Abstract
Background
Circulating Tumour Cells (CTCs) are pre-metastatic seeds released by solid tumours into the bloodstream or lymphatic system. Since CTCs can be detected in the blood of cancer patients, they possess the potential to be developed as a minimally invasive marker for improving cancer management. Nevertheless, clinical translation of CTCs is limited due to a lack of understanding of the role CTCs play in metastasis. This is mainly due to the rarity of CTCs (1-10 CTCs/mL) in peripheral blood, resulting in a lack of development of a robust method for their isolation. Currently, CellSearch is the only FDA-approved CTC enrichment technology that enriches CTCs that express the epithelial cell adhesion molecule (EpCAM) and excludes CD45-positive cells, a haematopoietic marker. Nevertheless, this method fails to enrich CTCs that have undergone epithelial-to-mesenchymal transition (EMT), which results in the loss of EpCAM expression. Further, CellSearch also misses out on CTC clusters (cCTC; CTC subpopulations more relevant to the process of metastasis).
Size-based techniques, on the other hand, can not only capture CTCs along the EMT axis but also have the ability to capture cCTCs effectively. The MetaCell CTC kit is a size-based CTC enrichment method that has previously been used to enrich and culture CTCs in vitro from a multitude of cancer types. To the best of my knowledge, there are no reports evaluating WBC depletion and CTC recovery rates with MetaCell, which are important parameters in gauging the performance of CTC enrichment technologies before attempting to enrich CTCs from cancer patient blood. Along with CTC enrichment, it was also essential to optimise the conditions required for expanding the rare CTC populations to enable further downstream analysis. Finally, the current markers used for CTC characterisation involve using two epithelial markers, EpCAM and cytokeratins (FDA approved). However, the metastatic cascade demands that CTCs possess phenotypic plasticity to metastasise successfully. This leads to CTCs existing in various EMT states, which contribute to the heterogeneity. Therefore, reliance on epithelial markers could miss out on these CTC populations and limit our understanding of the metastatic process. However, with the advent of single-cell RNA sequencing technologies, which generate transcriptomes at a single-cell level, there now exists a marker-free platform (as they do not rely on the expression of specific markers for CTC detection) that can be utilised for investigating the different CTC populations that exist in CRC patients.
Methods
I first explored the effect of various conditions, such as the presence or absence of serum and normoxia or hypoxia, on CRC spheroid growth. For this, I used HCT116 and DLD1 cells as CTC models and evaluated various parameters such as morphological examination, spheroid formation efficiency (SE), fold change in the spheroid area, and expression of hypoxia markers and EMT-related genes for spheroids grown in different conditions. Next, I evaluated MetaCell’s efficiency in enriching CRC (HCT116 and DLD1) cells spiked into healthy blood (cell recovery rates), followed by assessing the purity (WBC depletion) of the MetaCell enriched fraction. Next, I applied MetaCell to CRC patient blood to enrich and characterise CTCs using canonical markers (EpCAM and cytokeratins). Then, I explored the associations between various clinicopathological variables on CTC and cCTC enumeration and detection. Finally, I generated single-cell cDNA libraries of peripheral mononuclear blood cells (PBMCs) isolated from CRC patient blood using the 10X genomics protocol. Then, cDNA libraries were sequenced, and sequencing data was processed using CellRanger and Seurat packages to identify CTC-specific transcriptomic signatures.
Results
Parameters such as spheroid formation efficiency (SE) and fold change in spheroid area were evaluated to assess the study conditions that enabled spheroid formation and growth. The findings revealed that the presence of serum was conducive to spheroid growth, irrespective of normoxia or hypoxia. Further, the hypoxia markers I chose for my study were found to be upregulated in spheroids grown in normoxia compared to hypoxia. This suggested that hypoxia could not be successfully induced in spheroids, based on the expression pattern of hypoxia markers I chose for my study. Hence, I did not explore the expression of EMT genes in the spheroids as I would not be able to verify if hypoxia could aid upregulation of EMT genes. This led me to choose the combination of serum/normoxia for the remainder of my experiments. Next, I evaluated cell recovery and WBC depletion rates for MetaCell which revealed that MetaCell could enrich CRC cells with high purity. Applying MetaCell to CRC patients revealed that I could detect CTCs or cCTCs in 47.6% of CRC patients, irrespective of the AJCC stage. I also did not observe any correlation between clinicopathological variables and CTC detection, but CTC numbers were significantly associated with invasion, grade, MMR, and budding statuses of the primary tumour. With respect to cCTCs, only tumour budding status significantly correlated with cCTC enumeration. Next, the sequencing data was obtained from sequencing of cDNA libraries generated from PBMCs of CRC patients. This data was quality-checked and processed further to generate clusters (cells with similar gene expression). Clustering analysis generated 14 clusters. Then, differentially expressed genes (DEGs) were identified for each cluster, followed by investigating pathways enriched in each cluster. Two of the clusters showed enrichment of pathways related to EMT, which was not found in any of the other clusters.
Conclusion
In this study, I demonstrated MetaCell’s ability to enrich CTCs from CRC patient blood. The CTCs were characterised using the canonical CTC markers (EpCAM and cytokeratins). These markers can detect only epithelial CTCs while missing other relevant CTC populations, such as CTCs undergoing EMT. The limitation of these markers was demonstrated using scRNA-seq, a marker-free method. scRNA-seq data analysis revealed that pathways related to EMT were found to be enriched in CTCs. This suggests the presence of mesenchymal CTCs. However, these pathways have not been observed previously in CRC CTCs. Therefore, more studies need to be carried out to see if these pathways emerge again. This could lead to the development of markers beyond the epithelial markers that could be used for CRC CTC isolation.