Tumor radioresistance caused by radiation-induced changes of stem-like cell content and sub-lethal damage repair capability

Abstract

Cancer stem-like cells (CSCs) within solid tumors exhibit radioresistance, leading to recurrence and distant metastasis after radiotherapy. To experimentally study the characteristics of CSCs, radioresistant cell lines were successfully established using fractionated X-ray irradiation. The fundamental characteristics of CSCs in vitro have been previously reported; however, the relationship between CSC and acquired radioresistance remains uncertain. To efficiently study this relationship, we performed both in vitro experiments and theoretical analysis using a cell-killing model. Four types of human oral squamous carcinoma cell lines, non-radioresistant cell lines (SAS and HSC2), and radioresistant cell lines (SAS-R and HSC2-R), were used to measure the surviving fraction after single-dose irradiation, split-dose irradiation, and multi-fractionated irradiation. The SAS-R and HSC2-R cell lines were more positive for one of the CSC marker aldehyde dehydrogenase activity than the corresponding non-radioresistant cell lines. The theoretical model analysis showed that changes in both the experimental-based ALDH (+) fractions and DNA repair efficiency of ALDH (-) fractions (i.e., sub-lethal damage repair) are required to reproduce the measured cell survival data of non-radioresistant and radioresistant cell lines. These results suggest that the enhanced cell recovery in SAS-R and HSC2-R is important when predicting tumor control probability in radiotherapy to require a long dose-delivery time; in other words, intensity-modulated radiation therapy is ideal. This work provides a precise understanding of the mechanism of radioresistance, which is induced after irradiation of cancer cells.

Figure 1

Categories of cancer cell lines and the overview of the IMK model. (A) the biological categories for SAS, SAS-R, HSC2 and HSC2-R, (B) the parameters in the IMK model and their characteristics, and (C) surviving fraction of cancer cells estimated based on the IMK model. Note that f_p + f_s = 1. We assumed the increased fraction of CSCs and the high DNA repair (SLDR) capability acquired in the progeny cells after fractionated irradiation with total high dose as the characteristics of radioresistant cell lines.

Figure 2

Determination of (a + c) values for non-resistant cell lines. (A) is cell recovery curve of SAS and (B) is that of HSC2. The symbols are shown the experimental cell surviving fraction of fractionated radiations as a function of time intervals (h), while two dotted lines are initial slopes of dS/dτ and S(∞) expressed in Eq. (5), respectively. Using the values described in this figure, the SLDR rates of non-radioresisitant cell lines (SAS, HSC2) were determined.

Figure 3

The ALDH (+) population in four cell lines. The fraction of cells expressing ALDH activity was determined by flowcytometric analysis. The cells that tested positive for ALDH activity are shown in the scatter plot on the right. Representative cytogram of (AI) SAS and SAS-R cells, (BI) HSC2 cells and HSC2-R cells. DEAB (+) represents the negative control. (AII) and (BII) show the percentages of ALDH (+) cells subtracted from that of DEAB (+). Bracketed asterisks represent significant differences of P < 0.01 between the two groups.

Figure 4

Model parameter sets of non-radioresistant cell lines by the MCMC simulations. The dot plot and probability density histogram represent the posterior distributions of the model parameter sets of (A) SAS, and (B) HSC2. Blue represents the parameters of [α_0p*, β_0p*] for the progeny cells in the non-resistant cell populations, red represents the parameters of [α_0s, β_0s] for the CSCs in the non-resistant cell populations.

Figure 5

Cell surviving fraction of non-resistant and resistant cell lines after acute irradiation. (A) for SAS and SAS-R cell lines, and (B) for HSC2 and HSC2-R cell lines. The blue and red circle plots are the mean value of the experimental survival fraction of the non-resistant cells and resistant cells, respectively. The blue and red solid lines are the surviving fraction estimated by the IMK model considering the changes of CSCs fraction and SLDR rate.

Figure 6

Relative radiosensitivity for split-dose irradiation between the experiment and the model prediction. The circle plot is the experimental cell survival normalized by the cell survival at 4 Gy irradiation (i.e., non-interval irradiation). Solid line is the surviving fraction estimated by the IMK model.

Figure 7

Dose-rate effects measured by experiment and predicted by the IMK model. The logarithmic scale of the relation between dose rate and cell survival. (A) is for SAS and SAS-R at a constant dose of 10 Gy, and (B) is for HSC and HCS2-R at a constant dose of 6 Gy. Blue and red circle plots are the experimental cell survival of non-resistant cells (SAS and HSC2) and resistant cells (SAS-R and HSC2-R), respectively, while blue and red solid lines are the cell survival of non-resistant (SAS and HSC2) and resistant cells (SAS-R and HSC2-R) estimated by the IMK model.