Kristian Michael Hargadon

Lymph node invasion by tumor cells is an important process in the progression of melanoma and is a poor prognostic factor for patients with this cancer. Before they are able to spread to regional lymph nodes, though, melanoma cells must first adhere to lymphatic endothelium and transmigrate into the lymphatic vasculature. In order to study melanoma cell adhesion to lymphatic endothelial cells and the factors that regulate this process, we have developed an in vitro flow cytometry-based assay to measure melanoma cell attachment to lymphatic endothelial cells. This assay will be a useful tool for investigating the interactions that take place between melanoma cells and lymphatic endothelial cells during the adhesion process.

Cell migration and invasion are vital aspects of melanoma biology that are critical to the progression of this aggressive cancer. Numerous in vitro assays are available to study these hallmark properties of cancer, including two-dimensional wound healing (“scratch”) and transwell (Boyden chamber) assays, as well as three-dimensional microfluidic-based video microscopy assays and matrix/skin organ construct-based assays. Though each of these assays has unique advantages depending on the experimental question at hand, transwell assays are highly adaptable and thus particularly well-suited for investigating the role of specific stimulators/inhibitors of cancer cell migration and invasion. Traditionally, quantification of migrating/invading cells that are capable of transiting the Boyden chamber utilized in these assays is achieved by manual counting of cells that have reached the bottom chamber or by colorimetric/fluorometric detection of transited cells that were preloaded with specific dyes prior to assay initiation. These approaches, while commonly used, have inherent limitations in their design, including the introduction of quantification artifacts that arise from human error during direct cell counting or uneven/inconsistent cell labeling within/across experiments. To overcome these limitations, we have optimized a non-fluorescent flow cytometry-based assay for quantifying melanoma transwell migration and invasion, and we present herein an application of this assay to assess the role of the FOXC2 transcription factor in these processes.

Significant advances in our understanding of antitumor immunity over the last two decades have ushered in an exciting era of cancer immunotherapy. During this time, several strategies have been developed to elicit robust antitumor immune responses by CD8⁺ cytotoxic T lymphocytes. Despite the documented benefits of various approaches aimed at targeting these cells, however, there remains a substantial number of patients who either fail to respond to these regimens or who relapse after an initial response. Therefore, to gain insight into mechanisms of innate and acquired resistance to immune-based therapies for cancer, we must improve our understanding of factors that regulate the induction and maintenance of T cell responses against cancer antigens. In this regard, experimental systems that enable the study of cancer antigen-specific T cells are useful tools for investigating the activation and suppression of antitumor T lymphocytes. To this end, we describe herein a method for generating and characterizing model antigen-expressing melanoma cell lines, and we specifically validate this method using two variants of B16 murine melanoma that we engineered to express the OVA₂₅₇ epitope of chicken ovalbumin, a model neoantigen against which both natural and T cell receptor (TCR) transgenic OT-I CD8⁺ T cell responses can be monitored.

Recent advances in the treatment of metastatic melanoma have emerged only from advances in our understanding of melanoma development and progression at the cellular and molecular level. Despite the impact that such advances have made on the clinical management of this cancer over the last decade, additional insights into factors that promote melanoma progression and therapeutic resistance are needed to combat this disease. CRISPR-Cas9 gene editing technology is a powerful tool for studying gene function in a timely and cost-effective manner, enabling the manipulation of specific DNA sequences via a targeted approach. Herein, we describe a protocol for generating functional gene knockouts in melanoma cell lines by CRISPR-Cas9 gene editing, and we present an example application of this protocol for the successful knockout of the Foxc2 transcription factor-encoding gene in the B16-F1 murine melanoma cell line.

The forkhead box family transcription factor FOXC2 is an important oncogenic driver of several hallmarks of cancer progression. In addition to its well-characterized roles in tumor angiogenesis, metabolic adaptation, and the epithelial-mesenchymal transition that precedes invasion and metastasis, FOXC2 has recently been implicated in tumor immune evasion as well. This Perspective article highlights recent findings concerning the potential value of FOXC2 as a predictive biomarker for cancer immunotherapy and brings attention to data connecting FOXC2 dysregulation to critical determinants of anti-tumor immune outcome, including influences on the makeup of the tumor immune microenvironment and cancer cell responsiveness to type I and type II interferons. In summarizing the current state of knowledge surrounding the immunologic impact of FOXC2 dysregulation in cancer, this article also raises several important questions that will need to be addressed going forward as the field aims to better understand FOXC2′s role in tumor immune evasion so that these insights may be leveraged into translatable interventions that enhance the efficacy of cancer immunotherapy.

Tumor-draining lymph nodes function paradoxically not only as key sites for the priming and coordination of anti-tumor CD8+ T cell responses but also as regional hubs through which invading tumor cells can seed distant metastases. The quality of tumor-specific CD8+ T cells elicited at this site is therefore a critical determinant of the outcome of anti-tumor immunity and cancer progression. Recent studies have demonstrated the significance of CD8+ T cell dysfunction within tumor-draining lymph nodes, highlighting it as an important means of tumor immune escape that may arise early in the course of cancer progression. This review aims to bring attention to emerging data on this topic, with particular focus given to the implications that lymph-node-resident CD8+ T cell dysfunction has both for cancer immunotherapy and for pre-metastatic niche formation during early stages of cancer progression.

Background: The forkhead box family transcription factor FOXC2 has emerged as an important oncogene in cancers of epithelial origin, where it has been associated with several hallmarks of cancer progression. In this study, we provide clinical evidence of a role for FOXC2 in the progression of melanoma. Using bioinformatics‐based approaches, we aimed to gain insight into the oncogenic functions of this transcription factor in this cancer of non‐epithelial origin. Methods: We investigated multiple RNA‐sequencing datasets from biopsies of independent melanoma patient cohorts to identify genes whose expression correlated with that of the FOXC2 gene. These data were analysed using topology‐based Impact Pathway and high‐specificity pruning Gene Ontology approaches. Results: We identified novel biologic pathways and processes significantly impacted by FOXC2 ‐correlated genes, including those related to Rap1 signalling and collagen fibril organisation. For these novel pathways/processes, as well as others previously linked with the activity of FOXC2, our study also revealed specific FOXC2 ‐correlated genes associated with these biological phenomena. Conclusions: Together, our data offer novel insights into the oncogenic functions of FOXC2 in melanoma and other cancers.

Melanoma invasion of regional lymph nodes, a critical event in the progression of this disease, is well documented as a poor prognostic factor for patients with melanoma. Assessing lymph node involvement is therefore a routine part of the diagnostic workup for patients presenting with melanoma at a T stage of ≥T2a. In clinical settings, the status and degree of melanoma lymph node involvement is traditionally characterized by histopathological analysis of tissue obtained during a sentinel lymph node biopsy, a labor‐intensive and costly approach that requires technically challenging sample preparation and interpretation by a trained pathologist. Alternative approaches that might reduce the financial burden and turnaround time of a sentinel lymph node biopsy workup are therefore desirable. Likewise, the ability to accurately assess lymph node invasion by melanoma in experimental settings is necessary to gain new insights into mechanisms of distant metastasis and tumor‐associated immune suppression. With these applications in mind, we recently developed, and describe herein, a tissue‐specific qRT‐PCR‐based protocol for detecting melanoma cells within tumor‐draining lymph nodes. Using murine models of lymph‐node‐invasive and lymph‐node‐noninvasive melanoma cell lines and a melanin‐biosynthesis‐pathway‐specific Trp2 gene expression assay, we validated this method as a highly sensitive strategy for assessing lymph node involvement by melanoma. © 2025 Wiley Periodicals LLC.Basic Protocol 1: Growth and maintenance of murine melanoma cell culturesBasic Protocol 2: Extraction and quantification of RNA from murine melanoma culturesBasic Protocol 3: cDNA synthesis via reverse transcriptionBasic Protocol 4: Probe and primer design for TaqMan‐based qPCRBasic Protocol 5: qPCR analysis of tissue‐specific Trp2 gene expressionBasic Protocol 6: In vitro validation of qRT‐PCR Trp2 gene expression assay for detection of melanoma cells in murine whole lymph node preparationsBasic Protocol 7: In vivo validation of qRT‐PCR Trp2 gene expression assay for detection of melanoma cells in murine tumor‐draining lymph nodes

Background: Lymph node invasion by cancer cells is a poor prognostic factor and is often associated with anti‐tumor CD8+ T cell dysfunction. In this study, we investigated the role of lymph node invasion by melanoma cells in the induction of incomplete differentiation by tumor antigen‐specific CD8+ T cells.Aims: We aimed to determine whether lymph node invasion by melanoma cells is required for this specific form of anti‐tumor CD8+ T cell dysfunction.Methods and Results: We assessed lymph node invasion by the B16‐F1 and D5.1G4 murine melanoma cell lines and evaluated tumor antigen‐specific CD8+ T cell responses to these melanomas in the context of tumor‐free versus tumor‐involved lymph nodes. We demonstrate that CD8+ T cells recognizing antigen from established melanomas fail to acquire effector function, regardless of whether the tumor is stable or progressive. This CD8+ T cell dysfunction arises in the context of both tumor‐involved and tumor‐free lymph nodes draining established melanomas.Conclusions: Lymph node invasion by melanoma cells is not required for the induction of incomplete CD8+ T cell differentiation. These data and their implications for strategies to enhance CD8+ T cell responses against poorly immunogenic melanomas are discussed herein.