Single-cell resolution characterization of circulating tumor cell clusters

Metastatic dissemination is responsible for more than 90% of cancer-related deaths. However, the molecular features underlying the metastatic process are largely uncharacterized. Cancer cells that leave a primary tumor and enter the bloodstream are referred to as circulating tumor cells (CTCs). While extraordinarily rare compared to normal blood cells, their isolation and characterization offers a unique opportunity to study how metastasis occurs. Recent breakthrough developments in microfluidics technology have enabled a more detailed examination of blood samples, highlighting that tumor cells in circulation are organized as single and clustered CTCs, with the latter being associated with greater metastatic potential. Moreover, an additional subtype of CTCs was observed and characterized as white blood cells (WBCs) – associated CTCs with unknown prevalence and function.
The goal of my Ph.D. Thesis was to investigate CTCs in depth by implementing a novel approach in cancer metastasis research. For the first time, a single cell-resolution, parallel genome and transcriptome sequencing of single, clustered and WBC-associated CTCs of cancer patients and metastatic mouse models have been applied. This technique provided a mean for multiple observations including a breakthrough discovery – WBCs circulating in association with tumor cells have a crucial impact on their metastatic potential and these cell aggregates were described as the most metastatic CTC subtype. Moreover, single-cell RNA sequencing revealed a specific pattern of these WBCs, with neutrophils representing the majority of the cases. Additionally, cell-cell junction and cytokine-receptor pairs that define CTC-neutrophil clusters were described and proposed as key vulnerabilities of the metastatic process. Thus, the association between neutrophils and CTCs fuels cell cycle progression within the bloodstream and expands the metastatic potential of CTCs, providing a rationale for targeting this interaction in breast cancer.
Over the course of three and a half years, more than 160 patients with diverse cancer types agreed to donate blood for the ‘CTC Study’. The association between crucial clinical parameters and CTC characteristics have been evaluated in a selected group of 73 consecutive patients characterized by progressive invasive breast cancer, high tumor load, and treatment discontinuation. Among these patients, a correlation between treatment with the monoclonal antibody denosumab and the absence of CTCs were observed. Additionally, low red blood cell count was associated with the presence of CTCs, whereas high CA 15-3 tumor marker, high mean corpuscular volume, high white blood cell count and high mean platelet volume associated specifically with CTC clusters. These findings carry clinical applications, however further studies will be needed to validate the involvement of denosumab in the prevention of CTC generation.