Identification of universal biomarkers of metastatic melanoma in CTCs

Cutaneous melanoma is the most aggressive form of skin cancer and is notorious for its high metastatic propensity. Melanoma cells disseminate by gaining access to the bloodstream, where they are detectable as Circulating Tumor Cells (CTCs), a subpopulation that constitutes the seeds for metastases and thereby responsible for the vast majority of melanoma-related deaths. The detection and analysis of CTCs can therefore assist early patient prognoses and determine appropriate tailored treatments.

The main scientific objective of our research project is the identification of a universal (composite) biomarkers of metastatic melanoma CTCs, allowing their detection in liquid biopsies using a simple RTqPCR-based assay. To overcome the technical challenges highlighted above, we propose to take advantage of the power of mouse genetics, combined with lineage tracing and emerging single-cell profiling techniques.

We will use genetically engineered mouse models (GEMMs) of metastatic melanoma. Such models have been adopted as one of the workhorses of cancer research because they can be engineered to preserve the molecular and morphological characteristics as well as cellular heterogeneity of human cancers better than simpler cell line in vitro models and, importantly, tumorigenesis occurs spontaneously and in the presence of an intact immune system. They represent powerful platforms for studying the dynamics and mechanisms of cancer progression and therapy resistance. They are also amenable to facile genetic manipulations, lineage tracing/depletion, and functional and phenotypic readouts.

The Marine lab has contributed to the development and characterization of mouse models of melanoma that recapitulate key histopathological features of human melanoma development and allow for single-cell lineage tracing. The use of the tissue-specific conditional expression of a fluorescent reporter allele guarantees efficient isolation of cells of melanocytic lineage by Fluorescence-Activated Cell Sorting (FACS). Individual reporter-positive melanoma cells will be collected from different anatomical sites: primary tumors, blood, lymph nodes and distant metastases. The enclosed project is designed to capitalize on this know-how and established resources. It is obvious that understanding CTCs’ biology can only be properly achieved using methodologies that capture the magnitude and dynamics of both genetic and nongenetic intra-tumor heterogeneity in 4D (space and time) at the single-cell resolution. The advent of reliable single-cell profiling techniques is currently revolutionizing our understanding of individual (cancer) cell behaviors within complex cellular systems/populations, making the enclosed research program both feasible and timely.

The Marine lab has unique expertise in single-cell multi-Omics analytical tools, which allow to simultaneously profile the single cell’s genome, epigenome and transcriptome. Integrating lineage tracing with single-cell Multi-Omic profiling will provide a robust framework for deciphering CTCs biology and identifying specific (combinations of) biomarkers. Using these biomarkers, we will set up a multiparametric FACS-based method for the isolation of CTCs, the performance of which will first be assessed in spiking experiments. Cross-species transcriptome analyses will facilitate the development of a human melanoma-specific CTC biomarker composite, which will be tested on blood isolated from metastatic patient-derived xenograft (PDX) melanoma models. The Marine lab has contributed to the establishment of many melanoma PDX lines, which are hosted at TRACE, our institutional PDX platform. The Marine lab has used PDXs extensively and continues to invest to improve their clinical relevance. Finally, a simple RTqPCR assay will be developed to assess the presence of CTCs in the blood of human patients.

The proximity of the UZ Leuven hospitals will permit the validation of the assay. The ultimate long-term goal of this proposal is to establish putative correlations between melanoma CTC counts and clinical parameters in various clinical settings.We also aim at gaining new insights into the biology of melanoma CTCs and thereby the genetic and nongenetic mechanisms driving early metastatic dissemination. We will exploit our expertise in single-cell multi_omics to probe the extent of CTC heterogeneity and dissect the gene regulatory networks (GRNs) -including the master transcriptional regulators- at play in metastatic CTC populations. Ultimately, we anticipate that this knowledge may lead to the identification of CTC- vulnerabilities, yielding a catalog of potential therapeutic targets. The fundamental innovation of our research project will be the use of an integral in vivo model allowing the efficient and selective isolation of melanoma CTCs at multiple stages of the progression of the disease and their subsequent profiling using single-cell multi-omics methods. This will allow an exhaustive portraying of melanoma CTCs to an unprecedented resolution and depth.

Unfortunately, identification of CTCs in the blood of melanoma patients is a major challenge because of the lack of sufficiently sensitive and specific markers for their isolation using antibody-based methodologies. The markers used to isolate CTCs from epithelial cancers are indeed not expressed in melanoma CTCs. Physical separation techniques likewise are hindered by the low performance of their selection criteria. There is therefore the need to identify reliable melanoma CTC markers.

A keen interest in basic, clinical or translational cancer research is required. Prior experience in biobanking, histopathology and/or bioinformatics is desired.
Prior experience in wet lab technologies (cellular, molecular biology and/or animal/in vivo preclinical models) is a plus.

Medical background or training in the field of histopathology, cancer and metastasis would accelerate the integration of the MD in the project.