3.1 Effects of increasing RT doses on spheroid growth

The growth kinetics of F98 spheroids exposed to increasing doses of RT (0–20 Gy) is shown in Fig. 2a. RT as a single treatment was suboptimal up to 10 Gy, with spheroids usually reaching NTC volume after 14 days in culture. RT alone, however, did produce a growth delay compared to NTC at 8 and 10 Gy. However, radiation doses of 15 and 20 Gy were highly toxic, completely inhibiting spheroid growth even without RS. Assaying these spheroids, after 3 and 4 weeks of incubation, showed no sign of growth (data not shown).

Fig. 2

a Growth kinetics of F98 spheroids exposed to RT only at doses ranging from 0 to 20 Gy. b Direct measurement of FG-5FU released by fluorescence emission spectroscopy. The time course of cumulative release from FG-5FU after 2, 48, 72 h and assayed by drug fluorescence. FG loaded with 1 µg/mL of 5FU. The results are the average of 2 independent experiments and are shown as a % of the maximum release. Error bars represent standard deviation

3.2 Determination of FG-5FU drug release kinetics by fluorescence emission spectroscopy

The release profile of 5FU from FG was assayed using drug fluorescence. Here, 1 μg of 5FU was loaded into 0.4 mL of FG. Figure 2b illustrates the results, presenting the release ratio % in the supernatant at harvest intervals of 0.25, 2, 24, 48 and 72 h. The slow, controlled release from FG was demonstrated, with active drugs still being released after 48 h. Based on these results, FG drug harvesting was done at 48 h for all subsequent experiments.

3.3 RT effects on spheroid growth inhibition by 5FU and MGd as free or released drugs acting as RS

Figure 3a displays the effects of RT (10 Gy) on spheroid volume for 5FU as a free drug. A significant inhibiting effect was seen with RT in combination with increasing 5FU concentration (0.06–0.25 μg/mL). At a 5FU concentration of 0.25 μg/mL and RT, spheroid volume was 6% of control values after 14 days of growth. In the absence of RT, 5FU showed limited toxicity only at the highest concentration, 0.25 μg/mL.

Fig. 3

a Enhanced RT effects of 5FU as free RS over a range of concentrations, 0–0.25 μg/mL. b Effects on spheroid growth of FG-5FU, diluted 1:1, 2:1, 4:1. RT 0, 10 Gy. c Enhanced RT effects for MGd over a range of concentrations, 0–80 μg/mL. RT 0 and 10 Gy administered 24 h after RS. d FG- MGd diluted 1:1, 2:1, 4:1, and 8:1 with RT 10, 12 Gy Spheroid growth after 14 days. Each data point represents the average volume of 8 spheroids after 2 weeks in culture as a % of non-treated controls. Error bars, standard deviation. * Significant differences (p < 0.05) compared to controls

Loading of FG with 5FU (1 μg) or MGd (40 μg) was done as described in the materials and methods section. Supernatants were harvested after 48 h. As seen in Fig. 3b, FG-5FU combined with 10 Gy RT showed significant growth inhibiting effects compared to RT as a single treatment. At FG-5FU dilutions of 4:1, 2:1, and 1:1 combined with 10 Gy, RT resulted in spheroid volumes of 48%, 18%, and 6% of control volumes, respectively.

Similar results were obtained with MGd (Fig. 3c). MGd as a free drug combined with 10 Gy RT demonstrated significant efficacy at increasing MGd concentrations ranging from 20 to 80 μg/mL, although increasing MGd concentration above 20 μg/mL gave statistically non-significant results (p > 0.05). In contrast, MGd released from FG failed to inhibit spheroid growth combined with 10 Gy RT at all the FG-MGd dilutions examined (Fig. 3d). Based on these results for FG-MGd, all subsequent experiments were done using only 5FU as RS.

3.4 RS + RT kinetics of spheroid growth and light micrograft of spheroids

The kinetics of a typical spheroid growth pattern, following various forms of treatment, are illustrated in Fig. 4a. RT only (10 Gy) resulted in a significant delay in initial spheroid growth, but they did reach NTC volume after 14 days. When compared to the NTC, FG-5FU treated spheroids reached a volume of 80% of NTC but were still clearly in continuous growth. The most significant finding, however, was that the combined FG-5FU + RT treatment consistently inhibited spheroid growth. Spheroid volumes, at a 2:1 FG-5FU dilution and 10 Gy radiation dose, did not significantly differ from their initial volume on day 1 (p > 0.05). Figure 4b shows a light micrograph of a NTC and a FG-5FU + RT treated (10 Gy) spheroid following 14 days in culture. The FG-5FU + RT treated spheroid is the same size as it was at initiation (0.2 mm) compared to the NTC (0.8 mm). This represents a growth ratio of 64:1.

Fig. 4

a Growth kinetics of spheroid growth following treatment: RT only (10 Gy), FG-5FU only (2:1 dilution), and FG-5FU + RT (2:1 dilution, 10 Gy). Spheroid volume assayed after 4, 6, 10, and 14 days. Each data point represents the average volume of 16 spheroids, from 2 experiments, after 14 days in culture as a % of non-treated controls. Error bars, standard deviation. * Significant differences (p < 0.05) compared to controls. b Light micrograph of non-treated control (NTC) and FG-5FU + RT (10 Gy) treated spheroids

3.5 RT before and after 5FU addition

The effects of RT administration either before or after FG-5FU exposure were evaluated with a protocol where RT was given immediately before FG-5FU addition or where FG-5FU was first added to the spheroids 24 h before RT. The results are shown in Fig. 5, with Fig. 5a representing the RT before and Fig. 5b representing RT after FG-5FU addition. Comparing the two, spheroids subjected to RT prior to FG-5FU exhibited a greater reduction in tumor spheroid size post-treatment. Specifically, at FG-5FU dilution of 2:1, spheroids that were exposed to RT before FG-5FU showed a decrease to 10% of control values while those that underwent RT after FG-5FU addition exhibited a volume of 18% of controls. This was also the case for a 4:1 FG-5FU dilution.

Fig. 5

a RT before: RT immediately before FG-5FU. b RT after: FG-5FU added 24 h before RT. FG-5FU harvested at 48 h, diluted 1:1, 2:1, 4:1, and 8:1. Error bars, standard deviation. * Significant differences (p < 0.05) compared to controls

3.6 Single vs fractionated RT

The fractionation protocol was as follows: free 5FU or FG-5FU added on day 1 and refreshed on day 5. RT (6 Gy) done on day 2, 4, and 6 after spheroid formation. RT in single or fractionated doses for both free 5FU and for FG-5FU is shown in Fig. 6a and b, respectively. Radiation of 6 Gy as a single dose with free 5FU gave no spheroid growth inhibition (Fig. 6a). This was also the case for 6 Gy and FG-5FU which showed non-significant additional growth inhibition (p > 0.05) compared to that obtained with FG-5FU alone. In contrast, fractionated radiation of 6 Gy × 3 induced significant growth inhibition (p < 0.05) for free 5FU over the concentration range of 0.03–0.12 μg/mL and FG-5FU over a range of dilutions 4:1–1:1. A single RT dose of 18 Gy induced complete cessation of spheroid growth irrespective of the concentration of 5FU or the degree of dilution in FG-5FU.

Fig. 6

a Spheroids incubated with increasing concentrations of 5FU 0, 0.03, 0.06, and 0.12 μg/mL. Radiation 6, 18 Gy administered as single or 3 fractionated doses, 48 h between fractions. b FG-5FU diluted 1:1, 2:1, 4:1, and 8:1. Each data point represents the average volume of 16 spheroids, from 2 experiments, after 14 days in culture as a % of non-treated controls. Error bars, standard deviation. * Significant differences (p < 0.05) compared to controls

3.7 Synergistic effects of RS + RT

Since RS + RT is a technique that relies on the combination of RS and RT exposure, the resultant toxicity should show more than an additive effect of the single modalities of RS and RT. The degree of synergism was calculated both for free 5FU + RT and for FG-5FU + RT using the formula described in materials and methods. As evidenced from the calculated α values, shown in Table 1, both 5FU + RT and FG-5FU + RT demonstrated a significant synergistic effect (α > 1), particularly at a radiation levels 10 Gy, even at low 5FU and FG-5FU levels.

Table 1 Synergistic α values for 5FU free and 5FU FG for increasing RT dose