Patient Derived Cell Culture and Isolation of CD133⁺ Putative Cancer Stem Cells from Melanoma

Abstract

Despite improved treatments options for melanoma available today, patients with advanced malignant melanoma still have a poor prognosis for progression-free and overall survival. Therefore, translational research needs to provide further molecular evidence to improve targeted therapies for malignant melanomas. In the past, oncogenic mechanisms related to melanoma were extensively studied in established cell lines. On the way to more personalized treatment regimens based on individual genetic profiles, we propose to use patient-derived cell lines instead of generic cell lines. Together with high quality clinical data, especially on patient follow-up, these cells will be instrumental to better understand the molecular mechanisms behind melanoma progression.Here, we report the establishment of primary melanoma cultures from dissected fresh tumor tissue. This procedure includes mincing and dissociation of the tissue into single cells, removal of contaminations with erythrocytes and fibroblasts as well as primary culture and reliable verification of the cells' melanoma origin.Recent reports revealed that melanomas, like the majority of tumors, harbor a small subpopulation of cancer stem cells (CSCs), which seem to exclusively fuel tumor initiation and progression towards the metastatic state. One of the key markers for CSC identification and isolation in melanoma is CD133. To isolate CD133 + CSCs from primary melanoma cultures, we have modified and optimized the Magnetic-Activated Cell Sorting (MACS) procedure from Miltenyi resulting in high sorting purity and viability of CD133 + CSCs and CD133 -bulk, which can be cultivated and functionally analyzed thereafter.

Animal models and therapeutic molecular targets of cancer: utility and limitations

“… 4 , 5 On the way to personalized treatment protocols based on an individual’s genetic profile, the use of patient-derived primary cancer cell lines instead of generic cell lines has become a valuable in vitro system for developing cancer treatment regimes. 6 , 7 The advantages of in vitro cancer models are highly controlled conditions, homogeneity, discovery of molecular mechanisms, and reproducibility. The main limitations of two-dimensional in vitro cell culture cancer cell lines are selection of phenotypic and genotypic cells during adaptation to in vitro conditions, accumulation of mutations in cells over time in culture, a homogeneous population of cells, and isolation of cells from the tumor microenvironment.…”

Expression of CD133 Cancer Stem Cell Marker in Melanoma: A Systematic Review and Meta-Analysis

“…Among these markers, CD133 (also known as prominin-1/ AC133), a member of the pentaspan transmebrane glycoproteins with a molecular weight of 120 kDa, is considered to be the most important surface marker for identification of MSCs (5,12,17,(23)(24)(25)(26)(27)(28)(29). Monzani et al (17) proposed CD133 as a potential MSC marker for both in vivo and in vitro studies.…”

Carboxypeptidase A4 promotes proliferation and stem cell characteristics of hepatocellular carcinoma

“…CD133 was the first marker used for enriching CSCs in human solid tumours . CD133 expression has been used to enrich CSCs in solid cancers, including hepatocellular carcinoma, colon cancer, human melanoma and many others. ALDH1 activation and high CD44 expression have also been reported as markers to identify cell populations enriched for CSCs in breast cancer, gastric cancer and lung cancer .…”