Melanoma, 1st ed. 2021 Methods and Protocols Methods in Molecular Biology Series, Vol. 2265

1. Model Systems for the Study of Malignant Melanoma

            Randal K. Gregg

Part I: 2D Cell Culture-Based Approaches for Studying Melanoma Biology

2. Generation of Functional Gene Knockout Melanoma Cell Lines by CRISPR-Cas9 Gene Editing

            Kristian M. Hargadon, David Z. Bushhouse, Coleman E. Johnson, and Corey J. Willams

            Ewa Mazurkiewicz, Ewa Mrówczyńska, Aleksandra Simiczyjew, Dorota Nowak, and Antonina J. Mazur

4. Wound Healing Assay for Melanoma Cell Migration

            Juliano T. Freitas, Ivan Jozic, and Barbara Bedogni

5. A Fluorescence-Based Assay for Measuring Glucose Uptake in Living Melanoma Cells

            Jelena Grahovac, Marijana Pavlović, and Marija Vidosavljević

6. Analyzing Melanoma Cell Oxygen Consumption and Extracellular Acidification Rates Using Seahorse Technology

            Ashley V. Menk and Greg M. Delgoffe

7. Analysis of Melanoma Cell Glutamine Metabolism by Stable Isotope Tracing and Gas Chromatography-Mass Spectrometry

            David A. Scott

8. Determination of Cytotoxic Activities Against Melanoma Cells Using Flow Cytometry

            Guilan Shi and Richard Heller

9. In Vitro Differentiation of Tumor-Associated Macrophages from Monocyte Precursors with Modified Melanoma-Conditioned Medium

            Tao Wang and Russel E. Kaufman

10. A Flow Cytometric Assay for Investigating Melanoma Cell Adhesion to Lymphatic Endothelial Cells

            Kristian M. Hargadon and Coleman E. Johnson

Part II: 3D Cell Culture Systems for Studying Melanoma

11. An Approach to Study Melanoma Invasion and Crosstalk with Lymphatic Endothelial Cell Spheroids in 3D Using Immunofluorescence

            Sanni Alve, Silvia Gramolelli, and Päivi M. Ojala

12. Evaluating Melanoma Viability and Proliferation in 3D Microenvironments

            Vasanth Siruvallur Murali, Murat Can Cobanoglu, and Erik S. Welf

13. Preparation, Drug Treatment, and Immunohistological Analysis of Tri-Culture Spheroid 3D Melanoma-Like Models

            Maximilian E.A. Schäfer, Julia Klicks, Mathias Hafner, and Rüdiger Rudolf

14. Enrichment of Melanoma Cancer Stem Cells via Sphere Assays

            Nabanita Mukherjee, Karoline A. Lambert, David A. Norris, and Yiqun G. Shellman

Part III: Techniques for Isolating and Studying Circulating Melanoma Cells

15. Capture and Isolation of Circulating Melanoma Cells Using Photoacoustic Flowmetry

            Robert H. Edgar, Justin Cook, Madeline Douglas, Anie-Pier Samson, and John A. Viator

16. Multi-Marker Immunomagnetic Enrichment of Circulating Melanoma Cells

            Aaron B. Beasley, Emmanuel Acheampong, Weitao Lin, and Elin S. Gray

17. PD-L1 Detection on Circulating Melanoma Cells

            Joseph W. Po, Yafeng Ma, Bavanthi Balakrishnar, Daniel Brungs, Farhad Azimi, Adam Cooper, Erin Saricilar, Vinay Murthy, Paul de Souza, and Therese M. Becker

18. Transcript-Based Detection of Circulating Melanoma Cells

            Michael Morici, Weitao Lin, and Elin S. Gray

19. Isolation and Quantification of Plasma Circulating Tumor DNA from Melanoma Patients

            Gabriela Marsavela, Anna Reid, Elin S. Gray, and Leslie Calapre

20. Simultaneous BRAF^V600E Protein and DNA Aberration Detection in Circulating Melanoma Cells Using an Integrated Multi-Molecular Sensor

            Alain Wuethrich, Shuvashis Dey, Kevin M. Koo, Abu A.I. Sina, and Matt Trau

21. Single Cell Analysis of BRAF^V600E and NRAS^Q61R Mutation Status in Melanoma Cell Lines as Method Generation for Circulating Melanoma Cells

            Joseph W. Po, Yafeng Ma, Alison W.S. Luk, David Lynch, Bavanthi Balakrishnar, Daniel Brungs, Farhad Azimi, Adam Cooper, Erin Saricilar, Vinay Murthy, Paul de Souza, and Therese M. Becker

Part IV: Methods to Study Melanoma-Derived and Melanoma-Associated Exosomes

22. A Rapid Exosome Isolation Using Size Exclusion Chromatography (REIUS) Method for Exosome Isolation from Melanoma Cell Lines

            Shin La Shu, Cheryl L. Allen, Shawna Benjamin-Davalos, Marina Koroleva, Don MacFarland, Hans Minderman, and Marc S. Ernstoff

23. Immunoaffinity-Based Isolation of Melanoma Cell-Derived and T Cell-Derived Exosomes from Plasma of Melanoma Patients

            Sujan Kumar Mondal and Theresa L. Whiteside

24. An Immunocapture-Based Assay for Detecting Multiple Antigens in Melanoma-Derived Extracellular Vesicles

            Carmen Campos-Silva, Yaiza Cáceres-Martell, Sheila López-Cobo, María Josefa Rodriguez, Ricardo Jara, María Yáñez-Mó, and Mar Valés‐Gómez

25. Post-Lymphadenectomy Analysis of Exosomes from Lymphatic Exudate/Exudative Seroma of Melanoma Patients

            Susana García-Silva, Pilar Ximénez-Embún, Javier Muñoz, and Héctor Peinado

Part V: Assessing Melanoma Metastasis in Pre-Clinical and Clinical Settings

26. Fate Mapping of Cancer Cells in Metastatic Lymph Nodes Using Photoconvertible Proteins

            Ethel R. Pereira, Dmitriy Kedrin, and Timothy P. Padera

27. Detection of Melanoma Cells in Lymphatic Drainage (LD) after Lymph Nodes Dissection via Nested RT-PCR Analysis of Molecular Melanocytic Markers

28. A Clonogenic Assay to Quantify Melanoma Micrometastases in Pulmonary Tissue

            Fabrizio Mattei, Sara Andreone, and Giovanna Schiavoni

29. PET Imaging of Melanoma Using Melanin-Targeted Probe

            Xiaowei Ma and Zhen Cheng

30. Imaging and Isolation of Extravasation-Participating Endothelial and Melanoma Cells During Angiopellosis

            Tyler A. Allen and Ke Cheng

Part VI: Strategies for Diagnostic and Prognostic Biomarker Identification for Melanoma and Computational/Bioinformatic Approaches to OMICS Analysis of Melanoma

31. Salivary Proteomic Analysis of Canine Oral Melanoma by MALDI-TOF Mass Spectrometry and LC-Mass Spectrometry/Mass Spectrometry

            Sekkarin Ploypetch, Sittiruk Roytrakul, and Gunnaporn Suriyaphol

32. Detection of Uveal Melanoma by Multiplex Immunoassays of Serum Biomarkers

            Jin Song, Zhen Zhang, and Daniel W. Chan

33. Unbiased Microbiome and Metabolomic Profiling of Fecal Samples from Patients with Melanoma

            Ashley Bui, Yongbin Choi, Arthur E. Frankel, and Andrew Y. Koh

34. Assessment of Cell-Free microRNA by NGS Whole Transcriptome Analysis in Cutaneous Melanoma Patients’ Blood

            Kevin D. Tran, Rebecca Gross, Negin Rahimzadeh, Shanthy Chenathukattil, Dave S.B. Hoon, and Matias A. Bustos

35. High-Throughput Identification of miRNA-Target Interactions in Melanoma Using miR-CATCHv2.0

            Andrea Marranci, Romina D’Aurizio, Milena Rizzo, Catherine M. Greene, and Laura Poliseno

Part VII: Characterization of Melanoma-Associated Tumor-Infiltrating Lymphocytes and Tertiary Lymphoid Structures

36. Immunotyping and Quantification of Melanoma Tumor–Infiltrating Lymphocytes

            Max O. Meneveau, Zeyad T. Sahli, Kevin T. Lynch, Ileana S. Mauldin, and Craig L. Slingluff, Jr.

37. Single Cell Gene Expression, Clonality, and Feature Barcoding of Melanoma Tumor Infiltrating Lymphocytes

            Angela Pizzolla, Simon Keam, Criselle D’Souza, Timothy Semple, and Paul J. Neeson

38. Using Mass Cytometry to Analyze the Tumor-Infiltrating Lymphocytes in Human Melanoma

            Daniela Tantalo, Thu Nguyen, Han Xian Aw Yeang, Joe Zhu, Sean Macdonald, Minyu Wang, Harini de Silva, Criselle D’Souza, Angela Pizzolla, and Paul J. Neeson

39. Multiplex Immunohistochemistry Analysis of Melanoma Tumor Infiltrating Lymphocytes

            Thu Nguyen, Nikolce Kocovski, Sean Macdonald, Han Xian Aw Yeang, Minyu Wang, and Paul J. Neeson

40. Multiplex Immunofluorescence Histology for Immune Cell Infiltrates in Melanoma-Associated Tertiary Lymphoid Structures

            Ileana S. Mauldin, Adela Mahmutovic, Samuel J. Young, and Craig L. Slingluff, Jr.

Part VIII: Development and Evaluation of Therapeutic Strategies for Melanoma Treatment

41. The Use of Nanoparticles in the Delivery of Nucleic Acids for Melanoma Treatment

            Mohammad A. Obeid, Alaa A.A. Aljabali, Meriem Rezigue, Haneen Amawi, Hanin Alyamani, Shatha N. Abdeljaber, and Valerie A. Ferro

42. siRNA Delivery to Melanoma Cells with Cationic Niosomes

            Mohammad A. Obeid, Hanin Alyamani, Haneen Amawi, Alaa A.A. Aljabali, Meriem Rezigue, Shatha N. Abdeljaber, and Valerie A. Ferro

43. Controlled Delivery of Plasmid DNA to Melanoma Tumors by Gene Electrotransfer

            Richard Heller and Guilan Shi

44. Generation of Murine Chimeric Antigen Receptor T Cells for Adoptive T Cell Therapy for Melanoma

            Amorette Barber

45. Generation of Phosphopeptide-Specific T Cell Lines as Tools for Melanoma Immunotherapy

            Rebecca C. Obeng and Angela L. Ambakhutwala

Includes cutting-edge techniques

Provides step-by-step detail essential for reproducible results

Contains key implementation advice from the experts