As cancer progresses, tumor cells can travel through the blood circulation to form metastasis. These circulating tumor cells (CTC) can be used as a liquid biopsy for the real-time information they carry about the tumor. They can be isolated from blood using the EpCAM molecule for enrichment. The presence of EpCAM expressing CTC in patients can be used to evaluate and monitor the treatment effect and is related to a poor overall survival of the patient. In this thesis we investigated the presence of two subpopulations of CTC: EpCAMhigh and EpCAMlow CTC. In the EU FP7-Health program “CTC-Trap” we performed multiple patient studies and explored the potential of these CTC and other cancer biomarkers as a liquid biopsy. We analyzed multiple cancer biomarkers in a single tube of blood from metastatic cancer patients. The presence of EpCAMhigh CTC, EpCAMhigh tumor derived extracellular vesicles and circulating tumor DNA (ctDNA) were associated with poor overall survival, yet EpCAMlow CTC were not. This raises the question whether these EpCAMlow CTC are of cancerous origin or not. In one patient, we detected genetic aberrancies in EpCAMlow cells which were identical to the mutations present in the metastatic tumor of this patient. This suggests that the EpCAMlow cells are indeed of cancerous origin. Specific DNA mutations in ctDNA, present in the tumor, could be detected in patients with EpCAMhigh CTC, with EpCAMlow CTC and without any CTC. To improve detection of CTC, we classified all cell populations present after CTC EpCAM-enrichment with advanced image analysis, using the open source imaging program ACCEPT and Deep Learning segmentation. We improved the immunostaining and used an alternative light source and were thereby able to reduce the large population of unidentified cells to a small cell population that will need to be investigated further to unveil the presence of any CTC. To conclude, we investigated the presence of two CTC populations in metastatic cancer patients. The results presented in this thesis warrant an in-depth characterization of EpCAMlow and EpCAMhigh CTC to determine their value as a liquid biopsy and potential use in the clinic, to aid the patient with a fast and effective treatment for prolonged survival.