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Additional file 1: Table S1. Immune checkpoint and regulatory genes. Fig. S1. PRISM clinical trial study schema. Fig. S2. Cohort overview. Fig. S3. Deconvolution algorithms exhibit high concordance and an abundance of M2 macrophages in paediatric cancer. Fig. S4. Immunohistochemistry identifies paediatric patients with T-cell infiltrated tumours. Fig. S5. Prior treatment and steroid administration do not significantly affect T-cell infiltration. Fig. S6. Distribution of IPASS and T-cell clones are heterogenous across histologies. Fig. S7. IPASS correlations with additional markers of immune infiltration.

With the aim of preparing hybrid hydrogels suitable for use as patches for the local treatment of squamous cell carcinoma (SCC)-affected areas, curcumin (CUR) was loaded onto graphene oxide (GO) nanosheets, which were then blended into an alginate hydrogel that was crosslinked by means of calcium ions. The homogeneous incorporation of GO within the polymer network, which was confirmed through morphological investigations, improved the stability of the hybrid system compared to blank hydrogels. The weight loss in the 100–170 °C temperature range was reduced from 30% to 20%, and the degradation of alginate chains shifted to higher temperatures. Moreover, GO enhanced the stability in water media by counteracting the de-crosslinking process of the polymer network. Cell viability assays showed that the loading of CUR (2.5% and 5% by weight) was able to reduce the intrinsic toxicity of GO towards healthy cells, while higher amounts were ineffective due to the antioxidant/prooxidant paradox. Interestingly, the CUR-loaded systems were found to possess a strong cytotoxic effect in SCC cancer cells, and the sustained CUR release (~50% after 96 h) allowed long-term anticancer efficiency to be hypothesized.

BackgroundAggressive primary brain tumors such as glioblastoma are uniquely challenging to treat. The intracranial location poses barriers to therapy, and the potential for severe toxicity. Effective treatments for primary brain tumors are limited, and 5-year survival rates remain poor. Immune checkpoint inhibitor therapy has transformed treatment of some other cancers but has yet to significantly benefit patients with glioblastoma. Early phase trials of CAR-T cell therapy have demonstrated that this approach is safe and feasible, but with limited evidence of its effectiveness. The choices of appropriate target antigens for CAR-T cell therapy also remain limited.MethodsWe profiled an extensive biobank of patients’ biopsy tissues and patient-derived early passage glioma neural stem cell lines for GD2 expression using immunomicroscopy and flow cytometry. We then employed an approved clinical manufacturing process to make CAR-T cells from peripheral blood of glioblastoma and diffuse midline glioma patients and characterized their phenotype and function in vitro. Finally, we tested intravenously administered CAR-T cells in an aggressive intracranial xenograft model of glioblastoma and used multicolor flow cytometry, multicolor whole-tissue immunofluorescence and next-generation RNA sequencing to uncover markers associated with effective tumor control.ResultsHere we show that the tumor-associated antigen GD2 is highly and consistently expressed in primary glioblastoma tissue removed at surgery. Moreover, despite glioblastoma patients having perturbations in their immune system, highly functional GD2-specific CAR-T cells can be produced from their peripheral T cells using an approved clinical manufacturing process. Finally, after intravenous administration, GD2-CAR-T cells effectively infiltrated the brain and controlled tumor growth in an aggressive orthotopic xenograft model of glioblastoma. Tumor control was further improved using CAR-T cells manufactured with a clinical retroviral vector encoding an IL-15 transgene alongside the GD2-specific CAR. These CAR-T cells achieved a striking 50% complete response rate by bioluminescence imaging in established intracranial tumors. Markers associated with tumor control included those related to T-cell homing, infiltration, and cytotoxicity.ConclusionsTargeting GD2 using a clinically deployed CAR-T therapy has a sound scientific and clinical rationale as a treatment for glioblastoma and other aggressive primary brain tumors.What is already known on this topicGD2 is a tumor antigen of significant interest for targeting immunotherapy. A single preclinical study has shown the effectiveness of GD2-CAR-T cell therapy in an orthotopic xenograft model of diffuse midline glioma. Similarly, there is one previous preclinical study of GD2-CAR-T therapy in a orthotopic glioblastoma xenograft model but tumor control was achieved only following intracranial injection of CAR-T cells. Given that GD2-CAR-T therapy is already being evaluated clinically for other tumor indications, it is important to establish whether there is an acceptable rationale for its use in brain tumors.What this study addsThis is the first description of a GD2-targeted CAR-T cell therapy that shows antitumor effectiveness in a preclinical model of human glioblastoma following intravenous administration. It is also the first study to investigate the potential effects that the immune profile of glioblastoma patients may have on the feasibility of CAR-T cell manufacturing.How this study might affect research, practice, or policyThe results of this study have led to the initiation of an Australian phase 1 clinical trial program aiming to test GD2-specific CAR-T cells for the treatment of childhood and adult primary brain tumors. The study provides valuable insights into the microenvironmental factors that influence the effectiveness of CAR-T cell therapy for this type of tumor, paving the way for further optimization of CAR-T cell technology for treatment of aggressive primary brain tumors such as glioblastoma.

Therapeutic checkpoint antibodies blocking programmed death receptor 1/programmed death ligand 1 (PD-L1) signaling have radically improved clinical outcomes in cancer. However, the regulation of PD-L1 expression on tumor cells is still poorly understood. Here we show that intratumoral copper levels influence PD-L1 expression in cancer cells. Deep analysis of the The Cancer Genome Atlas database and tissue microarrays showed strong correlation between the major copper influx transporter copper transporter 1 (CTR-1) and PD-L1 expression across many cancers but not in corresponding normal tissues. Copper supplementation enhanced PD-L1 expression at mRNA and protein levels in cancer cells and RNA sequencing revealed that copper regulates key signaling pathways mediating PD-L1–driven cancer immune evasion. Conversely, copper chelators inhibited phosphorylation of STAT3 and EGFR and promoted ubiquitin-mediated degradation of PD-L1. Copper-chelating drugs also significantly increased the number of tumor-infiltrating CD8+ T and natural killer cells, slowed tumor growth, and improved mouse survival. Overall, this study reveals an important role for copper in regulating PD-L1 and suggests that anticancer immunotherapy might be enhanced by pharmacologically reducing intratumor copper levels. Significance: These findings characterize the role of copper in modulating PD-L1 expression and contributing to cancer immune evasion, highlighting the potential for repurposing copper chelators as enhancers of antitumor immunity.

The most exciting promise of cell micro-bioreactors is their expected ability to reproduce specific aspects of physiological environments in vitro, singularly or in combination, and to perform advanced biological studies to unravel novel interaction mechanisms; but this challenge is formidable. Moreover, the use of microfluidics is limited by the difficulty to adapt standard tissue culture protocols to miniaturised biochips and by the need of bulky external equipment such as liquid pumping systems. Here, we introduce an original microfluidic gradient generator for culturing cells in closed microchambers, based on: (1) automatic cell valving, (2) hydrostatic-pressure pumping, and (3) on-chip liquid reservoirs. The biochip is designed to be fully compatible with standard biological procedures and to operate with no external control units or cumbersome ancillary components. Remarkably, the use of integrated liquid reservoirs prevents air bubble formation and associated channel clogging. We detail the rationale behind the fluidic design, simulate and measure the chip fluid-dynamics by finite element simulations and microflow velocimetry, respectively. We finally exploit our chip to study the kinetics of an anti-cancer molecule, the catechin-dextran conjugate, on HeLa cells, demonstrating a very fast action. We argue that the presented fluidic concept and architecture are not limited to the present realization, but can be easily applied to other chip geometries and adapted to a number of biological studies. © 2014 the Partner Organisations.

Display Omitted We developed a tubeless microfluidic chip for cell co-culturing.On-chip cell filters were used to implement automated cell-loading.Topographically controlled co-cultures were realized by serial cell loadings.Cell migration within a closed microchamber was monitored for 72h. Lab-on-a-Chip devices exploit micro/nanoengineering techniques to develop miniaturized systems for handling and manipulating small volumes of fluids. Here, we demonstrate a microfluidic device for culturing cells in closed microchambers that is designed to be compatible with standard biological procedures. This approach facilitates the thermal and gaseous equilibration of all the components, preventing the nucleation of air bubbles. Serial asymmetrical loadings were performed to realize co-cultures with initial topographical organization. The migration of two different cell types was monitored for 72h by high-resolution fluorescence imaging.