B16F10 Melanoma Cells Research Articles

Abstract 2707: Therapeutic effects of WT1 silencing by respiratory administration of liposomal siRNA in a melanoma murine model

Abstract Introduction: Cancer is considered among the main causes of death worldwide and the number of cases keeps rising every year, as there is not an effective treatment to offer for most of them. The lungs represent a frequent target of primary and metastatic tumors due to his high oxygenation and large air spaces to grow. Melanoma is the most aggressive type of skin cancer showing a high affinity to metastasize into the lungs, being the overexpression of the WT1 protein associated with the development of this cancer, as well as several other types. Methods: In this study, we evaluated the effects of siRNA silencing the WT1 protein in both, in vitro in the B16F10 melanoma cell line, and in vivo in a murine model of pulmonary metastatic melanoma implementing a novel delivery strategy of neutral DOPC liposomal-siRNA system administrated via inhalation. Results: In vitro studies showed an effective silencing of WT1 protein (86%) in the siRNAsWT1 treated cells when compared with controls, resulting in a loss of viability (40%) and proliferation (49%) of the cells, induced G1 cell-cycle arrest (11%), a restriction of migration (87%) and invasion (100%) capacities, as well as an activation of apoptosis (12%). In vivo, we tested our delivery system obtaining an efficient biodistribution in lungs, kidney, liver and spleen. The respiratory administration of LWT1siRNA after two weeks of treatment resulted in silencing of WT1 protein (59%), achieving an important antitumor activity with a 44% reduction in tumor weight. In the survival study 80% of the L-WT1siRNA treated mice were alive after all the controls mice had died, delaying the death almost a week at the end of the study. Conclusions: This work demonstrates the efficacy of the neutral DOPC liposomal-siRNA delivery system by respiratory administration as a novel therapy to reduce the malignancy of pulmonary tumors and to increase survivability by silencing specific cancer biomarkers as WT1. Citation Format: Martin Ramos-Gonzalez, Eduardo Vázquez-Garza, Gerardo García-Rivas, Cristian Rodríguez-Aguayo, Arturo Chávez-Reyes. Therapeutic effects of WT1 silencing by respiratory administration of liposomal siRNA in a melanoma murine model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2707.

Abstract 6043: Manipulation of mitochondrial metabolism, through SIRT3 agonists, in a novel immunoreactive melanoma organoids model

Abstract The interplay between tumor cell metabolism and immune cells in the tumor microenvironment has emerged as a determinant factor in cancer progression and treatment. Sirtuin 3 (SIRT3) is a mitochondrial deacetylase that modulates ROS levels and governs many enzymes that regulate mitochondrial metabolism and homeostasis. SIRT3 has a dual role in cancer; it is regarded as an oncogene and tumor suppressor gene simultaneously. It has tumor-promoting action via keeping ROS levels low and favoring cell proliferation; on the contrary, it can trigger cell death under stress conditions by modulating tumor cell glycolysis. Recent data suggest a role for SIRT3 in cancer immunology with PD-L1 levels are found to be inversely associated with SIRT3 level in cervical cancer, and CD8+ T cells from SIRT3 knockout donor mice exhibited low GVHD in recipients. In this study, we explored the potential role of SIRT3 agonists in sensitizing the immunoreactivity of melanoma cells in a novel immunoreactive melanoma organoids model. We used the B16-F10 melanoma cell line spheroids to determine the effects of SIRT3 agonists on cellular viability, using an ATP assay. Validation of SIRT3 expression was determined via Western blot. Non-toxic amounts of SIRT3 agonists were also applied to melanoma organoids prepared from patient-derived metastatic melanoma from a lymph node. Preliminary results revealed a significant decrease in the viability of the organoids treated with R-propranolol (20µM) and Imipramine blue (0.25µM) agonists. We further investigated the effect of SIRT3 agonists R-propranolol and Imipramine blue on immunotherapy targeting PD-1, Pembrolizumab (100 nM) and Nivolumab (100 nM), and CTLA-4 inhibitor Ipilimumab (100 nM). We found a limited effect of Pembrolizumab and Nivolumab. Anti-tumor cytotoxicity was markedly increased in combination of Imipramine blue with Pembrolizumab and R-propranolol with Nivolumab. We propose that these effects are mediated through the activation of the endogenous immune cells and/or via the regulation of the PD-L1 and NKD2 ligand expression levels in the melanoma cells to become more sensitized to immune cells. Further studies will aim to validate these results in immunoreactive organoids from both mice and human patients. Our studies will shed light on the immune and metabolic axes and the use of specific agonists to develop new immune therapy treatment strategies for melanoma. Citation Format: Azza M. El-derby, Nadeem Wajih, Jack Arbiser, Konstantinous Votanopoulos, Shay Soker. Manipulation of mitochondrial metabolism, through SIRT3 agonists, in a novel immunoreactive melanoma organoids model. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6043.

Inhibitory Effects of Caulerpa racemosa, Ulva intestinalis, and Lobophora challengeriae on Tyrosinase Activity and α-MSH-Induced Melanogenesis in B16F10 Melanoma Cells.

Melanogenesis involves a synthesis of melanin pigment and is regulated by tyrosinase. The addition of whitening agents with tyrosinase-inhibiting properties in cosmetics is becoming increasingly important. In this study, the ethanolic extracts from twelve seaweeds were assessed for tyrosinase-inhibiting activity using mushroom tyrosinase and melanin synthesis in B16F10 melanoma cells. The highest mushroom tyrosinase inhibition (IC50) was observed with Lobophora challengeriae (0.15 ± 0.01 mg mL-1); treatment was more effective than kojic acid (IC50 = 0.35 ± 0.05 mg mL-1), a well-known tyrosinase inhibitor. Three seaweeds, Caulerpa racemosa, Ulva intestinalis, and L. challengeriae, were further investigated for their ability to reduce melanogenesis in B16F10 cells. The ethanolic extracts of C. racemosa, U. intestinalis, and L. challengeriae showed inhibitory effects by reducing melanin and intracellular tyrosinase levels in B16F10 cells treated with α-melanocyte stimulating hormone in a dose-dependent manner. C. racemosa (33.71%) and L. challengeriae (36.14%) at 25 µg mL-1 reduced melanin production comparable to that of kojic acid (36.18%). L. challengeriae showed a stronger inhibition of intracellular tyrosinase (decreased from 165.23% to 46.30%) than kojic acid (to 72.50%). Thus, ethanolic extracts from C. racemosa, U. intestinalis, and L. challengeriae can be good sources of natural tyrosinase inhibitors and therapeutic or cosmetic agents in the future.

Microenvironmental Changes in Mediastinal Fat-associated Lymphoid Clusters and Lungs in Early and Late Stages of Metastatic Lung Cancer Induction.

The prognosis of metastatic lung melanoma (MLM) has been reported to be poor. An increasing number of studies have reported the function of several immune cells in cancer regression. Although the function of mediastinal fat-associated lymphoid clusters (MFALCs) in the progression of inflammatory lung lesions has been previously reported, the association between MLM progression and MFALCs development has remained unexplored. Herein, we compared the microenvironmental changes in the lungs and MFALCs among phosphate-buffered saline (PBS) and cancer groups at early (1 week) and late (2 weeks) stages following the intravenous injection of B16-F10 melanoma cells into C57BL/6 mice. Except for lung CD4+ helper T-cells and Iba1+ macrophage populations of early stage, we observed a significant increase in the proliferating and immune cell (CD20+ B-lymphocytes, CD3+ T-lymphocytes, CD8+ cytotoxic T-cells, CD16+ natural killer (NK) cells populations, area of high endothelial venules, and lung lymphatic vessels in cancer groups at both the stages as compared with the PBS groups. Furthermore, a significant positive correlation was observed between immune cell populations in MFALCs and the lungs (B- and T-lymphocytes, and NK cells in both stages). Collectively, our findings suggest a promising cancer therapeutic strategy via targeting immune cells in MFALCs.

Anti-oxidant and Whitening Effect of Low Molecular Weight Kaki-tannin with Gamma irradiation

Purpose: This study aimed to investigate the use of purified Kaki-tannin obtained from a Korean natural product, Diospyros kaki Thumb. In order to confirm the potential application of kaki-tannin to cosmetics, the efficacy was evaluated by kakit-annin that molecular weight decreased by gamma irradiation.Methods: Kaki-tannin polymer was extracted by hydrothermal extraction from persimmon. The molecular weight of Kaki-tannin polymer reduced by gamma-ray irradiation, and then separated and purified. Further, the molecular weight of this polymer was reduced by gamma-ray irradiation; subsequently, it was separated and purified. Radical scavenging activities of this reduced molecular weight Kaki-tannin were examined by the 2,2′-diphenyl-1-picrylhydrazyl (DPPH) radical, 2,2′-azinobis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) diammonium salt radical cation, and hydroxyl radical scavenging assays. Moreover, the inhibitory effects of Kaki-tannin on tyrosinase and melanin synthesis in B16F10 melanoma cells were measured in vitro. Furthermore, the expression of tyrosinase, microphthalmia-associated transcription factor (MITF), tyrosinase-related protein (TRP)-1, and TRP-2 during melanogenesis was confirmed by western blotting.Results: Irradiated Kaki-tannin exhibited an antioxidant effect as well as suppressed tyrosinase activity and melanogenesis. The results of this study confirmed that the expression of tyrosinase, MITF, and TRP-2 decreased as the treatment of irradiated Kaki-tannin increased.Conclusion: In the present study, to incorporate high molecular weight Kaki-tannin in cosmetics, it was first irradiated with gamma rays in order to reduce its weight. This study confirmed that Kaki-tannin irradiated by gamma-ray has antioxidant and whitening effects as well as the potential to be used as a cosmetic material.

A 7-Hydroxy 4-Methylcoumarin Enhances Melanogenesis in B16-F10 Melanoma Cells.

The objectives of this study were to investigate the melanogenetic potentials of the naturally occurring 7-hydroxy coumarin derivatives 7-hydroxy 5,6-dimethoxycoumarin (7H-5,6DM), 7-hydroxy 6,8-dimethoxycoumarin (7H-6,8DM), 7-hydroxy 6-methoxycoumarin (7H-6M), and 7-hydroxy 4-methylcoumarin (7H-4M) in the melanogenic cells model for murine B16F10 melanoma cells. The initial results indicated that melanin production and intracellular tyrosinase activity were significantly stimulated by 7H-4M but not by 7H-5,6DM, 7H-6,8DM, or 7H-6M. Therefore, our present study further investigated the melanogenic effects of 7H-4M in B16-F10 cells, as well as its mechanisms of action. In a concentration-dependent manner, 7H-4M increased intracellular tyrosinase activity, leading to the accumulation of melanin without affecting the viability of B16-F10 cells. Our study further investigated the effects of 7H-4M on melanogenesis, including its ability to promote tyrosinase activity, increase melanin content, and activate molecular signaling pathways. The results indicate that 7H-4M effectively stimulated tyrosinase activity and significantly increased the expression of melanin synthesis-associated proteins, such as microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein-1 (TRP1), and TRP2. Based on our findings, we can conclude that 7H-4M has the ability to activate the melanogenesis process through the upregulation of cAMP-dependent protein kinase (PKA) and the cAMP response element-binding protein (CREB). Additionally, our study showed that 7H-4M induced melanogenic effects by downregulating the extracellular signal-regulated kinase (ERK) and the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt)/glycogen synthesis kinase-3β (GSK-3β) cascades, while upregulating the JNK and p38 signaling pathways. Finally, the potential of using 7H-4M in topical applications was tested through primary human skin irritation tests. During these tests, no adverse reactions were induced by 7H-4M. In summary, our results indicate that 7H-4M regulates melanogenesis through various signaling pathways such as GSK3β/β-catenin, AKT, PKA/CREB, and MAPK. These findings suggest that 7H-4M has the potential to prevent the development of pigmentation diseases.

Norcantharidin Nanoemulsion Development, Characterization, and In Vitro Antiproliferation Effect on B16F1 Melanoma Cells.

Melanoma is a highly lethal type of cancer that has had an increase in incidence in the last decades. Nevertheless, current therapies lack effectiveness and have highly disabling side effects, which calls for new therapeutic strategies. Norcantharidin (NCTD) is an acid derivative with potential antitumor activity isolated from natural blister beetles. However, its solubility limitations restrict its use. To address this issue, we developed an oil-in-water nanoemulsion using commonly available cosmetic ingredients, which increased NCTD solubility 10-fold compared to water. The developed nanoemulsion showed a good droplet size and homogeneity, with adequate pH and viscosity for skin application. In vitro drug release studies showed a sustained release profile, ideal for prolonged therapeutic effects. Accelerated stability studies proved that the formulation was reasonably stable under stress conditions, with particle separation fingerprints, instability index, particle size, and sedimentation velocity analyses being conducted. To assess the therapeutic potential of the developed formulation, in vitro studies were conducted on melanoma B16F1 cells; results showed an IC50 of 1.026 +/- 0.370 mg/kg, and the cells' metabolic activity decreased after exposure to the NCTD nanoemulsion. Hence, a new "easy-to-make" nanoformulation with therapeutic potential on melanoma cells was developed, as a possible adjuvant for future melanoma treatment.

Fermented Aronia melanocarpa Inhibits Melanogenesis through Dual Mechanisms of the PI3K/AKT/GSK-3β and PKA/CREB Pathways.

UV light causes excessive oxidative stress and abnormal melanin synthesis, which results in skin hyperpigmentation disorders such as freckles, sunspots, and age spots. Much research has been carried out to discover natural plants for ameliorating these disorders. Aronia melanocarpa contains various polyphenolic compounds with antioxidative activities, but its effects on melanogenesis have not been fully elucidated. In this study, we investigated the inhibitory effect of fermented Aronia melanocarpa (FA) fermented with Monascus purpureus on melanogenesis and its underlying mechanism in the B16F10 melanoma cell line. Our results indicate that FA inhibited tyrosinase activity and melanogenesis in alpha-melanocyte-stimulating hormone (α-MSH)-induced B16F10 cells. FA significantly downregulated the PKA/CREB pathway, resulting in decreased protein levels of tyrosinase, TRP-1, and MITF. FA also inhibited the transcription of MITF by increasing the phosphorylation levels of both GSK3β and AKT. Interestingly, we demonstrated that these results were owing to the significant increase in gallic acid, a phenolic compound of Aronia melanocarpa produced after the fermentation of Monascus purpureus. Taken together, our research suggests that Aronia melanocarpa fermented with Monascus purpureus acts as a melanin inhibitor and can be used as a potential cosmetic or therapeutic for improving hyperpigmentation disorders.

Therapeutic Effects of WT1 Silencing via Respiratory Administration of Neutral DOPC Liposomal-siRNA in a Lung Metastasis Melanoma Murine Model

The lungs represent a frequent target for metastatic melanoma as they offer a high-oxygen environment for tumor development. The overexpression of the WT1 protein has been associated with the occurrence of melanoma. In this study, we evaluated the effects of silencing the WT1 protein by siRNA in both in vitro in the B16F10 melanoma cell line and in vivo in a murine model of lung metastatic melanoma. We did this by implementing a novel respiratory delivery strategy of a neutral DOPC liposomal-siRNA system (L-siRNA). In vitro studies showed an effective silencing of the WT1 protein in the siRNAs' WT1-treated cells when compared with controls, resulting in a loss of the cell's viability and proliferation by inducing G1 arrest, the inhibition of the migration and invasion capacities of the cells, as well as the induction of apoptosis. In vivo, the respiratory administration of L-WT1 siRNA showed an efficient biodistribution on the lungs. After two weeks of treatment, the silencing of the WT1 protein resulted in an important antitumor activity that reduced the tumor weight. In the survival study, L-WT1 treatment could significantly delay the death of the animals. This work demonstrates the efficacy of the L-siRNA respiratory administration as a novel therapy to reduce pulmonary tumors and to increase survivability by silencing specific cancer oncogenes as WT1.

Unleashed Actin Assembly in Capping Protein-Deficient B16-F1 Cells Enables Identification of Multiple Factors Contributing to Filopodium Formation

Filopodia are dynamic, finger-like actin-filament bundles that overcome membrane tension by forces generated through actin polymerization at their tips to allow extension of these structures a few microns beyond the cell periphery. Actin assembly of these protrusions is regulated by accessory proteins including heterodimeric capping protein (CP) or Ena/VASP actin polymerases to either terminate or promote filament growth. Accordingly, the depletion of CP in B16-F1 melanoma cells was previously shown to cause an explosive formation of filopodia. In Ena/VASP-deficient cells, CP depletion appeared to result in ruffling instead of inducing filopodia, implying that Ena/VASP proteins are absolutely essential for filopodia formation. However, this hypothesis was not yet experimentally confirmed. Here, we used B16-F1 cells and CRISPR/Cas9 technology to eliminate CP either alone or in combination with Ena/VASP or other factors residing at filopodia tips, followed by quantifications of filopodia length and number. Unexpectedly, we find massive formations of filopodia even in the absence of CP and Ena/VASP proteins. Notably, combined inactivation of Ena/VASP, unconventional myosin-X and the formin FMNL3 was required to markedly impair filopodia formation in CP-deficient cells. Taken together, our results reveal that, besides Ena/VASP proteins, numerous other factors contribute to filopodia formation.

A Sulfur Containing Melanogenesis Substrate, N-Pr-4-S-CAP as a Potential Source for Selective Chemoimmunotherapy of Malignant Melanoma

N-propionyl-4-S-cysteaminylphenol (N-Pr-4-S-CAP) is a substrate for tyrosinase, which is a melanin biosynthesis enzyme and has been shown to be selectively incorporated into melanoma cells. It was found to cause selective cytotoxicity against melanocytes and melanoma cells after selective incorporation, resulting in the induction of anti-melanoma immunity. However, the underlying mechanisms for the induction of anti-melanoma immunity remain unclear. This study aimed to elucidate the cellular mechanism for the induction of anti-melanoma immunity and clarify whether N-Pr-4-S-CAP administration could be a new immunotherapeutic approach against melanoma, including local recurrence and distant metastasis. A T cell depletion assay was used for the identification of the effector cells responsible for N-Pr-4-S-CAP-mediated anti-melanoma immunity. A cross-presentation assay was carried out by using N-Pr-4-S-CAP-treated B16-OVA melanoma-loaded bone marrow-derived dendritic cells (BMDCs) and OVA-specific T cells. Administration of N-Pr-4-S-CAP induced CD8+ T cell-dependent anti-melanoma immunity and inhibited the growth of challenged B16F1 melanoma cells, indicating that the administration of N-Pr-4-S-CAP can be a prophylactic therapy against recurrence and metastasis of melanoma. Moreover, intratumoral injection of N-Pr-4-S-CAP in combination with BMDCs augmented the tumor growth inhibition when compared with administration of N-Pr-4-S-CAP alone. BMDCs cross-presented a melanoma-specific antigen to CD8+ T cells through N-Pr-4-S-CAP-mediated melanoma cell death. Combination therapy using N-Pr-4-S-CAP and BMDCs elicited a superior anti-melanoma effect. These results suggest that the administration of N-Pr-4-S-CAP could be a new strategy for the prevention of local recurrence and distant metastasis of melanoma.

Extract of Bletilla formosana callus elevates cellular antioxidative activity via Nrf2/HO-1 signaling pathway and inhibits melanogenesis in zebrafish

BackgroundBletilla species are endangered terrestrial orchids used in natural skin care formulas in Asia for a long history. In order to explore the bioactivity potential of Bletilla species as a cosmetic ingredient in a sustainable resource manner, the callus of Bletilla formosana (Hayata) Schltr. was established and extracted by an eco-friendly supercritical fluid CO2 extraction (SFE-CO2) method. The intracellular reactive oxygen species (ROS) scavenging activity and antioxidation-related gene expression of the callus extract were evaluated in both Hs68 fibroblast cells and HaCaT keratinocytes. The melanogenesis-inhibitory effect was investigated in B16F10 melanoma cells and in an in vivo zebrafish model. ResultsThe calli of B. formosana were propagated for 10–15 generations with a consistent yellow friable appearance and then subjected to SFE-CO2 extraction to obtain a yellow pasty extract. Obvious intracellular ROS scavenging activity of the extract was detected in both Hs68 and HaCaT cells with 64.30 ± 8.27% and 32.50 ± 4.05% reduction at the concentration of 250 μg/mL. Moreover, marked expression levels of heme oxygenase-1 (HO-1) and (NAD(P)H) quinone oxidoreductase-1 (NQO1) genes were detected after 6-h and 24-h treatments. These results indicate the cellular antioxidative activity of B. formosana callus extract was probably activated via the nuclear factor erythroid 2-related factor 2 (Nrf2)/HO-1 signaling pathway. Melanogenesis-inhibitory effect of the extract was observed in α-MSH stimuli-inducing B16F10 cells with 28.46% inhibition of intracellular melanin content at the concentration of 50 μg/ml. The effect was confirmed with in vivo zebrafish embryos that showed a relative pigmentation density of 80.27 ± 7.98% at the concentration of 100 μg/mL without toxicity. ConclusionOur results shed light on a sustainable utilization of Bletilla species as a potential ingredient for skin.

Artocarpus heterophyllus LAM. Stem Bark Inhibits Melanogenesis Through Regulation of ROS, cAMP and MAPK Pathways

Natural-based skin-lightening cosmeceutical products are attracting high popularity nowadays due to their relatively high bioavailability upon application. Artocarpus species have been highlighted with such potential, and our previous studies have reported that Artocarpus heterophyllusLam. stem bark extract exhibited a potent anti-melanogenic activity by reducing melanin content and inhibiting cellular tyrosinase activity in B16F10 melanoma cells. Hence, this study aimed to identify the bioactive fraction from A. heterophyllus Lam. stem bark and determine its anti-melanogenic mechanisms in B16F10 melanoma cells. Our results showed that a fraction (H-3) demonstrated the most pronounced anti-melanogenic effect at 12.00 µg/mL by reducing melanin content to 22.86 ± 2.90% and inhibiting cellular tyrosinase activity at treatment concentration 33-fold lower than kojic acid, without being cytotoxic against B16F10 melanoma cells. Moreover, treatment with H-3 for 24 and 48 h substantially scavenged intracellular reactive oxygen species (ROS) of hydrogen peroxide-challenged B16F10 melanoma cells by 1.8 and 4.4%, respectively. Based on the microarray profiling and qPCR analysis, H-3 downregulated Creb3l1, Creb3l2, Creb3l3, Mitf, Tyr, Tyrp1, and Dct genes in B16F10 melanoma cells, whereas the expression of Map3k20, Mapk14 (p38), and Foxo3 genes was markedly increased. Altogether, these results demonstrated that H-3 exhibited its anti-melanogenic activity in B16F10 melanoma cells through scavenging ROS and concurrent inhibition of the cAMP and activation of the p38/MAPK signaling pathways. These findings indicate that H-3 has the potential to be used as a skin lightening cosmeceutical agent in the treatment of skin hyperpigmentation.

Studies of Erk1/2 with Respect to Tumor Aggressiveness in B16f10 Murine Melanoma Cells

Melanoma is a very aggressive form of skin cancer and have the potential to spread from a small sized primary tumor and metastasizes to different locations like lungs, bones, lymph nodes, liver and even brain. Extracellular signal regulated kinase (ERK) molecule present in MAPK pathway plays a major role in different carcinogenic processes like cell migration, cell invasiveness by altering the extracellular matrix (ECM). Cell invasiveness is facilitated by matrix metalloproteins (MMPs) by means of degradation of gelatinase enzyme regulated by a family of receptor proteins called integrins. ECM ligand fibronectin causes increased secretion of MMPs, specially MMP2 and MMP9 facilitating invasiveness and metastasis in cancer cells. This study aims to find the modulatory role of ERK 1/ 2 with respect to tumor aggressiveness on both MMP2 and MMP9 on melanoma metastasis and invasion. ERK pathway was downregulated through administration of inhibitors and ERK 1/ 2 siRNA gene silencing the effect on MMP2 and MMP9 was studied. The studies were done in B16F10 melanoma cell line and mice model. Significant changes were found in levels of MMP2 and MMP9 after downregulating the ERK pathway and can therefore a correlation between ERK and MMP activities was concluded. Elucidating these molecular phenomena in melanoma, this result can help in developing and improving targeted cancer therapies with multiple options in drug discovery.

Comparison of direct and indirect low‐temperature plasma triggering immunogenic cell death in B16F10 melanoma

AbstractThe potential antitumor application of low‐temperature plasma (LTP) in immunotherapy has attracted wide attention from researchers. The induction of immunogenic cell death (ICD) is one of the possible mechanisms underlying this event. In this work, the immunogenicity of B16F10 melanoma cells treated with direct and indirect LTP protocols was investigated. The results revealed the concentrations of H2O2, NO, and NO2− increased as LTP treatment time was longer, while the cell viability decreased. By comparison, the direct treatment induced a stronger expression of ICD markers. In vivo, the LTP‐treated B16F10 reduced tumor burden in C57BL/6 mice. This study demonstrated both direct and indirect LTP treatments triggered immunogenic cell death and were expected to provide an effective immunotherapy strategy.

Experimental Melanoma Immunotherapy Model Using Tumor Vaccination with a Hematopoietic Cytokine.

Fms-like tyrosine kinase 3 ligand (Flt3L) is a hematopoietic cytokine that promotes the survival and differentiation of dendritic cells (DCs). It has been used in tumor vaccines to activate innate immunity and enhance antitumor responses. This protocol demonstrates a therapeutic model using cell-based tumor vaccine consisting of Flt3L-expressing B16-F10 melanoma cells along with phenotypic and functional analysis of immune cells in the tumor microenvironment (TME). Procedures for cultured tumor cell preparation, tumor implantation, cell irradiation, tumor size measurement, intratumoral immune cell isolation, and flow cytometry analysis are described. The overall goal of this protocol is to provide a preclinical solid tumor immunotherapy model, and a research platform to study the relationship between tumor cells and infiltrating immune cells. The immunotherapy protocol described here can be combined with other therapeutic modalities, such as immune checkpoint blockade (anti-CTLA-4, anti-PD-1, anti-PD-L1 antibodies) or chemotherapy in order to improve the cancer therapeutic effect of melanoma.

Experimental Melanoma Immunotherapy Model Using Tumor Vaccination with a Hematopoietic Cytokine

Fms-like tyrosine kinase 3 ligand (Flt3L) is a hematopoietic cytokine that promotes the survival and differentiation of dendritic cells (DCs). It has been used in tumor vaccines to activate innate immunity and enhance antitumor responses. This protocol demonstrates a therapeutic model using cell-based tumor vaccine consisting of Flt3L-expressing B16-F10 melanoma cells along with phenotypic and functional analysis of immune cells in the tumor microenvironment (TME). Procedures for cultured tumor cell preparation, tumor implantation, cell irradiation, tumor size measurement, intratumoral immune cell isolation, and flow cytometry analysis are described. The overall goal of this protocol is to provide a preclinical solid tumor immunotherapy model, and a research platform to study the relationship between tumor cells and infiltrating immune cells. The immunotherapy protocol described here can be combined with other therapeutic modalities, such as immune checkpoint blockade (anti-CTLA-4, anti-PD-1, anti-PD-L1 antibodies) or chemotherapy in order to improve the cancer therapeutic effect of melanoma.

Oleaginous Yeast Extracts and Their Possible Effects on Human Health

Four non-conventional oleaginous and pigmented yeast strains of Metschnikowia pulcherrima, Cystofilobasidium infirmominiatum, Phaffia rhodozyma, and Rhodotorula kratochvilovae were used in this study. Complex yeast extracts were prepared and tested for biological activity, safety, and effect on human health. In this paper, we measured the antioxidant activity and antimicrobial effect of yeast biomass as a whole and their extracts to compare the influence of carotenoids and other bioactive substances in the studied biomass. All yeast extracts exhibited a significant dose-dependent antimicrobial effect against both G+ and G- bacteria and had a strong antioxidant effect. No cytotoxicity in the mouse melanoma B16F1 cell line was found in concentrations up to 20% of rehydrated biomass in cell medium. All of the extracts were cytotoxic at a concentration of 5 mg of extract/g of dry biomass. All the pigmented yeast extracts showed some positive results for apoptosis of murine melanoma cell lines and are therefore strong candidates positively effect human health. Red yeast cell biomass is a prospective material with many attractive biological functions and can be used in the food industry, as a pharmaceutical material, or in the feed industry.

The pulse light mode enhances the effect of photobiomodulation on B16F10 melanoma cells through autophagy pathway.

Photobiomodulation (PBM) is the use of low irradiance light of specific wavelengths to generate physiological changes and therapeutic effects. However, there are few studies on the effects of PBM of different LED light modes on cells. Here, we investigated the difference of influence between continuous wave (CW) and pulse-PBM on B16F10 melanoma cells. Our results suggested that the pulse mode had a more significant PBM than the CW mode on B16F10 melanoma cells. Our study confirmed that ROS and Ca2+ levels in B16F10 melanoma cells treated with pulse-PBM were significantly higher than those in the control and CW-PBM groups. One mechanism that causes the difference in CW and pulse-PBM action is that pulse-PBM activates autophagy of melanoma cells through the ROS/OPN3/Ca2+ signaling pathway, and excessive autophagy activation inhibits proliferation and apoptosis of melanoma cells. Autophagy may be one of the reasons for the difference between pulse- and CW-PBM on melanoma cells. More importantly, melanoma cells responded to brief PBM pulses by increasing intracellular Ca2+ levels.

Efficient Synthesis of Chlorin e6 and Its Potential Photodynamic Immunotherapy in Mouse Melanoma by the Abscopal Effect.

Photodynamic therapy (PDT) can eradicate not only cancer cells but also stimulate an antitumor immune response. Herein, we describe two efficient synthetic methodologies for the preparation of Chlorin e6 (Ce6) from Spirulina platensis and address the phototoxic effect of Ce6 in vitro along with antitumor activity in vivo. Melanoma B16F10 cells were seeded and phototoxicity was monitored by the MTT assay. The C57BL/6 mice were subcutaneously inoculated on the left and right flank with B16F10 cells. The mice were intravenously injected with Ce6 of 2.5 mg/kg and then exposed to red light (660 nm) on the left flank tumors 3 h after the injection. The immune response was studied by analyzing Interferon-gamma (IFN-γ), tumor necrosis factor-alpha (TNF-α), and Interleukin-2 (IL-2) of the right flank tumors through qPCR. Our results revealed that the tumor was suppressed not only in the left flank but also in the right flank, where no PDT was given. The upregulated gene and protein expression of IFN-γ, TNF-α, and IL-2 revealed antitumor immunity due to Ce6-PDT. The findings of this study suggest an efficient methodology of Ce6 preparation and the efficacy of Ce6-PDT as a promising antitumor immune response.