Mouse Melanoma Cells Research Articles

Two Coffee Diterpenes, Kahweol and Cafestol, Inhibit Extracellular Melanogenesis: An In Vitro Pilot Study

Hyperpigmentation skin disorders are marked by an abnormal accumulation or export of melanin pigment synthesized within melanocytes and pose a significant aesthetic concern. The search for novel natural compounds that exhibit pharmacological potential for treating pigmentation disorders is growing. In this study, kahweol (KW) and cafestol (CFS), two structural analogs of coffee diterpenes, were evaluated and compared for their effects on melanogenesis using B16F10 mouse melanoma cells and primary human melanocytes derived from Asian and African American skin. To the best of our knowledge, there are no reports of the effects of KW and CFS on melanogenesis yet. We first screened nontoxic concentrations of both compounds using an MTS assay after 72 h incubations and subsequently tested their effects on melanin synthesis and export. Cellular tyrosinase activity and cell-free mushroom tyrosinase activity were assayed to study the mechanisms of melanogenesis suppression. Human melanocytes from a moderately pigmented donor (HEMn-MP cells) and from a darkly pigmented donor (HEMn-DP cells) were next examined, and effects on cellular viability, melanin content, cellular tyrosinase activity, and melanin export (quantitated via dendricity) were similarly examined for both compounds. Our results show that KW and CFS did not significantly affect intracellular melanin content but suppressed extracellular melanin in B16F10 cells and dendritic parameters in human melanocytes, indicating their unique capacity to target extracellular melanogenesis and melanin export. Although KW showed a greater extracellular melanogenesis inhibitory capacity in B16F10 cells, in both primary melanocyte cells, CFS emerged as a potent inhibitor of melanin export compared to KW. Together, these results reveal novel modes of action of both compounds and indicate a promise to use CFS as a novel candidate for treating hyperpigmentation disorders of the human skin for clinical and cosmetic use. Additional research is necessary to shed light on the molecular pathways and the efficacy of melanogenesis inhibition by CFS in 3D human skin equivalents and in vivo studies.

Chromosomal instability induced in cancer can enhance macrophage-initiated immune responses that include anti-tumor IgG.

Solid tumors generally exhibit chromosome copy number variation, which is typically caused by chromosomal instability (CIN) in mitosis. The resulting aneuploidy can drive evolution and associates with poor prognosis in various cancer types as well as poor response to T-cell checkpoint blockade in melanoma. Macrophages and the SIRPα-CD47 checkpoint are understudied in such contexts. Here, CIN is induced in poorly immunogenic B16F10 mouse melanoma cells using spindle assembly checkpoint MPS1 inhibitors that generate persistent micronuclei and diverse aneuploidy while skewing macrophages toward a tumoricidal 'M1-like' phenotype based on markers and short-term anti-tumor studies. Mice bearing CIN-afflicted tumors with wild-type CD47 levels succumb similar to controls, but long-term survival is maximized by SIRPα blockade on adoptively transferred myeloid cells plus anti-tumor monoclonal IgG. Such cells are the initiating effector cells, and survivors make de novo anti-cancer IgG that not only promote phagocytosis of CD47-null cells but also suppress tumor growth. CIN does not affect the IgG response, but pairing CIN with maximal macrophage anti-cancer activity increases durable cures that possess a vaccination-like response against recurrence.

Chromosomal instability induced in cancer can enhance macrophage-initiated immune responses that include anti-tumor IgG

Solid tumors generally exhibit chromosome copy number variation, which is typically caused by chromosomal instability (CIN) in mitosis. The resulting aneuploidy can drive evolution and associates with poor prognosis in various cancer types as well as poor response to T-cell checkpoint blockade in melanoma. Macrophages and the SIRPα-CD47 checkpoint are understudied in such contexts. Here, CIN is induced in poorly immunogenic B16F10 mouse melanoma cells using spindle assembly checkpoint MPS1 inhibitors that generate persistent micronuclei and diverse aneuploidy while skewing macrophages toward a tumoricidal ‘M1-like’ phenotype based on markers and short-term anti-tumor studies. Mice bearing CIN-afflicted tumors with wild-type CD47 levels succumb similar to controls, but long-term survival is maximized by SIRPα blockade on adoptively transferred myeloid cells plus anti-tumor monoclonal IgG. Such cells are the initiating effector cells, and survivors make de novo anti-cancer IgG that not only promote phagocytosis of CD47-null cells but also suppress tumor growth. CIN does not affect the IgG response, but pairing CIN with maximal macrophage anti-cancer activity increases durable cures that possess a vaccination-like response against recurrence.

Anti-apoptotic, anti-inflammatory, and anti-melanogenic effects of the ethanol extract of Picrasma quassioides (D. Don) Benn

Ethnopharmacological relevancePicrasma quassioides (D. Don) Benn is a vascular plant belonging to the genus Picrasma of Simaroubaceae family and grows in Korea, China, India, Taiwan, and Japan. Picrasma quassioides extract has been reported to have anti-inflammatory, anti-bacterial, and anti-cancer properties. Moreover, this plant has been also traditionally used to alleviate symptoms of eczema, atopic dermatitis, psoriasis, scabies, and boils in skin. Aim of the studyThe Pq-EE has been reported in Chinese pharmacopoeia for its pharmacological effects on skin. However, the detailed mechanism on alleviating skin conditions is not understood. Hence, we investigated the skin improvement potential of Pq-EE against skin damage. Materials and methodsWe used the human keratinocyte cell line (HaCaT) and mouse melanoma cell line (B16F10) to study the effects of Pq-EE on the epidermis. Additionally, in vitro antioxidant assays were performed using a solution that included either metal ions or free radicals. ResultsIn colorimetric antioxidant assays, Pq-EE inhibited free radicals in a dose-dependent manner. The Pq-EE did not affect cell viability and promoted cell survival under UVB exposure conditions in the MTT assay. The Pq-EE downregulated the mRNA levels of apoptotic factors. Moreover, MMP1 and inflammatory cytokine iNOS mRNA levels decreased with Pq-EE treatment. With regard to protein levels, caspases and cleaved caspases were more powerfully inhibited by Pq-EE than UVB-irritated conditions. p53 and Bax also decreased with Pq-EE treatment. The melanin contents and secretion were decreased at nontoxic concentrations of Pq-EE. The pigmentation pathway genes also were inhibited by treatment with Pq-EE. ConclusionsIn summary, we suggest the cell protective potential of Pq-EE against UVB and ROS, indicating its use in UV-protective cosmeceutical materials.

Pentoxifylline and Norcantharidin Modify p62 Expression in 2D and 3D Cultures of B16F1 Cells.

Three-dimensional cell cultures have improved the evaluation of drugs for cancer therapy, due to their high similarity to solid tumors. In melanoma, autophagy appears to show a dual role depending on the progression of the disease. p62 protein has been proposed for the evaluation of autophagic flux since its expression is an indicator of the state of autophagy. Pentoxifylline (PTX) and Norcantharidin (NCTD) are drugs that have been shown to possess anticancer effects. In this work, we used B16F1 mouse melanoma cells in two-dimensional (2D) monolayer cultures and three-dimensional (3D) spheroids to test the effect of PTX and NCTD over the p62 expression. We analyzed the effect on p62 expression through Western blot and immunofluorescence assays. Our results indicate that PTX decreases p62 expression in both cell culture models, while Norcantharidin increases its expression in 3D cultures at 24 h. Therefore, these drugs could have a potential therapeutic use for the regulation of autophagy in melanoma, depending on the state of evolution of the disease.

Experimental evidence that carbon-ion radiotherapy utilizes cytotoxic T lymphocyte-mediated anti-tumor immunity for shrinking tumors compared to X-ray therapy

The therapeutic efficacy of radiotherapy (RT) is primarily driven by two factors: biophysical DNA damage in cancer cells and radiation-induced anti-tumor immunity. However, Anti-tumor immune responses between X-ray RT (XRT) and carbon-ion RT (CIRT) remain unclear. In this study, we, employed mouse models to assess the immunological contribution, especially cytotoxic T-lymphocyte (CTL)-mediated immunity, to the therapeutic effectiveness of XRT and CIRT in shrinking tumors. We irradiated mouse intradermal tumors of B16F10-ovalbumin (OVA) mouse melanoma cells and 3LL-OVA mouse lung cancer cells with carbon-ion beams or X-rays in the presence or absence of CTLs. CTL removal was performed by administration of anti-CD8 monoclonal antibody (mAb) in mice. Based on tumor growth delay, we determined the tumor growth and regression curves. The enhancement ratio (ER) of the slope of regression lines in the presence of CTLs, relative to the absence of CTLs, indicates the dependency of RT on CTLs for shrinking mouse tumors, and the biological effectiveness (RBE) of CIRT relative to XRT were calculated. Tumor growth curves revealed that the elimination of CD8+ CTLs by administrating anti-CD8 mAb accelerated tumor growth compared to the presence of CTLs in both RTs. The ERs were larger in CIRT compared to XRT in the B16F10-OVA tumor models, but not in the 3LL-OVA models, suggesting a greater contribution of CTL-mediated anti-tumor immunity to tumor reduction in CIRT compared to XRT in the B16F10-OVA tumor model. In addition, the RBE values for both models were larger in the presence of CTLs compared to models without CTLs, suggesting that CIRT may utilize CTL-mediated anti-tumor immunity more than X-ray. The findings from this study suggest that although immunological contribution to therapeutic efficacy may vary depending on the type of tumor cell, CIRT utilizes CTL-mediated immunity to a greater extent compared to XRT.

Hyperosmotic cold shock mouse melanoma cells encapsulated with doxorubicin for targeted treatment of melanoma.

The primary treatment strategies for melanoma include surgical excision, chemotherapy, and radiotherapy. However, the efficacy of these treatments is often limited by drug resistance, recurrence, and severe side effects. Therefore, we aimed to develop a targeted drug delivery system capable of selectively locating tumor sites to minimize systemic toxicity and enhance therapeutic efficacy. This cell drug delivery system can also deliver chemotherapeutic drugs to the tumor microenvironment. We treated B16F10 cells with hyperosmotic cold shock (HCS) to obtain and characterize HCS cells. We then investigated the anti-tumor effects and immune activation capabilities of these cells and explored their potential as a targeted drug delivery system. HCS cells not only maintained an intact cellular structure and tumor antigens but also exhibited high expression of the homologous melanoma-associated antigen glycoprotein 100. These cells demonstrated an exceptional capacity for loading and releasing doxorubicin, which has chemotherapeutic anti-tumor effects. HCS cells can precisely target the tumor microenvironment to minimize systemic toxicity, inducing an immune response by activating CD3+ and CD4+ T cells. HCS cells are non-carcinogenic, with both cellular and tumor antigens intact; thus, they are suitable drug delivery carriers. Our findings highlight the potential of HCS cells for carrying doxorubicin because of their high drug-loading efficiency, effective tumor-targeting and anti-tumor effects. Therefore, our results will facilitate the development of melanoma treatments that have higher efficacy than those in the literature.

The Antioxidant and Whitening Effect of Miracle Fruit (Synsepalum dulcificum) Seed and Leaf Methanol Extracts

The seed and leaf 70% methanol extracts of miracle fruit (Synsepalum dulcificum) were investigated for its whitening activity for application as a functional ingredient in cosmetic products. At the leaf extract concentration of 100 mg/ml, the DPPH radical scavenging activity was found to be 94.58%, the ABTS+ radical scavenging activity was 36.62%. The total polyphenolic content of seed and leaf extract was 18.76 mgGAE/g, 71.42 mgGAE/g, respectively. The seed and leaf extracts reduced both tyrosinase activity and L-DOPA oxidation in a concentration-dependent manner. In B16F10 mouse melanoma cells, leaf extract inhibited 30% of melanin synthesis at 100 mg/ml. Miracle fruit seed and leaf extracts also inhibited the expression of melanogenesis genes such as microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein (TRP)-1. Moreover, reverse transcription-polymerase chain reaction of the extract showed that the mRNA expression of MITF, tyrosinase, and TRP-1 was decreased. The data suggested a protective role for the Synsepalum dulcificum seed and leaf extracts against melanogenesis.

Yemeni Sidr Honey Inhibits Cell Proliferation and Promotes Apoptosis in Human Cancer and Mouse-Derived Cell Lines

Honey has become popular as a potential treatment for several ailments, including cancer. Honeys from different parts of the world have been shown to have different anti-proliferative, immune-modulatory, and anti-inflammatory actions. Yemeni Sidr honey (YSH) is world-renowned for its anti-inflammatory activity and has been suggested to have anti-cancer activity, although empirical evidence is lacking. We tested three YSH samples by HPLC to show they contained similar sugars and an overlapping group of phenolic and flavonoid components, as described previously. YSH’s apoptotic and anti-proliferative activities were measured in in vitro models of cancer growth. The treatment of breast cancer cell lines (MDA-MB-231 and MCF-7), a cervical cancer cell line (HeLa), and mouse melanoma cells (B16-BL6) with 1% (w/v) YSH in media for 48–72 h almost completely inhibited cell proliferation and promoted cell apoptosis. In contrast, a non-malignant HBL-100 cell line was more resistant to treatment with YSH. This suggests that YSH may be a good candidate as an anti-cancer treatment, which requires further study.

Abstract 5524: Identification of PAEP as an immune activity modulator to mediate the immune resistance in tumor metastasis

Abstract Most cancer-related deaths are associated with the complex phenotypic behavior of metastasis, a process in which tumor cells spread from their initial site to distant sites. To successfully metastasize, the tumor cell will be required to make numerous adjustments and survive a series of stages involving intravasation, circulation in the blood or lymph system, extravasation, and growth at distant organs, and overcome the challenges of immunosurveillance. The tumor microenvironment is the ecosystem that surrounds and feeds the tumor. The molecular mechanism by which the metastatic tumors survive and escape from immune attack in the tumor microenvironment remains unclear. To study the molecular mechanism of interaction between metastatic tumors and the immune system in metastasis, we established immune-resistant metastatic models. We identified that PAEP, a progestogen-associated endometrial protein, is significantly upregulated in immune-resistant metastatic tumors compared with non-immune-resistant tumors. PAEP is a glycoprotein that inhibits cell immune function and plays an essential role in the pregnancy process. To examine the function of PAEP in tumor metastasis, we first introduced PAEP into non-immune resistant and poorly metastatic melanoma and rhabdomyosarcoma (RMS) cells. We found that overexpression of PAEP significantly promotes tumor metastatic potential of both melanoma and RMS cells in immunocompetent mice. Interestingly, there was no difference in metastasis between the control and overexpression groups in immunodeficient mice. These data suggest that PAEP may mediate tumor metastasis relating to host immunity. To further confirm the function of PAEP in tumor metastasis, we use CRISPR/Cas9 technology to knockout endogenous PAEP and will test the metastatic potential in different hosts. We will also investigate the molecular mechanisms using RNA sequencing and Digital Spatial Profiling (DSP) to analyze the metastatic samples. Our study will uncover a new mechanism for how tumors survive in the microenvironment and identify a novel target for the treatment of metastatic diseases. Citation Format: Rand Gabriel M. Buenaventura, Weiping Chen, Shuling Zhang, Beverly Mock, Glenn Merlino, Yanlin Yu. Identification of PAEP as an immune activity modulator to mediate the immune resistance in tumor metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5524.

Abstract 1217: Defining the anti-tumor activity and immune effects of the RASMULTI(ON) inhibitor RMC-7977 in preclinical models of NRAS-mutant melanoma

Abstract Introduction: Mutations in the small GTPase NRAS are the second most common oncogenic driver in cutaneous melanoma, being found in ~25% of cases. Targeted therapy strategies are currently lacking for NRAS-mutant melanoma. Here, we characterized the anti-tumor activity and immune effects of RMC-7977, a RASMULTI(ON) preclinical tool compound, representative of the investigational agent RMC-6236, in preclinical mouse and human models of NRAS-mutant melanoma. Methods: The in vitro potency of RMC-7977 was determined using mouse and human NRAS-mutant melanoma cell lines. Recovery of signaling was quantified by Western Blot and receptor tyrosine kinase (RTK) arrays. In vivo anti-tumor activity of RMC-7977 was determined in two syngeneic mouse melanoma models (SW1 NRASG13D and NRAS#5 NRASQ61R), and tumor immune infiltrate quantified using multiplexed IHC and flow cytometry. Mechanisms of resistance were defined using RNA-seq. Results: RMC-7977 had good activity in vitro against human cancer cell lines and in mouse models of NRAS-mutant melanoma. Time-course studies demonstrated RMC-7977 rapidly inhibited phospho-ERK signaling, which then began to recover after 8 hr of treatment. There was also evidence of adaptive receptor tyrosine kinase signaling by 48 hr. In syngeneic mouse models of NRAS-mutant melanoma, RMC-7977 led to a rapid inhibition of tumor growth, increased expression of tumor MHC class I and PD-L1 and an increase in CD4+ and CD8+ T cell infiltrate into tumors. Therapeutic escape was associated with decreased CD4+ and CD8+ T cell infiltration and increased numbers of immunosuppressive MDSCs. Cells collected from tumors resistant to RMC-7977 in vivo did not show any shift in sensitivity to the inhibitor when re-challenged in vitro, suggesting that the resistance observed in vivo resulted from mechanisms specific to the tumor microenvironment, for example adaptive RTK signaling or immune escape. RNA-seq and IHC analysis suggested that resistance was associated with decreased MHC expression and antigen presentation, as well as reduced co-stimulation of T cells. Combination studies of RMC-7977 with anti-PD-1 are ongoing to identify potential synergistic interactions in these preclinical models. Conclusion: RMC-7977 has anti-tumor activity in syngeneic mouse models of NRAS-mutant melanoma, associated with a robust immune response. Citation Format: Larissa A. Carvalho, Manali S. Phadke, Keiran S. M. Smalley. Defining the anti-tumor activity and immune effects of the RASMULTI(ON) inhibitor RMC-7977 in preclinical models of NRAS-mutant melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1217.

Abstract 417: Chromosomal instability leads to durable tumor suppression upon macrophage-checkpoint disruption, with induction of anti-tumor IgG

Abstract Solid tumors generally exhibit chromosome copy number variation caused by chromosomal instability (CIN) in mitosis, and the resulting aneuploidy associates with poor prognosis in various cancer types and poor Tcell checkpoint blockade response in melanoma. In such contexts, however, macrophages and the SIRPa-CD47 checkpoint are understudied. Here, CIN is induced pharmacologically in poorly immunogenic B16F10 mouse melanoma cells, generating micronuclei plus diverse aneuploidy and skewing macrophages towards an anti-cancer phenotype based on markers and short-term tumor studies. Mice bearing CIN-afflicted tumors with wild-type CD47 levels survive only slightly longer compared to chromosomally stable controls, but long-term survival can be maximized when anti-tumor IgG opsonization is combined with adoptive transfer of macrophages with SIRPa blockade or with CD47 knockout of the B16F10. Multi-epitope, de novo anti-cancer IgG in survivors promote phagocytosis of CD47 knockout B16F10 cells by macrophages and suppress tumoroids in vitro and growth of tumors in vivo. An unexpected benefit of pairing CIN with maximal macrophage anti-cancer activity is thus an anti-cancer vaccination-like response that can lead to durable cures and potentiate cell-mediated acquired immunity. Citation Format: Dennis E. Discher. Chromosomal instability leads to durable tumor suppression upon macrophage-checkpoint disruption, with induction of anti-tumor IgG [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 417.

Abstract 7472: Melanoma-intrinsic expression of signal-regulatory protein alpha (SIRPα) confers tumor cells resistance to anti-tumor adaptive immunity

Abstract Introduction: Melanoma patient survival rates have significantly improved recently owing to the development of both targeted therapies and immunotherapies. However, there are still around 50% of patients relapse. The limited response to immunotherapies and global rise in melanoma incidence, with around 290,000 cases newly diagnosed each year, highlight the need for identifying new biomarkers, modulatory pathways, and risk factors for melanoma development. Earlier studies have characterized that aberrant expression of CD47 (a.k.a. “don’t eat me” signal) is one of the mechanisms that tumors exploit to avoid innate immune surveillance. CD47 blockade has been demonstrated to restore myeloid effector function by reinvigorating phagocytosis of tumor cells, which further enhances anti-tumor T cell responses. However, melanoma tumors are resistant to CD47 blockade. Interestingly, we and others have discovered that human and mouse melanoma cells express high levels of SIRPα, the receptor for CD47, which is typically expressed in myeloid compartments. Experimental Procedure: To investigate the role of tumor SIRPα, we designed gRNAs to CRISPR out the SIRPα N-terminal region, where it interacts with CD47, in B16 melanoma and generated SIRPα+/+ (WT) and SIRPα-/- (KO) B16 tumors. We conducted multiple in vitro and in vivo experiments to investigate whether tumor SIRPα played a role in tumor growth and modulated anti-tumor immunity. The preclinical tumor model we used is B16-F10 melanoma and the murine strains we used are the immune-competent (C57BL/6j; B6) and the immune-deficient (RAGnull) mice. For phenotypic and functional assessments, we performed analysis using flow cytometry, immunohistochemistry (IHC) and single cell RNA-sequencing (scRNA-seq) derived from live-sorted CD45+ tumor infiltrating lymphocytes (TILs) of SIRPα-WT and SIRPα-KO B16 tumors. Summary of Unpublished Data Our preliminary data in a preclinical B16-F10 melanoma model revealed that surface SIRPα expression is necessary for optimal B16-F10 melanoma growth in immune-competent mice, but not in immune-deficient RAGnull mice, suggesting a potential crosstalk between tumor SIRPα and the host adaptive immune system. Intriguingly, we found that selective SIRPα deletion in melanoma cells results in a significant increase of immune infiltrates within melanoma tumors that further suppresses tumor growth. Concluding Remarks: SIRPα is required for optimal melanoma development by suppressing anti-tumor adaptive immunity. Ethics Approval: All mice were maintained in microisolator cages and treated in accordance with the NIH and American Association of Laboratory Animal Care regulations. All mouse procedures and experiments for this study were approved by the Weill Cornell Medicine Institutional Animal Care and Use Committee. Citation Format: Chien-Huan Weng, Chana Schochet, Linda Hamadene, Shabnam Eghbali, Sébastien Monette, Levi Mangarin, Cailian Liu, Fadi Samaan, Jedd D. Wolchok, Taha Merghoub. Melanoma-intrinsic expression of signal-regulatory protein alpha (SIRPα) confers tumor cells resistance to anti-tumor adaptive immunity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7472.

Abstract 2788: Immunological targeting of integrin β1high/β3high melanoma

Abstract We previously reported two integrin β4 (ITGB4)-targeted immunologic strategies, e.g., ITGB4 protein-pulsed dendritic cells (ITGB4-DC) for vaccination and adoptive transfer of anti-CD3/anti-ITGB4 bispecific antibody (ITGB4 BiAb)-armed tumor draining lymph node (TDLN) T cells. Both immunotherapies significantly demonstrated efficacy in inhibiting local tumor growth and lung metastases. We found these therapeutic efficacies were closely associated with the T cell as well as humoral anti-integrin β4high cancer stem cells (CSCs) in breast (4T1) and head and neck squamous cell carcinoma (SCC7) tumor models. Recently, we further found that integrin subunits β1 (ITGB1) and β3 (ITGB3) are expressed at significantly high level in melanoma cells. In culture cell lines, integrin β1 and β3 are highly expressed on approximately 98% and 95% of the two mouse melanoma cell lines F10 and D5. We confirmed the expression of these two integrin subunits in newly harvested fresh melanoma tumors in vivo, which is very similar to the findings in cell lines. We then assessed ITGB1 and/or ITGB3-pulsed dendritic cell (DC) vaccines using F10 mouse model and demonstrated that ITGB1-DC or ITGB3-DC vaccine both exhibited significant antitumor efficacy inhibiting tumor growth, and ITGB1-DC + ITGB3-DC showed an additive/synergistic anti-tumor effect. Notably, we found that ITGB1 and ITGB3 loaded on separate DCs demonstrated more potent antitumor efficacy than loaded on the same DCs as vaccine. In addition, anti-PD-L1 significantly augmented the antitumor efficacy of ITGB1-DC/ITGB3-DC vaccine to inhibit tumor growth. Considering the crucial role of CSCs in tumor initiation, progression, metastasis, and recurrence, we further evaluated the expression of ITGB1 and ITGB3 on F10 and D5 ALDHhigh CSCs. We detected significantly higher expression of ITGB1 on ALDHhigh F10 CSCs (83.3%) and ALDHhigh D5 CSCs (87.1%) vs. ALDHlow F10 non-CSCs (0.2%) and ALDHlow D5 non-CSCs (2.0%), suggesting that ITGB1 and ITGB3-targeted vaccine-conferred anti-melanoma effects may have significantly involved targeting of ITGB1high/ITGB3high melanoma CSCs. Furthermore, recent studies suggested that bone marrow (BM)-DCs expressing dysfunctional integrin β2 (ITGB2) exhibited more potent antigen presentation function than WT BM-DCs. We found that ITGB2 highly (63.3%) expressed on the surface of BM-DC, but not on F10 or D5 tumor cells. On-going experiments include the investigation of experimental evidence to verify that ITGB1 and ITGB3-targeted vaccines have induced targeting of ITGB1high/ITGB3high melanoma CSCs; and both the cellular and humoral mechanisms underlining the anti- ITGB1high/ITGB3high melanoma CSC immunity. At the same time, generation and use of ITGB2 deficient DCs for the vaccine preparation to improve the efficacy of DC vaccine targeting integrin ITGB1high/ITGB3high melanoma and melanoma CSCs warranty further investigation. Citation Format: Feng Wang, Ying Xin, James J. Moon, Alfred E. Chang, Max Wicha, Qiao Li. Immunological targeting of integrin β1high/β3high melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2788.

Abstract 6748: Intramuscular delivery of tumor associated antigen DNA vaccines elicits strong cellular immune response, delays tumor growth and prolongs survival

Abstract The development of effective cancer vaccines that can trigger strong and enduring memory T cell responses against specific tumor associated antigens (TAAs) is crucial for advancing immunotherapy. DNA-based cancer vaccines have shown promise in generating precise and long-lasting immune responses. In particular DNA vaccines are capable of inducing both humoral and cellular immune responses. In this study, we aimed to investigate the effectiveness of DNA vaccines targeting the mouse Tyrosine-related protein-2 (Trp2) and human New York esophageal squamous cell carcinoma 1 (NY-ESO-1) TAAs in mouse melanoma model. These DNA vaccines were formulated with a functionalized polymer designed to protect DNA from degradation and improve its bioavailability. DNA vaccines targeting both mouse Trp2 (pVACTR) and human NYESO-1 (pVACNY) were constructed. A mouse melanoma cell line (B16F10-NY) overexpressing human NYESO-1 was also generated and demonstrated to induce tumors in C57BL/6 mice. Female C57BL/6 mice received two doses of intramuscular (i.m.) injection of formulated pVACTR or pVACNY or a combination thereof, formulated with the synthetic functionalized polymer, with a 3-week interval. The DNA vaccination induced a potent T cell response, as evidenced by ELISPOT assays showing strong IFN-γ secreting T cells against NYESO-1 and Trp2. Flow cytometry analysis of spleenocytes showed strong IFN-γ and TNF-α secreting CD4 T cells against NYESO-1. We further evaluated the prophylactic and therapeutic efficacy of DNA vaccines targeting both Trp2 and NYESO-1 using a murine syngeneic model. Animals were challenged with a lethal dose of B16F10-NY two weeks after the second immunization exhibited a significant (p<0.0001) delay in tumor progression and a substantial increase in survival compared to the mock control group. In the therapeutic testing, animals challenged with B16F10-NY and subsequently vaccinated at 4, 11, and 25 days also demonstrated delayed tumor growth and prolonged survival. These findings indicate that formulated DNA vaccine delivered via i.m. triggers robust T cell responses and exhibit both prophylactic and therapeutic efficacy, leading to significantly prolonged survival and delayed tumor growth. A DNA-based vaccine independent of viral vector or device has potential to elicit potent anti-tumor responses with better safety and compliance. Citation Format: Kempaiah Rayavara, John Henderson, Meredyth Kinsella, Jessica Kim, Majed Matar, Subeena Sood, Olivia Signer, Chelsey Bellmon, Corinne Le Goff, Khursheed Anwer, Jean Boyer. Intramuscular delivery of tumor associated antigen DNA vaccines elicits strong cellular immune response, delays tumor growth and prolongs survival [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6748.

ZnAl2O4:Er3+ Upconversion Nanophosphor for SPECT Imaging and Luminescence Modulation via Defect Engineering.

This work reports an "all-in-one" theranostic upconversion luminescence (UCL) system having potential for both diagnostic and therapeutic applications. Despite considerable efforts in designing upconversion nanoparticles (UCNPs) for multimodal imaging and tumor therapy, there are few reports investigating dual modality SPECT/optical imaging for theranostics. Especially, research focusing on in vivo biodistribution studies of intrinsically radiolabeled UCNPs after intravenous injection is of utmost importance for the potential clinical translation of such formulations. Here, we utilized the gamma emission from 169Er and 171Er radionuclides for the demonstration of radiolabeled ZnAl2O4:171/169Er3+ as a potent agent for dual-modality SPECT/optical imaging. No uptake of radio nanoformulation was detected in the skeleton after 4 h of administration, which evidenced the robust integrity of ZnAl2O4:169/171Er3+. Combining the therapeutics using the emission of β- particulates from 169Er and 171Er will be promising for the radio-theranostic application of the synthesized ZnAl2O4:169/171Er3+ nanoformulation. Cell toxicity studies of ZnAl2O4:1%Er3+ nanoparticles were examined by an MTT assay in B16F10 mouse melanoma cell lines, which demonstrated good biocompatibility. In addition, we explored the mechanism of UCL modulation via defect engineering by Bi3+ codoping in the ZnAl2O4:Er3+ upconversion nanophosphor. The UCL color tuning was successfully achieved from the red to the green region as a function of Bi3+ codoping concentrations. Further, we tried to establish a correlation of UCL tuning with the intrinsic oxygen and cation vacancy defects as a function of Bi3+ codoping concentrations with the help of electron paramagnetic resonance (EPR) and positron annihilation lifetime spectroscopy (PALS) studies. This study contributes to building a bridge between nature of defects and UC luminescence that is crucial for the design of advanced UCNPs for theranostics.

Tyrosinase regulates the motility of human melanoma cell line A375 through its hydroxylase enzymatic activity

Melanoma, originating from melanocytes, is a highly aggressive tumor. Tyrosinase is involved in melanin production in melanocytes, and its overexpression is noted in malignant melanomas. However, the role of tyrosinase in melanomas remains unclear. Therefore, this study aimed to evaluate the potential functions of tyrosinase in the human melanoma cell line A375. The expression level of tyrosinase in A375 cells was undetectable. However, markedly increased expression level was observed in the mouse melanoma cell line B16F10 and the human melanoma cell line WM266-4. Subsequently, we investigated the effect of ectopic tyrosinase expression on A375 cell motility using wound-healing assay. The overexpression of tyrosinase resulted in enhanced cell migration in both stable and transient tyrosinase expression cells. The levels of filamentous actin were decreased in tyrosinase-expressing A375 cells, suggesting that tyrosinase regulates cell motility by modulating actin polymerization. Histidine residues in tyrosinase are important for its enzymatic activity for synthesizing melanin. Substitution of these histidine residues to alanine residues mitigated the promotion of tyrosinase-induced A375 cell metastasis. Furthermore, melanin treatment enhanced A375 cell metastasis and phosphorylation of Cofilin. Thus, our findings suggest that tyrosinase increases the migration of A375 cells by regulating actin polymerization through its enzymatic activity.

Antioxidant and Anti-Melanogenesis Effects of Teucrium chamaedrys L. Cell Suspension Extract and Its Main Phenylethanoid Glycoside in B16-F10 Cells

Teucrium chamaedrys L. is a typical European–Mediterranean species of the genus Teucrium. Among the phenolic compounds belonging to phenylethanoid glycosides (PGs), teucrioside (TS) is only found in this species, and it was previously demonstrated to be produced by in vitro-elicited cell cultures at levels higher than those found in leaves. However, T. chamaedrys cell suspension extracts (Cell-Ex) and pure TS have not been investigated yet for any biological effects. In this study, we evaluated the antioxidant and anti-melanogenesis activity of both Cell-Ex and TS in B16-F10 mouse melanoma cells. The results showed that Cell-Ex inhibited the reactive oxygen species formation evoked in B16-F10 cells by tert-butyl hydroperoxide and 5 J/cm2 of UVA, as well as the melanin increase stimulated by α-MSH or 20 J/cm2 of UVA. In parallel, a TS concentration equivalent to that present in Cell-Ex recorded the same biological effect profile, suggesting the main contribution of TS to the antioxidant and anti-melanogenic properties of Cell-Ex. Both Cell-Ex and TS also modulated the melanogenesis pathway through their ability to inhibit the tyrosinase activity both in a cell-free system and in B16-F10 cells stimulated by α-MSH. These results support the potential cosmeceutical use of Cell-Ex for protection against photooxidative damage and hyperpigmentation.

Comparative Study of the Effects of Curcuminoids and Tetrahydrocurcuminoids on Melanogenesis: Role of the Methoxy Groups

Curcuminoids are naturally occurring yellow-colored compounds that, when hydrogenated to remove their conjugated double bond, become colorless and are referred to as tetrahydrocurcuminoids. Curcuminoids consist of pure curcumin (PC) in major amounts and demethoxycurcumin (DC) and bisdemethoxycurcumin (BDC) in minor amounts. Tetrahydrocurcuminoids similarly consist mainly of tetrahydrocurcumin (THC), along with minor amounts of tetrahydrodemethoxycurcumin (THDC) and tetrahydrobisdemethoxycurcumin (THBDC). Previous studies have shown the inhibitory effects of PC, DC, and BDC on melanin production, but there are contradictory findings about THC. In addition, there are currently no reports on the effects of THDC and THBDC on melanogenesis. Our previous report described that, in contrast to PC, which suppressed melanin production, THC stimulated melanin production in B16F10 and MNT-1 cells; this effect was ascribed to the loss of the conjugated heptadiene moiety of PC. However, whether this finding can be generalized to the two curcumin derivatives (DC and BDC), such that THDC and THBDC might also stimulate melanogenesis, has not been addressed. Herein, a comparative study of six curcumin derivatives (PC, DC, BDC, THC, THDC, and THBDC) was undertaken to identify their effects on melanogenesis with the goal of elucidating the structure–activity relationships (SARs) focused on assessing the two regions of the parent curcumins’ structure: (i) the hydrogenation of the two double bonds bridging the phenyl rings to the β-diketone moiety, and (ii) the effect of the ortho-methoxy substituent (-OCH3) on the two phenyl rings. To determine the direct effects of the six compounds, antioxidant activity and tyrosinase activity were assessed in cell-free systems before cellular experiments utilizing the B16F10 mouse melanoma cells, MNT-1 human melanoma cells, and primary cells. Evaluations were made on cytotoxicity, melanin concentration, and cellular tyrosinase activity. The results showed that BDC inhibited melanogenesis in B16F10 and MNT-1 cells. However, it was ineffective in primary human melanocytes, while THBDC continued to exhibit anti-melanogenic capacity in normal human melanocytes. Moreover, these findings provide a novel perspective into the role of the methoxy groups of PC on the biological effects of melanogenesis and also confirm that the removal of the conjugated double bonds abolishes the anti-melanogenic capacity of PC and DC only, but not BDC, as THBDC maintained anti-melanogenic activity that was greater than BDC. However, the outcome is contingent upon the specific kind of cell involved. To the best of our knowledge, this work presents novel findings indicating that the anti-melanogenic capacity of the colored BDC is not only intact but enhanced after its hydrogenation as observed in THBDC. The findings show potential for using colorless THBDC as a pharmacological candidate to diminish the increased pigmentation characteristic of skin hyperpigmentation disorders. Future pharmacological therapeutics that incorporate pure THBDC or THBDC-enriched extracts, which retain both a colorless appearance and potent anti-melanogenic activity, can be applied to compounds for anti-melanoma therapeutics where the demand for nontoxic novel molecules is desired for established efficacies.

Targeting HDAC6 improves anti-CD47 immunotherapy

BackgroundCancer cells can overexpress CD47, an innate immune checkpoint that prevents phagocytosis upon interaction with signal regulatory protein alpha (SIRPα) expressed in macrophages and other myeloid cells. Several clinical trials have reported that CD47 blockade reduces tumor growth in hematological malignancies. However, CD47 blockade has shown modest results in solid tumors, including melanoma. Our group has demonstrated that histone deacetylase 6 inhibitors (HDAC6is) have immunomodulatory properties, such as controlling macrophage phenotype and inflammatory properties. However, the molecular and cellular mechanisms controlling these processes are not fully understood. In this study, we evaluated the role of HDAC6 in regulating the CD47/SIRPα axis and phagocytosis in macrophages.MethodsWe tested the role of HDAC6is, especially Nexturastat A, in regulating macrophage phenotype and phagocytic function using bone marrow-derived macrophages and macrophage cell lines. The modulation of the CD47/SIRPα axis and phagocytosis by HDAC6is was investigated using murine and human melanoma cell lines and macrophages. Phagocytosis was evaluated via coculture assays of macrophages and melanoma cells by flow cytometry and immunofluorescence. Lastly, to evaluate the antitumor activity of Nexturastat A in combination with anti-CD47 or anti-SIRPα antibodies, we performed in vivo studies using the SM1 and/or B16F10 melanoma mouse models.ResultsWe observed that HDAC6is enhanced the phenotype of antitumoral M1 macrophages while decreasing the protumoral M2 phenotype. In addition, HDAC6 inhibition diminished the expression of SIRPα, increased the expression of other pro-phagocytic signals in macrophages, and downregulated CD47 expression in mouse and human melanoma cells. This regulatory role on the CD47/SIRPα axis translated into enhanced antitumoral phagocytic capacity of macrophages treated with Nexturastat A and anti-CD47. We also observed that the systemic administration of HDAC6i enhanced the in vivo antitumor activity of anti-CD47 blockade in melanoma by modulating macrophage and natural killer cells in the tumor microenvironment. However, Nexturastat A did not enhance the antitumor activity of anti-SIRPα despite its modulation of macrophage populations in the SM1 tumor microenvironment.ConclusionsOur results demonstrate the critical regulatory role of HDAC6 in phagocytosis and innate immunity for the first time, further underscoring the use of these inhibitors to potentiate CD47 immune checkpoint blockade therapeutic strategies.