Robust formation of optimal single spheroids towards cost-effective in vitro three-dimensional tumor models.

While useful for fundamental in vitro studies, monolayer cell cultures are not physiologically relevant. Spheroids, a complex three-dimensional (3D) structure, more closely resemble in vivo tumor growth. Spheroids allow the results obtained relating to proliferation, cell death, differentiation, metabolism, and various antitumor therapies to be more predictive of in vivo outcomes. In the protocol herein, a rapid and high-throughput method is discussed for the generation of single spheroids using various cancer cell lines, including brain cancer cells (U87 MG, SEBTA-027, SF188), prostate cancer cells (DU-145, TRAMP-C1), and breast cancer cells (BT-549, Py230) in 96-round bottom-well plates. The proposed method is associated with significantly low costs per plate without requiring refining or transferring. Homogeneous compact spheroid morphology was evidenced as early as 1 day after following this protocol. Proliferating cells were traced in the rim, while dead cells were found to be located inside the core region of the spheroid using confocal microscopy and the Incucyte® live imaging system. H&E staining of spheroid sections was utilized to investigate the tightness of the cell packaging. Through western blotting analyses, it was revealed that a stem cell-like phenotype was adopted by these spheroids. This method was also used to obtain the EC50 of the anticancer dipeptide carnosine on U87 MG 3D culture. This affordable, easy-to-follow five-step protocol allows for the robust generation of various uniform spheroids with 3D morphology characteristics.

Controlled release of carnosine from poly(lactic-co-glycolic acid) beads using nanomechanical magnetic trigger towards the treatment of glioblastoma.

Nanometer scale rods of superparamagnetic iron oxide have been encapsulated, along with the anti-cancer therapeutic carnosine, inside porous poly(lactic-co-glycolic acid) microbeads with a uniform morphology, synthesised using microfluidic arrays. The sustained and externally triggered controlled release from these vehicles was demonstrated using a rotating Halbach magnet array, quantified via liquid chromatography, and imaged in situ using magnetic resonance imaging (MRI) and scanning electron microscopy (SEM). In the absence of the external magnetic trigger, the carnosine was found to be released from the polymer in a linear profile; however, over 50% of the drug could be released within 30 minutes of exposure to the rotating magnetic field. In addition, the release of carnosine embedded on the surface of the nano-rods was delayed if it was mixed with the iron oxide nano rods before the encapsulation. These new drug delivery vesicles have the potential to pave the way towards the safe and triggered release of onsite drug delivery, as part of a theragnostic treatment for glioblastoma.

Synthesis and Functionalisation of Superparamagnetic Nano-Rods towards the Treatment of Glioblastoma Brain Tumours.

The complete removal of glioblastoma brain tumours is impossible to achieve by surgery alone due to the complex finger-like tentacle structure of the tumour cells and their migration away from the bulk of the tumour at the time of surgery; furthermore, despite aggressive chemotherapy and radiotherapy treatments following surgery, tumour cells continue to grow, leading to the death of patients within 15 months after diagnosis. The naturally occurring carnosine dipeptide has previously demonstrated activity against in vitro cultured glioblastoma cells; however, at natural physiological concentrations, its activity is too low to have a significant effect. Towards realising the full oncological potential of carnosine, the dipeptide was embedded within an externally triggered carrier, comprising a novel nano rod-shaped superparamagnetic iron oxide nanoparticle (ca. 86 × 19 × 11 nm) capped with a branched polyethyleneimine, which released the therapeutic agent in the presence of an external magnetic field. The new nano-carrier was characterized using electron microscopy, dynamic light scattering, elemental analysis, and magnetic resonance imaging techniques. In addition to cytotoxicity studies, the carnosine carrier's effectiveness as a treatment for glioblastoma was screened in vitro using the U87 human glioblastoma astrocytoma cell line. The labile carnosine (100 mM) suppresses both the U87 cells' proliferation and mobility over 48 h, resulting in significant reduction in migration and potential metastasis. Carnosine was found to be fully released from the carrier using only mild hyperthermia conditions (40 °C), facilitating an achievable clinical application of the slow, sustained-release treatment of glioblastoma brain tumours that demonstrates potential to inhibit post-surgery metastasis with the added benefit of non-invasive monitoring via MRI.

Cancer Vaccines: Adjuvant Potency, Importance of Age, Lifestyle, and Treatments.

Although the discovery and characterization of multiple tumor antigens have sparked the development of many antigen/derived cancer vaccines, many are poorly immunogenic and thus, lack clinical efficacy. Adjuvants are therefore incorporated into vaccine formulations to trigger strong and long-lasting immune responses. Adjuvants have generally been classified into two categories: those that 'depot' antigens (e.g. mineral salts such as aluminum hydroxide, emulsions, liposomes) and those that act as immunostimulants (Toll Like Receptor agonists, saponins, cytokines). In addition, several novel technologies using vector-based delivery of antigens have been used. Unfortunately, the immune system declines with age, a phenomenon known as immunosenescence, and this is characterized by functional changes in both innate and adaptive cellular immunity systems as well as in lymph node architecture. While many of the immune functions decline over time, others paradoxically increase. Indeed, aging is known to be associated with a low level of chronic inflammation-inflamm-aging. Given that the median age of cancer diagnosis is 66 years and that immunotherapeutic interventions such as cancer vaccines are currently given in combination with or after other forms of treatments which themselves have immune-modulating potential such as surgery, chemotherapy and radiotherapy, the choice of adjuvants requires careful consideration in order to achieve the maximum immune response in a compromised environment. In addition, more clinical trials need to be performed to carefully assess how less conventional form of immune adjuvants, such as exercise, diet and psychological care which have all be shown to influence immune responses can be incorporated to improve the efficacy of cancer vaccines. In this review, adjuvants will be discussed with respect to the above-mentioned important elements.