Journal: Scientific Reports

Article Title: Remodeling tumor microenvironment by liposomal codelivery of DMXAA and simvastatin inhibits malignant melanoma progression

doi: 10.1038/s41598-021-01284-5

Figure Lengend Snippet: Effects of the combined administration of free and liposome-encapsulated SIM and DMXAA on the growth of s.c . B16.F10 murine melanoma. Mice received two i.v . injections of therapeutic agents at day 11 and day 14, after cancer cell inoculation. Tumor growth curves were presented in panels ( A ), ( C ), and ( E ). Tumor volumes after different treatments at day 15 (when mice were killed) were presented in panels ( B ), ( D ), and ( F ). Control—LCL-treated group; SIM—experimental group treated with 5 mg/kg free SIM; LCL-SIM—experimental group treated with 5 mg/kg SIM as liposome-encapsulated form; DMXAA—experimental group treated with 14 mg/kg free DMXAA; LCL-DMXAA—experimental group treated with 14 mg/kg DMXAA as liposome-encapsulated form; SIM + DMXAA—experimental group treated with 5 mg/kg free SIM and 14 mg/kg free DMXAA; LCL-SIM + LCL-DMXAA—experimental group treated with 5 mg/kg SIM and 14 mg/kg DMXAA as liposome-encapsulated forms. Results were expressed as mean ± SD of tumor volumes of 5 mice. One way ANOVA test with Bonferroni correction for multiple comparisons was performed to analyze the differences between the effects of the treatments on tumor growth ( ns , P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001). The graphs were generated using GraphPad Prism 9.2.0.332 (Serial number: GPS-2216002-E###-#####, MachineID: 3383874FBDD) ( https://www.graphpad.com/ ).

Article Snippet: The graphs were generated using GraphPad Prism 9.2.0.332 (Serial number: GPS-2216002-E###-#####, MachineID: 3383874FBDD) ( https://www.graphpad.com/ ).

Techniques: Generated

Journal: Scientific Reports

Article Title: Remodeling tumor microenvironment by liposomal codelivery of DMXAA and simvastatin inhibits malignant melanoma progression

doi: 10.1038/s41598-021-01284-5

Figure Lengend Snippet: Effects of LCL-SIM and LCL-DMXAA single and combined treatment on the mRNA expression levels of enzymes involved in arginine metabolism in TME. ( A ), ( B ) Effects of liposomal SIM and DMXAA administration on the expression levels of iNOS and ARG-1. mRNA was quantified by RT-qPCR and the results were expressed as fold change based on the Ct calculations. Control—LCL-treated group was used as calibrator. Results were expressed as mean ± SD of three independent measurements. Control—LCL-treated group; LCL-SIM—experimental group treated with 5 mg/kg SIM as liposome-encapsulated form; LCL-DMXAA—experimental group treated with 14 mg/kg DMXAA as liposome-encapsulated form; LCL-SIM + LCL-DMXAA—experimental group treated with 5 mg/kg SIM and 14 mg/kg DMXAA as liposome-encapsulated forms. ( ns , P > 0.05; *, P < 0.05; **, P < 0.01; ***, P < 0.001). The graphs were generated using GraphPad Prism 9.2.0.332 (Serial number: GPS-2216002-E###-#####, MachineID: 3383874FBDD) ( https://www.graphpad.com/ ).

Article Snippet: The graphs were generated using GraphPad Prism 9.2.0.332 (Serial number: GPS-2216002-E###-#####, MachineID: 3383874FBDD) ( https://www.graphpad.com/ ).

Techniques: Expressing, Quantitative RT-PCR, Generated

Journal: Scientific Reports

Article Title: Remodeling tumor microenvironment by liposomal codelivery of DMXAA and simvastatin inhibits malignant melanoma progression

doi: 10.1038/s41598-021-01284-5

Figure Lengend Snippet: Effects of different treatments on angiogenic and inflammatory protein production and neovascularization in the tumor microenvironment. ( A ) Protein levels after different treatments were compared with the levels of the same proteins in control—LCL-treated group. Data are expressed as average % of reduction (–) of protein levels ranging from 0% (white) to − 100% (black) or stimulation (+) of production of proteins ranging from 0% (white) to + 100% (red) compared with the levels of the same proteins in control group. Immunohistochemical scores of ( B ) angiogenesis antigen CD31, ( C ) pan macrophage marker F4/80 and ( D ) M1 macrophage marker iNOS, in B16.F10 murine melanoma sections treated with free or liposomal SIM or/and DMXAA. The scores for immunoreaction intensities, obtained from minimum 10 random fields/condition, were analyzed by using rank-based nonparametric Kruskall-Wallis test with Dunn’s test for multiple comparisons ( ns , P > 0.05; *, P < 0.05). Control—LCL-treated group; LCL-SIM—experimental group treated with 5 mg/kg SIM as liposome-encapsulated form; LCL-DMXAA—experimental group treated with 14 mg/kg DMXAA as liposome-encapsulated form; LCL-SIM + LCL-DMXAA—experimental group treated with 5 mg/kg SIM and 14 mg/kg DMXAA as liposome-encapsulated forms. The graphs were generated using GraphPad Prism 9.2.0.332 (Serial number: GPS-2216002-E###-#####, MachineID: 3383874FBDD) ( https://www.graphpad.com/ ). Multi-panel figure assembly and heatmap were generated using Microsoft® Office Home and Student 2019 MSO 64-bit (product ID: 00,405–57,743-39,916-AA493) ( https://www.microsoft.com/ ).

Article Snippet: The graphs were generated using GraphPad Prism 9.2.0.332 (Serial number: GPS-2216002-E###-#####, MachineID: 3383874FBDD) ( https://www.graphpad.com/ ).

Techniques: Immunohistochemical staining, Marker, Generated

Journal: Scientific Reports

Article Title: Remodeling tumor microenvironment by liposomal codelivery of DMXAA and simvastatin inhibits malignant melanoma progression

doi: 10.1038/s41598-021-01284-5

Figure Lengend Snippet: Effects of liposome-encapsulated SIM and DMXAA on intratumoral levels of key invasion and metastasis promoters. ( A ), ( C ) Western blot analysis showing the effects of different treatments on the intratumor levels of HIF-1α and pAP-1 c-Jun, respectively. β-actin was used as loading control. ( B ), ( D ) Protein levels of HIF-1α and pAP-1 c-Jun in lysates from treated groups expressed as percentage from Control—LCL-treated group. Data represent the mean ± SD of two independent measurements. One way ANOVA test with Bonferroni correction for multiple comparisons was performed to analyze the differences between the effects of the treatments on apoptotic proteins ( ns , P > 0.05; *, P < 0.05; ***, P < 0.001). ( E ), ( F ), ( G )—The effects of different treatments on the activity of microenvironmental matrix metalloproteinases. ( E ) Gelatin zymography analysis of tumor lysates from mice treated with various liposome-encapsulated SIM and DMXAA therapies. Coomassie blue staining highlights gelatinolytic activity corresponding to MMP-9 and MMP-2 (pro-froms and active forms). ( F ), ( G ) Percentage of MMP-9 and MMP-2 activity in tumor lysates from mice treated with single and combined SIM and DMXAA liposomal therapies compared to control. Data represent the mean ± SD of two independent measurements. One way ANOVA test with Dunnett correction was performed to analyze the differences between the effects of various treatments on MMP-9 and MMP-2 levels compared to control untreated group ( ns , P > 0.05; *, P < 0.05; **, P < 0.01). Control—LCL-treated group; LCL-SIM—experimental group treated with 5 mg/kg SIM as liposome-encapsulated form; LCL-DMXAA—experimental group treated with 14 mg/kg DMXAA as liposome-encapsulated form; LCL-SIM + LCL-DMXAA—experimental group treated with 5 mg/kg SIM and 14 mg/kg DMXAA as liposome-encapsulated forms. The uncropped images of HIF-1α, pAP-1 c-Jun and β-actin blots, as well as the uncropped gel showing MMP-9 and MMP-2 gelatinolytic activity are presented in Supplementary Information file. The graphs were generated using GraphPad Prism 9.2.0.332 (Serial number: GPS-2216002-E###-#####, MachineID: 3383874FBDD) ( https://www.graphpad.com/ ). Images were processed using open source IrfanView graphic viewer 64-bit Version 4.58 ( https://www.irfanview.com/ ) and multi-panel figure assembly was generated using Microsoft® Office Home and Student 2019 MSO 64-bit (product ID: 00405-57743-39916-AA493) ( https://www.microsoft.com/ ).

Article Snippet: The graphs were generated using GraphPad Prism 9.2.0.332 (Serial number: GPS-2216002-E###-#####, MachineID: 3383874FBDD) ( https://www.graphpad.com/ ).

Techniques: Western Blot, Activity Assay, Zymography, Staining, Generated

Journal: Scientific Reports

Article Title: Remodeling tumor microenvironment by liposomal codelivery of DMXAA and simvastatin inhibits malignant melanoma progression

doi: 10.1038/s41598-021-01284-5

Figure Lengend Snippet: Effects of different liposomal treatments with SIM and DMXAA on tumor oxidative stress parameters. ( A ) MDA concentration expressed as nmoles MDA/mg protein; ( B ) Catalase activity expressed as U/mg protein; ( C ) TAC expressed as μmoles Trolox/mg protein. All parameters were measured in tumor lysates from mice treated with LCL-SIM and LCL-DMXAA as single or combined therapy. Data represent the mean ± SD of duplicate measurements ( ns , P > 0.05; *, P < 0.05; **, P < 0.01). Control—LCL-treated group; LCL-SIM—experimental group treated with 5 mg/kg SIM as liposome-encapsulated form; LCL-DMXAA—experimental group treated with 14 mg/kg DMXAA as liposome-encapsulated form; LCL-SIM + LCL-DMXAA—experimental group treated with 5 mg/kg SIM and 14 mg/kg DMXAA as liposome-encapsulated forms. The graphs were generated using GraphPad Prism 9.2.0.332 (Serial number: GPS-2216002-E###-#####, MachineID: 3383874FBDD) ( https://www.graphpad.com/ ).

Article Snippet: The graphs were generated using GraphPad Prism 9.2.0.332 (Serial number: GPS-2216002-E###-#####, MachineID: 3383874FBDD) ( https://www.graphpad.com/ ).

Techniques: Concentration Assay, Activity Assay, Generated

Journal: Scientific Reports

Article Title: Remodeling tumor microenvironment by liposomal codelivery of DMXAA and simvastatin inhibits malignant melanoma progression

doi: 10.1038/s41598-021-01284-5

Figure Lengend Snippet: Effects of liposome-encapsulated SIM and DMXAA on intratumoral levels of apoptotic proteins. ( A ), ( B ) Western blot analysis showing the effects of different treatments on the intratumor levels of Bax and Bcl-xL, respectively. β-actin was used as loading control. ( C ), ( D ) Protein levels of Bax and Bcl-xL in lysates from treated groups expressed as percentage from control—LCL-treated group. ( E ) The ratio of % expression levels of Bax/Bcl-xL compared to Control. Data represent the mean ± SD of two independent measurements. One way ANOVA test with Bonferroni correction for multiple comparisons was performed to analyze the differences between the effects of the treatments on the apoptotic proteins ( ns , P > 0.05; *, P < 0.05; **, P < 0.01). Control—LCL-treated group; LCL-SIM—experimental group treated with 5 mg/kg SIM as liposome-encapsulated form; LCL-DMXAA—experimental group treated with 14 mg/kg DMXAA as liposome-encapsulated form; LCL-SIM + LCL-DMXAA—experimental group treated with 5 mg/kg SIM and 14 mg/kg DMXAA as liposome-encapsulated forms. The uncropped images of Bax, Bcl-xL and β-actin blots are presented in Supplementary Information file. The graphs were generated using GraphPad Prism 9.2.0.332 (Serial number: GPS-2216002-E###-#####, MachineID: 3383874FBDD) ( https://www.graphpad.com/ ). Images were processed using open source IrfanView graphic viewer 64-bit Version 4.58 ( https://www.irfanview.com/ ) and multi-panel figure assembly was generated using Microsoft® Office Home and Student 2019 MSO 64-bit (product ID: 00405-57743-39916-AA493) ( https://www.microsoft.com/ ).

Article Snippet: The graphs were generated using GraphPad Prism 9.2.0.332 (Serial number: GPS-2216002-E###-#####, MachineID: 3383874FBDD) ( https://www.graphpad.com/ ).

Techniques: Western Blot, Expressing, Generated