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.
Metastatic cancer cells exploit Epithelial-mesenchymal-transition (EMT) to enhance their migration, invasion, and resistance to treatments. Recent studies highlight that elevated levels of copper are ...implicated in cancer progression and metastasis. Clinical trials using copper chelators are associated with improved patient survival; however, the molecular mechanisms by which copper depletion inhibits tumor progression and metastasis are poorly understood. This remains a major hurdle to the clinical translation of copper chelators. Here, we propose that copper chelation inhibits metastasis by reducing TGF-β levels and EMT signaling. Given that many drugs targeting TGF-β have failed in clinical trials, partly because of severe side effects arising in patients, we hypothesized that copper chelation therapy might be a less toxic alternative to target the TGF-β/EMT axis.
Our cytokine array and RNA-seq data suggested a link between copper homeostasis, TGF-β and EMT process. To validate this hypothesis, we performed single-cell imaging, protein assays, and in vivo studies. Here, we used the copper chelating agent TEPA to block copper trafficking. Our in vivo study showed a reduction of TGF-β levels and metastasis to the lung in the TNBC mouse model. Mechanistically, TEPA significantly downregulated canonical (TGF-β/SMAD2&3) and non-canonical (TGF-β/PI3K/AKT, TGF-β/RAS/RAF/MEK/ERK, and TGF-β/WNT/β-catenin) TGF-β signaling pathways. Additionally, EMT markers of MMP-9, MMP-14, Vimentin, β-catenin, ZEB1, and p-SMAD2 were downregulated, and EMT transcription factors of SNAI1, ZEB1, and p-SMAD2 accumulated in the cytoplasm after treatment.
Our study suggests that copper chelation therapy represents a potentially effective therapeutic approach for targeting TGF-β and inhibiting EMT in a diverse range of cancers.
Abstract Glioblastoma, a highly aggressive brain tumor with a dismal prognosis necessitates novel therapeutic approaches as current standard treatments yield limited success. Despite the ...transformative impact of immune checkpoint blockade in various malignancies, its efficacy in glioblastoma remains elusive. Similarly, chimeric antigen receptor T (CAR-T) cell therapy, while successful in hematological malignancies, faces challenges in solid tumors. This study addresses the immunosuppressive barriers hindering the effectiveness of immunotherapies in glioblastoma. Highlighting copper's pivotal role in glioblastoma, our research group demonstrated its elevation and influence on PD-L1 expression. Given the success of copper chelators in treating Wilson's Disease, in which neurological manifestations are alleviated following treatment, we hypothesized that copper chelation therapy could be a promising strategy for glioblastoma, especially when combined with immunotherapies. Flow cytometry analysis revealed a significant increase in the tumor-associated antigen GD2 following treatment with the copper chelator trientine (TETA), suggesting its potential synergy with GD2-targeted immunotherapies. In an immunocompetent murine glioblastoma model, combination therapy with GD2-targeted CAR-T cells and TETA effectively slowed tumor growth, demonstrating enhanced efficacy. Additionally, TETA as a monotherapy significantly improved survival compared to the control. The combination of TETA with anti-PD1 antibody therapy also exhibited promising results in inhibiting tumor growth and subsequently prolonging survival, with some mice demonstrating complete tumor regression. Importantly, TETA decreased copper levels in the brains of the mice, indicating the drug can act on the brain, a crucial therapeutic hurdle that is hampering the efficacy of novel therapies in glioblastoma. Further exploration of immune cell populations using OPAL Multiplex immunohistochemistry following TETA, anti-PD1, and GD2-targeted CAR-T cell treatments will underscore the potential of copper chelation in reshaping the tumor microenvironment. Our findings suggest that copper chelation, with agents like TETA, can be repurposed as a viable anticancer therapy when combined with immunotherapies. This study provides a compelling rationale for further pre-clinical validation, offering a potential breakthrough in the quest for effective glioblastoma treatments. Citation Format: Tyler Shai-Hee, Toni Rose Jue, Riccardo Cazzoli, Jourdin R. Rouaen, Filip Michniewicz, Jessica Bell, Tessa Gargett, Michael P. Brown, Orazio Vittorio. Priming of the glioblastoma tumour microenvironment via copper chelation to enhance the efficacy of immunotherapies abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1418.
Abstract Neuroblastoma is the most frequent extra-cranial pediatric cancer with high-risk disease possessing a dismal five-year survival rate <50%. Antibody-based therapies targeting the surface ...disialoganglioside GD2 have shown promising anti-tumor activity in patients however efficacy is strongly hampered by immunosuppressive mechanisms present in the tumor microenvironment. Neuroblastomas typically feature low mutational burdens, limited major histocompatibility complex class I expression, increased immune checkpoint markers, sparse immune infiltration, and soluble mediators which drastically limit anti-tumor immunity. Given the emerging link between elevated copper levels and tumoral immune evasion, we explored copper chelation as a therapeutic strategy using the agents tetraethylenepentamine (TEPA) and the clinically approved analogue triethylenetetramine (TETA). Using the immunocompetent TH-MYCN model, we performed single-cell RNA sequencing supported by OPAL multiplex immunohistochemistry and cytokine profiling of resected tumors to assess cellular and molecular changes occurring after one week of TEPA treatment. Therapy was observed to successfully deplete intratumoral copper and reinvigorate anti-tumor immunity with increased infiltration and activity of pro-inflammatory immune cells, and was specifically found to induce egress and N1-polarisation of neutrophils. Further mechanistic studies in vitro revealed the sequestration of copper by neuroblastoma cells attenuated neutrophil function, which could be successfully reversed upon TEPA treatment. Findings propose a novel mechanism of immune evasion, highlighting copper chelation as a therapeutic strategy to counteract immunosuppression. Using the TH-MYCN model, TEPA was employed as a priming agent to effectively potentiate anti-GD2 antibody therapy and achieve durable tumor control. Importantly, this was associated with increased Fc-receptor-bearing natural killer and myeloid cells, which elicit tumor clearance via antibody-dependent cellular cytotoxicity. Currently indicated for the treatment of copper overload disorder Wilson’s disease, TETA was evaluated for potential repurposing as a novel immunomodulatory agent. Using the syngeneic NXS2 model, TETA exhibited an exceptional safety profile and combination therapy achieved durable tumor control with no relapses occurring after treatment cessation. This result was similarly associated with the infiltration of pro-inflammatory immune cells, including N1 neutrophils. Collectively, this study credentials copper chelation as a non-toxic strategy to disrupt the immunosuppressive tumor microenvironment and reinvigorate anti-tumour immunity. Findings provide crucial translational evidence for repurposing TETA to potentiate anti-GD2 antibody therapy and improve neuroblastoma patient responses. Citation Format: Jourdin R. Rouaen, Antonella Salerno, Fabio Luciani, Jayne Murray, Tyler Shai-Hee, Nicodemus Tedla, Michelle Haber, Toby N. Trahair, Orazio Vittorio. Copper chelation overcomes the immunosuppressive neuroblastoma microenvironment to potentiate anti-GD2 antibody therapy abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2673.
Metastatic cancer cells exploit Epithelial-mesenchymal-transition (EMT) to enhance their migration, invasion, and resistance to treatments. Recent studies highlight that elevated levels of copper are ...implicated in cancer progression and metastasis. Clinical trials using copper chelators are associated with improved patient survival; however, the molecular mechanisms by which copper depletion inhibits tumor progression and metastasis are poorly understood. This remains a major hurdle to the clinical translation of copper chelators. Here, we propose that copper chelation inhibits metastasis by reducing TGF-beta levels and EMT signaling. Given that many drugs targeting TGF-beta have failed in clinical trials, partly because of severe side effects arising in patients, we hypothesized that copper chelation therapy might be a less toxic alternative to target the TGF-beta/EMT axis. Our cytokine array and RNA-seq data suggested a link between copper homeostasis, TGF-beta and EMT process. To validate this hypothesis, we performed single-cell imaging, protein assays, and in vivo studies. Here, we used the copper chelating agent TEPA to block copper trafficking. Our in vivo study showed a reduction of TGF-beta levels and metastasis to the lung in the TNBC mouse model. Mechanistically, TEPA significantly downregulated canonical (TGF-beta/SMAD2&3) and non-canonical (TGF-beta/PI3K/AKT, TGF-beta/RAS/RAF/MEK/ERK, and TGF-beta/WNT/beta-catenin) TGF-beta signaling pathways. Additionally, EMT markers of MMP-9, MMP-14, Vimentin, beta-catenin, ZEB1, and p-SMAD2 were downregulated, and EMT transcription factors of SNAI1, ZEB1, and p-SMAD2 accumulated in the cytoplasm after treatment. Our study suggests that copper chelation therapy represents a potentially effective therapeutic approach for targeting TGF-beta and inhibiting EMT in a diverse range of cancers.
Abstract Diffuse Midline Gliomas (DMG) are incurable pediatric brain tumors driven by histone mutation H3K27M, globally reducing H3K27 trimethylation altering the epigenome and deregulating gene ...expression. These mutations often partner with and are strongly integrated into kinase signaling and metabolic alterations, driving aggressive tumor growth with many mechanisms which can compensate for one another and therefore evade traditional single agent therapies. Copper over-accumulation has been linked to epigenetic, kinase and metabolic dysregulation in various cancers and neurological disorders. Copper chelators are clinically approved for use in pediatric Wilson’s Disease patients, improve neurological symptoms and are under clinical investigation for several cancers. We propose copper chelators may simultaneously target these mechanisms in DMG. We performed transcriptomic interrogation of patient datasets, in vitro investigations in a panel of DMG cell lines and an in vivo investigation using an orthotopic xenograft model of DIPG (SU-DIPGVI-Luc). Copper chelator tetraethylenepentamine-pentachloride (TEPA) was used to treat DMG cells. Transcriptomic (RNA-Seq), proteomic, phospho-proteomic and metabolomic (Mass Spectrometry) investigations, western blots and immunofluorescent microscopy determined efficacy and mechanism. In vivo investigation consisted of survival study with concurrent luminescence imaging, histology, and copper assay. We demonstrate copper chaperone transcript expression is increased in DMG patient samples compared to non-DMG samples. In vitro, apoptosis and growth assays demonstrated H3K27M DMG were more sensitive to TEPA than H3-WT DMG. In H3K27M DMG, TEPA downregulated ATP, GTP and SAM metabolism which corresponded with downregulated kinase signaling and altered H3K27 trimethylation and DNA methylation patterns around cell cycle genes. Affected genes included those regulating centrosome segregation and ATP synthesis. Further transcriptomic and proteomic investigations highlighted TEPA downregulated G2-M phase kinase pathways, with immunofluorescence demonstrating TEPA reduced expression of mitotic marker p-H3S10 and epigenetic methylators EZH2 and DNMT1. In vivo, TEPA reduced brain copper accumulation, eliminated tumors and increased survival in an orthotopic patient-derived xenograft DMG model. Collectively, our data indicates TEPA induced multi-modal targeting of M-phase cell cycle progression through epigenetic, kinase signaling and metabolic mechanisms. Efficacy was observed both in vitro and in vivo, providing a promising basis for future investigation of copper chelators in DMG. We propose copper-chelation is a viable therapeutic strategy for DMG patients warranting clinical investigation. Citation Format: Filip Michniewicz, Riccardo Cazzoli, Jessica Bell, Federica Saletta, Ensieh Poursani, Jourdin R. Rouaen, Tyler Shai-Hee, Vu Pham, Toni Rose Jue, Matthew Biery, Liesl Bramberger, Terry Lim, Aaminah Khan, Federico M. Giorgi, Daniele Mercatelli, Chelsea Mayoh, Giuseppe Cirillo, Alexander Macmillan, Renee Whan, Christopher T. Barlow, Maria Tsoli, Nicholas Vitanza, Scott Berry, Pouya Faridi, David Ziegler, Orazio Vittorio. A comprehensive approach: Copper chelation therapy modulates epigenetic, kinase signaling and metabolic pathways in diffuse midline gliomas (DMG) abstract. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 525.
PDF
