679. Heat Stress Elevate Cell Population That Express Cancer Stem Cell Markers in Different Types of Cancer Cell Line

Abstract

Discovery of cancer stem cells (CSC) as driving force behind cancer cells tumorigenesis and heterogeneity in solid tumors led to a paradigm shift. The recognition of non-CSC to CSC transient introduces the cancer cell plasticity concept. This behavior appeared to be brought into existence majorly by the hostility of tumor microenvironment and stress imposed by hypoxia, pH, and nutrient depletion in the tumor niche. This recognition draw attention to the correlation between cancer hallmarks such as metastasis, chemotherapy, radiotherapy resistant and the transition of non-CSC to CSC by determination of CSC markers in cells subpopulations. Different types of cancer cell lines were shown to have a subset of cells expressing CSC markers such as CD44, ALDH, C133, Oct 3/4 with tumorigenicity in vivo. Recently, metabolic stress in a long-term nutrient deprivation showed to induces conversion of non-CSC to CSC like state. Additionally the hypoxic microenvironment was shown to upgraded stem-like properties of gastric cancer cells. Since it was recognized that metabolic alterations in tumors often combined by hyperthermia, we hypothesized that hyperthermia could be one of the stress components imposed by microenvironment that may manipulate non-CSC to CSC status and we asked if this elevation in temperature would affect phenotypic characteristics or up-regulate the genes responsible for stress resistant and the genes of cancer stem cell markers. To answer this, three different types of cancer cell lines were investigated by incubating in elevated temperature without CO2 supplement. They were rhebdomyosarcoma cell line (RD), cervical carcinoma cell line (HeLa), and mice mammary adenocarcenoma (AMN3). In all experiments cell lines were seeded in 12 well tissue culture plates and incubated at 37°C with 5% CO2 until they reach absolute confluent monolayer, after that subsets of plates incubated either at 40°C or 37°C without CO2 for 24 or 48 hr. Cultivation of cell lines in RPMI1640 at 37°C for 24hr without CO2 served as control untreated experiments. We followed changes in cells viability, phenotypic alterations, cologencity, and genetic markers of heat resistant (HSP90-beta and HIF), genetic markers of cancer stem cells (CD44, ALDH, Oct4, and CD133) by RT- qPCR. Results showed that upon incubation at 40°C for 24 hr or 48 hr without CO2 supplement cells were detached floated and acquired spherical shapes and aggregated. Noticeably the formed aggregation resembled to some extent the cell spheres that induced in cancer stem cells at sphere forming assay (fig.1,A,Bfig.1,A,B). The count of viable floated cells for RD, HeLa, and AMN3 cells were 3.93E–6, 2.1E–6, and 5.7E–6 cell/ml at 40°C for 48 hr respectively, and generally it was temperature and time dependant (Fig 1,CFig 1,C). All viable floated cells from the cell lines investigated were capable to form colonies in colony forming assay, the plating efficiency for RD, HeLa, and AMN3 cells were 48%, 64%, and 57% after incubation at 37°C for 10 days with CO2 supplement respectively (Fig.1,DFig.1,D). Up regulation of heat stress gene HSP90-beta and HIF was detected in floated cells derived from all experiment and time of incubation. This was combined by up regulation of CSC markers CD44, ALDH, Oct4 and CD133 in floated cells derived from all experiment. These results may indicate a shift of non-CSC to CSC during heat stress and the increment of cancer stem cell population in the cell line under investigation which need more careful investigation.View Large Image | Download PowerPoint Slide