ScRNA-seq analysis of tumor endothelial cells and cancer cells to unravel vessel co-option, a resistance mechanism to anti-angiogenic therapy

Endothelial cells (ECs) line the blood vessels and play diverse roles in several biomechanical and biochemical functions such as angiogenesis, regulation of vessel permeability, hemostasis, leukocyte transmigration, vasomotor regulation and immunomodulation. In tumors, sprouting angiogenesis is often considered the most significant mechanism of tumor vascularization and thus became the main target for anti-angiogenic therapy (AAT). During sprouting angiogenesis, vessel growth occurs by proliferation and migration of ECs from preexisting vessels. In addition to angiogenic stimuli including vascular endothelial growth factor (VEGF, a target of current AAT), the host lab revealed EC metabolism as key driver of the tumor angiogenesis. For instance, most glycolytic genes, including the glycolytic stimulator PFKFB3, are transcriptionally upregulated in tumor ECs, and silencing of PFKFB3 in tumor-bearing mice was shown to reduce cancer cell intravasation and metastasis by normalizing the typically aberrant, tortuous tumor vessels. While previous studies predominantly focused on the role of central carbon metabolism in tumor angiogenesis, recently the host lab developed EC-specific genome scale metabolic models (built on bulk and single-cell transcriptomic data from ECs) to identify additional metabolic genes potentially important for angiogenesis. While numerous novel EC-specific metabolic targets were in silico predicted to be essential for biomass production (as readout of EC proliferation/angiogenesis), data about the validation of the functional role of these genes in ECs in vitro or in vivo are however scarce. It thus remains unknown which target genes are functionally relevant and /or therapeutically attractive.

Besides sprouting angiogenesis, tumors can also secure their blood and nutrient supply by means of alternative vascularization mechanisms, such as vessel splitting (intussusceptive angiogenesis), vascular mimicry, and vessel co-option (VCO). As opposed to sprouting angiogenesis, during VCO, there is no new blood vessel formation but instead, cancer cells hijack pre-existing blood vessels to grow and invade the healthy tissue. Although tumor VCO is often proposed as resistance mechanism against AAT, it remains poorly understood and barely studied. It is thus important to (a) develop suitable animal models of tumor VCO, and to (b) study the molecular mechanisms underlying this process and identify the cell types involved. 

Besides their role in angiogenesis, ECs are also known to modulate inflammation, by regulating immune cell trafficking, activation status, and function. ECs are the first cells to interact with immune cells, which must cross the EC layer to migrate from these vessels into peripheral tissues. Moreover, ECs are in direct contact with circulating pathogens. These features place them in the ideal position as the primary defense line to contribute to immunological responses. The inflammatory process is mainly the result of a balance between pro- and anti-inflammatory molecules, secreted by various immune cells, but also other cell types, which are involved in the positive or negative regulation of cell chemotaxis, migration, and proliferation. Imbalance between pro- and anti-inflammatory molecules can lead to sustained and chronic inflammation or to an insufficient immune response and in turn, mediates a wide spectrum of diseases such as cardiovascular diseases, diabetes mellitus, or cancer. In tumors, often an increase of anti-inflammatory molecules can be observed, which leads to accumulation of immunosuppressive immune cells and inhibition of anti-tumor immune cells, thereby exacerbating tumor growth. Moreover, interactions between different cell types, either directly, or indirectly, impact the tumor immune environment. For instance, the typically aberrant, tortuous tumor vasculature can restrict the access of cytotoxic T-lymphocytes to the tumor core, either by directly binding and modulating T-lymphocytes, or indirectly via secretion of ant-inflammatory molecules or via hypoxia-driven mechanisms; and thereby prevent them from exerting their anti-tumor effects and promote tumor growth. Despite multiple indications of EC-immune interactions potentially driving tumorigenesis, targeting EC immune functions has not yet been probed as anti-cancer therapy. In fact, only the angiogenic properties of tumor ECs have been in focus of therapeutical approaches. Thus, targeting immunoregulatory functions of ECs, either in combination with current immunotherapy treatments, or even in parallel with AAT are promising and thus far unexplored avenues for future cancer treatments. 

Interestingly, cancer, as well as several other diseases are characterized by both angiogenesis and inflammation. In fact, several studies indicate a positive correlation between inflammation and angiogenesis, but whether these mechanisms can also impact each other negatively remains to be elucidated.

During my PhD, I aim to further study the link between tumor vascularization and inflammation. To better understand how AAT may impact tumor inflammation, I first compared the transcriptome of ECs and immune cells during sprouting angiogenesis to those in AAT-induced VCO. 

Secondly, I sought to find molecular drivers in ECs, which may impact both immune, as well as angiogenic properties of these cells. Here, I exploited data generated by EC-specific genome scale metabolic models and probed the role of Gene X[1] (a mitochondrial metabolite transporter identified as a key regulator of EC biomass production (suggesting a role in EC proliferation/angiogenesis)) in ECs during choroid neovascularization (CNV), and non-small cell lung carcinoma (NSCLC).

[1] Due to patenting reasons, the identity of Gene X currently cannot be disclosed yet.