Efficient isolation, culture, purification, and stem cell expression profiles of primary tumor cells derived from uterine cervical squamous cell carcinoma

Abstract

ProblemSince not too many human uterus cervical squamous cell carcinoma (CSCC) cell lines in existence, efficient isolation, culture, and purification protocols for primary CSCC cells were optimized as a tool for the study of uterus CSCC.Method of studyThe protocols for partial multiple enzymatic digestion and explant cell culture were combined and then the resulting mixed cell component cultures were purified by magnetic‐activated cell sorting. Colony‐forming assay was utilized for detection of cell carcinogenesis potential, and immunofluorescence was used to detect protein expression of CSCC. Finally, flow cytometry (FCM) was performed to analyze cancer stem cells (CSCs) phenotypic markers as well as programmed cell death ligand 1(PD‐L 1).ResultsFreshly isolated cells containing tumor cells and cancer‐associated fibroblasts (CAFs) efficiently proliferate to 85% confluence on a 6 cm petri dish in 5‐7 days. Anti‐epithelial cell adhesion molecule antibody (EpCAM) microbeads were used to successfully separate a homogeneous subpopulation of epithelial tumor cells. Both EpCAM+ and EpCAM− cell subpopulations were able to be passaged more than 30 times. Proportions of tumor cell populations expressed CSCs markers such as CD133, CD24, aldehyde dehydrogenase 1 (ALDH1), and CD44. The vimentin+ & EpCAM− population, defined with CAFs, could express CD146 mesenchymal stem cells marker. Meanwhile, PD‐L 1 was identified in most subpopulation of CD44+ cells at low passage numbers.ConclusionEfficient isolation, culture, and purification protocols for primary CSCC cells were successfully built. Additionally, the profiling of CSCs cell markers might provide promising therapeutic targets and clinic strategies.

Upregulation of NOD1 and NOD2 contribute to cancer progression through the positive regulation of tumorigenicity and metastasis in human squamous cervical cancer

“…The cells were treated with Tri-DAP, MDP (InvivoGen, USA), ML-130 (TargetMol, USA), CXCR1/2 inhibitor (Reparixin, Med Chem Express, USA), NF-κB inhibitor (EVP4593, Sellect, USA), or ERK inhibitor (SCH772984, Sellect, USA) as required. Primary CSCC cells were isolated from patient samples as previously described [ 58 ]. Briefly, the specimens were minced into 1-mm 3 pieces in 6-cm petri dishes, and sequentially digested with 0.05% trypsin containing EDTA (Lonza, Walkersville, MD, USA) and 0.2% type I collagenase (Sigma-Aldrich Corp., St Louis, MO, USA) at 37 °C with constant shaking.…”

ZEB2 facilitates peritoneal metastasis by regulating the invasiveness and tumorigenesis of cancer stem-like cells in high-grade serous ovarian cancers

“…A time-dependent method was used to discard tumour stromal cells [ 44 ]. Cultivated tumour cells reaching 80–90% confluence were trypsinised.…”

Infection by High-Risk Human Papillomaviruses, Epithelial-to-Mesenchymal Transition and Squamous Pre-Malignant or Malignant Lesions of the Uterine Cervix: A Series of Chained Events?

“…As for physiological EMT, that also accompanies SCC onset or progression occurs gradually, explaining why cells with intermediate phenotypes between the fully epithelial and the decidedly mesenchymal ones can be found in the squamous lesions of the uterine cervix [88][89][90][91][92][93][94][95][96][97]. Such a variety of phenotypes could depend on the multitude of EMT promoters that may be present in the cervix of HPV-infected women, and on the temporal sequence according to which cervical epithelial cells are exposed to these factors.…”