It is evident also that in order to maintain suitable fidelity to the original tumors, the cancer-initiating cells, as well as other cell types used for genomic and proteomic profiling, should be isolated from a large spectrum of primary and metastatic tumors, not from the established cancer cell lines. However, it is notoriously difficult to establish primary cell lines and particularly CIC lines from fresh tumor specimens. First, there are objective difficulties in the isolation of pure cell populations from heterogeneous tumor tissues. Tumor impurity and multiclonality are well documented problems. At the molecular level, there are currently no definitive markers to prove the malignant or nonmalignant nature of cells, as well as to accurately distinguish between normal and cancer stem cells. Growing evidence also suggests that CICs might NSI-189 represent a heterogeneous subpopulation of the tumor-initiating cells. Nevertheless, a combination of multiple approaches and multiple cell surface markers followed by a thorough functional characterization of the isolated cell phenotypes may enable the purification of the most functionally significant, i.e. tumor-and metastasis-initiating and the most drug resistant cells. Several colorectal cell lines of different cellular, biochemical and molecular characteristics have been established during the last two decades. Here we report the establishment of a novel CIC-enriched, BRL-54443 highly tumorigenic colorectal cancer cell line isolated from liver metastasis of a CRC patient. Dissociated cell suspensions from 13 freshly resected colorectal carcinomas of various histological grades and 3 liver metastases were tested for clonogenic and tumorigenic potential in vivo and in vitro as described in the Methods section. Two specimens were severely contaminated with bacteria, and despite repeated treatments with antibiotics, the primary colonies were also contaminated and therefore discarded. Two primary cultures developed from primary and metastatic tumor specimens of patients previously treated with chemotherapy underwent profound cell death after 1�C2 days in culture and did not show any viable cells during the next week of observation. The other nine specimens contained a subpopulation of fast-adherent cells to the type I collagen, which initially proliferated in serum-free stem cell medium and induced floating spheroids, which are characteristic of CICs, as well as loose multicellular aggregates in 3D cultures.