Metastatic melanoma, a highly lethal form of skin cancer, presents significant clinical challenges due to limited therapeutic options and high metastatic capacity. Recent studies have demonstrated that cancer dissemination can occur earlier, before the diagnosis of the primary tumor. The progress in understanding the kinetics of cancer dissemination is limited by the lack of animal models that accurately mimic disease progression. We have established a xenograft model of human melanoma that spontaneously metastasizes to lymph nodes and lungs. This model allows precise monitoring of melanoma progression and is suitable for the quantitative and qualitative analysis of circulating tumor cells (CTCs). We have validated a flow cytometry-based protocol for CTCs enumeration and isolation. We could demonstrate that (i) CTCs were detectable in the bloodstream from the fourth week after tumor initiation, coinciding with the lymph node metastases appearance, (ii) excision of the primary tumor accelerated the formation of metastases in lymph nodes and lungs as early as one-week post-surgery, accompanied by the increased numbers of CTCs, and (iii) CTCs change their surface protein signature. In summary, we present a model of human melanoma that can be effectively utilized for future drug efficacy studies.

Centre for Inflammation Research University of Edinburgh Institute for Regeneration and Repair Edinburgh Scotland

Department of Cytokinetics Institute of Biophysics of the Czech Academy of Sciences Brno Czech Republic

Department of Experimental Biology Faculty of Science Masaryk University Brno Czech Republic

Institute of Biostatistics and Analyses Faculty of Medicine Masaryk University Brno Czech Republic

International Clinical Research Center St Anne's University Hospital Brno Brno Czech Republic

See more in PubMed

Rebecca VW, Somasundaram R, Herlyn M. Pre-clinical modeling of cutaneous melanoma. Nat. Commun. 2020;11:2858. doi: 10.1038/s41467-020-15546-15549. PubMed DOI PMC

Fund, W. C. R. Skin cancer. https://www.wcrf.org/dietandcancer/skin-cancer/ (2021).

Jenkins RW, Fisher DE. Treatment of advanced melanoma in 2020 and beyond. J. Investig. Dermatol. 2021;141:23–31. doi: 10.1016/j.jid.2020.03.943. PubMed DOI PMC

Massagué J, Obenauf AC. Metastatic colonization by circulating tumour cells. Nature. 2016;529:298–306. doi: 10.1038/nature17038. PubMed DOI PMC

Alix-Panabières C, Pantel K. Liquid biopsy: From discovery to clinical application. Cancer Discov. 2021;11:858–873. doi: 10.1158/2159-8290.Cd-20-1311. PubMed DOI

Aya-Bonilla CA, et al. Detection and prognostic role of heterogeneous populations of melanoma circulating tumour cells. Br. J. Cancer. 2020;122:1059–1067. doi: 10.1038/s41416-020-0750-9. PubMed DOI PMC

Kiniwa Y, et al. Usefulness of monitoring circulating tumor cells as a therapeutic biomarker in melanoma with BRAF mutation. BMC Cancer. 2021;21:287. doi: 10.1186/s12885-021-08016-y. PubMed DOI PMC

Kang YT, et al. Isolation of circulating tumor cells to diagnose melanoma and evaluate the efficacy of surgical resection using melanoma-specific microsystem. Adv. Nanobiomed. Res. 2022 doi: 10.1002/anbr.202100083. PubMed DOI PMC

Alix-Panabières C, Pantel K. Challenges in circulating tumour cell research. Nat. Rev. Cancer. 2014;14:623–631. doi: 10.1038/nrc3820. PubMed DOI

Overwijk WW, Restifo NP. B16 as a mouse model for human melanoma. Curr. Protoc. Immunol. 2001 doi: 10.1002/0471142735.im2001s39. PubMed DOI PMC

Pérez-Guijarro E, Day C-P, Merlino G, Zaidi MR. Genetically engineered mouse models of melanoma. Cancer. 2017;123:2089–2103. doi: 10.1002/cncr.30684. PubMed DOI PMC

Kuzu OF, Nguyen FD, Noory MA, Sharma A. Current state of animal (mouse) modeling in melanoma research. Cancer Growth Metastas. 2015;8:81–94. doi: 10.4137/cgm.S21214. PubMed DOI PMC

Xu L, et al. Gene expression changes in an animal melanoma model correlate with aggressiveness of human melanoma metastases. Mol. Cancer Res. 2008;6:760–769. doi: 10.1158/1541-7786.Mcr-07-0344. PubMed DOI PMC

Cruz-Munoz W, Man S, Xu P, Kerbel RS. Development of a preclinical model of spontaneous human melanoma central nervous system metastasis. Cancer Res. 2008;68:4500–4505. doi: 10.1158/0008-5472.Can-08-0041. PubMed DOI

Kucerova L, Skolekova S, Demkova L, Bohovic R, Matuskova M. Long-term efficiency of mesenchymal stromal cell-mediated CD-MSC/5FC therapy in human melanoma xenograft model. Gene Ther. 2014;21:874–887. doi: 10.1038/gt.2014.66. PubMed DOI

Franken NA, Rodermond HM, Stap J, Haveman J, van Bree C. Clonogenic assay of cells in vitro. Nat. Protoc. 2006;1:2315–2319. doi: 10.1038/nprot.2006.339. PubMed DOI

Grill A, Kiouptsi K, Karwot C, Jurk K, Reinhardt C. Evaluation of blood collection methods and anticoagulants for platelet function analyses on C57BL/6J laboratory mice. Platelets. 2020;31:981–988. doi: 10.1080/09537104.2019.1701185. PubMed DOI

Hosseini H, et al. Early dissemination seeds metastasis in breast cancer. Nature. 2016;540:552–558. doi: 10.1038/nature20785. PubMed DOI PMC

Klein CA. Parallel progression of primary tumours and metastases. Nat. Rev. Cancer. 2009;9:302–312. doi: 10.1038/nrc2627. PubMed DOI

Krall JA, et al. The systemic response to surgery triggers the outgrowth of distant immune-controlled tumors in mouse models of dormancy. Sci. Transl. Med. 2018;10:3464. doi: 10.1126/scitranslmed.aan3464. PubMed DOI PMC

Tohme S, Simmons RL, Tsung A. Surgery for cancer: a trigger for metastases. Cancer Res. 2017;77:1548–1552. doi: 10.1158/0008-5472.Can-16-1536. PubMed DOI PMC

Yu M, et al. Circulating breast tumor cells exhibit dynamic changes in epithelial and mesenchymal composition. Science. 2013;339:580–584. doi: 10.1126/science.1228522. PubMed DOI PMC

Kozovska Z, Gabrisova V, Kucerova L. Malignant melanoma: Diagnosis, treatment and cancer stem cells. Neoplasma. 2016;63:510–517. doi: 10.4149/neo_2016_403. PubMed DOI

Gires O, Pan M, Schinke H, Canis M, Baeuerle PA. Expression and function of epithelial cell adhesion molecule EpCAM: Where are we after 40 years? Cancer Metastas. Rev. 2020;39:969–987. doi: 10.1007/s10555-020-09898-3. PubMed DOI PMC

Lenart S, et al. Trop2: Jack of all trades, master of none. Cancers Basel. 2020;12:3328. doi: 10.3390/cancers12113328. PubMed DOI PMC

Remsik J, et al. Trop-2 plasticity is controlled by epithelial-to-mesenchymal transition. Carcinogenesis. 2018 doi: 10.1093/carcin/bgy095. PubMed DOI

Patton EE, et al. Melanoma models for the next generation of therapies. Cancer Cell. 2021;39:610–631. doi: 10.1016/j.ccell.2021.01.011. PubMed DOI PMC

Savoia P, Zavattaro E, Cremona O. Clinical implications of acquired BRAF inhibitors resistance in melanoma. Int. J. Mol. Sci. 2020;21:9730. doi: 10.3390/ijms21249730. PubMed DOI PMC

Tangella LP, Clark ME, Gray ES. Resistance mechanisms to targeted therapy in BRAF-mutant melanoma: A mini review. Biochim. Biophys. Acta BBA Gen. Subj. 2021;1865:129736. doi: 10.1016/j.bbagen.2020.129736. PubMed DOI

Quail DF, Joyce JA. Microenvironmental regulation of tumor progression and metastasis. Nat. Med. 2013;19:1423–1437. doi: 10.1038/nm.3394. PubMed DOI PMC

Dotse E, Lim KH, Wang M, Wijanarko KJ, Chow KT. An immunological perspective of circulating tumor cells as diagnostic biomarkers and therapeutic targets. Life. 2022;12:323. doi: 10.3390/life12020323. PubMed DOI PMC

Pearson T, et al. Non-obese diabetic–recombination activating gene-1 (NOD–Rag 1 null) interleukin (IL)-2 receptor common gamma chain (IL 2 rγnull) null mice: A radioresistant model for human lymphohaematopoietic engraftment. Clin. Exp. Immunol. 2008;154:270–284. doi: 10.1111/j.1365-2249.2008.03753.x. PubMed DOI PMC

Shultz LD, Brehm MA, Garcia-Martinez JV, Greiner DL. Humanized mice for immune system investigation: Progress, promise and challenges. Nat. Rev. Immunol. 2012;12:786–798. doi: 10.1038/nri3311. PubMed DOI PMC

Lawrence R, Watters M, Davies CR, Pantel K, Lu Y-J. Circulating tumour cells for early detection of clinically relevant cancer. Nat. Rev. Clin. Oncol. 2023;20:487–500. doi: 10.1038/s41571-023-00781-y. PubMed DOI PMC

Goodale D, Phay C, Postenka CO, Keeney M, Allan AL. Characterization of tumor cell dissemination patterns in preclinical models of cancer metastasis using flow cytometry and laser scanning cytometry. Cytometry A. 2009;75:344–355. doi: 10.1002/cyto.a.20657. PubMed DOI

Schmidt CM, et al. Characterization of spontaneous metastasis in an aggressive breast carcinoma model using flow cytometry. Clin. Exp. Metastasis. 1999;17:537–544. doi: 10.1023/a:1006719800907. PubMed DOI

Gray ES, et al. Circulating melanoma cell subpopulations: Their heterogeneity and differential responses to treatment. J. Investig. Dermatol. 2015;135:2040–2048. doi: 10.1038/jid.2015.127. PubMed DOI PMC

Hall CS, et al. Circulating tumor cells in Stage IV melanoma patients. J. Am. Coll. Surg. 2018;227:116–124. doi: 10.1016/j.jamcollsurg.2018.04.026. PubMed DOI

Joshi P, et al. Enrichment of circulating melanoma cells (CMCs) using negative selection from patients with metastatic melanoma. Oncotarget. 2014;5:2450–2461. doi: 10.18632/oncotarget.1683. PubMed DOI PMC

Padera TP, et al. Lymphatic metastasis in the absence of functional intratumor lymphatics. Science. 2002;296:1883–1886. doi: 10.1126/science.1071420. PubMed DOI

Mohammed SI, Torres-Luquis O, Walls E, Lloyd F. Lymph-circulating tumor cells show distinct properties to blood-circulating tumor cells and are efficient metastatic precursors. Mol. Oncol. 2019;13:1400–1418. doi: 10.1002/1878-0261.12494. PubMed DOI PMC

Krall JA, et al. The systemic response to surgery triggers the outgrowth of distant immune-controlled tumors in mouse models of dormancy. Sci. Transl. Med. 2018;10:3464. doi: 10.1126/scitranslmed.aan3464. PubMed DOI PMC

Hynes RO. Metastatic potential: Generic predisposition of the primary tumor or rare, metastatic variants—or both? Cell. 2003;113:821–823. doi: 10.1016/S0092-8674(03)00468-9. PubMed DOI

Eddy K, Shah R, Chen S. Decoding melanoma development and progression: Identification of therapeutic vulnerabilities. Front. Oncol. 2021;10:8. doi: 10.3389/fonc.2020.626129. PubMed DOI PMC

Boiko AD, et al. Human melanoma-initiating cells express neural crest nerve growth factor receptor CD271. Nature. 2010;466:133–137. doi: 10.1038/nature09161. PubMed DOI PMC

Redmer T, et al. The nerve growth factor receptor CD271 is crucial to maintain tumorigenicity and stem-like properties of melanoma cells. PLoS One. 2014;9:e92596. doi: 10.1371/journal.pone.0092596. PubMed DOI PMC

Gorges TM, et al. Circulating tumour cells escape from EpCAM-based detection due to epithelial-to-mesenchymal transition. BMC Cancer. 2012;12:178. doi: 10.1186/1471-2407-12-178. PubMed DOI PMC