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Considerables avances se han vivido en los últimos años, tanto en el conocimiento de la biología del melanoma como en el desarrollo de nuevas terapias médicas que han acreditado ser muy activas, capaces no sólo de inducir respuestas clínicas y radiológicas, sino también de controlar la enfermedad a largo plazo. Más aún, los resultados actualizados de los ensayos fase 3 randomizado en pacientes con enfermedad avanzada o de alto riesgo, han hecho a muchos investigadores especular sobre la posibilidad de que se haya conseguido incluso curar a algunos pacientes. No se puede obviar que los resultados actuales se han precedido de numerosos años de fracasos en la investigación médica con quimioterapia y bioquimioterapia, de varias décadas de investigación y refinamiento en las técnicas quirúrgicas que demostraron ser curativas para la enfermedad localizada y, literalmente, de centurias de perplejidad, ante una enfermedad desconcertante por su comportamiento y agresividad clínica. Presentamos una breve revisión histórica del conocimiento del melanoma, desde una perspectiva médico quirúrgica, con el objetivo que despertar en el lector el interés por avanzar en el conocimiento del pasado y sobre todo del futuro de una patología tumoral, que hasta hace muy poco no tenía opciones terapéuticas, pero que ha abierto las puertas para la innovación terapéutica en oncología.

Background The usefulness of sentinel lymph node biopsy (SLNB) in staging cutaneous melanoma has been proven. Therefore, different tracers have been used to identify the sentinel lymph nodes (SLNs). The use of isotopic tracers together with radioactivity detectors allowed a much more precise and direct approach to the SLNs. However, not all centres have access to a Nuclear Medicine department hindering sentinel lymph node detection (SLND) and consequently, other markers such as ferromagnetic tracers have been evaluated looking for the same advantages and effectiveness as isotopic tracers. Ferromagnetic tracers have proven their usefulness in other cancer entities such as breast, prostate and thyroid cancer. The objective was to assess the detection and concordance rates between isotopic and ferromagnetic techniques for SLNB in cutaneous melanoma. Method Isotopic SLNB technique and ferromagnetic tracer were compared for cutaneous melanoma in a non-inferiority multicentre prospective study carried out in six Spanish hospitals. Results A total of 60 patients were recruited and 133 lymph nodes removed. The detection rate was slightly higher with ferromagnetic tracer in head-neck and trunk melanomas, and with isotopic tracer in limbs. The patients' and nodes' concordance rates between both techniques for ex vivo samples were 95% and 86% for head-neck and trunk tumours and 97% and 93% for limbs tumours, respectively. The concordance rates for involved nodes were 100% and 88.2% for patients and nodes, respectively. Conclusion The intraoperative detection and biopsy of SLN in cutaneous melanoma using a ferromagnetic was a reliable alternative method to the isotopic technique in cutaneous melanomas.

Additional file 9: Figure S9. The figure shows the full original and uncropped images for the western blots presented in the indicated figures. The identification of corresponding protein bands was based on the expected molecular weight as indicated in the main figure.

Additional file 6: Figure S6. Adam10 gene knockout using the CRISPR/Cas9 system in B16/F10 cells. The schematic diagram shows the guide RNA (gRNA) targeting site on exon 3 for clone F4 and exon 2 for clone B6 of the mouse Adam10 gene. Protospacer adjacent motif (PAM) sequences are also presented. The figure also shows Sanger sequencing analysis of PCR fragments amplified from gRNA target regions (the inserted nucleotide is in blue) and protein sequence in wild type (WT) and knockout (KO) cells. Protein expression in WT and two selected clones (F4 and B6) was assayed by western blot. Full blots are shown in Fig. S9. Histogram represents protein quantification.

Additional file 2: Figure S2. IR modulates the immune responsiveness of melanoma cells. a Effects of IR (10 Gy) on HLA-I proteins assayed by confocal microscopy (the scale bar, 15 μm, refers to both panels). b Effects of IR on HLA-I proteins assayed by flow cytometry (IR, 10 Gy, 48 h). c Effects of IR on HLA-I proteins assayed by qRT-PCR (IR, 10 Gy); *p

Additional file 7: Figure S7. Quantification of both MITF and MLANA in flow cytometry experiments depicted in Fig. 2a. *p

Additional file 4: Figure S4. B16/F10-MITF-KO cells were able to form tumors in different experimental models. a Luciferase imaging of R2G2 mice injected in the tail vein with B16/F10 or B16/F10-MITF-KO cells. Picture are representative of n = 3 per group. Color scale was set to Min = 1.00e6; Max = 1.00e7 for all pictures. b Luciferase imaging after subcutaneous injection of indicated melanoma cells induced tumors in C57BL/6 mice. Picture are representative of n = 3 per group. c Luciferase imaging of athymic mice injected in the tail vein with B16/F10-luc2-puromycin cells. Cells were irradiated ex vivo (10 Gy; 24 h). (n = 4).

Additional file 3: Figure S3. MITF gene knockout using the CRISPR/Cas9 system in B16/F10-luc2 and IGR37 cells. a, b The schematic diagrams show the guide RNA (gRNA) targeting site on exon 3 of the Mitf mouse gene and exon 2 of MITF human gene. Protospacer adjacent motif (PAM) sequences are also presented. The figures also show Sanger sequencing analysis of PCR fragments amplified from gRNA target regions (the inserted nucleotide is in blue) and protein sequence in wild type (WT) and knockout (KO) cells. c Protein expression in WT and selected clones was assayed by western blot. Full blots are shown in Fig. S9. Histograms represent protein quantification. d Morphological aspect of WT and knockout melanoma cells used in this study. Bars, 100 μm. e Functional characterization (proliferation and apoptosis) of knockout cells compared to their respective WT cells. MITF knockout in melanoma cells did not induce significant apoptosis (ns, no significant). Treatment of indicated cells with doxorubicin (1 μM; 24 h) was included as an apoptosis positive control; *p

Additional file 8: Figure S8. Densitometry analysis of Western blot data presented in the indicated figures.

Additional file 5: Figure S5. Relationship between MITF and ADAM10 in different melanoma cell lines. a The ChIP-Seq data in 501mel cells [40] indicate the sequence of binding of MITF (E-Box) to the ADAM10 promoter. b The analysis of the normalized expression data (RNA-Seq) shows a positive correlation for the expression of MITF and ADAM10 in melanoma. c Relationship of MITF and ADAM10 mRNAs in different melanoma cell lines. The mRNA profiles have been calculated in relation to the actin of each sample.

Additional file 1: Figure S1. Depletion of CD8+ T lymphocytes. C57BL/6 mice were treated intraperitoneally with a dose (250 μg) of IgG isotype control b or anti-CD8 (clone 2.43) a, c monoclonal antibodies every 3 days. After four doses, peripheral blood was collected and stained to test the presence of CD8+ cytotoxic T lymphocytes among lymphoid cells (R− 1) by flow cytometry. Data shows a complete clearance of T CD8+ cells in a representative experiment out of two with similar results. The remaining blood cell counts (granulocyte, monocytes, NK and B lymphocytes) were unchanged (data not shown).