Dissecting the role of the P2X7 receptor in vesicles-mediated intercellular communication of colon carcinoma

Background Extracellular vesicles (EV) play an oncogenic role in tumor-stromal crosstalk, immune evasion, metastasis, and drug resistance. However, the mechanisms underlying their increased biogenesis in cancer remain unclear, posing a challenge for targeted therapeutic interventions. The tumor microenvironment (TME) is enriched in extracellular ATP (eATP), a nucleotide that we recently demonstrated can trigger the release of a particular subset of EV by activating the P2X7 receptor: P2X7-VS. P2X7-VS include various subpopulations (e.g., microvesicles, exosomes, mitovesicles) and exhibit unique molecular content compared to spontaneously released EV. Hypothesis Our preliminary data indicate that P2X7-VS released by cancer cells promote colon carcinoma (CRC) metastasis in vivo. Additionally, immune and enteric glial cells secrete P2X7-VS, which contain mitochondria. We, therefore, hypothesize that the high levels of eATP present in the TME via P2X7 receptor activation drive EV release from multiple cell types within the CRC TME. Consequently, P2X7 inhibition could block the release of a complex mix of pro-tumor EV, representing a promising strategy for preventing and treating CRC metastasis. Aims The project's primary goals are to characterize the content of P2X7-VS coming from different cellular sources and their ability to influence eATP and adenosine levels, immune-mediated tumor eradication or promotion, enteric glial intervention, and preconditioning of the metastatic niche. Experimental Design We will investigate P2X7-VS release mechanisms, their content, and their impacts on eATP levels and purinergic signaling. We will follow the vesicles in vivo to dissect their role in preparing a favorable metastatic niche and influencing organ microenvironment composition, focusing on macrophages, innate lymphocytes, and enteric glia. We will analyze the role played by P2X7 activation and P2X7-VS in the mitochondrial exchange between cancer and colon resident cells. Finally, we will comprehensively investigate the effects of P2X7-VS and P2X7 antagonism in primary tumors and liver metastasis explants from CRC patients. These samples will undergo digital spatial profiling transcriptomic analysis to characterize changes in mRNA expression across thousands of genes within distinct tumor and tissue cellular subpopulations. The key findings from the transcriptomic analysis will be validated in a cohort of 100 CRC patients. Expected Results We expect to demonstrate that vesicle release, driven by the high ATP concentration in the TME, will favor the crosstalk between CRC cells and colon resident cells, make specific organs prone to host metastatic CRC, and increase ATP and adenosine levels in the tissues that P2X7-VS reach. We also foresee proving that P2X7 blockade will effectively prevent the pro-tumorigenic effects of P2X7-VS. Finally, we will establish a big dataset of transcripts affected by both P2X7-VS and P2X7 antagonists in surgery-derived human CRC samples. Impact On Cancer Since EV also sustain a series of physiological functions, effective anticancer strategies targeting them must selectively inhibit cancer-specific vesicular release mechanisms to achieve therapeutic efficacy. If our hypothesis is validated, P2X7 blockade will emerge as a promising tumor-specific EV-targeting therapy. Moreover, its clinical applicability in CRC patients will be facilitated by the prior administration of P2X7 antagonists in humans, where they have demonstrated good tolerability and minimal side effects.