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. 2021 Dec 30;14:10397–10416. doi: 10.2147/IJGM.S336105

Immune Score Indicator for the Survival of Melanoma Patients Based on Tumor Microenvironment

Xuchao Ning 1, Renzhi Li 2, Bin Zhang 3, Yue Wang 4, Ziyi Zhou 1, Zanzan Ji 5, Xiajie Lyu 6, Zhenyu Chen 1,✉
PMCID: PMC8724722  PMID: 35002296

Abstract

Background

Tumor microenvironment (TME) refers to the cellular environment where tumors exist, including immune cells, fibroblasts, stromal cells, chemokines, etc. TME is closely related to the prognosis of various tumors; nevertheless, limited studies have established predictive prognosis models based on TME. This work aims to construct a survival prediction model for melanoma patients based on TME.

Methods

Data of 482 melanoma patients were extracted from The Cancer Genome Atlas (TCGA) database. Based on the infiltration of immune cells (Immune score), stromal cells (Stromal score), and tumor purity (Estimate score), the “Estimate” algorithm was used to construct 3 scores for each patient. To identify the differentially expressed genes (DEGs), Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were conducted using DAVID database and visualized using the R software. The STRING database was used to construct the protein-protein interaction (PPI) network and functional modules. FGD2 expression was confirmed via Western Blotting and quantitative reverse transcription PCR (RT-qPCR) analyses.

Results

Patients with higher immune scores estimate scores showed better OS than those with lower scores. All three scores were related to age and primary tumor stage. Further, DEGs between patients with high immune/stromal scores and low immune/stromal scores were screened. Eventually, 10 down-regulated DEGs and 201 up-regulated DEGs were identified as TME associated genes. Out of these, the FGD2 gene demonstrated close association with survival and was confirmed in the included melanoma patients.

Conclusion

In summary, TME is closely associated with the prognosis of melanoma patients. Besides, genes including FGD2 promote the TME-mediated regulation of melanoma.

Keywords: tumor microenvironment, the cancer genome atlas, melanoma, FGD2

Introduction

The tumor microenvironment (TME) is the initial internal environment where tumor cells proliferate. The main cell types in TME include stromal cells (fibroblasts, endothelial cells, and many more) and immune cells (T cells, B cells, etc.). Accumulating studies indicate that the tumor microenvironment regulates tumor immunosuppression, drug resistance, tumor invasion, metastasis, and growth.1,2

In the past decades, significant treatment efforts of cancers targeted tumor cells; nevertheless, with the growing research importance of TME, there has been a gradual shift in the concept of cancer treatment. Unlike the adaptive mutation and acquired drug resistance produced by tumor cell accumulation, the immunotherapy approach targeting TME is stable As the most promising therapy in various cancers, immune checkpoint inhibitors (ICIs) are based on the immune escape in TME. Immune checkpoints are molecules producing costimulatory or inhibitory signals in the immune response, thus regulating the host immune response. Recent studies focused on the immune checkpoint PD-1 and its ligand PD-L1 signal axis. PD-L1, highly expressed in tumors, binds to PD-1 on the surface of T cells, inducing their depletion, thereby causing immune escape of tumor cells. Thus, the treatment of PD-1 or PD-L1 monoclonal antibodies to rescue the suppression of TME on T cells restores the normal activation of T cells.3,4 Although the stromal cells in TME are not as important as immune cells in tumor immunotherapy, they regulate anti-tumor therapy. Commonly used methods minimize matrix hardness and fibrosis, thereby promoting immune cell infiltration and drug delivery.5,6 Besides the therapeutic effect, TME mediates the prediction of cancer progression and response to immunotherapy. Reportedly, a Tumor Inflammation Signature (TIS) based on the expression of 18-gene signatures demonstrate satisfactory performance in predicting adaptive immune response.7 In digestive system cancers, a prognostic immune score based on 22 types of immune cells shows satisfactory performance in predicting the survival of patients.8

Melanoma is a tumor produced by melanocytes in the skin and other organs with high malignancy. Its early diagnosis and treatment are crucial for prevention. Melanoma incidence has increased at an annual rate of about 3% to 7%, hence one of the fastest-growing malignant tumors in recent years. The primary risk factors for melanoma include a history of long-term sun exposure, UV exposure history, local chronic injury, or irritation. Meanwhile, melanoma is cancer with highly activated TME.9 As such, our research seeks to understand the prediction role of TME in melanoma and molecular mechanisms underlying TME regulation.

Materials and Methods

Data Acquisition and Score Construction

The data were obtained from the TCGA (The results here are in whole or part based upon data generated by the TCGA Research Network: https://www.cancer.gov/tcga). database. Transcriptome data of 482 melanoma patients were identified and downloaded from the TCGA database using the R package “TCGA-Assembler”. Relevant clinical characteristics were also obtained and are shown in Table 1.

Table 1.

Relevant clinical characteristics of melanoma patients

Id Futime Fustat Age Gender Stage T M N
TCGA-DA-A95Z 396 0 87 MALE Stage IV TX M1a N0
TCGA-FS-A1ZF 470 1 78 FEMALE Stage IIC T4b M0 N0
TCGA-D3-A2J8 1992 1 48 MALE Stage IB T2a M0 N0
TCGA-ER-A2NC 1333 1 50 MALE Stage IB T2a M0 N0
TCGA-RP-A693 10 0 77 MALE Stage IV TX M1c NX
TCGA-EB-A82C 17 0 70 FEMALE Stage IIC T4b M0 N0
TCGA-W3-AA1R 3379 1 71 MALE Stage II T3 M0 N0
TCGA-EE-A2GU 2884 0 65 FEMALE Stage IA T1a M0 N0
TCGA-BF-A1PZ 853 0 71 FEMALE Stage IIB T4a M0 N0
TCGA-FS-A1ZQ 4062 1 31 MALE I/II NOS TX M0 N0
TCGA-EE-A20I 412 1 79 MALE Stage IV TX M1c N0
TCGA-FR-A44A 5299 0 29 FEMALE Stage II T3a M0 N0
TCGA-D3-A3C6 1766 1 54 FEMALE Stage IB T2a M0 N0
TCGA-EE-A3AE 1658 0 52 FEMALE Stage IA T1a M0 N0
TCGA-GN-A262 4255 0 47 FEMALE unknow unknow unknow unknow
TCGA-ER-A2NE 613 1 39 MALE Stage 0 Tis M0 N0
TCGA-D3-A51R 1941 0 60 MALE Stage IIA T3a M0 N0
TCGA-EB-A97M 414 0 66 MALE Stage IIC T4b M0 N0
TCGA-WE-A8ZQ 1923 0 48 MALE Stage IIA T3a M0 N0
TCGA-ER-A42K 394 1 40 FEMALE Stage IIIC T4b M0 N3
TCGA-EE-A2MT 2166 0 45 MALE Stage IB T2a M0 N0
TCGA-DA-A960 804 0 73 MALE Stage IIB T3b M0 N0
TCGA-XV-AAZY 405 0 76 FEMALE Stage IIIC T4 M0 N3
TCGA-EE-A2M6 3932 0 61 MALE Stage I T1 M0 N0
TCGA-GN-A264 3587 1 60 MALE unknow unknow unknow unknow
TCGA-ER-A19O 1 56 MALE Stage IIIB T3b M0 N1b
TCGA-D9-A6E9 301 0 75 FEMALE Stage IIIA T3a M0 N1
TCGA-EE-A2MN 1446 1 58 MALE Stage I T2 M0 N0
TCGA-DA-A1I4 1093 1 51 MALE Stage IIIC T3b M0 N2b
TCGA-EE-A3AB 3733 0 30 MALE Stage III T0 M0 N2a
TCGA-DA-A3F8 1319 0 39 MALE Stage IIIB T2a M0 N2b
TCGA-BF-AAP6 325 0 55 MALE Stage III T4b M0 N2
TCGA-FS-A1ZD 1628 1 63 MALE Stage IIA T2b M0 N0
TCGA-D9-A4Z3 505 0 73 FEMALE Stage IIIC T4b M0 N1b
TCGA-D3-A8GB 938 1 48 MALE Stage IIIB T3a M0 N1b
TCGA-DA-A95V 2193 0 83 FEMALE Stage IIC T4b unknow N0
TCGA-EE-A2A5 1195 1 43 MALE Stage IB T2a M0 N0
TCGA-D9-A3Z4 519 1 54 MALE Stage IIIC T4b M0 N3
TCGA-FR-A8YE 3176 0 41 MALE Stage IA T1a M0 N0
TCGA-EE-A2GC 2051 0 82 MALE Stage IIB T3b M0 N0
TCGA-EE-A29G 2192 1 53 MALE Stage IIIA T4a M0 N2a
TCGA-EE-A29S 1864 1 79 MALE Stage IIA T3a M0 N0
TCGA-D3-A3MO 284 1 47 MALE Stage III TX M0 N2c
TCGA-WE-A8ZO 2145 0 73 FEMALE Stage IIIB T3a M0 N1b
TCGA-BF-A9VF 440 0 77 MALE Stage IIC T4b M0 N0
TCGA-YD-A89C 210 0 43 FEMALE Stage IA T1a M0 NX
TCGA-EE-A2GT 1365 0 77 MALE Stage IIA T3a M0 N0
TCGA-HR-A5NC 0 0 90 FEMALE unknow T4 M0 NX
TCGA-ER-A19G 9188 0 48 FEMALE unknow unknow M0 N0
TCGA-D3-A3CB 5065 0 39 MALE I/II NOS T2 M0 N0
TCGA-EB-A44P 741 0 58 FEMALE Stage IIC T4b M0 N0
TCGA-EB-A6R0 608 1 58 FEMALE Stage IIC T4b M0 N0
TCGA-D3-A8GD 718 0 63 FEMALE Stage IIIC T4b M0 N3
TCGA-ER-A197 424 1 83 FEMALE Stage IIIB T4b M0 N1a
TCGA-EE-A29X 545 1 58 FEMALE Stage IB T2a M0 N0
TCGA-YD-A9TA 1496 0 75 MALE unknow unknow unknow unknow
TCGA-EE-A2GE 5286 0 44 MALE Stage I T2 M0 N0
TCGA-EB-A57M 472 1 56 MALE Stage IIIB T4b M0 N1
TCGA-EB-A85J 360 0 66 FEMALE Stage IIB T4a M0 N0
TCGA-D3-A2JB 5110 1 70 FEMALE Stage 0 Tis M0 N0
TCGA-D3-A1QB 2912 0 75 FEMALE Stage III T0 M0 N2c
TCGA-D3-A2JE 841 1 75 FEMALE Stage IIIC TX M0 N3
TCGA-DA-A3F5 6873 1 45 MALE Stage I T1a M0 N0
TCGA-EE-A2M5 659 1 49 MALE Stage I T2 M0 N0
TCGA-D3-A2JA 3514 0 68 MALE Stage IIIA T2a M0 N1a
TCGA-ER-A19H 4634 1 40 MALE unknow unknow M0 N0
TCGA-EE-A3JA 1618 1 44 MALE Stage IB T2a M0 N0
TCGA-FS-A4F8 5318 1 52 MALE Stage I T1 M0 N0
TCGA-WE-A8ZR 274 1 49 MALE Stage IIIC T4b M0 N1b
TCGA-EE-A3JD 832 1 70 MALE Stage III TX M0 N2b
TCGA-Z2-AA3S 2950 0 58 MALE Stage IA T1a M0 N0
TCGA-ER-A198 1544 1 45 MALE unknow unknow M0 NX
TCGA-ER-A42L 4533 0 49 MALE Stage II T3 M0 N0
TCGA-FR-A7U8 847 0 50 MALE Stage IIIC TX M0 N3
TCGA-EB-A41B 291 0 76 FEMALE Stage IIC T4b M0 N0
TCGA-EB-A44O 81 0 69 MALE Stage IIB T4a M0 N0
TCGA-GN-A267 1960 1 38 MALE Stage IIIA T4a M0 N1a
TCGA-EE-A2MI 6225 1 43 MALE Stage IIB T4 M0 N0
TCGA-ER-A19N 1341 1 47 MALE unknow unknow unknow unknow
TCGA-EB-A3XC 650 0 74 MALE Stage IIC T4b M0 N0
TCGA-EE-A2M7 877 1 66 MALE Stage II T3a M0 N0
TCGA-EB-A42Z 441 0 49 MALE Stage IIIC T4b M0 N1b
TCGA-D3-A8GI 1780 1 68 MALE Stage IA T1a M0 N0
TCGA-FR-A728 583 0 54 FEMALE Stage IIIB T4b M0 N2a
TCGA-D3-A8GQ 884 1 66 MALE Stage II T3 M0 N0
TCGA-DA-A1I5 4107 0 27 FEMALE Stage IV T1a M1c N0
TCGA-EE-A2GK 1665 0 46 FEMALE Stage I T1 M0 N0
TCGA-BF-A5EO 703 0 65 MALE Stage IIC T4b M0 N0
TCGA-ER-A3EV 1429 1 55 MALE Stage III T4 M0 N0
TCGA-EB-A4OY 977 0 65 FEMALE Stage IIIB T4b M0 N1a
TCGA-D3-A3CF 746 1 61 FEMALE Stage IIIC T4b M0 N3
TCGA-XV-AB01 403 0 54 FEMALE Stage II T3 M0 NX
TCGA-EE-A29E 1940 0 54 MALE Stage IIIB T3a M0 N1b
TCGA-DA-A1IC 2071 1 81 MALE Stage IIIB T3a M0 N2c
TCGA-EE-A180 2889 1 69 MALE Stage III T4a M0 N0
TCGA-D3-A5GS 553 0 58 MALE Stage IV T1b M1c N1b
TCGA-EE-A3AC 1948 0 47 MALE Stage III T0 M0 N2b
TCGA-FS-A1ZZ 822 1 54 FEMALE Stage IIB T3b M0 N0
TCGA-WE-A8K1 1492 0 74 MALE Stage IIIC T3b M0 N3
TCGA-FR-A8YC 1059 1 78 MALE Stage IIB T3b M0 N0
TCGA-FS-A1Z7 237 1 19 MALE Stage IIIC T4b M0 N1b
TCGA-FS-A1ZK 728 1 68 MALE Stage II T4 M0 N0
TCGA-D3-A3CC 2644 0 69 FEMALE Stage IIC T4b M0 N0
TCGA-WE-A8JZ 731 0 70 MALE Stage IIIB T4b M0 N1a
TCGA-ER-A19M 1857 1 36 MALE Stage IB T2a M0 N0
TCGA-FS-A1ZN 730 1 43 MALE Stage IIIA T4b M0 N1a
TCGA-D3-A8GL 2711 1 43 MALE Stage IIIB T2a M0 N1b
TCGA-EB-A5UN 1792 0 49 MALE Stage IIC T4b M0 NX
TCGA-EE-A17X 907 1 54 MALE Stage IA T1a M0 N0
TCGA-EE-A2GD 10346 1 58 FEMALE Stage IIB T4 M0 N0
TCGA-EB-A3XE 180 0 77 FEMALE Stage IIA T3a M0 N0
TCGA-FR-A726 0 0 90 MALE Stage IIC T4b M0 N0
TCGA-D9-A4Z2 190 1 50 MALE Stage IIIC T4b M0 N3
TCGA-FS-A4FC 1655 1 75 FEMALE Stage IIA T3a M0 N0
TCGA-XV-AAZW 393 1 62 FEMALE Stage II T4 M0 N0
TCGA-EB-A1NK 1039 0 48 MALE Stage IIC T4b M0 N0
TCGA-EE-A2GJ 2270 0 83 MALE Stage IA T1a M0 N0
TCGA-EE-A20B 4070 0 66 FEMALE Stage II T3 M0 N0
TCGA-EE-A2MK 5487 0 18 FEMALE Stage III T4a M0 N0
TCGA-GF-A3OT 301 0 58 FEMALE Stage IIIC T3 M0 N3
TCGA-FS-A1ZG 295 1 60 FEMALE Stage IIIC T4b M0 N2b
TCGA-EE-A182 447 1 84 FEMALE Stage IIIC T4b M0 N1b
TCGA-FS-A1Z3 636 1 72 FEMALE Stage IV TX M1 N0
TCGA-ER-A19W 4507 1 48 FEMALE unknow unknow unknow unknow
TCGA-ER-A19P 4930 1 47 FEMALE unknow unknow M0 N0
TCGA-ER-A19C 1487 1 77 MALE Stage I T2a M0 NX
TCGA-BF-A5ES 490 0 76 FEMALE Stage IIC T4b M0 N0
TCGA-D9-A1X3 551 0 63 MALE unknow T4b unknow N2b
TCGA-D3-A1Q7 4053 0 42 FEMALE Stage IB T1b M0 N0
TCGA-EB-A4P0 326 1 82 MALE Stage IIC T4b M0 N0
TCGA-D3-A3MV 1378 0 38 FEMALE Stage IIIB T2b M0 N2a
TCGA-EE-A2GM 2296 0 70 FEMALE Stage IIC T4b M0 N0
TCGA-FR-A7U9 571 0 63 FEMALE Stage IIIC T3b M0 N3
TCGA-FS-A1ZB 1486 1 57 MALE Stage II T3a M0 N0
TCGA-QB-A6FS 220 0 49 MALE Stage IIIC T0 M0 N3
TCGA-WE-A8ZY 1506 1 62 MALE Stage IIA T3a M0 NX
TCGA-EE-A2MF 8174 1 39 FEMALE Stage I T2 M0 N0
TCGA-EB-A4XL 777 0 56 FEMALE Stage IIC T4b M0 NX
TCGA-EE-A185 151 1 55 FEMALE Stage IIIC T4b M0 N3
TCGA-GN-A4U3 3708 0 30 MALE Stage III T3a M0 N1a
TCGA-EB-A3XB 796 0 63 MALE Stage II T4 M0 NX
TCGA-EE-A2A0 1424 1 77 FEMALE Stage IIA T3a M0 N0
TCGA-DA-A3F3 319 1 52 MALE Stage IIIB T0 M0 N2b
TCGA-EE-A3AF 420 1 48 FEMALE Stage IIIC T0 M0 N3
TCGA-D3-A3BZ 3976 0 63 MALE Stage IIB T4a M0 N0
TCGA-ER-A3ET 2829 1 64 FEMALE Stage IIIA T3a M0 N1a
TCGA-RP-A694 21 0 71 MALE Stage IV TX M1c NX
TCGA-EE-A29C 2402 1 20 MALE Stage IB T2a M0 N0
TCGA-EE-A2MH 516 1 66 MALE Stage III T4a M0 N0
TCGA-EB-A5UM 779 0 48 FEMALE Stage IIC T4b M0 N0
TCGA-EE-A2MC 1871 1 73 MALE Stage I T2 M0 N0
TCGA-BF-A1PV 14 0 74 FEMALE Stage IIC T4b M0 N0
TCGA-GF-A6C8 62 0 62 FEMALE Stage IIB T3b M0 NX
TCGA-XV-A9VZ 0 0 90 FEMALE Stage II T4 M0 N0
TCGA-GN-A4U7 317 1 56 FEMALE Stage IIIC T2b M0 N3
TCGA-DA-A95W 1136 0 52 MALE Stage IIIB TX M0 N1b
TCGA-D3-A3C3 0 unknow FEMALE I/II NOS TX M0 N0
TCGA-D3-A51K 1002 0 51 MALE Stage IIIB Tis M0 N2b
TCGA-D3-A2JD 361 1 58 MALE Stage IIIC T4b M0 N1b
TCGA-EB-A5VU 321 1 56 MALE Stage IIIB T4b M0 N1
TCGA-WE-A8ZN 1794 0 57 MALE Stage IIB T4a M0 NX
TCGA-FS-A1ZH 996 1 71 FEMALE Stage IV T3b M1c N2c
TCGA-D3-A51F 1695 0 51 MALE Stage IIIC T4b M0 N1b
TCGA-BF-AAP2 405 0 62 MALE Stage IIB T3b M0 N0
TCGA-ER-A19L 4000 1 35 MALE unknow unknow unknow unknow
TCGA-W3-A825 1917 1 60 FEMALE Stage II T3 M0 N0
TCGA-GN-A265 2948 0 53 MALE unknow unknow unknow unknow
TCGA-D3-A2JG 3453 1 30 FEMALE Stage IIIA T3a M0 N1a
TCGA-EB-A5SG 2076 0 57 FEMALE unknow unknow unknow unknow
TCGA-W3-A828 3683 1 66 MALE Stage II T3 M0 N0
TCGA-FS-A1ZA 843 1 45 FEMALE Stage IIIB T4b M0 N2c
TCGA-OD-A75X 9061 1 49 MALE unknow TX M0 NX
TCGA-EB-A5SE 401 1 73 MALE Stage IIB T3b M0 NX
TCGA-D3-A8GK 5177 0 45 MALE Stage IIA T3a M0 N0
TCGA-BF-AAP4 335 0 61 MALE Stage IIC T4b M0 N0
TCGA-EE-A2MP 7563 0 34 FEMALE Stage I T2 M0 N0
TCGA-FW-A5DY 587 0 48 FEMALE Stage III T3 unknow N1
TCGA-EB-A5KH 619 1 55 MALE Stage III T0 M0 N1
TCGA-EE-A2MM 5107 1 63 FEMALE Stage I T2 M0 N0
TCGA-EB-A5FP 454 1 65 FEMALE Stage IV T4b M1b NX
TCGA-FS-A1ZR 347 1 36 MALE Stage II T2 M0 N0
TCGA-WE-A8ZT 359 0 25 FEMALE Stage IV T3b M1b N1b
TCGA-D3-A2J6 1321 1 65 MALE Stage IIB T3b M0 N0
TCGA-EE-A29Q 2030 1 70 FEMALE Stage IIB T3b M0 N0
TCGA-EE-A2GS 2470 1 28 FEMALE Stage IB T2a M0 N0
TCGA-D3-A1QA 2765 0 55 MALE Stage IB T2a M0 N0
TCGA-D9-A1JW 111 0 82 MALE unknow T1a M0 N2a
TCGA-ER-A2ND 710 1 57 FEMALE Stage IIIC T1b M0 N3
TCGA-GN-A26C 821 1 77 MALE Stage IIIC T4b M0 N2b
TCGA-EE-A2ME 3141 1 51 MALE Stage I T2 M0 N0
TCGA-WE-AAA3 651 0 84 FEMALE Stage IIIC T4b M0 N2b
TCGA-BF-A3DM 601 0 63 MALE Stage IIA T2b M0 N0
TCGA-D3-A3MU 1209 0 53 MALE Stage IIIA T3a M0 N2a
TCGA-FR-A3YN 2828 0 44 MALE Stage IB T2a M0 N0
TCGA-D9-A3Z3 678 0 39 FEMALE Stage IIIB T3a M0 N1b
TCGA-EE-A3J7 1949 0 43 MALE Stage I T2 M0 N0
TCGA-D3-A2JF 1888 0 74 MALE Stage IA T1a M0 N0
TCGA-EE-A29L 79 1 78 MALE Stage IIIC T4b M0 N3
TCGA-FS-A4FB 813 1 46 FEMALE Stage III T2 M0 N1a
TCGA-D3-A5GN 4129 0 15 FEMALE Stage I T1 M0 N0
TCGA-XV-AAZV 412 0 56 FEMALE Stage II T4 M0 N0
TCGA-D9-A149 1663 0 65 FEMALE unknow TX M0 N1b
TCGA-DA-A1HW 1096 1 37 FEMALE Stage IIIB T1a M0 N1b
TCGA-ER-A2NB 857 1 57 MALE Stage IIIB T4b M0 N2
TCGA-D3-A1Q3 507 1 64 MALE Stage IIC T4b M0 N0
TCGA-D3-A2J7 3136 1 67 MALE Stage IIIC T3b M0 N1b
TCGA-ER-A2NG 1490 1 43 FEMALE Stage IIIC T3b M0 N3
TCGA-FS-A1ZE 1413 1 40 MALE Stage IIC T4b M0 N0
TCGA-DA-A95X 2249 0 62 MALE Stage IB T2a M0 N0
TCGA-FS-A4FD 2454 1 39 MALE Stage IIIC T2 M0 N3
TCGA-GN-A26D 1460 1 72 FEMALE Stage IIC T4b unknow N0
TCGA-3N-A9WC 2022 0 82 MALE Stage IIA T2b M0 NX
TCGA-D3-A1Q6 2184 1 55 MALE Stage III T4 M0 N1b
TCGA-ER-A2NF 877 1 53 MALE Stage IIIB T3b M0 N3
TCGA-FS-A1Z0 6164 1 32 FEMALE Stage IA T1a M0 N0
TCGA-BF-A1Q0 831 0 80 MALE Stage IIC T4b M0 N0
TCGA-EE-A2GR 1301 1 78 MALE Stage II T4 M0 N0
TCGA-WE-AAA0 1229 0 47 MALE Stage IA T1a M0 N0
TCGA-EE-A29P 1716 0 73 FEMALE Stage IIC T4b M0 N0
TCGA-WE-A8K5 1860 1 65 MALE Stage IV T2a M1c N3
TCGA-YG-AA3N 306 0 67 MALE Stage IIC T4b M0 N0
TCGA-DA-A1IB 1235 1 69 FEMALE Stage IIIC T2b M0 N2b
TCGA-EB-A430 1 83 MALE Stage IIC T4b M0 N0
TCGA-BF-A1PX 282 1 56 MALE Stage IIIB T4b M0 N2a
TCGA-FS-A4F2 1525 1 46 FEMALE Stage IIC T4b M0 N0
TCGA-GN-A8LN 772 0 68 MALE Stage IIC T4b M0 NX
TCGA-EB-A299 378 0 63 MALE Stage IIA T2b M0 N0
TCGA-EE-A2MU 1620 0 71 MALE Stage IA T1a M0 N0
TCGA-ER-A199 279 1 86 FEMALE Stage IIIC T4b M0 N3
TCGA-BF-AAP8 447 0 58 MALE Stage IIC T4b M0 N0
TCGA-ER-A194 1354 1 77 MALE unknow unknow M0 N0
TCGA-EB-A5UL 891 0 71 MALE Stage III TX M0 N1
TCGA-EE-A29H 1966 0 59 FEMALE Stage IA T1a M0 N0
TCGA-D3-A51N 688 0 56 FEMALE Stage IV T0 M1c N3
TCGA-EB-A5SH 1643 0 60 FEMALE Stage III T4 M0 N0
TCGA-EE-A2MJ 2927 1 60 MALE Stage III T4b M0 N0
TCGA-RP-A690 6 0 66 FEMALE unknow unknow unknow unknow
TCGA-EE-A29B 2588 1 67 MALE Stage IIB T3b M0 N0
TCGA-QB-AA9O 549 1 73 MALE Stage IIIC TX M0 N3
TCGA-EB-A550 264 1 75 FEMALE Stage IIC T4b M0 NX
TCGA-FS-A1ZJ 1441 1 75 FEMALE Stage I T2 M0 N0
TCGA-EB-A3HV 39 0 37 MALE Stage IIC T4b M0 N0
TCGA-3N-A9WB 518 1 71 MALE Stage IA T1a M0 NX
TCGA-W3-AA21 3195 1 26 MALE Stage I T2 M0 N0
TCGA-D3-A8GC 2421 1 48 MALE Stage IIIC TX M0 N3
TCGA-FS-A1ZT 1617 0 55 MALE Stage III T2 M0 N1b
TCGA-EE-A181 1026 1 82 FEMALE Stage II T3 M0 N0
TCGA-D3-A8GP 4638 0 77 MALE Stage III T2 M0 N2c
TCGA-BF-AAP0 454 0 40 FEMALE Stage IV T4 M1 NX
TCGA-DA-A1I8 1640 1 63 FEMALE Stage IIC T4b M0 N0
TCGA-D3-A5GO 4195 0 61 MALE Stage II T4 M0 N0
TCGA-D3-A51T 818 0 59 FEMALE Stage IIIC T4b M0 N1b
TCGA-ER-A19F 802 1 82 MALE unknow unknow M0 N0
TCGA-EB-A44R 315 1 52 MALE Stage IIIB TX M0 N2b
TCGA-FS-A1Z4 854 1 62 MALE Stage I T1 M0 N0
TCGA-FR-A3YO 0 unknow FEMALE I/II NOS T2 M0 N0
TCGA-BF-AAP1 409 0 86 MALE Stage IIC T4b M0 N0
TCGA-D9-A3Z1 468 1 66 MALE Stage IIIC T2a M0 N3
TCGA-EB-A6L9 1109 0 55 MALE Stage IIIC TX M0 N3
TCGA-ER-A42H 426 1 76 MALE unknow unknow unknow unknow
TCGA-ER-A19S 1505 0 81 FEMALE unknow unknow unknow unknow
TCGA-ER-A1A1 3196 0 58 MALE Stage IIIC TX M0 N3
TCGA-DA-A1I1 6768 0 55 MALE Stage III T0 M0 N2a
TCGA-D3-A3C1 0 unknow MALE I/II NOS TX M0 N0
TCGA-EB-A82B 390 0 58 FEMALE Stage III T4b M0 N2
TCGA-EE-A29A 1927 1 68 MALE Stage IIIA T3a M0 N1a
TCGA-EB-A431 568 0 34 MALE Stage IIC T4b M0 N0
TCGA-FS-A4F5 874 1 77 FEMALE Stage IB T2a M0 N0
TCGA-EB-A42Y 721 1 73 FEMALE Stage IIC T4b M0 N0
TCGA-D3-A2JK 368 1 24 MALE Stage IIIC T4b M0 N2b
TCGA-D3-A51J 4414 0 19 MALE Stage III T0 M0 N1b
TCGA-WE-A8ZX 1089 0 45 MALE Stage IIIB TX M0 N1b
TCGA-EE-A29T 11252 0 51 FEMALE unknow TX M0 NX
TCGA-ER-A19J 196 1 54 MALE Stage IV TX M1 N3
TCGA-W3-AA1W 6666 0 64 MALE Stage II T3 M0 N0
TCGA-BF-A1PU 387 0 46 FEMALE Stage IIC T4b M0 N0
TCGA-EB-A3XF 278 0 57 MALE Stage IIC T4b M0 N0
TCGA-GN-A4U9 673 1 71 MALE Stage IIIC T2b M0 N3
TCGA-EB-A4IS 774 0 77 MALE Stage IIB T3b M0 NX
TCGA-FS-A4F0 2367 0 67 FEMALE Stage IIB T4a M0 N0
TCGA-BF-A5EP 335 0 75 FEMALE Stage IIIC T4b M0 N3
TCGA-EB-A41A 0 0 90 MALE Stage IIC T4b M0 N0
TCGA-ER-A193 955 1 62 MALE Stage IIB T3b M0 N0
TCGA-D3-A2JO 2010 0 50 FEMALE Stage IIIC TX M0 N3
TCGA-LH-A9QB 11217 0 24 FEMALE unknow unknow unknow unknow
TCGA-D3-A3CE 1832 1 74 FEMALE Stage III T0 M0 N1b
TCGA-D3-A5GL 3826 0 74 MALE Stage IB T2a M0 N0
TCGA-EE-A3J5 1124 1 71 MALE Stage III T4a M0 N1
TCGA-EE-A29D 425 1 87 MALE Stage IIIC T3b M0 N1b
TCGA-EE-A2A6 2620 0 43 MALE Stage IA T1a M0 N0
TCGA-D3-A51E 5318 0 39 FEMALE I/II NOS T2 M0 N0
TCGA-EE-A2GH 6699 0 34 MALE Stage I T2 M0 N0
TCGA-EE-A2A2 1814 0 71 MALE Stage IIIC T4b M0 N1b
TCGA-GN-A9SD 1807 1 59 FEMALE Stage IA T1a M0 NX
TCGA-EE-A183 818 1 48 MALE Stage 0 Tis M0 N0
TCGA-EE-A17Z 263 1 57 MALE Stage IIB T4a M0 N0
TCGA-GF-A6C9 480 0 78 MALE Stage IIIB unknow unknow unknow
TCGA-D9-A4Z5 218 0 68 MALE Stage IIB T4a M0 N0
TCGA-D3-A1Q1 504 1 79 FEMALE Stage IIIC T1b M0 N3
TCGA-EB-A3Y7 326 1 86 FEMALE Stage IIIB T3a M0 N2c
TCGA-ER-A3PL 1010 0 30 MALE Stage IV T3b M1a N0
TCGA-D3-A5GU 3808 0 36 MALE Stage IB T1b M0 N0
TCGA-EE-A2GP 423 1 80 MALE Stage IIIB T4b M0 N1a
TCGA-FS-A1YW 6598 1 52 MALE Stage IB T1b M0 N0
TCGA-D3-A2JN 2022 1 46 FEMALE Stage III T0 M0 N1b
TCGA-FS-A1ZC 10870 1 51 MALE I/II NOS TX M0 N0
TCGA-EE-A2MS 4942 0 72 MALE Stage II T3a M0 N0
TCGA-W3-A824 6940 0 63 MALE Stage I T2 M0 N0
TCGA-FS-A1ZW 1505 0 65 MALE Stage IIIB T2b M0 N1a
TCGA-D9-A1JX 216 1 80 FEMALE unknow TX M0 NX
TCGA-EE-A3JB 6138 0 60 FEMALE Stage III T3a M0 N1
TCGA-EE-A2GI 1482 0 39 MALE Stage IA T1a M0 N0
TCGA-EE-A3JH 4086 0 54 MALE Stage IB T2 M0 N0
TCGA-D3-A2JP 1812 0 37 MALE Stage IIIC T0 M0 N3
TCGA-ER-A19Q 1548 1 37 FEMALE unknow unknow M0 N0
TCGA-FR-A8YD 1103 1 56 FEMALE Stage IIC T4b M0 N0
TCGA-BF-A3DJ 464 0 36 FEMALE Stage IIIB T4b M0 N1
TCGA-EE-A20F 2785 0 53 MALE Stage I T1 M0 N0
TCGA-EE-A3AG 1265 1 25 MALE Stage III T0 M0 N2c
TCGA-EE-A29V 787 1 85 MALE Stage IIIC T3b M0 N1b
TCGA-EE-A20H 5118 1 56 MALE Stage I T2 M0 N0
TCGA-ER-A19E 396 1 36 FEMALE Stage IB T2a M0 N0
TCGA-GN-A4U5 1156 0 61 FEMALE Stage IB T2a M0 NX
TCGA-EE-A3J3 5237 1 42 MALE Stage IB T2 M0 N0
TCGA-FW-A3TU 1691 1 72 FEMALE unknow unknow unknow unknow
TCGA-EE-A2MD 1438 1 52 MALE Stage II T3a M0 N0
TCGA-EE-A2GB 1803 0 51 MALE Stage IIIB T2b M0 N1a
TCGA-XV-A9W5 392 0 51 MALE I/II NOS T2 M0 N0
TCGA-GN-A8LL 650 1 68 FEMALE Stage IIC T4b M0 NX
TCGA-BF-A5ER 327 0 63 MALE Stage IIC T4b M0 N0
TCGA-BF-AAOX 444 0 83 MALE Stage IIC T4b M0 N0
TCGA-EB-A44Q 422 0 51 FEMALE Stage IIIC TX M0 N3
TCGA-BF-AAP7 318 0 76 FEMALE Stage IIC T4b M0 N0
TCGA-Z2-A8RT 839 0 42 FEMALE Stage IIB T3b M0 N0
TCGA-D3-A1Q8 854 1 33 MALE Stage IV T0 M1b N3
TCGA-EE-A2M8 601 1 54 FEMALE Stage III T3a M0 N1
TCGA-EB-A553 226 0 62 MALE Stage IIC T4b M0 N0
TCGA-BF-A3DN 717 0 81 FEMALE Stage IIIC T3b M0 N3
TCGA-ER-A3ES 7514 1 25 MALE unknow unknow unknow unknow
TCGA-EB-A85I 362 0 66 MALE Stage IIC T4b M0 N0
TCGA-FR-A69P 478 0 34 FEMALE Stage IIIC TX unknow N3
TCGA-EE-A3AD 875 1 50 MALE Stage III T0 M0 N1b
TCGA-EB-A24D 645 0 72 MALE Stage IIIB T4a M0 N2b
TCGA-D9-A4Z6 561 1 54 MALE Stage IIIC T3b M0 N1b
TCGA-FR-A3R1 685 0 69 MALE Stage IIC T4b M0 N0
TCGA-FS-A1ZY 824 1 71 MALE Stage IIB T3b M0 N0
TCGA-FW-A3I3 531 0 59 FEMALE Stage IV unknow M1 N0
TCGA-EB-A4IQ 636 1 42 FEMALE Stage IIIB T4b M0 N1
TCGA-ER-A19K 469 1 79 FEMALE Stage IIC T4b M0 N0
TCGA-FW-A3TV 411 0 57 FEMALE Stage IIIB T1 M0 N2b
TCGA-EE-A2GN 3106 1 67 MALE Stage IIA T2b M0 N0
TCGA-FR-A7UA 1164 0 65 FEMALE Stage IB T2a M0 N0
TCGA-DA-A3F2 1032 1 55 MALE Stage IIIB T4a M0 N2b
TCGA-Z2-AA3V 486 0 57 FEMALE Stage IA T1a M0 N0
TCGA-FR-A2OS 368 1 49 FEMALE Stage IIC T4b M0 N0
TCGA-EE-A2MQ 1315 1 70 FEMALE Stage IIIA T3a M0 N2a
TCGA-FR-A729 6716 0 38 FEMALE Stage I T1 M0 N0
TCGA-FS-A1YY 6953 1 55 FEMALE Stage IIA T3a M0 N0
TCGA-BF-A3DL 769 0 84 FEMALE Stage IIIB T3b M0 N2
TCGA-YG-AA3P 439 0 63 FEMALE Stage IIB T4a M0 N0
TCGA-DA-A1I7 2703 0 62 MALE Stage IIIB T0 M0 N2b
TCGA-WE-A8K4 614 0 85 MALE Stage IIB T4a M0 NX
TCGA-EE-A2MR 4088 0 61 MALE Stage I T2 M0 N0
TCGA-EB-A3Y6 126 0 56 FEMALE Stage IIC T4b M0 N0
TCGA-BF-AAOU 476 0 73 FEMALE Stage IIC T4b M0 N0
TCGA-ER-A19D 383 1 46 FEMALE Stage IB T2a M0 N0
TCGA-D3-A1Q9 961 1 72 MALE Stage IIIB T4b M0 N2a
TCGA-D3-A2JC 2639 0 53 FEMALE Stage III T0 M0 N2b
TCGA-DA-A1HV 2329 0 75 FEMALE Stage IIIB T0 M0 N2b
TCGA-EE-A2GL 2423 0 40 FEMALE Stage IIA T3a M0 N0
TCGA-ER-A19T 270 1 51 MALE Stage IV T4a M1a N3
TCGA-D3-A2JH 1280 0 68 MALE Stage IB T1b M0 N0
TCGA-GN-A268 1910 1 83 FEMALE Stage IIB T4a M0 N0
TCGA-WE-A8K6 546 0 79 MALE Stage IIIB TX M0 N1b
TCGA-GF-A2C7 21 0 48 MALE Stage IIC T4b M0 N0
TCGA-EE-A2ML 6590 1 35 MALE Stage II T3a M0 N0
TCGA-D3-A1Q4 3408 0 53 FEMALE Stage IIIC T2b M0 N1b
TCGA-D3-A51G 0 unknow MALE Stage 0 Tis M0 N0
TCGA-EE-A2A1 3527 0 46 MALE Stage IB T2a M0 N0
TCGA-GN-A269 170 1 70 MALE Stage IIIC T4b M0 N3
TCGA-D3-A8GN 4897 0 27 FEMALE I/II NOS TX M0 N0
TCGA-D3-A8GJ 7342 0 18 MALE Stage II T3 M0 N0
TCGA-D3-A3ML 422 1 70 MALE Stage IIIA T3a M0 N2a
TCGA-W3-AA1Q 2101 1 57 MALE Stage III TX M0 N1
TCGA-HR-A2OG 7 0 50 FEMALE unknow unknow unknow unknow
TCGA-EE-A3AA 3781 0 47 MALE Stage III T0 M0 N2a
TCGA-FS-A4F4 2028 1 64 MALE Stage II T3a M0 N0
TCGA-EE-A29M 1729 0 33 FEMALE Stage IB T2a M0 N0
TCGA-WE-AAA4 760 0 56 FEMALE Stage IIIC TX M0 N3
TCGA-DA-A1I2 5370 1 45 MALE Stage III T4b M0 N2b
TCGA-WE-A8ZM 3082 0 70 MALE Stage IIIB TX M0 N1b
TCGA-FS-A1ZU 808 1 70 FEMALE Stage IIC T4b M0 N0
TCGA-D3-A2JL 5219 0 43 FEMALE I/II NOS TX M0 N0
TCGA-EB-A4OZ 620 0 41 FEMALE Stage IIIC T4a M0 N3
TCGA-ER-A196 1785 0 64 FEMALE Stage IIC T4b M0 N0
TCGA-FW-A5DX 640 0 71 MALE Stage IIIC T4a unknow N3
TCGA-EB-A6QZ 352 1 76 FEMALE Stage IIA T3a M0 N0
TCGA-D3-A8GS 3564 1 52 MALE Stage I T1 M0 N0
TCGA-DA-A95Y 430 1 68 MALE Stage IIC T4b M0 N0
TCGA-EE-A2GO 3857 0 66 FEMALE Stage II T3b M0 N0
TCGA-EE-A29W 5932 0 42 MALE Stage 0 Tis M0 N0
TCGA-EE-A29N 566 1 78 MALE I/II NOS TX M0 N0
TCGA-EB-A551 590 0 78 FEMALE Stage IIIC T4b M0 N2b
TCGA-D3-A2J9 723 1 75 MALE Stage IIIC T4b M0 N3
TCGA-EE-A3JE 1562 0 75 MALE Stage IIIB T3b M0 N1a
TCGA-EE-A17Y 828 1 69 MALE Stage IIIB T3b M0 N1a
TCGA-D3-A3C8 1409 0 58 FEMALE Stage IIIC TX M0 N3
TCGA-D3-A3C7 1429 0 57 FEMALE Stage III T0 M0 N1b
TCGA-EE-A2MG 3139 1 23 MALE Stage I T2 M0 N0
TCGA-D3-A1Q5 3424 1 60 MALE I/II NOS TX M0 N0
TCGA-EB-A24C 632 0 56 MALE unknow T4b M0 NX
TCGA-XV-A9W2 417 0 81 MALE Stage I T1 M0 N0
TCGA-D9-A6EC 2359 0 56 MALE Stage IIIA T3a M0 N1
TCGA-BF-A5EQ 323 0 63 MALE Stage IIC T4b M0 N0
TCGA-W3-AA1V 1280 1 63 MALE Stage II T3 M0 N0
TCGA-FS-A1ZP 2273 1 52 MALE Stage II T3 M0 N0
TCGA-GN-A4U4 1197 0 73 MALE Stage IIA T2b M0 NX
TCGA-D3-A8GE 804 0 26 MALE Stage IV TX M1b N0
TCGA-EE-A3J8 1044 1 59 MALE Stage IIIA T4a M0 N1a
TCGA-EB-A5SF 369 1 78 FEMALE Stage IIC T4b M0 NX
TCGA-GF-A769 1070 1 39 MALE Stage IIC T4b M0 NX
TCGA-D3-A8GM 3259 1 73 MALE Stage IIB T3b M0 N0
TCGA-FS-A1ZM 3080 0 74 MALE Stage III T2 M0 N2c
TCGA-YD-A9TB 0 unknow FEMALE unknow unknow unknow unknow
TCGA-EE-A3AH 4222 1 30 MALE Stage II T3b M0 N0
TCGA-GN-A266 308 1 45 MALE unknow unknow unknow unknow
TCGA-EB-A5VV 214 0 74 FEMALE Stage IIIB T3b M0 N1
TCGA-EB-A3XD 1160 0 53 FEMALE Stage IIC T4b M0 NX
TCGA-EE-A29R 440 0 48 FEMALE Stage IIIC T3b M0 N1b
TCGA-3N-A9WD 395 1 82 MALE Stage IIIA T2a M0 N1a
TCGA-EE-A20C 4601 1 59 MALE Stage 0 Tis M0 N0
TCGA-D3-A8GV 5101 1 25 MALE I/II NOS TX M0 N0
TCGA-ER-A19A 2365 0 79 MALE Stage IV TX M1 N0
TCGA-ER-A2NH 1264 0 49 MALE Stage IIIC T3a M0 N3
TCGA-EE-A3J4 3869 1 72 MALE Stage II T3a M0 N0
TCGA-D9-A148 4609 0 40 MALE unknow TX M1b N3
TCGA-FS-A1ZS 4526 0 54 MALE Stage I T2 M0 N0
TCGA-ER-A19B 2993 1 42 MALE unknow TX M0 N0
TCGA-GN-A8LK 1524 1 70 MALE Stage IB T1b unknow NX
TCGA-W3-AA1O 122 1 85 MALE Stage III TX M0 N2
TCGA-RP-A6K9 0 unknow FEMALE unknow unknow unknow unknow
TCGA-WE-AA9Y 370 0 37 MALE Stage IIIC T2a M0 N3
TCGA-EE-A3JI 4648 1 48 MALE Stage I T2 M0 N0
TCGA-EB-A6QY 382 0 71 MALE Stage IIC T4b M0 N0
TCGA-GF-A4EO 591 0 74 FEMALE Stage IIIC T0 M0 N3
TCGA-D3-A5GR 5424 0 23 FEMALE Stage III T1b M0 N1
TCGA-D9-A6EG 698 1 56 MALE Stage IIIA T4a M0 N1
TCGA-DA-A1I0 620 1 63 MALE Stage IV T4b M1a N3
TCGA-FW-A3R5 1124 0 68 MALE Stage III TX M0 N2
TCGA-D3-A5GT 487 0 43 MALE Stage IIIC T2b M0 N3
TCGA-EE-A184 2073 1 72 MALE Stage IB T2a M0 N0
TCGA-YG-AA3O 1154 1 62 MALE unknow unknow unknow unknow
TCGA-GN-A4U8 1487 0 51 MALE unknow unknow unknow unknow
TCGA-RP-A695 0 unknow MALE Stage IV TX M1c NX
TCGA-FS-A4F9 1035 0 80 MALE Stage IIIC T4b M0 N3
TCGA-EB-A44N 205 1 59 MALE Stage IIC T4b M0 N0
TCGA-D9-A6EA 766 0 70 MALE Stage IIIC T4a M0 N3
TCGA-GN-A263 467 1 24 MALE Stage IV T4b M1c N3
TCGA-EB-A51B 931 0 53 MALE Stage IIC T4b M0 NX
TCGA-D3-A3MR 3151 0 42 MALE Stage III T0 M0 N1b
TCGA-GN-A26A 988 1 63 FEMALE Stage IIIA T3a M0 N1a
TCGA-DA-A1HY 4407 0 42 MALE Stage III T2b M0 N1
TCGA-D3-A8GO 1 unknow FEMALE I/II NOS T2 M0 N0
TCGA-FS-A1YX 1478 1 39 FEMALE Stage I T2 M0 N0
TCGA-HR-A2OH 2004 1 46 FEMALE Stage IIIB T3b M0 N2a
TCGA-D3-A51H 1714 0 60 MALE Stage IIIC T1b M0 N3
TCGA-ER-A195 1078 1 46 MALE unknow TX M0 N0
TCGA-IH-A3EA 524 0 61 MALE Stage IIC T4b M0 N0
TCGA-D3-A8GR 3943 1 54 FEMALE Stage 0 Tis M0 N0
TCGA-DA-A1IA 2005 1 32 FEMALE Stage IIIB T2a M0 N1b
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The TME score was analyzed using the R package “Estimate”; this algorithm was also used to obtain the three scores, including stromal score, immune score, and estimate score. A higher stromal score and immune score indicated higher infiltration of stromal and immune cells. The estimated score was the sum of the stromal and immune scores. A higher estimate score indicated lower purity of tumor cells.

Screening of Differentially Expressed Genes (DEGs)

The R software “Limma” package was used to normalize the expression of mRNAs based on transcript data derived from the TCGA database. Further, the “DEGseq” package was utilized to screen the DEGs between different groups. P<0.05 and Fold-change>1.5 or Fold-change<-1.5 were set as the screening filters of DEGs.

Gene Ontology, KEGG Pathway, and Gene Set Enrichment Analyses

For Gene Ontology (GO) and KEGG pathway analyses, all the screened DEGs were uploaded to the Database for Annotation Visualization and Integrated Discovery (DAVID, david.ncifcrf.gov/) online tool. Besides, concrete pathways and annotations were obtained using the above-mentioned tool and further visualized using the R software. GSEA database (http://software.broadinstitute.org/gsea/index.jsp) built-in standard datasets were used for gene set enrichment analysis (GSEA) analysis.

Protein Extraction and Western Blotting Analyses

Melanoma tissues samples were extracted from patients diagnosed with melanoma by (three independent) experienced physicians (based on Chinese guidelines for diagnosis and treatment of melanoma). The tissues of each group were digested and lysed using the 100ul RIPA lysate. After complete lysis, the lysate was centrifuged at 4 °C for 15 minutes. The supernatants were collected as the total protein extract. Then, the BCA assay was performed to quantify the proteins (Thermo Fisher Scientific, Waltham, MA, USA). Exactly 20μg proteins were then loaded and separated by 10% SDS-PAGE gels. The proteins were transferred to the PVDF membranes (0.45 mm, Merck Millipore, Billerica, MA, USA). The PVDF membranes were blocked with 5% bovine albumin (BSA) at room temperature for 1 h, then overnight incubated with FGD2 and GAPDH rabbit polyclonal antibodies (1:4000, Abcam, UK) at 4°C. The secondary antibodies were used at a dilution of 1:4000 and incubated at room temperature for 1 h. Eventually, the bands were visualized using the ECL reagents (Merck Millipore).

RNA Extraction, Reverse Transcription, and Quantitative PCR (RT-qPCR)

Melanoma tissues samples were extracted from patients diagnosed with melanoma by (three independent) experienced physicians (based on Chinese guidelines for diagnosis and treatment of melanoma). The total RNA was extracted using the Trizol Reagent (Invitrogen) from tissues based on the manufacturer’s instructions (Trizol, chloroform, and isopropanol were added in turn; the supernatant was centrifuged and quantified by absorbance value of 260nm and stored at - 80 °C). Subsequently, a reverse transcription kit (Takara Bio, Inc., Otsu, Japan) was used to reverse-transcribe RNA into cDNA in a 20ul system. Subsequently, the cDNA was used as a template, detected by the SYBR Green (Takara Bio) and ABI 7900HT Real‑Time PCR system (Applied Biosystems Life Technologies, Foster City, CA, USA). The primers used are shown in Table S1. The comparative cycle threshold values (2‑ΔΔCt) were used to analyze the final results.

Statistical Analysis

The IBM SPSS 19.0 software was used for statistical analyses of all experimental data. Data were expressed as mean ± sd. Graphpad Prism version 7.0 software was used to visualize the statistical results. T-test was used to compare data between two groups, whereas One-way ANOVA was used to compare data between multiple groups; LSDt-test was used for pairwise comparison within the group. Overall Survival (OS) curves were drawn through the Kaplan–Meier analysis. The difference with P < 0.05 was considered statistically significant.

Results

Construction of Tumor Microenvironment Score

In total, transcript data of 482 melanoma patients were extracted from the TCGA-SKCM database; the R software “Limma” package was used for data standardization. The “Estimate” package was utilized to obtain three TME scores for each patient, respectively. Notably, higher stromal and immune scores indicated higher infiltration of stromal cells and immune cells. Estimate scores were the sum of the stromal score and immune score. A higher estimate score indicated lower purity of tumor cells. Patients with higher immune and estimate scores displayed better OS than those with lower scores (Figure 1).

Figure 1.

Figure 1

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Construction of tumor microenvironment score. (A) Kaplan-Meier analysis for the survival of patients based on the stromal score; (B) Kaplan-Meier analysis for the survival of patients based on the immune score; (C) Kaplan-Meier analysis for the survival of patients based on the estimated score. Patients were divided by the median of all these three score systems.

Tumor Microenvironment Score is Associated with Age and Tumor Size

The relationship between TME scores (stromal score: Figure 2A, immune score: Figure 2B, estimate score: Figure 2C) and clinical features of patients (age, gender, pathological stages, etc.) was analyzed. Interestingly, higher TME scores were closely related to younger age (Figure 2 left panel) and earlier primary tumor stage (Figure 2 right panel).

Figure 2.

Figure 2

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Tumor microenvironment score associated with age and tumor size (A–C) The relationship between TME score and clinical features analyzed using One-way ANOVA.

Screening for Tumor Microenvironment Associated Genes

To evaluate the molecular mechanisms underlying the relationship between TME and survival, the patients were divided into two groups based on the median of the stromal and immune scores, respectively (Figure 3A, B, C and D). DEGs were screened between the two groups and further intersected based on stromal score and immune score. Consequently, 10 down-regulated DEGs and 201 up-regulated DEGs were identified. These DEGs were closely related to the TME, hence defined as TME associated genes (Figure 3E and F).

Figure 3.

Figure 3

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Screening for tumor microenvironment associated genes. (A) Heatmap of DEGs between patients with high and low stromal scores; (B) Volcano map of DEGs between patients with high and low stromal scores; (C) Heatmap and of DEGs between patients with high and low immune scores; (D) Volcano map of DEGs between patients with high and low immune scores; (E) Downregulated genes of the intersection of DEGs derived from immune and stromal scores; (F) Upregulated genes of the intersection of DEGs derived from immune and stromal scores.

Gene Ontology, KEGG Pathway, and Protein-Protein Interaction Analyses of Tumor Microenvironment Associated Genes

Furthermore, GO and KEGG analyses were performed based on the TME associated genes. As a result, TME associated genes were closely related to T cell activation, cytokine-cytokine receptor interaction, etc. (Figure 4A and B). Moreover, a PPI network for TME associated genes was constructed, and Top20 hub-genes were calculated using the Cytoscape software (Figure 4C and D). The association of all DEGs and survival was analyzed through the Cox and Kaplan-Meier analyses. Consequently, 138 genes were confirmed to be associated with the survival of melanoma patients (Table S2). Further, 12 intersected genes were finally obtained between the Top 20 hub-genes and 138 survival-associated genes (Figure 4E). Among them, FGD2 showed the smallest q-value, hence was selected for subsequent analyses (Figure 4F).

Figure 4.

Figure 4

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Gene ontology, KEGG pathway, and protein-protein interaction analysis of tumor microenvironment associated genes. (A) Gene ontology (GO) analysis of TME associated genes; (B) KEGG pathway analysis of TME associated genes; (C) protein-protein interaction (PPI) analysis of TME associated genes; (D) Identified of Top 20 hub-genes via the Cytoscape software; (E) Intersection of Top 20 hub-genes and 138 survival associated genes; (F) Survival analysis of 12 intersected genes based on Cox method and visualized by forest map.

FGD2 is Associated with the Progression of Melanoma

FGD2 was found to be associated with the progression of pan-cancer, including adrenocortical carcinoma (ACC), bladder urothelial carcinoma (BLCA), and so on (Figure 5A). Further, were analyzed the association of FGD2 and clinical features. Consequently, higher expression of FGD2 indicated better survival (Figure 5B) and earlier primary tumor stage (Figure 5C). Nonetheless, FGD2 expression was not associated with lymph nodes metastasis, distant metastasis, pathological stage, and age (Figure 5D–G).

Figure 5.

Figure 5

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FGD2 associated with the progression of melanoma. (A) The association of FGD2 with survival in pan-cancer; (B) The association of FGD2 with survival in melanoma performed by Kaplan-Meier analysis; (C–G) The association of FGD2 with clinical features of melanoma patients analyzed by One-way ANOVA.

Validation of FGD2 in Clinical Specimens

To further verify the FGD2 expression, melanoma specimens and paired non-tumor skin tissues were used to perform Western blotting and RT-qPCR analyses (Figure 6A and B). As expected, FGD2 expression was significantly downregulated in melanoma compared to that in paired normal tissues (P<0.001). Also, FGD2 associated pathways were examined through GSEA analysis. As a result, FGD2 was associated with IL6-JAK, KRAS, TNF-α pathways, etc. All these pathways were closely related to the progression of melanoma and TME (Figure 6C). The relationship between FGD2 and various types of immune cells was assessed through ssGSEA analysis. As a consequence, FGD2 expression was closely related to the infiltration of T cells, B cells, and so on (Figure 6D and E). Generally, we confirmed the FGD2 downregulation in melanoma. Besides, downregulated FGD2 may modulate the TME by regulating the infiltration of immune cells. (WB original pictures are shown in the Supplementary Material)

Figure 6.

Figure 6

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Validation of FGD2 in clinical specimens. (A) FGD2 expression in melanoma and paired non-tumor skin tissues validated by Western Blotting analysis (n=10). N: Normal tissues; T: Tumor tissues; (B) FGD2 expression of in melanoma and paired non-tumor skin tissues validated by RT-qPCR analysis (analyzed by Student’s t-test); (C) FGD2 associated pathways assessed by GSEA analysis; (D) The correlation of FGD2 and various types of immune cells assessed by ssGSEA analysis; (E) Top 6 FGD2 associated immune cells derived from the ssGSEA analysis. All experiments were conducted in triplicate.

Discussion

The tumor microenvironment is vital in the development of various tumors. Several studies have reported the role of part cells or factors in the TME of melanoma, including immune cells, immune checkpoints, etc.10,11 Nevertheless, limited information is available on the regulatory mechanisms of TME as a whole.

CIBERSORT is a gene expression-based deconvolution algorithm developed to examine the proportion of stromal and cells in tumor samples.12 Because of its excellent performance, CIBERSORT has been utilized in TME research.13 Based on this algorithm, we calculated three TME scores for each patient, respectively. Stromal and immune scores indicated the infiltration of stromal and immune cells. The estimated score is the sum of stromal and immune scores indicating lower purity of tumor cells. In this scoring system, patients with higher immune and estimate scores demonstrated better survival. This also meant that patients with high infiltration of immune cells displayed better survival. Similar to other solid tumors, melanoma comprises a large number of immune cells, which potentially reflects tumor response. TME with high immune infiltration revealed strong antigenicity and can easily be detected by the immune system. Nonspecific innate immune mechanisms (including phagocytes, natural killer cells, etc.) and specific acquired immune mechanisms (including CD4 + T cells, CD8 + T cells) are involved in the process of tumor cell clearance.14 Further, we analyzed the relationship between estimate score and clinical features. As a result, a higher estimate score was related to younger age and earlier primary tumor stage. That is, highly immune infiltration in the early stage inhibits tumor progression. With the secretion of cytokines in the TME, immune cells are inhibited, immune escape occurs, causing tumor progression.15

We screened DEGs between patients with different TME scores to establish the related mechanisms underlying the regulation of TME. These DEGs were enriched in the T cell activation, cytokine-cytokine receptor interaction, and so on. T cells participate in killing tumors and the effective recognition of tumor cells is the premise of this role. In the TME, tumor cells exhibit selective inhibitory ligands and receptors, which regulate the function of T cells. In recent years, pharmacological modulators of these pathways (known as immune checkpoint therapy, specifically monoclonal antibody forms against PD-1 and CTLA-4) have been widely studied and utilized as novel immunotherapeutic agents against melanoma.16 Considering the early success of immune checkpoint therapy, the development of immunotherapy targeting other costimulatory receptors activating the anti-tumor immune response is seemingly a convincing treatment approach.17

In subsequent analyses, we verified that FGD2 may be the hub-gene of TME regulation in melanoma. Additionally, FGD2 was closely related to the progression of melanoma. Patients with high FGD2 expression demonstrated better survival. The protein encoded by this gene is a member of the guanine nucleotide exchange factors (GEFs) family which regulate cytoskeleton-dependent membrane rearrangements by activating the cell division cycle 42 (CDC42) protein. This gene is expressed in B lymphocytes, macrophages, and dendritic cells. In the B lymphocyte lineage, FGD2 levels change with the developmental stage. In both mature splenic and immature bone marrow B cells, FGD2 expression is suppressed upon activation through the B cell antigen receptor.18 Also, previous research approved FGD2 as a biomarker for head and neck squamous cell carcinoma.19 However, the roles of FGD2 in the response of tumors remain unclear. Through GSEA analysis, we established that FGD2 may regulate immune infiltration of various types of immune cells, including T cells, B cells, etc. Future studies should explore the role of FGD2 in the immune response of tumors.

In conclusion, we established a relationship between TME and the survival of melanoma patients. Consequently, we discovered a novel FGD2 gene that potentially regulates the TME in melanoma.

Acknowledgments

The results here are in part based upon data generated by the TCGA Research Network: https://www.cancer.gov/tcga.

Funding Statement

This study was supported by the Application of excipients loaded with platelet plasma in wound healing (Grant No.3457) and Qingdao Science and Technology Development Plan (Grant No.1651).

Data Sharing Statement

The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Ethics Approval and Consent to Participate

All patients provided and signed informed consents. The study was approved by the Ethical Committee of the Affiliated Hospital of Qingdao University and experiments were performed as per the Ethical Committee’s guidelines and regulations. All procedures involving human participants were performed based on the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards.

Author Contributions

All authors made a significant contribution to the work reported, including in the conception, study design, execution, data acquisition, analysis, and interpretation. Also, the authors participated in drafting, revising, or critically reviewing the article; provided final approval of the version to be published; agreed on the journal to which the article has been submitted, and remain accountable for all aspects of the work. Xuchao Ning and Renzhi Li equally contributed to this paper.

Disclosure

The authors declare no conflicts of interest concerning the publication of this article.

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