B16F10 Melanoma Research Articles

VE-cadherin junction dynamics in initial lymphatic vessels promotes lymph node metastasis.

The endothelial junction component vascular endothelial (VE)-cadherin governs junctional dynamics in the blood and lymphatic vasculature. Here, we explored how lymphatic junction stability is modulated by elevated VEGFA signaling to facilitate metastasis to sentinel lymph nodes. Zippering of VE-cadherin junctions was established in dermal initial lymphatic vessels after VEGFA injection and in tumor-proximal lymphatics in mice. Shape analysis of pan-cellular VE-cadherin fragments revealed that junctional zippering was accompanied by accumulation of small round-shaped VE-cadherin fragments in the lymphatic endothelium. In mice expressing a mutant VEGFR2 lacking the Y949 phosphosite (Vegfr2 Y949F/Y949F ) required for activation of Src family kinases, zippering of lymphatic junctions persisted, whereas accumulation of small VE-cadherin fragments was suppressed. Moreover, tumor cell entry into initial lymphatic vessels and subsequent metastatic spread to lymph nodes was reduced in mutant mice compared with WT, after challenge with B16F10 melanoma or EO771 breast cancer. We conclude that VEGFA mediates zippering of VE-cadherin junctions in initial lymphatics. Zippering is accompanied by increased VE-cadherin fragmentation through VEGFA-induced Src kinase activation, correlating with tumor dissemination to sentinel lymph nodes.

Development of Recombinant Oncolytic rVSV-mIL12-mGMCSF for Cancer Immunotherapy.

Anti-cancer therapy based on oncolytic viruses (OVs) is a targeted approach that takes advantage of OVs' ability to selectively infect and replicate in tumor cells, activate the host immune response, and destroy malignant cells over healthy ones. Vesicular stomatitis virus (VSV) is known for its wide range of advantages: a lack of pre-existing immunity, a genome that is easily amenable to manipulation, and rapid growth to high titers in a broad range of cell lines, to name a few. VSV-induced tumor immunity can be enhanced by the delivery of immunostimulatory cytokines. The targeted cytokine delivery to tumors avoids the significant toxicity associated with systemic delivery while also boosting the immune response. To demonstrate this enhanced effect on both tumor growth and survival, a novel recombinant VSV (rVSV)-mIL12-mGMCSF, co-expressing mouse IL-12 (interleukin-12) and GM-CSF (granulocyte-macrophage colony-stimulating factor), was tested alongside rVSV-dM51-GFP (rVSV-GFP) that was injected intratumorally in a syngeneic in vivo C57BL/6 mouse model infused subcutaneously with B16-F10 melanoma cells. The pilot study tested the effect of two viral injections 4 days apart and demonstrated that treatment with the two rVSVs resulted in partial inhibition of tumor growth (TGII of around 40%) and an increased survival rate in animals from the treatment groups. The effect of the two VSVs on immune cell populations will be investigated in future in vivo studies with an optimized experimental design with multiple higher viral doses, as a lack of this information presents a limitation of this study.

Infection of tumor cells with Salmonella typhimurium mimics immunogenic cell death and elicits tumor-specific immune responses.

Some properties of Salmonella-infected cells overlap with immunogenic cell death. In this study, we demonstrated that intracellular infection of melanoma with Salmonella typhimurium induced high immunogenicity in melanoma cells, leading to antitumor effects with melanoma-antigen-specific T-cell responses. Murine B16F10 melanoma cells were infected with tdTomato-expressing attenuated S. typhimurium (VNP20009; VNP-tdT), triggering massive cell vacuolization. VNP-tdT-infected B16F10 cells were phagocytosed efficiently, which induced the activation of antigen-presenting cells with CD86 expression in vitro. Subcutaneous coimplantation of uninfected and VNP-tdT-infected B16F10 cells into C57BL/6 mice significantly suppressed tumor growth compared with the implantation of uninfected B16F10 cells alone. Inoculation of mice with VNP-tdT-infected B16F10 cells elicited the proliferation of melanoma-antigen (gp100)-specific T cells, and it protected the mice from the second tumor challenge of uninfected B16F10 cells. These results suggest that Salmonella-infected tumor cells acquire effective adjuvanticity, leading to ideal antitumor immune responses.

Ethyl Acetate Fraction from a Catalpa ovata G. Don Extract Inhibits ɑ-MSH-Induced Melanogenesis through the cAMP/CREB Pathway.

The whitening effect of reducing skin pigmentation is one of the most important goals of cosmetics. The purpose of this study was to determine whether Catalpa ovata extract and its fractions have potential as natural skin-lightening agents. Initially, we screened various fractions of Catalpa ovata extract using an in vitro antioxidant assay. Then, the inhibitory effects of C. ovata extract and its fraction on melanogenesis and the related mechanisms were investigated in B16F1 melanoma cells. The results showed that the ethyl acetate fraction (EF) from C. ovata extract markedly inhibited melanin synthesis in a dose-dependent manner at non-toxic concentrations. Furthermore, EF downregulated both the protein and mRNA levels of tyrosinase, which is a specific enzyme that catalyzes the conversion of tyrosine into melanin. We also found that EF decreased the microphthalmia-associated transcription factor (MITF) at the protein and mRNA levels. EF increased the phosphorylation of ERK and suppressed the phosphorylation of JNK and p38 in ɑ-MSH-induced B16F1 cells. These results indicate that EF can regulate the MAPK pathway. In addition, EF has an anti-melanogenic effect via the downregulation of intracellular cyclic-AMP (cAMP). Nineteen major compounds of EF were identified using LC-MS/MS. Taken together, these results suggest that EF may be a potential anti-melanogenic agent for use in skin-whitening cosmetics and in topical treatments for hyperpigmentation disorders.

Anti-skin ageing activities of rice (Oryza sativa) bran soft and hard waxes in cultured skin cells.

Rice (Oryza sativa) bran waxes, the by-products of rice bran oil manufacturing, are widely used as inactive components in several preparations. Nevertheless, the function of rice bran waxes against skin ageing has never been reported. This study aimed to investigate thermal property and fatty acid profile of rice bran waxes, including rice bran soft (RBS) and hard (RBH) waxes, and the activities against skin ageing in cultured skin cells. Thermal property and fatty acid profile of rice bran waxes were analysed by differential scanning calorimetry and gas chromatography-mass spectrometry, respectively. The cytotoxicity assay of waxes was performed in B16F10 melanoma cells, human skin fibroblasts and co-culture cells of HaCaT cells and human skin fibroblasts. The non-cytotoxic concentrations of waxes were evaluated for their activities against skin ageing, including melanogenesis assay, antioxidant activity, collagen content analysis, matrix metalloproteinase-1 and matrix metalloproteinase-2 inhibitory assay and anti-inflammatory activity. Thermal property indicated the endotherm peaks with melting temperatures at 40.89 ± 0.27°C and 69.64 ± 0.34°C for RBS and RBH, respectively. The main fatty acids in RBS were oleic (31.68 ± 0.75%) and linoleic acids (27.19 ± 0.40%), whereas those in RBH were palmitic (36.24 ± 1.08%) and stearic acids (35.21 ± 4.51%). The cytotoxicity assay in single cells and co-culture cells showed the non-cytotoxicity of RBS (0.0001-1 mg/mL) and RBH (0.0001-0.1 mg/mL). The anti-skin ageing activities of 1 mg/mL RBS and 0.1 mg/mL RBH included the melanogenesis inhibition by suppression of tyrosinase and tyrosinase-related protein-2 enzymes, the antioxidant activity by cellular protection against cell damage and cell death, the collagen stimulation, the matrix metalloproteinase-1 and matrix metalloproteinase-2 suppression and the anti-inflammation. The study results suggest that RBS and RBH can potentially be applied as the functional ingredients in formulations against skin ageing as well as provide the superior benefit on skin moisturization.

SON-1210 - a novel bifunctional IL-12 / IL-15 fusion protein that improves cytokine half-life, targets tumors, and enhances therapeutic efficacy.

The potential synergy between interleukin-12 (IL-12) and IL-15 holds promise for more effective solid tumor immunotherapy. Nevertheless, previous clinical trials involving therapeutic cytokines have encountered obstacles such as short pharmacokinetics, limited tumor microenvironment (TME) targeting, and substantial systemic toxicity. To address these challenges, we fused single-chain human IL-12 and native human IL-15 in cis onto a fully human albumin binding (FHAB) domain single-chain antibody fragment (scFv). This novel fusion protein, IL12-FHAB-IL15 (SON-1210), is anticipated to amplify the therapeutic impact of interleukins and combination immunotherapies in human TME. The molecule was studied in vitro and in animal models to assess its pharmacokinetics, potency, functional characteristics, safety, immune response, and efficacy. SON-1210 demonstrated robust binding affinity to albumin and exhibited the anticipated in vitro activity and tumor model efficacy that might be expected based on decades of research on native IL-12 and IL-15. Notably, in the B16F10 melanoma model (a non-immunogenic, relatively "cold" tumor), the murine counterpart of the construct, which had mouse (m) and human (h) cytokine sequences for the respective payloads (mIL12-FHAB-hIL15), outperformed equimolar doses of the co-administered native cytokines in a dose-dependent manner. A single dose caused a marked reduction in tumor growth that was concomitant with increased IFNγ levels; increased Th1, CTL, and activated NK cells; a shift in macrophages from the M2 to M1 phenotype; and a reduction in Treg cells. In addition, a repeat-dose non-human primate (NHP) toxicology study displayed excellent tolerability up to 62.5 µg/kg of SON-1210 administered three times, which was accompanied by the anticipated increases in IFNγ levels. Toxicokinetic analyses showed sustained serum levels of SON-1210, using a sandwich ELISA with anti-IL-15 for capture and biotinylated anti-IL-12 for detection, along with sustained IFNγ levels, indicating prolonged kinetics and biological activity. Collectively, these findings support the suitability of SON-1210 for patient trials in terms of activity, efficacy, and safety, offering a promising opportunity for solid tumor immunotherapy. Linking cytokine payloads to a fully human albumin binding domain provides an indirect opportunity to target the TME using potent cytokines in cis that can redirect the immune response and control tumor growth.

Treatment with Viscum album potencies induces reduction of MCP-1 and ROs levels in murine melanoma cells.

Melanoma is responsible for most skin cancer deaths. It has a high metastatic potential, complex treatment, and a hopeless prognosis. Viscum album extract (Va) is a complementary treatment in cancer, with in vitro and in vivo cytotoxic effects on different tumor types. However, its ultra-diluted homeopathic preparations' effects and mechanisms on cancer cells need further study. Thus, the aim of present work was to investigate cytokine profile and ROs production of melanoma cells after treatment with ultradiluted VA collected from host tree Abies alba (VaAa) and Quercus rubur (VaQr)during Swiss summer. For this, potencies of Va in different homeopathic scales were prepared using mother tincture (MT) from VaAa and VaQr submitted to homeopathic ethanolic maceration and then a subsequent dynamization process. Then, B16F0 melanoma cells cultivated in 24-well plates were treated with MT or 12DH, 200CH or 5LM potencies of VaAa or VaQr for 24, 48 or 72 hours. After these periods, culture supernatants were collected to assess the levels of MCP-1, INF-γ, TNF-α, IL-10, IL-12p70 and IL-6 cytokines and, ROs production. Results demonstrated significant reduction of MCP-1 and ROs levels in B16F10 melanoma cells treated with Va (VaQr, preferentially) at 12DH or 5LM potencies when compared to untreated or carboplatin-treated cells. In conclusion, we demonstrated for the first time, the in vitro effects of Va potencies prepared in different homeopathic scales in relation to cytokines and ROs production in cell melanoma cells.

Exposure to phenols reduces melanogenesis in B16F10 cells and zebrafish

Phenols, ubiquitous environmental contaminants found in water, soil, and air, pose risks to organisms even at minimal concentrations, and many are classified as hazardous pollutants. Skin pigmentation is a natural shield against ultraviolet-induced DNA damage and oxidative stress, pivotal in reducing skin cancer incidences. Studies on B16F10 melanoma cells and zebrafish offer valuable insights into potential therapeutic avenues for melanoma in the context of phenol exposure. Upon phenol treatment, there was a marked decrease in melanin content and melanogenesis-associated protein expression, such as tyrosinase and the microphthalmia-associated transcription factor (MITF) in these melanoma cells. Additionally, phenols led to diminished p38 phosphorylation, amplified extracellular signal-regulated kinase (ERK) phosphorylation, and curtailed melanin expression in zebrafish. These observations underscore the detrimental impact of phenols on melanogenesis and propose a mechanism of action centered on the ERK/p38 signaling pathway. Consequently, our data spotlight the adverse effects of phenols on melanogenesis."

Assessment of the photocatalytic performance and cytotoxic effects of barium sulfate nanoparticles synthesized with a one-step hydrothermal method

The article introduces a novel method for the production of barium sulfate nanoparticles (BaSO4-NPs), which was executed utilizing a particular methodology that entailed the reaction between barium chloride and sodium sulfate. FT-IR, PXRD, UV–Vis, FESEM/EDX, TEM, and PSA analyses were employed to approve the fabrication of BaSO4-NPs. The XRD pattern showed that the NPs had a higher occurrence of the orthorhombic phase with the Pnma space group. Furthermore, the synthesis of BaSO4-NPs was confirmed utilizing FTIR analysis, which displayed their strong bands. The FESEM pictures of BaSO4-NPs exhibited spherical morphology and uniform distribution. The proficiency of photocatalysis by BaSO4-NPs was evaluated in the degradation of Methylene Blue (MB) dye. After 150 min, the observed degradation percentage was 89%. In the current research, the cytotoxicity of the BaSO4-NPs was studied on normal mouse fibroblast NIH3T3 cell and cancer mouse melanoma B16F0 cell lines and the IC50 value was 823.8 µg/mL for cancer B16F0 cells and the results revealed a slight reduction in the viability of cancer cells compared to normal cells.

Impact of cell-free dna from blood serum of mice with metastatic melanoma on the enhancement of oncogenic properties of melanoma cells

Currently, a significant increase in the levels of circulating cell-free DNA (cfDNA) in the blood of patients is a generally recognized marker of the development of oncological diseases. Although tumor-associated cfDNA has been well studied, its biological functions remain unclear. In this work, we investigated the effect of cfDNA isolated from the blood serum of mice with B16-F10 metastatic melanoma on the properties of B16-F10 melanoma cells in vitro. It was found that the profile of cfDNA isolated form blood serum of mice with melanoma differs significantly from the cfDNA isolated from blood serum of healthy mice, and is similar concerning the abundance of oncogenes and mobile genetic elements (MGE) to the genomic DNA of B16 cells. It was shown that cfDNA of mice with melanoma penetrated into B16 cells, resulting to the increase in the abundance of oncogenes and MGE fragments, and caused the 5-fold increased mRNA level of the secreted DNase Dnase1l3 and a slight increase of the mRNA level of the Jun, Fos, Ras, and Myc oncogenes. cfDNA of healthy mice caused increasing of mRNA level of the intracellular regulatory DNase EndoG and a 4-fold increase of mRNA level of Fos and Ras oncogenes, which are well-known triggers of a large number of signal cascades, from apoptosis inhibition to increased tumor cell proliferation. Thus, it is obvious that the circulating cfDNA of tumor origin is able to penetrate into cells and, despite the fact that no changes were found in the level of viability and migration activity of tumor cells, cfDNA, even with a single exposure, can cause changes at the cellular level that increase the oncogenicity of recipient cells.

BDE-209 exposure in murine melanoma (B16–F1) cells modulates tumor malignancy and progression in vivo

Melanoma is a type of skin cancer considered aggressive due to its high metastatic ability and rapid progression to other tissues and organs. BDE-209 (2,2′,3,3′,4,4′,5,5′,6,6′-decabromodiphenyl ether) is an additive used as a flame retardant and classified as a persistent organic pollutant that has a high bioaccumulation capacity due to its lipophilic nature. This substance has already been detected in rivers, air, soil, plants and even in different human biological samples, such as plasma, umbilical cord blood and breast milk, revealing a great concern to human populations. Thus, in the current study we investigated whether prior exposure of murine melanoma B16–F1 cells to BDE-209 modulates in vivo progression and malignancy of melanoma. B16–F1 cells were cultured and exposed in vitro to BDE-209 (0.01, 0.1 e 1 nM) for 15 days and then inoculated, via caudal vein, in C57BL/6 mice for experimental metastasis analysis after 20 days. Inoculation of BDE-209-exposed cells resulted in 82% increase of metastasis colonized area in the lungs of mice, downregulation of tumor suppressors genes, such as Timp3 and Reck, decrease of lipid peroxidation and increase of systemic and local inflammatory response. These findings are related to melanoma progression. Additionally, the histopathological analysis revealed greater number of focal points of metastases in the lungs and invasiveness of metastases to the mice brain (89%). The results showed that exposure to BDE-209 may alter the phenotype of B16–F1 cells, worsening their metastatic profile. Current data showed that BDE-209 may interfere with the prognosis of melanoma by modulating cells with less invasiveness capacity to a more aggressive profile.

Synergistic impact of arbutin and kaempferol-7-O-α-l-rhamnopyranoside from Nephelium lappaceum L. on whitening efficacy and stability of cosmetic formulations

Four flavonoid glycosides, namely quercetin-3-O-rhamnoside (1), kaempferol-3-O-β-d-glucopyranosyl (2), kaempferol-7-O-α-l-rhamnopyranoside (3), and kaempferol-3-O-β-d-glucopyranosyl-7-O-α-l-rhamnopyranoside (4), from Nephelium lappaceum L. seeds were evaluated for their efficacy against melanin inhibition in B16F10 melanoma cells and tyrosinase inhibition. Among them, kaempferol-7-O-α-l-rhamnopyranoside (3) displayed the highest potency in both activities without any significant cytotoxicity. The combination of compound 3 and arbutin in specific proportions demonstrated a synergistic effect (CI < 1) in inhibiting melanin production in B16F10 cells and tyrosinase inhibition. Additionally, a cosmetic formulation containing compound 3 and arbutin as active ingredients exhibited favorable stability under accelerated storage conditions. Quantitative analysis indicated that compound 3 and arbutin levels in the formulation were above 90% after one month of storage. Determination of the formulation's shelf life using the Q10 method, estimating it to be around 5.2 months from the date of manufacture. The synergy between arbutin and kaempferol-7-O-α-l-rhamnopyranoside (3) extracted from N. lappaceum substantially enhances both the whitening effectiveness and the stability of cosmetic formulations.

Aerobic exercise training mitigates tumor growth and cancer-induced splenomegaly through modulation of non-platelet platelet factor 4 expression

Exercise training reduces the incidence of several cancers, but the mechanisms underlying these effects are not fully understood. Exercise training can affect the spleen function, which controls the hematopoiesis and immune response. Analyzing different cancer models, we identified that 4T1, LLC, and CT26 tumor-bearing mice displayed enlarged spleen (splenomegaly), and exercise training reduced spleen mass toward control levels in two of these models (LLC and CT26). Exercise training also slowed tumor growth in melanoma B16F10, colon tumor 26 (CT26), and Lewis lung carcinoma (LLC) tumor-bearing mice, with minor effects in mammary carcinoma 4T1, MDA-MB-231, and MMTV-PyMT mice. In silico analyses using transcriptome profiles derived from these models revealed that platelet factor 4 (Pf4) is one of the main upregulated genes associated with splenomegaly during cancer progression. To understand whether exercise training would modulate the expression of these genes in the tumor and spleen, we investigated particularly the CT26 model, which displayed splenomegaly and had a clear response to the exercise training effects. RT-qPCR analysis confirmed that trained CT26 tumor-bearing mice had decreased Pf4 mRNA levels in both the tumor and spleen when compared to untrained CT26 tumor-bearing mice. Furthermore, exercise training specifically decreased Pf4 mRNA levels in the CT26 tumor cells. Aspirin treatment did not change tumor growth, splenomegaly, and tumor Pf4 mRNA levels, confirming that exercise decreased non-platelet Pf4 mRNA levels. Finally, tumor Pf4 mRNA levels are deregulated in The Cancer Genome Atlas Program (TCGA) samples and predict survival in multiple cancer types. This highlights the potential therapeutic value of exercise as a complementary approach to cancer treatment and underscores the importance of understanding the exercise-induced transcriptional changes in the spleen for the development of novel cancer therapies.

Paris polyphylla saponins II inhibits invasive, migration and epithelial-mesenchymal transition of melanoma cells through activation of autophagy

Malignant melanoma is a kind of malignant tumor derived from normal epidermal melanocytes or original nevus cells. It has a high degree of malignancy, rapid progress, dangerous condition, and poor prognosis. In recent years, the innovation of traditional Chinese medicine has broadened the scope and effect of tumor treatment. It is a hotspot and breakthrough to find new anti-tumor invasion and migration drugs from natural plants or traditional Chinese medicine. This study explored the role of PPII in promoting autophagy to inhibit EMT of melanoma cells, the role of the PI3K/Akt signaling pathway in the invasion and migration of melanoma cells induced by PPII. We found that PPII effectively inhibited the proliferation, invasion and migration of melanoma B16 and B16F10 in vitro, and induced autophagy. We also established the xenograft tumor and metastatic tumor model of C57BL/6 mice with B16F10 cells. Results showed that PPII effectively inhibited the growth of transplanted tumors, induced autophagy and inhibited the expression level of EMT related protein; Metastasis experiment showed that PPII inhibited the invasion and migration of B16F10, the effect of inhibiting lung metastasis is the most significant. Further mechanism studies showed that the inhibition of PPII on melanoma invasion and migration is related to its induction of autophagy and then inhibition of EMT.

Metabolic tagging of extracellular vesicles and development of enhanced extracellular vesicle based cancer vaccines

As key mediators of cellular communication, extracellular vesicles (EVs) have been actively explored for diagnostic and therapeutic applications. However, effective methods to functionalize EVs and modulate the interaction between EVs and recipient cells are still lacking. Here we report a facile and universal metabolic tagging technology that can install unique chemical tags (e.g., azido groups) onto EVs. The surface chemical tags enable conjugation of molecules via efficient click chemistry, for the tracking and targeted modulation of EVs. In the context of tumor EV vaccines, we show that the conjugation of toll-like receptor 9 agonists onto EVs enables timely activation of dendritic cells and generation of superior antitumor CD8+ T cell response. These lead to 80% tumor-free survival against E.G7 lymphoma and 33% tumor-free survival against B16F10 melanoma. Our study yields a universal technology to generate chemically tagged EVs from parent cells, modulate EV-cell interactions, and develop potent EV vaccines.

Block Copolymer‐Stabilized Metal–Organic Framework Hybrids Loading Pd Nanoparticles Enable Tumor Remission Through Near‐Infrared Photothermal Therapy

Metal–organic frameworks (MOFs), such as the magnetic resonance imaging‐fit MIL‐100 based on Fe, are gaining significant attention as versatile theranostics with high‐loading capability. Moreover, as MOFs can be engineered to target tumors, there is much interest in applying them for precise pin‐point treatment of cancer. Herein, Pd nanoparticles within MIL‐100(Fe) are generated to create MOFs with remarkable photothermal conversion properties for cancer therapy. The Pd‐loaded MIL‐100(Fe) (Pd@MIL‐100(Fe)) are stabilized with biocompatible block copolymers to generate MOFs with PEGylated surfaces. This is achieved by directly mixing poly(ethylene glycol)‐poly(L‐aspartic acid) (PEG‐p(Asp)) or dopamine‐modified PEG‐p(Asp) (PEG‐p(Asp‐Dopa)) block copolymers with the MOFs in aqueous conditions. The resulting block copolymer‐stabilized MOF hybrids are stable in physiological conditions. Particularly, the Pd@MIL‐100(Fe)/PEG‐p(Asp‐Dopa) hybrids show enhanced blood circulation and increased accumulation in B16F10 melanoma. Furthermore, when irradiated with 808 nm light, the Pd@MIL‐100(Fe)/PEG‐p(Asp‐Dopa) hybrids rapidly increase the temperature to 50 °C, enabling tumor remission. The surface‐stabilized Pd@MIL‐100(Fe)/polymer hybrids open viable opportunities for innovating MOF/polymer hybrid‐based approaches for drug delivery.

Abstract A005: Synergistically Engaging a b-Selective IL-2 Agonist with PD1/PDL-1 Blockade in a Bifunctional Superkine, MDNA223

Abstract Introduction: Combination treatment with a long-acting IL-2 superkine (MDNA109FEAA-Fc; aka MDNA19) and anti-PD1 or anti-CTLA4 has shown clear evidence of synergy in blunting tumor growth. To further leverage this synergism, we designed MDNA223, a BiSKIT (Bifunctional SuperKine for ImmunoTherapy) comprising MDNA109FEAA with enhanced affinity for IL-2Rb and no binding to IL-2Ra, fused to either human or mouse anti-PD1. We present in vitro and in vivo characterization of MDNA223 and demonstrate its enhanced IL-2R agonism and effective PD1/PDL-1 blockade in multiple “hot” and “cold” syngeneic tumor models. Experimental Procedure: In vitro studies included human PBMCs and cell-based reporter assays. In vivo studies were conducted in CT26, B16F10, E0771 and TRAMP-C1 tumor models by intraperiotonneal (IP) administration. Intratumoral treatment was performed in a CT26 bilateral tumor model with or without a STING agonist. Analysis of tumor infiltrating lymphocytes (TILs) was performed using flow cytometry. Results: In studies with human PBMCs, MDNA223 showed increased p-STAT5 signaling in CD8+ T cells and reduced activity in Tregs compared to rhIL-2, consistent with its enhanced receptor selectivity. MDNA223 and anti-PD1 were similarly potent at PD1/PDL-1 blockade in a cell-based reporter assay. In mice, MDNA223 induced proliferation (based on Ki67 expression) and expansion of peripheral CD8+ T cells for over 7 days. IP treatment with MDNA223 resulted in significantly greater inhibition of CT26, B16F10, E0771 and TRAMP-C1 tumor growth when compared to combination of MDNA19 with anti-PD1 at equimolar dosage. This observation is consistent with the proposed cis-binding mechanism of MDNA223 to both, IL-2R and PD1 on the same effector immune cell. In a bilateral CT26 tumor model, IT treatment with MDNA223 not only inhibited the treated tumor but also exhibited an abscopal effect by slowing growth of the untreated tumor implanted on the opposite flank. TILs analysis of B16F10 melanoma following a single dose of MDNA223 showed increased infiltration of CD8+ T cells over Tregs compared to single agent treatment with MDNA19 or anti-PD1 as well as their combination. Treatment with MDNA223 also resulted in a larger population of functional effector CD8+ T cells expressing low levels of the Tim-3 exhaustion marker and high levels of the cytotoxic Granzyme-B protease. Conclusion: MDNA223 demonstrated superior therapeutic activity over the combination of MDNA19 and anti-PD1 in both immunologically ‘hot’ (CT26) and ‘cold’ (B16F10, E0771 and TRAMP-C1) tumor models. Additional studies with variants of MDNA223 capable of tumor selective targeting and/or conditional activation within the tumor micro-environment are in progress to maximize activity at the tumor site with tunable peripheral stimulation. Citation Format: Aanchal Sharma, Minh D To, Hardeep Kataria, Qian Liu, Rosemina Merchant, Fahar Merchant. Synergistically Engaging a b-Selective IL-2 Agonist with PD1/PDL-1 Blockade in a Bifunctional Superkine, MDNA223 [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr A005.

Abstract B001: Harnessing invariant natural killer T cell cytotoxicity for cancer therapy

Abstract Cytotoxic cells are major effectors in adoptive cell transfer (ACT) cancer therapies. Although autologous conventional T cells are commonly used in ACT therapies, other types of cells, such as invariant natural killer T (iNKT) cells, have become attractive for the design of off-the-shelf ACT therapies. Unlike conventional T cells, iNKT cells react to glycolipid antigens presented by CD1d, a non-polymorphic MHC I-like molecule. Based on their cytokine profile and transcription factor expression, three main helper iNKT subsets have been described in mice: iNKT1, iNKT2, and iNKT17. In addition, the cytotoxic response of iNKT cells has been reported, but this function is less characterized. In a previous transcriptomic study, we have shown that the iNKT1 subset is heterogeneous and it comprises two main clusters: iNKT1b, enriched in genes related to T helper 1, and iNKT1c, characterized by a cytotoxic gene profile. Here, we demonstrated that iNKT1c cells exhibit higher in vitro specific killing activity against EL4 tumor cells compared to iNKT1b cells. We identified IL-15 as one of the factors that boosts the cytotoxic functions of iNKT1c cells. Moreover, adoptive cell transfer of iNKT1c cells in mice previously injected with B16F10 melanoma cells resulted in reduced metastatic lung nodules. We also explored the presence of a distinct cytotoxic iNKT cell subset in humans by spectral flow cytometry and clustering analysis. From PBMCs of healthy donors, we identified a population of CD57+Tbethi iNKT cells that show a cytotoxic profile. Interestingly, this population can also be found infiltrating tumors in lung cancer patients. This study unraveled bona fide cytotoxic iNKT cells and provides insights for the design of future cancer therapies. Citation Format: Carolina de Amat Herbozo, Stephanie Wong, Meggie Kuypers, Jessica Matthews, Thomas Baranek, Sarah Crome, Adrian Sacher, Christophe Paget, Thierry Mallevaey. Harnessing invariant natural killer T cell cytotoxicity for cancer therapy [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Tumor Immunology and Immunotherapy; 2023 Oct 1-4; Toronto, Ontario, Canada. Philadelphia (PA): AACR; Cancer Immunol Res 2023;11(12 Suppl):Abstract nr B001.

Higher N2 gas-flow rate ratios in O2- and Ar-containing feed gas mixtures enhance the cytotoxic effects of radical-activated medium against murine melanoma B16F10 cells

Radical-activated medium (RAM), produced from cold atmospheric plasma irradiation of liquid cell culture medium, is of significant interest for its potential application in cancer treatment. In this study, murine melanoma B16F10 cells were used to examine the anticancer activity of RAMs generated from various plasma feed gas mixtures. RAMs generated from plasma feed gases containing O2 decreased cell viability, which was enhanced by N2. Mitochondrial membrane potentials decreased, whereas caspase-9 and caspase-3 activity increased, suggesting an induction of mitochondria-mediated apoptosis. Interestingly, caspase-3 activity was enhanced when N2 was present in the plasma feed gas. Moreover, only RAM generated from plasma feed gases containing N2 and O2 activated caspase-12 and c-Jun N-terminal kinase, which suggests an induction of endoplasmic reticulum-mediated apoptosis. Overall, the results suggest that N2 in the feed gas during RAM production enhances its anticancer activity by triggering both endoplasmic reticulum and mitochondria-mediated cell death.

Aldehyde dehydrogenase 2-mediated aldehyde metabolism promotes tumor immune evasion by regulating the NOD/VISTA axis

BackgroundAldehyde dehydrogenase 2 (ALDH2) is a crucial enzyme involved in endogenous aldehyde detoxification and has been implicated in tumor progression. However, its role in tumor immune evasion remains unclear.MethodsHere, we...