Intra-tumor hypoxia drives the formation of breast cancer circulating tumor cell clusters

Abstract

Breast cancer implicates the formation of a tumor in the breast tissue which, in 20-30% of the cases, will spread to distant organs such as lungs, bones, brain and liver through the shedding of cells in the blood vasculature, referred to as circulating tumor cells (CTCs). CTCs are shed as single cells, multicellular aggregates (CTC clusters) or in association with immune or stromal cells, and are considered to be metastatic precursors in several cancer types, including breast cancer. The metastatic dissemination is responsible of 90% of cancer-related deaths. Thus, understanding the causes of CTCs generation is crucial for better patients’ prognosis. Several factors have been described to promote CTCs and metastasis formation such as mutations in driver genes and microenvironmental stress. Among the latter group, intra-tumor hypoxia has emerged as a potent factor for breast cancer metastasis. Interestingly, while anti-angiogenic or vascular normalizing treatments have been introduced to ameliorate the hypoxia response, therapies have consistently failed to prolong survival of breast cancer patients. The goal of my PhD thesis was to understand what are the drivers and mechanisms of CTCs generation from breast tumors in preclinical models of breast cancer by directly interrogating single CTCs and CTC clusters at a molecular level. Through the establishment of a system to dynamically label hypoxia throughout disease progression, we observe that the majority of CTC clusters undergoes hypoxia, and that hypoxia endows CTC clusters of a higher metastatic potential compared to single CTCs. Interestingly, single-cell RNA-sequencing of hypoxic versus normoxic clusters reveled a gene expression pattern that predicts a worse outcome in breast cancer patients. Strikingly, we found that vascular endothelial growth factor (VEGF) targeting leads to primary tumor shrinkage but it increases intra-tumor hypoxia, resulting in a higher CTC clusters shedding rate and metastasis formation. Conversely, a pro-angiogenic approach increases primary tumor size yet it dramatically suppresses the formation of CTC clusters and metastasis. Thus, intra-tumor hypoxia leads to the formation of clustered CTCs with high metastatic ability, and a pro-angiogenic therapy suppresses metastasis formation through prevention of CTC clusters generation. During the course of my PhD, we also established a platform to dissect the characteristics of CTCs at the single-cell level, aiming towards a better understanding of CTC biology. We created a pipeline for precise analysis of single CTCs and CTC clusters which includes CTC immunostaining and micromanipulation, ex vivo culture to assess proliferative and survival capabilities of individual cells, and in vivo metastasis-formation assays. Additionally, we provide a protocol to achieve the dissociation of CTC clusters into individual cells and the investigation of intra-cluster heterogeneity. With these approaches, we precisely quantify survival and proliferative potential of single CTCs and individual cells within CTC clusters, leading to the observation that cells within clusters display better survival and proliferation in ex vivo cultures compared to single cells. Overall, our workflow offers a platform to dissect the characteristics of CTCs at the single cell level, aiming towards the identification of metastasis-relevant pathways and a better understanding of CTC biology.