Melanoma Model Research Articles

Antiproliferative effects of LY-2183240 combined with various chemotherapeutic drugs in an isobolographic in vitro model of malignant melanoma

Despite a great progress in identifying treatment options for patients with malignant melanoma, novel therapies tend to be costly and, in some cases, produce adverse effects forcing the melanoma patients to withdraw drugs. There is a strong need for less expensive drugs with a more favorable spectrum of anticancer actions. This study was designed to assess whether LY-2183240 (a potent inhibitor of both, anandamide cellular reuptake and fatty acid amide hydrolase (FAAH), an enzyme that degrades anandamide) has antiproliferative and cytotoxic effects on various human malignant melanoma cell lines (primary A375 and FM55P, metastatic SK-MEL28 and FM55M2) when administered alone or in combination with docetaxel, paclitaxel, mitoxantrone and cisplatin via the MTT assay. The MTT, LDH and BrdU assays were used to evaluate the potency and safety of LY-2183240, whereas isobolographic analysis of interactions was applied to characterize the interactions of LY-2183240 with the studied chemotherapeutics (docetaxel, paclitaxel, mitoxantrone and cisplatin). The isobolography confirmed that the combinations of LY-2183240 with docetaxel, paclitaxel and mitoxantrone produced additive interactions in all the tested melanoma cell lines. Only two antagonistic interactions for LY-2183240 combined with cisplatin in the A375 and FM55P cell lines were observed by the MTT assay. In conclusion, LY-2183240 can be considered an add-on drug for the treatment of melanoma, when combined with docetaxel, paclitaxel, or mitoxantrone, but not with cisplatin.

High-throughput screening identifies ibuprofen as an sEV PD-L1 inhibitor for synergistic cancer immunotherapy

Programmed death-ligand 1 (PD-L1) on tumor-derived small extracellular vesicles (sEVs) limits therapeutic effectiveness by interacting with the PD-1 receptor on host immune cells. Targeting the secretion of sEV PD-L1 has emerged as a promising strategy to enhance immunotherapy. However, the lack of small-molecule inhibitors poses a challenge for clinical translation. In this study, we developed a target and phenotype dual-driven high-throughput screening (TAP-HTS) strategy that combined virtual screening with nanoflow-based experimental verification. We identified ibuprofen (IBP) as a novel inhibitor that effectively targeted sEV PD-L1 secretion. IBP disrupted the biogenesis and secretion of PD-L1+ sEVs in tumor cells by physically interacting with a critical regulator of sEV biogenesis, hepatocyte growth factor-regulated tyrosine kinase substrate (HRS). Notably, the mechanism of action of IBP is distinct from its commonly known targets, cyclooxygenases (COX1 or COX2). Administration of IBP stimulated antitumor immunity and enhanced the efficacy of anti-PD-1 therapy in melanoma and oral squamous cell carcinoma (OSCC) mouse models. To address potential adverse effects, we further developed an IBP gel for topical application, which demonstrated remarkable therapeutic efficacy when combined with anti-PD-1 treatment. The discovery of this specific small inhibitor provides a promising avenue for establishing durable, systemic antitumor immunity.

Development and validation of prognostic nomogram in pediatric melanoma: a population-based study.

The aim of the study is to use the Surveillance, Epidemiology, and End Results (SEER) database to develop a useful clinical nomogram that uses prognosis prediction for pediatric melanoma patients. We obtained clinical information on pediatric melanoma patients from the SEER database between 2000 and 2018. Each patient was split into a training cohort or a validation cohort at random. Results between various subgroups were compared using Kaplan-Meier analyses. We created a nomogram to calculate the probability of survival for pediatric patients with melanoma. The performance of nomograms was assessed using calibration and discrimination. To assess the clinical use of this newly created model, decision curve analysis was also performed. In this study, a total of 890 eligible patients were chosen at random and allocated to 70% of training cohorts (n = 623) and 30% of validation cohorts (n = 267). After applying the chosen various components to create a nomogram, validated indexes showed that the nomogram had a strong capacity for discrimination. The training set's and validation set's C-index values were 0.817 and 0.832, respectively. The calibration plots demonstrated a strong correlation between the observation and the forecast. The model has a good clinical net benefit for pediatric melanoma patients, according to the clinical decision curve. In conclusion, we created an effective survival prediction model for pediatric melanoma. This nomogram is accurate and useful for clinical decision-making. Still, more external confirmation is required.

An ultra-long-acting L-asparaginase synergizes with an immune checkpoint inhibitor in starvation-immunotherapy of metastatic solid tumors

Metastasis stands as the primary contributor to mortality associated with tumors. Chemotherapy and immunotherapy are frequently utilized in the management of metastatic solid tumors. Nevertheless, these therapeutic modalities are linked to serious adverse effects and limited effectiveness in preventing metastasis. Here, we report a novel therapeutic strategy named starvation-immunotherapy, wherein an immune checkpoint inhibitor is combined with an ultra-long-acting L-asparaginase that is a fusion protein comprising L-asparaginase (ASNase) and an elastin-like polypeptide (ELP), termed ASNase-ELP. ASNase-ELP's thermosensitivity enables it to generate an in-situ depot following an intratumoral injection, yielding increased dose tolerance, improved pharmacokinetics, sustained release, optimized biodistribution, and augmented tumor retention compared to free ASNase. As a result, in murine models of oral cancer, melanoma, and cervical cancer, the antitumor efficacy of ASNase-ELP by selectively and sustainably depleting L-asparagine essential for tumor cell survival was substantially superior to that of ASNase or Cisplatin, a first-line anti-solid tumor medicine, without any observable adverse effects. Furthermore, the combination of ASNase-ELP and an immune checkpoint inhibitor was more effective than either therapy alone in impeding melanoma metastasis. Overall, the synergistic strategy of starvation-immunotherapy holds excellent promise in reshaping the therapeutic landscape of refractory metastatic tumors and offering a new alternative for next-generation oncology treatments.

Blockade of glucose-6-phosphate dehydrogenase induces immunogenic cell death and accelerates immunotherapy

BackgroundEnhanced glucose metabolism has been reported in many cancers. Glucose-6-phosphate dehydrogenase (G6PD) is a rate-limiting enzyme involved in the pentose phosphate pathway, which maintains NADPH levels and protects cells from...

Polymersomes with splenic avidity target red pulp myeloid cells for cancer immunotherapy.

Regulating innate immunity is an emerging approach to improve cancer immunotherapy. Such regulation requires engaging myeloid cells by delivering immunomodulatory compounds to hematopoietic organs, including the spleen. Here we present a polymersome-based nanocarrier with splenic avidity and propensity for red pulp myeloid cell uptake. We characterized the in vivo behaviour of four chemically identical yet topologically different polymersomes by in vivo positron emission tomography imaging and innovative flow and mass cytometry techniques. Upon intravenous administration, relatively large and spherical polymersomes accumulated rapidly in the spleen and efficiently targeted myeloid cells in the splenic red pulp. When loaded with β-glucan, intravenously administered polymersomes significantly reduced tumour growth in a mouse melanoma model. We initiated our nanotherapeutic's clinical translation with a biodistribution study in non-human primates, which revealed that the platform's splenic avidity is preserved across species.

Dual Adjuvant-Loaded Peptide Antigen Self-Assembly Potentiates Dendritic Cell-Mediated Tumor Immunotherapy.

Clinical translation of current cancer vaccine research has been hampered by limited antitumor immune responses due to inefficient antigen delivery and presentation, suboptimal DC and T cell activation. Biomaterial-based nanovaccine offers targeted antigen delivery, protection from degradation in vivo, and prolonged tumor therapeutic efficacy. This study introduces a lipid-coated deoxycholic acid-survivin nanoassembly (DA-L-DSA). Survivin, overexpressed in several cancer cells and involved in cancer cell growth and immune evasion, is selected as a tumor-associated antigen. An major histocompatibility complex class I binding epitope of survivin is engineered into the nanoassembly. R848, TLR 7/8 agonist, and SD-208, TGF-beta receptor1 kinase inhibitor, are coencapsulated into the nanoassembly as potent adjuvants to boost DC maturation and enhance antigen presentation. The DA-L-DSA effectively stimulates the maturation of dendritic cells, migrates into lymph nodes, and enhances T-cell activation and Th1 response. A substantial influx of cytotoxic T lymphocytes into primary tumors is observed in a murine melanoma model and demonstrates anti-metastatic effects in a spontaneous breast cancer metastasis model. Furthermore, DA-L-DSA exhibits a remarkable synergistic effect in the combination therapy with immune checkpoint inhibitors alleviating immunosuppressive tumor microenvironment. Taken together, these findings suggest DA-L-DSA as a promising immuno-therapeutic platform that could be applicable to diverse intractable cancers.

Combination of oncolytic Maraba virus with immune checkpoint blockade overcomes therapy resistance in an immunologically cold model of advanced melanoma with dysfunctional T-cell receptor signalling

BackgroundOver the past decade, cancer immunotherapies have revolutionized the treatment of melanoma; however, responses vary across patient populations. Recently, baseline tumor size has been identified as an independent prognostic factor...

Naturally-derived phenethyl isothiocyanate modulates apoptotic induction through regulation of the intrinsic cascade and resulting apoptosome formation in human malignant melanoma cells

Malignant melanoma is the most aggressive type of skin cancer with increasing incidence rates worldwide. On the other hand, watercress is a rich source of phenethyl isothiocyanate (PEITC), among others, which has been widely investigated for its anticancer properties against various cancers. In the present study, we evaluated the role of a watercress extract in modulating apoptotic induction in an in vitro model of human malignant melanoma consisting of melanoma (A375, COLO-679, COLO-800), non-melanoma epidermoid carcinoma (A431) and immortalized, non-tumorigenic keratinocyte (HaCaT) cells. Moreover, the chemical composition of the watercress extract was characterized through UPLC MS/MS and other analytical methodologies. In addition, cytotoxicity was assessed by the alamar blue assay whereas apoptosis was determined, initially, by a multiplex activity assay kit (measuring levels of activated caspases −3, −8 and −9) as well as by qRT-PCR for the identification of major genes regulating apoptosis. In addition, protein expression levels were evaluated by western immunoblotting. Our data indicate that the extract contains various phytochemicals (e.g. phenolics, flavonoids, pigments, etc.) while isothiocyanates (ITCs; especially PEITC) were the most abundant. In addition, the extract was shown to exert a significant time- and dose-dependent cytotoxicity against all malignant melanoma cell lines while non-melanoma and non-tumorigenic cells exhibited significant resistance. Finally, expression profiling revealed a number of genes (and corresponding proteins) being implicated in regulating apoptotic induction through activation of the intrinsic apoptotic cascade. Overall, our data indicate the potential of PEITC as a promising anti-cancer agent in the clinical management of human malignant melanoma.

1H and 31P Magnetic Resonance Spectroscopic Metabolomic Imaging: Assessing Mitogen-Activated Protein Kinase Inhibition in Melanoma.

The MAPK signaling pathway with BRAF mutations has been shown to drive the pathogenesis of 40-60% of melanomas. Inhibitors of this pathway's BRAF and MEK components are currently used to treat these malignancies. However, responses to these treatments are not always successful. Therefore, identifying noninvasive biomarkers to predict treatment responses is essential for personalized medicine in melanoma. Using noninvasive 1H magnetic resonance spectroscopy (1H MRS), we previously showed that BRAF inhibition reduces lactate and alanine tumor levels in the early stages of effective therapy and could be considered as metabolic imaging biomarkers for drug response. The present work demonstrates that these metabolic changes observed by 1H MRS and those assessed by 31P MRS are also found in preclinical human melanoma models treated with MEK inhibitors. Apart from 1H and 31P MRS, additional supporting in vitro biochemical analyses are described. Our results indicate significant early metabolic correlations with response levels to MEK inhibition in the melanoma models and are consistent with our previous study of BRAF inhibition. Given these results, our study supports the potential clinical utility of noninvasive MRS to objectively image metabolic biomarkers for the early prediction of melanoma's response to MEK inhibition.

Sphinganine recruits TLR4 adaptors in macrophages and promotes inflammation in murine models of sepsis and melanoma

After recognizing its ligand lipopolysaccharide, Toll-like receptor 4 (TLR4) recruits adaptor proteins to the cell membrane, thereby initiating downstream signaling and triggering inflammation. Whether this recruitment of adaptor proteins is dependent solely on protein-protein interactions is unknown. Here, we report that the sphingolipid sphinganine physically interacts with the adaptor proteins MyD88 and TIRAP and promotes MyD88 recruitment in macrophages. Myeloid cell-specific deficiency in serine palmitoyltransferase long chain base subunit 2, which encodes the key enzyme catalyzing sphingolipid biosynthesis, decreases the membrane recruitment of MyD88 and inhibits inflammatory responses in in vitro bone marrow-derived macrophage and in vivo sepsis models. In a melanoma mouse model, serine palmitoyltransferase long chain base subunit 2 deficiency decreases anti-tumor myeloid cell responses and increases tumor growth. Therefore, sphinganine biosynthesis is required for the initiation of TLR4 signal transduction and serves as a checkpoint for macrophage pattern recognition in sepsis and melanoma mouse models.

A laponite-based immunologically active gel delivery system for long-acting tumor vaccine

Traditional bolus vaccines typically require multiple doses, which complicates the vaccination process and may cause missed shots, leading to sub-optimal immunity and reduced vaccine effectiveness. Herein, a gel-based long-acting vaccine system with self-adjuvant properties based on laponite was constructed to simplify vaccination procedures and improve vaccine effectiveness. Firstly, the gel system could recruit multiple types of immune cells to form immune niches. Secondly, it could achieve sustained delivery of antigens to lymph nodes by active transport and passive drainage. Then, the gel system triggered the formation of a large number of germinal centers, which elicited enhanced and durable humoral immune responses, as well as strong cellular immune responses. As a result, it eventually showed good prophylactic and therapeutic effects in a variety of tumor models including melanoma, colorectal cancer and peritoneal metastasis models. By further combining the immunoadjuvant CpG ODN and cytokine IL-12, the effect of the gel-vaccine could be further enhanced. In a murine peritoneal metastasis model of colorectal carcinoma, a single administration of the gel-vaccine resulted in complete tumor eradication in 8/9 mice. In summary, this study developed an immunologically active gel-vaccine system. And as a robust and versatile vaccine platform, by loading different antigens and adjuvants, this gel-vaccine system is expected to realize its better therapeutic potential.

Localized in vivo gene editing of murine cancer-associated fibroblasts.

Discovering the role of fibroblasts residing in the tumor microenvironment (TME) requires controlled, localized perturbations because fibroblasts play critical roles in regulating immunity and tumor biology at multiple sites. Systemic perturbations can lead to unintended, confounding secondary effects, and methods to locally genetically engineer fibroblasts are lacking. To specifically investigate murine stromal cell perturbations restricted to the TME, we developed an adeno-associated virus (AAV)-based method to target any gene-of-interest in fibroblasts at high efficiency (>80%). As proof of concept, we generated single (sKO) and double gene KOs (dKO) of Osmr , Tgfbr2 , and Il1r1 in cancer-associated fibroblasts (CAFs) and investigated how their cell states and those of other cells of the TME subsequently change in mouse models of melanoma and pancreatic ductal adenocarcinoma (PDAC). Furthermore, we developed an in vivo knockin-knockout (KIKO) strategy to achieve long-term tracking of CAFs with target gene KO via knocked-in reporter gene expression. This validated in vivo gene editing toolbox is fast, affordable, and modular, and thus holds great potential for further exploration of gene function in stromal cells residing in tumors and beyond.

Therapeutic Anti-Tumor Efficacy of DC-Based Vaccines Targeting TME-Associated Antigens Is Improved When Combined with a Chemokine-Modulating Regimen and/or Anti-PD-L1.

We previously reported that dendritic cell (DC)-based vaccines targeting antigens expressed by tumor-associated vascular endothelial cells (VECs) and pericytes effectively control tumor growth in translational mouse tumor models. In the current report, we examined whether the therapeutic benefits of such tumor blood vessel antigen (TBVA)-targeted vaccines could be improved by the cotargeting of tumor antigens in the s.c. B16 melanoma model. We also evaluated whether combination vaccines incorporating anti-PD-L1 checkpoint blockade and/or a chemokine-modulating (CKM; IFNα + TLR3-L [rintatolimod] + Celecoxib) regimen would improve T cell infiltration/functionality in tumors yielding enhanced treatment benefits. We report that DC-peptide or DC-tumor lysate vaccines coordinately targeting melanoma antigens and TBVAs were effective in slowing B16 growth in vivo and extending survival, with superior outcomes observed for DC-peptide-based vaccines. Peptide-based vaccines that selectively target either melanoma antigens or TBVAs elicited a CD8+ T cell repertoire recognizing both tumor cells and tumor-associated VECs and pericytes in vitro, consistent with a treatment-induced epitope spreading mechanism. Notably, combination vaccines including anti-PD-L1 + CKM yielded superior therapeutic effects on tumor growth and animal survival, in association with the potentiation of polyfunctional CD8+ T cell reactivity against both tumor cells and tumor-associated vascular cells and a pro-inflammatory TME.

Engineered bacteria-mediated augmentation of sialoglycan degradation strategy and immunogenic chemotherapy to boost effective cancer therapy

Sialic acid, as the new immunosuppressive molecule, is positively correlated with tumor progression, metastasis, and drug resistance. Although targeting desialylation strategy has promising implications for regulating immunosuppressive environment and sensitizing chemotherapy, delivery strategies that integrate the amplifying effects of desialylation and immunogenic chemotherapy are still unexplored. Here, we engineered an Escherichia coli Nissle 1917 hybrid (SSE@ZIF-90/Oxa) to integrate the co-delivery of sialidase and immunogenic cell death inducers which were expected to boost strong antitumor response. In the tumor-hypoxic environment, engineered SSE bacteria exert hypoxia-responsive sialidase expression and periodic lysis to release sialidase, disrupting the sialoglycan-mediated immune suppression. ZIF-90/Oxa was hybridized on the surface of SSE to further induce ICD effect to recruit more infiltration of immune cells. In melanoma models in male mice, this SSE@ZIF-90/Oxa facilitated the co-delivery of sialidase and Oxa both deeply penetrated into and across tumor, suppressed the tumor, and prolonged the survival time of mice, by markedly increasing mature DCs, TNF-α+ CD8+ T cells, and M1 macrophages, while reducing PD-1+CD8+ T cells and Tregs, indicating effective activation of anti-tumor immune cells. Thus, our study provides an intelligent biohybrid therapeutic platform for boosting the combined effect of desialylation-based immunotherapy and immunogenic chemotherapy.

Biofabrication and Monitoring of a 3D Printed Skin Model for Melanoma.

There is an unmet need for in vitro cancer models that emulate the complexity of human tissues. 3D-printed solid tumor micromodels based on decellularized extracellular matrices (dECMs) recreate the biomolecule-rich matrix of native tissue. Herein a 3D in vitro metastatic melanoma model that is amenable for drug screening purposes and recapitulates features of both the tumor and the skin microenvironment is described. Epidermal, basement membrane, and dermal biocompatible inks are prepared by means of combined chemical, mechanical, and enzymatic processes. Bioink printability is confirmed by rheological assessment and bioprinting, and bioinks are subsequently combined with melanoma cells and dermal fibroblasts to build complex 3D melanoma models. Cells are tracked by confocal microscopy and surface-enhanced Raman spectroscopy (SERS) mapping. Printed dECMs and cell tracking allow modeling of the initial steps of metastatic disease, and may be used to better understand melanoma cell behavior and response to drugs.

Development of a prognostic risk model of uveal melanoma based on N7-methylguanosine-related regulators

BackgroundUveal melanoma (UVM) stands as the predominant type of primary intraocular malignancy among adults. The clinical significance of N7-methylguanosine (m7G), a prevalent RNA modifications, in UVM remains unclear.MethodsPrimary information from 80 UVM patients were analyzed as the training set, incorporating clinical information, mutation annotations and mRNA expression obtained from The Cancer Genome Atlas (TCGA) website. The validation set was carried out using Gene Expression Omnibus (GEO) database GSE22138 and GSE84976. Kaplan–Meier and Cox regression of univariate analyses were subjected to identify m7G-related regulators as prognostic genes.ResultA prognostic risk model comprising EIF4E2, NUDT16, SNUPN and WDR4 was established through Cox regression of LASSO. Evaluation of the model’s predictability for UVM patients’ prognosis by Receiver Operating Characteristic (ROC) curves in the training set, demonstrated excellent performance Area Under the Curve (AUC) > 0.75. The high-risk prognosis within the TCGA cohort exhibit a notable worse outcome. Additionally, an independent correlation between the risk score and overall survival (OS) among UVM patients were identified. External validation of this model was carried out using the validation sets (GSE22138 and GSE84976). Immune-related analysis revealed that patients with high score of m7G-related risk model exhibited elevated level of immune infiltration and immune checkpoint gene expression.ConclusionWe have developed a risk prediction model based on four m7G-related regulators, facilitating effective estimate UVM patients’ survival by clinicians. Our findings shed novel light on essential role of m7G-related regulators in UVM and suggest potential novel targets for the diagnosis, prognosis and therapy of UVM.

Exploring the Surface: Sampling of Potential Skin Cancer Biomarkers Kynurenine and Tryptophan, Studied on 3D Melanocyte and Melanoma Models.

Early detection of cancer via biomarkers is vital for improving patient survival rates. In the case of skin cancers, low-molecular-weight biomarkers can penetrate the skin barrier, enabling non-invasive sampling at an early stage. This study focuses on detecting tryptophan (Trp) and kynurenine (Kyn) on the surface of reconstructed 3D melanoma and melanocyte models. This is examined in connection with IDO-1 and IL-6 expression in response to IFN-γ or UVB stimulation, both crucial factors of the melanoma tumor microenvironment (TME). Using a polystyrene scaffold, full-thickness human skin equivalents containing fibroblasts, keratinocytes, and melanocytes or melanoma cells were developed. The samples were stimulated with IFN-γ or UVB, and Trp and Kyn secretion was measured using HPLC-PDA and HPLC-MS. The expression of IDO-1 and IL-6 was measured using RT-qPCR. Increased Trp catabolism to Kyn was observed in IFN-γ-stimulated melanoma and melanocyte models, along with higher IDO-1 expression. UVB exposure led to significant changes in Kyn levels but only in the melanoma model. This study demonstrates the potential of skin surface Trp and Kyn monitoring to capture TME metabolic changes. It also lays the groundwork for future in vivo studies, aiding in understanding and monitoring skin cancer progression.

Oncolytic Peptide-Nanoplatform Drives Oncoimmune Response and Reverses Adenosine-Induced Immunosuppressive Tumor Microenvironment.

The application of oncolytic peptides has become a powerful approach to induce complete and long-lasting remission in multiple types of carcinomas, as affirmed by the appearance of tumor-associated antigens and adenosine triphosphate (ATP) in large quantities, which jumpstarts the cancer-immunity cycle. However, the ATP breakdown product adenosine is a significant contributor to forming the immunosuppressive tumor microenvironment, which substantially weakens peptide-driven oncolytic immunotherapy. In this study, a lipid-coated micelle (CA@TLM) loaded with a stapled oncolytic peptide (PalAno) and an adenosine 2A receptor (A2AR) inhibitor (CPI-444) is devised to enact tumor-targeted oncolytic immunotherapy and to overcome adenosine-mediated immune suppression simultaneously. The CA@TLM micelle accumulates in tumors with high efficiency, and the acidic tumor microenvironment prompts the rapid release of PalAno and CPI-444. Subsequently, PalAno induces swift membrane lysis of tumor cells and the release of antigenic materials. Meanwhile, CPI-444 blocks the activation of the immunosuppressive adenosine-A2AR signaling pathway. This combined approach exhibits pronounced synergy at stalling tumor growth and metastasis in animal models for triple-negative breast cancer and melanoma, providing a novel strategy for enhanced oncolytic immunotherapy.

Differential impact of genetic deletion of TIGIT or PD-1 on melanoma-specific T-lymphocytes

ABSTRACT Immune checkpoint (IC) blockade and adoptive transfer of tumor-specific T-cells (ACT) are two major strategies to treat metastatic melanoma. Their combination can potentiate T-cell activation in the suppressive tumor microenvironment, but the autoimmune adverse effects associated with systemic injection of IC blockers persist with this strategy. ACT of tumor-reactive T-cells defective for IC expression would overcome this issue. For this purpose, PD-1 and TIGIT appear to be relevant candidates, because their co-expression on highly tumor-reactive lymphocytes limits their therapeutic efficacy within the tumor microenvironme,nt. Our study compares the consequences of PDCD1 or TIGIT genetic deletion on anti-tumor properties and T-cell fitness of melanoma-specific T lymphocytes. Transcriptomic analyses revealed down-regulation of cell cycle-related genes in PD-1KO T-cells, consistent with biological observations, whereas proliferative pathways were preserved in TIGITKO T-cells. Functional analyses showed that PD-1KO and TIGITKO T-cells displayed superior antitumor reactivity than their wild-type counterpart in vitro and in a preclinical melanoma model using immunodeficient mice. Interestingly, it appears that TIGITKO T-cells were more effective at inhibiting tumor cell proliferation in vivo, and persist longer within tumors than PD-1KO T-cells, consistent with the absence of impact of TIGIT deletion on T-cell fitness. Taken together, these results suggest that TIGIT deletion, over PD-1 deletion, in melanoma-specific T-cells is a compelling option for future immunotherapeutic strategies.