In Situ Nanotransformable Hydrogel for Chemo-Photothermal Therapy of Localized Tumors and Targeted Therapy of Highly Metastatic Tumors.

Metastasis is one of the predisposing factors for cancer-related mortalities worldwide. Patients with advanced cancers (stage IV) receive palliative care with minimal possibility of achieving complete remission. Antibody-based therapeutic modalities are capable of targeting tumors that are confined to a particular location but are ineffective in targeting distant secondary tumors. In the current study, we have developed a smart nano-transforming hydrogel (NTG) that transforms in situ to polymeric nanoparticles (PA NPs) of 100-150 nm when injected subcutaneously. These nanoparticles targeted the primary and secondary metastatic tumors for up to ∼5 and ∼3 days, respectively. The in situ-formed PA NPs also demonstrated a pH-responsive drug release resulting in about ∼80% release within 100 h at 5.8 pH. When tested in vivo, substantial inhibition of lung metastases was observed compared to chemotherapy, thus demonstrating the efficiency of nanotransforming hydrogels in targeting and inhibiting primary and secondary metastatic tumors.

PD-1 Blockade on Tumor Microenvironment-Resident ILC2s Promotes TNF-α Production and Restricts Progression of Metastatic Melanoma.

While pulmonary ILC2s represent one of the major tissue-resident innate lymphoid cell populations at steady state and are key drivers of cytokine secretion in their occupational niche, their role in pulmonary cancer progression remains unclear. As the programmed cell death protein-1 (PD-1) plays a major role in cancer immunotherapy and immunoregulatory properties, here we investigate the specific effect of PD-1 inhibition on ILC2s during pulmonary B16 melanoma cancer metastasis. We demonstrate that PD-1 inhibition on ILC2s suppresses B16 tumor growth. Further, PD-1 inhibition upregulates pulmonary ILC2-derived TNF-α production, a cytotoxic cytokine that directly induces cell death in B16 cells, independent of adaptive immunity. Together, these results highlight the importance of ILC2s and their anti-tumor role in pulmonary B16 cancer progression during PD-1 inhibitory immunotherapy.

CRISPR activation screen in mice identifies novel membrane proteins enhancing pulmonary metastatic colonisation.

Melanoma represents ~5% of all cutaneous malignancies, yet accounts for the majority of skin cancer deaths due to its propensity to metastasise. To develop new therapies, novel target molecules must to be identified and the accessibility of cell surface proteins makes them attractive targets. Using CRISPR activation technology, we screened a library of guide RNAs targeting membrane protein-encoding genes to identify cell surface molecules whose upregulation enhances the metastatic pulmonary colonisation capabilities of tumour cells in vivo. We show that upregulated expression of the cell surface protein LRRN4CL led to increased pulmonary metastases in mice. Critically, LRRN4CL expression was elevated in melanoma patient samples, with high expression levels correlating with decreased survival. Collectively, our findings uncover an unappreciated role for LRRN4CL in the outcome of melanoma patients and identifies a potential therapeutic target and biomarker.

Green does not always mean go: A sulfated galactan from Codium isthmocladum green seaweed reduces melanoma metastasis through direct regulation of malignancy features.

Melanoma is the most lethal form of skin cancer, with a worldwide increase in incidence. Despite the increased overall survival of metastatic melanoma patients given recent advances in targeted and immunotherapy, it still has a poor prognosis and available treatment options carry diverse severe side effects. Polysaccharides from seaweed have been shown to exert antitumor activities. Here we show in vitro and in vivo antitumor activities of a sulfated homogalactan (named 3G4S) from Codium isthmocladum seaweed in the B16-F10 murine melanoma cell line. 3G4S did not induce cytotoxicity or proliferation changes; however, it was able to reduce solid tumor growth and metastasis, while not inducing side effects in mice. B16-F10 cells traits related to the metastatic cascade were also impaired by 3G4S, reducing cell invasion, colony-forming capacity and membrane glycoconjugates. Therefore, 3G4S shows promising antitumor activities without the commonly associated drawbacks of cancer treatments and can be further explored.

Dendrimer-based micelles with highly potent targeting to sites of active bone turnover for the treatment of bone metastasis.

Bone-drug targeting therapies using nanoparticles based on targeting ligands remain challenging due to their uptake clearance at non-target sites such as the liver, kidney, and spleen. Furthermore, the distribution sites of nanoparticles in bones have not been fully investigated, thus halting the development of more effective bone metastasis treatment strategies. In this study, we developed nanoparticles self-assembled from cholesterol-terminated, polyethylene glycol-conjugated, aspartic acid (Asp)-modified polyamidoamine dendrimer (Asp-PAMAM-Micelles) with targeting to active bone turnover sites associated with bone metastasis pathogenesis. On analysis through whole-body single photon emission computed tomography/computed tomography (SPECT/CT) imaging, 111In-Asp-PAMAM-Micelles showed high specificity to active bone turnover sites (especially the joints in the lower limbs, shoulder, and pelvis) after intravenous injection in mice. The lower limb bone uptake clearance for 111In-Asp-PAMAM-Micelles encapsulating paclitaxel (PTX) was 3.5-fold higher than that for 111In-unmodified PAMAM-Micelles (PTX). 3H-PTX encapsulated Asp-PAMAM-Micelles effectively accumulated in the lower limb bones in a similar manner as the 111In-Asp-PAMAM-Micelles (PTX). In a bone metastatic tumor mouse model, the tumor growth in the lower limb bones was significantly inhibited by injection of Asp-PAMAM-Micelles (PTX) compared to unmodified PAMAM-Micelles (PTX). Our results demonstrate that Asp-PAMAM-Micelles are sophisticated drug delivery systems for highly potent targeting to active bone turnover sites.

Ephrin-B2-EphB4 communication mediates tumor-endothelial cell interactions during hematogenous spread to spinal bone in a melanoma metastasis model.

Metastases account for the majority of cancer deaths. Bone represents one of the most common sites of distant metastases, and spinal bone metastasis is the most common source of neurological morbidity in cancer patients. During metastatic seeding of cancer cells, endothelial-tumor cell interactions govern extravasation to the bone and potentially represent one of the first points of action for antimetastatic treatment. The ephrin-B2-EphB4 pathway controls cellular interactions by inducing repulsive or adhesive properties, depending on forward or reverse signaling. Here, we report that in an in vivo metastatic melanoma model, ephrin-B2-mediated activation of EphB4 induces tumor cell repulsion from bone endothelium, translating in reduced spinal bone metastatic loci and improved neurological function. Selective ephrin-B2 depletion in endothelial cells or EphB4 inhibition increases bone metastasis and shortens the time window to hind-limb locomotion deficit from spinal cord compression. EphB4 overexpression in melanoma cells ameliorates the metastatic phenotype and improves neurological outcome. Timely harvesting of bone tissue after tumor cell injection and intravital bone microscopy revealed less tumor cells attached to ephrin-B2-positive endothelial cells. These results suggest that ephrin-B2-EphB4 communication influences bone metastasis formation by altering melanoma cell repulsion/adhesion to bone endothelial cells, and represents a molecular target for therapeutic intervention.

Evaluation of [225Ac]Ac-DOTA-anti-VLA-4 for targeted alpha therapy of metastatic melanoma.

Very late antigen 4 (VLA-4; also called integrin α4ß1) is overexpressed in melanoma tumor cells with an active role in tumor growth, angiogenesis, and metastasis, making VLA-4 a potential target for targeted alpha therapy (TAT). METHODS: An anti-VLA-4 antibody was conjugated to DOTA for [225Ac]Ac-labeling and DTPA for [111In]In-labeling. The resulting agents, [225Ac]Ac- or [111In]In-labeled anti-VLA-4 were evaluated in vitro, including binding affinity, internalization, and colony formation assays as well as in vivo biodistribution studies. In addition, the therapeutic efficacy of [225Ac]Ac-DOTA-anti-VLA-4 was evaluated in a disseminated disease mouse model of melanoma. RESULTS: [111In]In-DTPA-anti-VLA-4 demonstrated high affinity for VLA-4 (Kd = 5.2 ± 1.6 nM). [225Ac]Ac-DOTA-anti-VLA-4 was labeled with an apparent molar activity of 3.5 MBq/nmol and > 95% radiochemical purity. Colony formation assays demonstrated a decrease in the surviving fraction of B16F10 cells treated with [225Ac]Ac-DOTA-anti-VLA-4 compared to control. Biodistribution studies demonstrated accumulation in the VLA-4-positive tumor and VLA-4 rich organs. Therapeutic efficacy studies demonstrated a significant increase in survival in mice treated with [225Ac]Ac-DOTA-anti-VLA-4 as compared to controls. CONCLUSION: The work presented here demonstrated that [225Ac]Ac-DOTA-anti-VLA-4 was effective as a treatment in mice with disseminated disease, but potentially has dose limiting hematopoietic toxicity. Preliminary studies presented here also supported the potential to overcome this limitation by exploring a pre-loading or blocking dose strategy, to optimize the targeting vector to help minimize the absorbed dose to VLA-4 rich organs while maximizing the dose delivered to VLA-4-positive melanoma tumor cells.

Dimethyl fumarate suppresses metastasis and growth of melanoma cells by inhibiting the nuclear translocation of NF-κB.

BACKGROUND: Malignant melanoma is among the deadliest forms of skin cancers, and its incidence has been increasing over the past decades. In malignant melanoma, activation of the nuclear factor kappa B (NF-κB) promotes survival, migration, and invasion of cancer cells. Anti-NF-κB agents for treating metastatic melanoma would be beneficial, but no such drug is approved as either monotherapy or adjuvant therapy. Dimethyl fumarate (DMF) is an approved anti-inflammatory drug already in clinical use for psoriasis and multiple sclerosis. OBJECTIVE: We investigated the anti-tumour effect of DMF treatment in metastatic melanoma in vitro and in vivo. METHODS: The cell viability was assessed via trypan blue exclusion assay. The migration and invasion was analyzed in a Boyden chamber assay. The anti-metastatic effects and anti-tumour activity of DMF was determined in an in-vivo model. The expressions of NF-κB pathway and NF-κB regulatory proteins were detected via western blotting. RESULTS: DMF decreased the cell viability, migration and invasion in vitro. In addition, DMF inhibited spontaneous metastasis and tumour growth. Mechanistically, DMF prevented the nuclear translocation of NF-κB, whereas no changes were observed in the phosphorylation levels of inhibitor of kappa B (IκB). In addition, DMF inhibited the expression of matrix metalloproteinases (MMPs) and very late antigens (VLAs). Furthermore, DMF treatment decreased the expression of Survivin and Bcl-extra large (Bcl-XL) proteins. CONCLUSION: Our results suggest that DMF as a novel inhibitor of NF-κB may be a potential therapeutic agent for metastatic melanoma.

Antibody targeting tumor-derived soluble NKG2D ligand sMIC reprograms NK cell homeostatic survival and function and enhances melanoma response to PDL1 blockade therapy.

BACKGROUND: Melanoma patients who have detectable serum soluble NKG2D ligands either at the baseline or post-treatment of PD1/PDL1 blockade exhibit poor overall survival. Among families of soluble human NKG2D ligands, the soluble human MHC I chain-related molecule (sMIC) was found to be elevated in melanoma patients and mostly associated with poor response to PD1/PDL1 blockade therapy. METHODS: In this study, we aim to investigate whether co-targeting tumor-released sMIC enhances the therapeutic outcome of PD1/PDL1 blockade therapy for melanoma. We implanted sMIC-expressing B16F10 melanoma tumors into syngeneic host and evaluated therapeutic efficacy of anti-sMIC antibody and anti-PDL1 antibody combination therapy in comparison with monotherapy. We analyzed associated effector mechanism. We also assessed sMIC/MIC prevalence in metastatic human melanoma tumors. RESULTS: We found that the combination therapy of the anti-PDL1 antibody with an antibody targeting sMIC significantly improved animal survival as compared to monotherapies and that the effect of combination therapy depends significantly on NK cells. We show that combination therapy significantly increased IL-2Rα (CD25) on NK cells which sensitizes NK cells to low dose IL-2 for survival. We demonstrate that sMIC negatively reprograms gene expression related to NK cell homeostatic survival and proliferation and that antibody clearing sMIC reverses the effect of sMIC and reprograms NK cell for survival. We further show that sMIC/MIC is abundantly present in metastatic human melanoma tumors. CONCLUSIONS: Our findings provide a pre-clinical proof-of-concept and a new mechanistic understanding to underscore the significance of antibody targeting sMIC to improve therapeutic efficacy of anti-PD1/PDL1 antibody for MIC/sMIC+ metastatic melanoma patients.

Chemo-immunotherapy with doxorubicin prodrug and erythrocyte membrane-enveloped polymer nano-vaccine enhances antitumor activity.

There are plenty of evidences to show that combining of chemotherapy and immunotherapy should be a very potent anti-cancer therapeutic method. In this research, we evaluated the anti-tumor activity of doxorubicin prodrug combined with erythrocyte membrane-enveloped polymer nano-vaccine against tumor in vitro and in vivo. We also analyzed the immune cell populations to investigate the effect in the tumor microenvironment and explored the inhibitory of lung metastasis. Our study showed that chemo-immunotherapy could inhibit the growth of melanoma tumor in mice. This combination approach promoted a stronger immune response by up-regulating the expression of dendritic cells and cytotoxic T cells in lymph nodes and increased the secretion of cytokines. It also restricted the immunosuppressive environment through inhibiting expression of regulatory T cells. It suggested that combination of prodrug and nano-vaccine achieved better anti-tumor effect than monotherapy, which should be widely valued and further studied to establish in a more economical and efficient way, expected to apply in the future clinical practice of cancer treatment.

Frontline Science: Kindlin-3 is essential for patrolling and phagocytosis functions of nonclassical monocytes during metastatic cancer surveillance.

Nonclassical monocytes maintain vascular homeostasis by patrolling the vascular endothelium, responding to inflammatory signals, and scavenging cellular debris. Nonclassical monocytes also prevent metastatic tumor cells from seeding new tissues, but whether the patrolling function of nonclassical monocytes is required for this process is unknown. To answer this question, we utilized an inducible-knockout mouse that exhibits loss of the integrin-adaptor protein Kindlin-3 specifically in nonclassical monocytes. We show that Kindlin-3-deficient nonclassical monocytes are unable to patrol the vascular endothelium in either the lungs or periphery. We also find that Kindlin-3-deficient nonclassical monocytes cannot firmly adhere to, and instead "slip" along, the vascular endothelium. Loss of patrolling activity by nonclassical monocytes was phenocopied by ablation of LFA-1, an integrin-binding partner of Kindlin-3. When B16F10 murine melanoma tumor cells were introduced into Kindlin-3-deficient mice, nonclassical monocytes showed defective patrolling towards tumor cells and failure to ingest tumor particles in vivo. Consequently, we observed a significant, 4-fold increase in lung tumor metastases in mice possessing Kindlin-3-deficient nonclassical monocytes. Thus, we conclude that the patrolling function of nonclassical monocytes is mediated by Kindlin-3 and essential for these cells to maintain vascular endothelial homeostasis and prevent tumor metastasis to the lung.

Screening of plants from the Brazilian Atlantic Forest led to the identification of Athenaea velutina (Solanaceae) as a novel source of antimetastatic agents.

Plant biodiversity is a source of potential natural products for the treatment of many diseases. One of the ways of discovering new drugs is through the cytotoxic screening of extract libraries. The present study evaluated 196 extracts prepared by maceration of Brazilian Atlantic Forest trees with organic solvents and distilled water for cytotoxic and antimetastatic activity. The MTT assay was used to screen the extract activity in MCF-7, HepG2 and B16F10 cancer cells. The highest cytotoxic extract had antimetastatic activity, as determined in in vitro assays and melanoma murine model. The organic extract of the leaves of Athenaea velutina (EAv) significantly inhibited migration, adhesion, invasion and cell colony formation in B16F10 cells. The phenolic compounds and flavonoids in EAv were identified for the first time, using flow injection with electrospray negative ionization-ion trap tandem mass spectrometry analysis (FIA-ESI-IT-MSn ). EAv markedly suppressed the development of pulmonary melanomas following the intravenous injection of melanoma cells to C57BL/6 mice. Stereological analysis of the spleen cross-sections showed enlargement of the red pulp area after EAv treatment, which indicated the activation of the haematopoietic system. The treatment of melanoma-bearing mice with EAv did not result in liver damage. In conclusion, these findings suggest that A velutina is a source of natural products with potent antimetastatic activity.

Pericyte dysfunction due to Shb gene deficiency increases B16F10 melanoma lung metastasis.

Intravasation, vascular dissemination and metastasis of malignant tumor cells require their passage through the vascular wall which is commonly composed of pericytes and endothelial cells. We currently decided to investigate the relative contribution of these cell types to B16F10 melanoma metastasis in mice using an experimental model of host Shb gene (Src homology 2 domain-containing protein B) inactivation. Conditional inactivation of Shb in endothelial cells using Cdh5-CreERt2 resulted in decreased tumor growth, reduced vascular leakage, increased hypoxia and no effect on pericyte coverage and lung metastasis. RNAseq of tumor endothelial cells from these mice revealed changes in cellular components such as adherens junctions and focal adhesions by gene ontology analysis that were in line with the observed effects on leakage and junction morphology. Conditional inactivation of Shb in pericytes using Pdgfrb-CreERt2 resulted in decreased pericyte coverage of small tumor vessels with lumen, increased leakage, aberrant platelet-derived growth factor receptor B (PDGFRB) signaling and a higher frequency of lung metastasis without concomitant effects on tumor growth or oxygenation. Flow cytometry failed to reveal immune cell alterations that could explain the metastatic phenotype in this genetic model of Shb deficiency. It is concluded that proper pericyte function plays a significant role in suppressing B16F10 lung metastasis.

In Vivo Effects of Human Bone Marrow Mesenchymal Stromal Cells on the Development of Experimental B16 Melanoma in Mice.

Experiments on F1(CBA×C57BL/6) mice with experimental metastatic melanoma B16 F10 showed that single intravenous injection of xenogeneic bone marrow mesenchymal stromal cells (BM-MSC) in a dose of 106 cells/mouse significantly increased 100-day survival rate of tumor-bearing animals. In contrast, administration of BM-MSC in a dose of 2×106 cells/ mouse reduced survival rates in comparison with the biocontrol (injection of B16 cells alone, 5×105 cells/mouse). This phenomenon can be related to in vivo participation of BM-MSC in reprogramming of resident tissue macrophages, including tumor microenvironment, towards pro- (M1) or anti-inflammatory (M2) phenotype. This is indirectly confirmed by the data on switching from activation to inhibition of ROS-producing activity of blood mononuclears and peritoneal macrophages in tumor-bearing mice in the test of luminol-dependent zymosaninduced chemiluminescence.

Anti-tumor immune response varies among individuals: A gene expression profiling of mouse melanoma.

Melanoma is amongst the most aggressive malignant tumors. The purpose of this study is to detect the tumor microenvironment systematically using multi-omics analyses and to propose strategies for precision medicine. Multiple factors of tumor microenvironment contribute to the drug resistance and immune surveillance failure. Here we analyzed genome mutations and characterized the immune state of tumor microenvironments in mouse melanoma by whole exome sequencing (WES) and RNA sequencing (RNA-Seq) approaches. Somatic mutation analysis revealed 35.1% novel mutations in mouse tumors when compared with B16F10 cell line, provided a basis for multi-site sequencing for accurate neoantigen selection. Mutation cluster, gene expression comparison, and gene ontology (GO) analyses by R packages proved DNA repair damage, inflammation, slower cell division, and metabolic change in tumor microenvironment. Further analyses of T-cell receptor (TCR) sequences, immune signaling pathway activation, tumor infiltrated immune cells and chemokine expression revealed extensive difference of antitumor immune response among individuals. Our study revealed the characteristics of tumor microenvironment with mouse melanoma model, suggested the need of comprehensive genome mutations and personal immune state analyses for cancer precision medicine.

Gene expression signatures of site-specificity in cancer metastases.

We have previously shown that metastases are generally characterized by a core program of gene expression that induces the oxidative energy metabolism, activates vascularization/tissue remodeling, silences extracellular matrix interactions, and alters ion homeostasis. This core program distinguishes metastases from their originating primary tumors as well as from their target host tissues. We hypothesized that organ preference is reflected in additional, site-selective components within the metastatic gene expression programs. Expanding our prior analysis of 653 human gene expression profiles plus data from a murine model, we find that the release from the primary tumor is associated with a suppression of functions that are important for the identity of the organ of origin, such as a down-regulation of steroid hormone responsiveness in the disseminated foci derived from prostate cancer. Metastases adjust to their target microenvironment by up-regulating-even overexpressing-genes and genetic programs that are characteristic of that organ. Finally, alterations in RNA and protein processing as well as immune deviation are common. In the clinic, metastases are mostly treated with the chemotherapy protocols devised for their primary tumors. Adjustments that account for the gene expression differences between primary and metastatic cancers have the potential to improve the currently dismal success rates.

Cancer Targeted Gene Therapy for Inhibition of Melanoma Lung Metastasis with eIF3i shRNA Loaded Liposomes.

Eukaryotic translation initiation factors 3i (eIF3i) is a proto-oncogene that is overexpressed in various tumors, reducing its expression by eIF3i shRNA is a promising strategy to inhibit tumor growth or metastasis. Tumor cell is the target of eIF3i shRNA so that tumor-site accumulation could be important for fulfilling its therapeutic effect. Thus, the iRGD modified liposome (R-LP) was rationally synthesized to enhance the antitumor effect by active targeted delivery of eIF3i shRNA to B16F10 melanoma cells. R-LP encapsulating eIF3i shRNA gene (R-LP/sheIF3i) were prepared by a film dispersion method. The transfection experiment proves that R-LP could effectively transfect B16F10 cells. R-LP/sheIF3i notably restrained the migration, invasion, and adhesion of melanoma cells in vitro. In a mouse model of lung metastasis, R-LP/sheIF3i administered by intravenous injection suppressed pulmonary metastasis of melanoma by dramatically downregulated eIF3i expression and subsequently inhibiting tumor neovascularization and tumor cells proliferation in vivo. Our results provide a basis for tumor cells targeting strategies to reduce the expression of eIF3i by RNAi in the treatment of tumor metastasis.

An inhalable nanoparticulate STING agonist synergizes with radiotherapy to confer long-term control of lung metastases.

Mounting evidence suggests that the tumor microenvironment is profoundly immunosuppressive. Thus, mitigating tumor immunosuppression is crucial for inducing sustained antitumor immunity. Whereas previous studies involved intratumoral injection, we report here an inhalable nanoparticle-immunotherapy system targeting pulmonary antigen presenting cells (APCs) to enhance anticancer immunity against lung metastases. Inhalation of phosphatidylserine coated liposome loaded with STING agonist cyclic guanosine monophosphate-adenosine monophosphate (NP-cGAMP) in mouse models of lung metastases enables rapid distribution of NP-cGAMP to both lungs and subsequent uptake by APCs without causing immunopathology. NP-cGAMP designed for enhanced cytosolic release of cGAMP stimulates STING signaling and type I interferons production in APCs, resulting in the pro-inflammatory tumor microenvironment in multifocal lung metastases. Furthermore, fractionated radiation delivered to one tumor-bearing lung synergizes with inhaled NP-cGAMP, eliciting systemic anticancer immunity, controlling metastases in both lungs, and conferring long-term survival in mice with lung metastases and with repeated tumor challenge.

Repurposing of the anti-helminthic drug niclosamide to treat melanoma and pulmonary metastasis via the STAT3 signaling pathway.

The incidence of melanoma is increasing rapidly worldwide. Additionally, new and effective candidates for treating melanoma are needed because of the increase in drug resistance and the high metastatic potential of this cancer. The STAT3 signaling pathway plays a pivotal role in pathogenesis of melanoma, making STAT3 a promising anticancer target for melanoma therapy. Niclosamide, an FDA-approved anti-helminthic drug, has been identified as a potent STAT3 inhibitor that suppresses STAT3 phosphorylation at Tyr705 and its transcript activity. In this study, we evaluated the biological activities of niclosamide in melanoma in vitro and in vivo. Niclosamide potently inhibited the growth of four melanoma cell lines and induced the apoptosis of melanoma cells via the mitochondrial apoptotic pathway. Further, western blot analysis indicated that cell apoptosis was correlated with activation of Bax and cleaved caspase-3 and decreased expression of Bcl-2. Moreover, niclosamide markedly impaired melanoma cell migration and invasion, reduced phosphorylated STAT3Tyr705 levels, and inhibited matrix metalloproteinase-2 and -9 expression. Additionally, in a xenograft model of A375, intraperitoneal administration of niclosamide inhibited tumor growth and tumor weight in a dose-dependent manner without obvious side effects. Histological and immunohistochemical analyses revealed a decrease in Ki-67-positive cells and p-STAT3Try705-positive cells and increase in cleaved caspase-3-positive cells. Notably, niclosamide significantly inhibited pulmonary metastasis in a B16-F10 melanoma lung metastasis model, including the number of lung metastatic nodules and lung/body coefficient. Importantly, a marked reduction in myeloid-derived suppressor cells (Gr1+CD11b+) infiltration in the pulmonary metastasis tissue was observed. Taken together, these results demonstrate that niclosamide is a promising candidate for treating melanoma.

Thymoquinone Enhances the Effect of Gamma Knife in B16-F10 Melanoma Through Inhibition of Phosphorylated STAT3.

BACKGROUND: Patients with brain metastasis from melanoma have a dismal prognosis with poor survival time. Gamma Knife (GK) is an effective treatment to control brain metastasis from melanoma. Thymoquinone (TQ) has emerged as a potential therapeutic option due to its antiproliferative effects on various cancers. The purpose of the study was to assess the effect of GK on B16-F10 melanoma cells in vitro and intracerebral melanoma in vivo, and its synergistic effect in combination with TQ. METHODS: The effects of GK and combination treatment of GK and TQ were studied on B16-F10 melanoma cells by evaluating cytotoxicity with an adenosine triphosphate assay, apoptosis by acridine orange staining, and genotoxicity by comet assay. Western blot analysis was performed to investigate the expression of STAT3, p-STAT3 (Tyr705), JAK2, p-JAK2, caspase-3, Bax, Bcl-2, survivin, and ß-actin. Expression of inflammatory cytokines was assessed by enzyme-linked immunosorbent assay. GK alone and in combination with TQ was assessed in an established intracerebral melanoma tumor in mice. RESULTS: The effects of GK on cytotoxicity, genotoxicity, and apoptosis were enhanced by TQ in B16-F10 melanoma cells. GK induced apoptosis through inhibition of p-STAT3 expression, which in turn regulated pro- and antiapoptotic proteins such as caspase-3, Bax, Bcl-2, and survivin. Adding TQ to GK irradiation further enhanced this apoptotic effect of GK irradiation. GK was shown to reduce the levels of tumor-related inflammatory cytokines in B16-F10 melanoma cells. This effect was more pronounced when TQ was added to GK irradiation. GK with 15 Gy increased the survival of mice with intracerebral melanoma compared with untreated mice. However, despite the additive effect of TQ in addition to GK irradiation on B16-F10 melanoma cells in vitro, TQ did not add any significant survival benefit to GK treatment in mice with intracerebral melanoma. CONCLUSIONS: Our findings suggest that TQ would be a potential therapeutic agent in addition to GK to enhance the antitumor effect of irradiation. Further studies are required to support our findings.