Chick Chorioallantoic Membrane (CAM) Assays of tumour aggregates from both mono and co-cultures, at day 15. Vessels with less than 15 mm of diameter, growing in a wheel shape manner towards the inoculation area, were quantified as newly formed vessels. Data are mean ± SD from 18 eggs. ** indicate significant difference with p < 0.001 by a t- Test (two-tailed p-value). M.F. Estrada et al. / Biomaterials 78 (2016) 50e61 59 (IMI grant agreement n 115188), resources composed of financial contribution from EU e FP7 and EFPIA companies in kind contribution. We also acknowledge support from MINECO/FEDER project BIO2014-59614-JIN and from Fundaçao para a Ci ~ encia e Tecnologia ^ (FCT), Portugal (EXPL/BBB-IMG/0363/2013); MFE is the recipient of a PhD fellowship from FCT (SFRH/BD/52208/2013).