Enhancing radiation response by a second-generation TRAIL receptor agonist using a new in vitro organoid model system

Abstract

Background: For many cancer types, including colorectal carcinoma (CRC), combined modality treatments have shown to improve outcome, but are frequently associated with significant toxicity, illustrating the need for new therapeutic approaches. Based on preclinical data, TRAIL receptor agonists appeared to be promising agents for cancer therapy especially in combination with DNA damaging regimens. Here, we present the combination of the second-generation TRAIL receptor agonist APG-880 with radiation in a new and clinically relevant 3D model system.

Methods: To investigate the effect of APG-880 in combination with radiation we performed short-term cytotoxicity and long-term clonogenic survival assays in established CRC cell lines, and in tumor organoids derived from colon cancer patients.

Results: APG-880 is a potent inducer of apoptosis in CRC cell lines and in patient-derived CRC organoids. Furthermore, a supra-additive effect on cytotoxicity was found when APG-880 and radiation were combined simultaneously, with combination indices around 0.7. Lastly, in the long-term survival assays, we demonstrated a radiosensitizing effect of APG-880 with dose enhancement factors between 1.3 and 1.5.

Conclusions: In a new, clinically relevant CRC-organoid model system we demonstrated a more than additive combined effect between the second-generation TRAIL receptor agonist APG-880 and radiation.

Keywords: Clonogenic survival; Colorectal carcinoma; Organoids; Radiation; TRAIL receptor agonist.

Fig. 1

CRC cell lines and organoids express DR4 and DR5. Western blot analysis of DR4 and DR5 expression in HT29 and HCT116 cells (A). In (B), (C) and (D), CRC organoids express cell surface DR4 and DR5. Endogenous DR4 and DR5 expression on the cell surface of three different colorectal carcinoma patient derived organoids, ITO17, ITO60 and ITO77 analyzed by live-cell flow cytometry. (B) Negative control of organoid ITO60 consisting of secondary antibody only, in (C) anti-DR4 fluorescent staining of ITO60 and in (D) anti DR5 fluorescent staining of ITO60. (E) Quantification of the percentage DR4 and DR5 positive cells in the different organoids analyzed. J16 cells serve as a negative control for anti-DR4 as well as a positive control for anti-DR5.

Fig. 2

APG-880 induces DNA fragmentation. Staining with Hoechst demonstrated typical apoptotic body formation visualized by fluorescence microscopy. (A) Nuclei of HT29 control cells. (B) HT29 cells treated with 100 ng/ml APG-880. (C) Organoids ITO60 control. (D) Organoid ITO60 treated with 100 ng/ml APG-880. (E) and (F) Magnification of a cutout of respectively (C) and (D).

Fig. 3

APG-880 induces apoptosis in a dose-dependent manner in CRC cell lines. Apoptosis induction by APG-880 HCT116 (A) and HT29 (B) as read-out by counting cells displaying DNA fragmentation.

Fig. 4

Radiation induces apoptosis in a dose-dependent manner in CRC cell lines. Apoptosis induction at 1, 4, 7, 24, 48 h after radiation in HCT116 (A) and 24, 48, 72 h after radiation in HT29 (B).

Fig. 5

APG-880 and radiation decrease clonogenic survival in cell lines. (A) HCT116 (spheres) and HT29 (squares) clonogenic survival curves following increases doses of radiation (n = 3–4 experiments, performed in triplicate), error bars represent SEM. (B) Clonogenic survival graph of colon carcinoma cell lines HT29 and HCT116 upon APG-880 treatment. Graph shows the results of 1–3 experiments in triplicate, error bars represent SD.

Fig. 6

Colon cancer cell lines show a more than additive effect when exposed to the combination of radiation and APG-880. In graphs (A) and (B) combination experiments are shown where radiation and APG-880 were applied concurrently and apoptosis was determined after 24 h for HCT116 and 48 h for HT29. Data represent mean values ± SEM of an average of 3 independent experiments, performed in duplicate. Combination indices were calculated from these graphs. (C), (D), (E): Growing organoids were exposed to APG-880 for 6 days. Graph represents the average of 2 independent experiments in triplicate, error bars represent SEM.

Fig. 7

APG-880 combined with radiation decreases clonogenic survival in HT29. Clonogenic survival curves of HT29 cells in the absence (solid line) or presence of 100 ng APG-880 (normalized dashed line) are shown. Graphs show representative curves of 4 experiments in triplicate, error bars represent SEM. DEF37 = 1.3.

Fig. 8

APG-880 combined with radiation decreases clonogenic survival in organoids. Clonogenic survival curves of colon carcinoma derived organoids ITO60 in the absence (solid line) or presence of 100 ng/ml APG-880 (normalized dashed line) are shown. Graphs show representative curves of 3 experiments in triplicate, error bars represent SEM. DEF37 = 1.5.