TRP-2 / gp100 DNA vaccine and PD-1 checkpoint blockade combination for the treatment of intracranial tumors.

Intracranial tumors present a significant therapeutic challenge due to their physiological location. Immunotherapy presents an attractive method for targeting these intracranial tumors due to relatively low toxicity and tumor specificity. Here we show that SCIB1, a TRP-2 and gp100 directed ImmunoBody® DNA vaccine, generates a strong TRP-2 specific immune response, as demonstrated by the high number of TRP2-specific IFNγ spots produced and the detection of a significant number of pentamer positive T cells in the spleen of vaccinated mice. Furthermore, vaccine-induced T cells were able to recognize and kill B16HHDII/DR1 cells after a short in vitro culture. Having found that glioblastoma multiforme (GBM) expresses significant levels of PD-L1 and IDO1, with PD-L1 correlating with poorer survival in patients with the mesenchymal subtype of GBM, we decided to combine SCIB1 ImmunoBody® with PD-1 immune checkpoint blockade to treat mice harboring intracranial tumors expressing TRP-2 and gp100. Time-to-death was significantly prolonged, and this correlated with increased CD4+ and CD8+ T cell infiltration in the tissue microenvironment (TME). However, in addition to PD-L1 and IDO, the GBM TME was found to contain a significant number of immunoregulatory T (Treg) cell-associated transcripts, and the presence of such cells is likely to significantly affect clinical outcome unless also tackled.

A Holistic Approach to Hard-to-Treat Cancers: The Future of Immunotherapy for Glioblastoma, Triple Negative Breast Cancer, and Advanced Prostate Cancer.

Immunotherapy represents an attractive avenue for cancer therapy due to its tumour specificity and relatively low frequency of adverse effects compared to other treatment modalities. Despite many advances being made in the field of cancer immunotherapy, very few immunotherapeutic treatments have been approved for difficult-to-treat solid tumours such as triple negative breast cancer (TNBC), glioblastoma multiforme (GBM), and advanced prostate cancer (PCa). The anatomical location of some of these cancers may also make them more difficult to treat. Many trials focus solely on immunotherapy and have failed to consider or manipulate, prior to the immunotherapeutic intervention, important factors such as the microbiota, which itself is directly linked to lifestyle factors, diet, stress, social support, exercise, sleep, and oral hygiene. This review summarises the most recent treatments for hard-to-treat cancers whilst factoring in the less conventional interventions which could tilt the balance of treatment in favour of success for these malignancies.

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.

Immune Escape in Glioblastoma Multiforme and the Adaptation of Immunotherapies for Treatment.

Glioblastoma multiforme (GBM) is the most frequently occurring primary brain tumor and has a very poor prognosis, with only around 5% of patients surviving for a period of 5 years or more after diagnosis. Despite aggressive multimodal therapy, consisting mostly of a combination of surgery, radiotherapy, and temozolomide chemotherapy, tumors nearly always recur close to the site of resection. For the past 15 years, very little progress has been made with regards to improving patient survival. Although immunotherapy represents an attractive therapy modality due to the promising pre-clinical results observed, many of these potential immunotherapeutic approaches fail during clinical trials, and to date no immunotherapeutic treatments for GBM have been approved. As for many other difficult to treat cancers, GBM combines a lack of immunogenicity with few mutations and a highly immunosuppressive tumor microenvironment (TME). Unfortunately, both tumor and immune cells have been shown to contribute towards this immunosuppressive phenotype. In addition, current therapeutics also exacerbate this immunosuppression which might explain the failure of immunotherapy-based clinical trials in the GBM setting. Understanding how these mechanisms interact with one another, as well as how one can increase the anti-tumor immune response by addressing local immunosuppression will lead to better clinical results for immune-based therapeutics. Improving therapeutic delivery across the blood brain barrier also presents a challenge for immunotherapy and future therapies will need to consider this. This review highlights the immunosuppressive mechanisms employed by GBM cancers and examines potential immunotherapeutic treatments that can overcome these significant immunosuppressive hurdles.

Targeting cellular pathways in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is a debilitating disease that is associated with poor prognosis, short median patient survival and a very limited response to therapies. GBM has a very complex pathogenesis that involves mutations and alterations of several key cellular pathways that are involved in cell proliferation, survival, migration and angiogenesis. Therefore, efforts that are directed toward better understanding of GBM pathogenesis are essential to the development of efficient therapies that provide hope and extent patient survival. In this review, we outline the alterations commonly associated with GBM pathogenesis and summarize therapeutic strategies that are aimed at targeting aberrant cellular pathways in GBM.