GABA regulates electrical activity and tumor initiation in melanoma

Oncogenes can initiate tumors only in certain cellular contexts, which is referred to as oncogenic competence. In melanoma, whether cells in the microenvironment can endow such competence remains unclear. Using a combination of zebrafish transgenesis coupled with human tissues, we demonstrate that GABAergic signaling between keratinocytes and melanocytes promotes melanoma initiation by BRAFV600E. GABA is synthesized in melanoma cells, which then acts on GABA-A receptors in keratinocytes. Electron microscopy demonstrates specialized cell–cell junctions between keratinocytes and melanoma cells, and multielectrode array analysis shows that GABA acts to inhibit electrical activity in melanoma/keratinocyte cocultures. Genetic and pharmacologic perturbation of GABA synthesis abrogates melanoma initiation in vivo. These data suggest that GABAergic signaling across the skin microenvironment regulates the ability of oncogenes to initiate melanoma. Significance: This study shows evidence of GABA-mediated regulation of electrical activity between melanoma cells and keratinocytes, providing a new mechanism by which the microenvironment promotes tumor initiation. This provides insights into the role of the skin microenvironment in early melanomas while identifying GABA as a potential therapeutic target in melanoma

Synthesis, Characterisation, and Preliminary Anti-Cancer Photodynamic Therapeutic \u3ci\u3eIn Vitro\u3c/i\u3e Studies of Mixed-Metal Binuclear Ruthenium(II)-Vanadium(IV) Complexes

We report the synthesis and characterisation of mixed-metal binuclear ruthenium(II)-vanadium(IV) complexes, which were used as potential photodynamic therapeutic agents for melanoma cell growth inhibition. The novel complexes, [Ru(pbt)2(phen2DTT)](PF6)2•1.5H2O 1 (where phen2DTT = 1,4-bis(1,10-phenanthrolin-5-ylsulfanyl)butane-2,3-diol and pbt = 2-(2\u27-pyridyl)benzothiazole) and [Ru(pbt)2(tpphz)](PF6)2•3H2O 2 (where tpphz = tetrapyrido[3,2-a:2′,3′-c:3″,2″-h:2‴,3‴-j]phenazine) were synthesised and characterised. Compound 1 was reacted with [VO(sal-L-tryp)(H2O)] (where sal-L-tryp = N-salicylidene-L-tryptophanate) to produce [Ru(pbt)2(phen2DTT)VO(sal-L-tryp)](PF6)2•5H2O 4; while [VO(sal-L-tryp)(H2O)] was reacted with compound 2 to produce [Ru(pbt)2(tpphz)VO(sal-L-tryp)](PF6)2•6H2O 3. All complexes were characterised by elemental analysis, HRMS, ESI MS, UV-visible absorption, ESR spectroscopy, and cyclic voltammetry, where appropriate. In vitro cell toxicity studies (with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay) via dark and light reaction conditions were carried out with sodium diaqua-4,4\u27,4”,4”\u27tetrasulfophthalocyaninecobaltate(II) (Na4[Co(tspc)(H2O)2]), [VO(sal-L-tryp)(phen)]•H2O, and the chloride salts of complexes 3 and 4. Such studies involved A431, human epidermoid carcinoma cells; human amelanotic malignant melanoma cells; and HFF, non-cancerous human skin fibroblast cells. Both chloride salts of complexes 3 and 4 were found to be more toxic to melanoma cells than to non-cancerous fibroblast cells, and preferentially led to apoptosis of the melanoma cells over non-cancerous skin cells. The anti-cancer property of the chloride salts of complexes 3 and 4 was further enhanced when treated cells were exposed to light, while no such effect was observed on non-cancerous skin fibroblast cells. ESR and 51V NMR spectroscopic studies were also used to assess the stability of the chloride salts of complexes 3 and 4 in aqueous media at pH 7.19. This research illustrates the potential for using mixed-metal binuclear ruthenium(II)-vanadium(IV) complexes fighting skin cancer

Preliminary Anti-Cancer Photodynamic Therapeutic \u3ci\u3eIn Vitro\u3c/i\u3e Studies With Mixed-Metal Binuclear Ruthenium(II)-Vanadium(IV) Complexes

We report the synthesis and characterisation of mixed-metal binuclear ruthenium(II)–vanadium(IV) complexes, which were used as potential photodynamic therapeutic agents for melanoma cell growth inhibition. The novel complexes, [Ru(pbt)2(phen2DTT)](PF6)2·1.5H2O 1 (where phen2DTT = 1,4-bis(1,10-phenanthrolin-5-ylsulfanyl)butane-2,3-diol and pbt = 2-(2′-pyridyl)benzothiazole) and [Ru(pbt)2(tpphz)](PF6)2·3H2O 2 (where tpphz = tetrapyrido[3,2-a:2′,3′-c:3′′,2′′-h:2′′′,3′′′-j]phenazine) were synthesised and characterised. Compound 1 was reacted with [VO(sal-L-tryp)(H2O)] (where sal-L-tryp = N-salicylidene-L-tryptophanate) to produce [Ru(pbt)2(phen2DTT)VO(sal-L-tryp)](PF6)2·5H2O 4; while [VO(sal-L-tryp)(H2O)] was reacted with compound 2 to produce [Ru(pbt)2(tpphz)VO(sal-L-tryp)](PF6)2·6H2O 3. All complexes were characterised by elemental analysis, HRMS, ESI MS, UV-visible absorption, ESR spectroscopy, and cyclic voltammetry, where appropriate. In vitro cell toxicity studies (with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) colorimetric assay) via dark and light reaction conditions were carried out with sodium diaqua-4,4′,4′′,4′′′ tetrasulfophthalocyaninecobaltate(II) (Na4[Co(tspc)(H2O)2]), [VO(sal-L-tryp)(phen)]·H2O, and the chloride salts of complexes 3 and 4. Such studies involved A431, human epidermoid carcinoma cells; human amelanotic malignant melanoma cells; and HFF, non-cancerous human skin fibroblast cells. Both chloride salts of complexes 3 and 4 were found to be more toxic to melanoma cells than to non-cancerous fibroblast cells, and preferentially led to apoptosis of the melanoma cells over non-cancerous skin cells. The anti-cancer property of the chloride salts of complexes 3 and 4 was further enhanced when treated cells were exposed to light, while no such effect was observed on non-cancerous skin fibroblast cells. ESR and 51V NMR spectroscopic studies were also used to assess the stability of the chloride salts of complexes 3 and 4 in aqueous media at pH 7.19. This research illustrates the potential for using mixed-metal binuclear ruthenium(II)–vanadium(IV) complexes to fight skin cancer

TCR signal strength defines distinct mechanisms of T cell dysfunction and cancer evasion

T cell receptor (TCR) signal strength is a key determinant of T cell responses. We developed a cancer mouse model in which tumor-specific CD8 T cells (TST cells) encounter tumor antigens with varying TCR signal strength. High-signal-strength interactions caused TST cells to up-regulate inhibitory receptors (IRs), lose effector function, and establish a dysfunction-associated molecular program. TST cells undergoing low-signal-strength interactions also up-regulated IRs, including PD1, but retained a cell-intrinsic functional state. Surprisingly, neither high- nor low-signal-strength interactions led to tumor control in vivo, revealing two distinct mechanisms by which PD1hi TST cells permit tumor escape; high signal strength drives dysfunction, while low signal strength results in functional inertness, where the signal strength is too low to mediate effective cancer cell killing by functional TST cells. CRISPR-Cas9-mediated fine-tuning of signal strength to an intermediate range improved anti-tumor activity in vivo. Our study defines the role of TCR signal strength in TST cell function, with important implications for T cell-based cancer immunotherapies

Supplementary Video 3 from GABA Regulates Electrical Activity and Tumor Initiation in Melanoma

3D rendering of gephyrin positive clusters in melanoma/keratinocyte co-cultures</p

Supplementary Video 1 from GABA Regulates Electrical Activity and Tumor Initiation in Melanoma

3D rendering of melanoma/keratinocyte contacts in zebrafish embryos</p

Supplementary Table 1 from GABA Regulates Electrical Activity and Tumor Initiation in Melanoma

LOPAC Screen results in melanoma keratinocyte co-cultures</p

Supplementary Video 2 from GABA Regulates Electrical Activity and Tumor Initiation in Melanoma

3D rendering of melanoma/switched keratinocyte contacts and stained vesicles in human melanoma/keratinocyte co-cultures</p

Supplementary Figures S1-S17 from GABA Regulates Electrical Activity and Tumor Initiation in Melanoma

Combined supplementary figures and figure legends. Supplementary Fig. 1: Switching requires direct contact between nascent melanoma cells and keratinocytes in vivo. Supplementary Fig. 2: Switching requires direct contact between melanoma cells and keratinocytes in vitro. Supplementary Fig. 3: Switching does not involve cell fusion between keratinocytes and melanoma cells. Supplementary Fig. 4: Exosome-like vesicles are transferred from melanoma cells to keratinocytes. Supplementary Fig. 5: GABAergic signaling is active in skin and melanoma cells. Supplementary Fig. 6: Melanoma cells express GAD1 and produce GABA. Supplementary Fig. 7: Disruption of GABA signaling blocks melanoma/keratinocyte communication. Supplementary Fig. 8: Specialized cell-cell junction signatures are enriched in melanoma cells and keratinocytes. Supplementary Fig. 9: Melanoma vesicles are present near specialized GABAergic cell-cell junctions in co-cultures. Supplementary Fig. 10: gad activation correlates with higher tumor burden in melanoma. Supplementary Fig. 11: GABA producing enzymes are expressed in zebrafish melanoma cells. Supplementary Fig. 12: Disruption of GABA synthesis blocks tumor initiation in melanoma. Supplementary Fig. 13: GAD expression/GABA treatment is pro-tumorigenic in a non cellautonomous manner. Supplementary Fig. 14: Switched keratinocytes are pro-tumorigenic in melanoma. Supplementary Fig. 15: LIF expression in keratinocytes drives melanoma cell proliferation. Supplementary Fig. 16: Zebrafish melanoma scRNAseq identifies GABA and LIF associated components in the TME. Supplementary Fig. 17: MYCN drives LIF expression in keratinocytes.</p