Differential response of esophageal cancer cells to particle irradiation

Abstract

Background: Radiation therapy is a mainstay in the treatment of esophageal cancer (EC) patients, and photon radiotherapy has proved beneficial both in the neoadjuvant and the definitive setting. However, regarding the still poor prognosis of many EC patients, particle radiation employing a higher biological effectiveness may help to further improve patient outcomes. However, the influence of clinically available particle radiation on EC cells remains largely unknown.

Methods: Patient-derived esophageal adenocarcinoma and squamous cell cancer lines were treated with photon and particle irradiation using clinically available proton (¹H), carbon (¹²C) or oxygen (¹⁶O) beams at the Heidelberg Ion Therapy Center. Histology-dependent clonogenic survival was calculated for increasing physical radiation doses, and resulting relative biological effectiveness (RBE) was calculated for each radiation modality. Cell cycle effects caused by photon and particle radiation were assessed, and radiation-induced apoptosis was measured in adenocarcinoma and squamous cell EC samples by activated caspase-3 and sub-G1 populations. Repair kinetics of DNA double strand breaks induced by photon and particle radiation were investigated.

Results: While both adenocarcinoma EC cell lines demonstrated increasing sensitivities for ¹H, ¹²C and ¹⁶O radiation, the two squamous cell carcinoma lines exhibited a more heterogeneous response to photon and particle treatment; average RBE values were calculated as 1.15 for ¹H, 2.3 for ¹²C and 2.5 for ¹⁶O irradiation. After particle irradiation, squamous cell EC samples reacted with an increased and prolonged block in G2 phase of the cell cycle compared to adenocarcinoma cells. Particle radiation resulted in an incomplete repair of radiation-induced DNA double strand breaks in both adenocarcinoma and squamous cell carcinoma samples, with the levels of initial strand break induction correlating well with the individual cellular survival after photon and particle radiation. Similarly, EC samples demonstrated heterogeneous levels of radiation-induced apoptosis that also corresponded to the observed cellular survival of individual cell lines.

Conclusions: Esophageal cancer cells exhibit differential responses to irradiation with photons and ¹H, ¹²C and ¹⁶O particles that were independent of tumor histology. Therefore, yet unknown molecular markers beyond histology may help to establish which esophageal cancer patients benefit from the biological effects of particle treatment.

Keywords: Carbon ion radiation; Esophageal cancer; Heavy ion radiotherapy; Proton radiation.

Fig. 1

Esophageal cancer cell lines show heterogeneous survival after photon and particle irradiation. Clonogenic survival curves of OE19 and OE33 adenocarcinoma and KYSE270 and KYSE410 squamous cell carcinoma cell lines after photon (X), proton (¹H), carbon ion (¹²C) and oxygen ion (¹⁶O) irradiation (mean ± SD of 3 independent experiments). The table depicts the relative biological effectiveness (RBE) for ¹H, ¹²C and ¹⁶O radiation at 10% survival

Fig. 2

Particle irradiation results in a prolonged block in the G2 phase. Percentage of cells in G2/M phase of four EC cell lines at 24 h after irradiation with biologically isoeffective doses of photons (X), protons (¹H) and heavy ions (¹²C, ¹⁶O) at a low dose level (left bars) and a high dose level (right bars) (mean and SD of n = 3 replicate samples). For clarity the low dose and high dose data is shown separately; the control groups are identical in both parts because for each radiation modality both dose levels were applied in the same experiment with one common control group per time point. *p < 0.05, **p < 0.01 (two-sided Student’s t-test between the indicated groups). For test comparing different radiation modalities were the control levels were subtracted from the

Fig. 3

Particle irradiation leads to heterogeneous increases in apoptosis induction of esophageal cancer cells. Percentage of apoptotic EC cells accessed by the sub-G1 fraction and cellular caspase-3 activation measured at 96 h after irradiation with biologically isoeffective doses of photons (X), protons (¹H) and heavy ions (¹²C, ¹⁶O) (mean and SD of n = 3 replicate samples). *p < 0.05, **p < 0.01 (two-sided Student’s t-test against unirradiated controls)

Fig. 4

Esophageal cancer cells incompletely repair DNA double strand breaks induced by particle irradiation. Normalized γH2AX levels at 2 and 24 h after irradiation with biologically isoeffective doses of photons (X), protons (¹H) and heavy ions (¹²C, ¹⁶O) (mean and SD of n = 3 replicate samples). Values were corrected for cell cycle-specific differences as detailed in Materials and Methods. *p < 0.05, **p < 0.01 (two-sided Student’s t-test against unirradiated controls)