Alpha-particle emitting 213Bi-anti-EGFR immunoconjugates eradicate tumor cells independent of oxygenation

Abstract

Hypoxia is a central problem in tumor treatment because hypoxic cells are less sensitive to chemo- and radiotherapy than normoxic cells. Radioresistance of hypoxic tumor cells is due to reduced sensitivity towards low Linear Energy Transfer (LET) radiation. High LET α-emitters are thought to eradicate tumor cells independent of cellular oxygenation. Therefore, the aim of this study was to demonstrate that cell-bound α-particle emitting (213)Bi immunoconjugates kill hypoxic and normoxic CAL33 tumor cells with identical efficiency. For that purpose CAL33 cells were incubated with (213)Bi-anti-EGFR-MAb or irradiated with photons with a nominal energy of 6 MeV both under hypoxic and normoxic conditions. Oxygenation of cells was checked via the hypoxia-associated marker HIF-1α. Survival of cells was analysed using the clonogenic assay. Cell viability was monitored with the WST colorimetric assay. Results were evaluated statistically using a t-test and a Generalized Linear Mixed Model (GLMM). Survival and viability of CAL33 cells decreased both after incubation with increasing (213)Bi-anti-EGFR-MAb activity concentrations (9.25 kBq/ml-1.48 MBq/ml) and irradiation with increasing doses of photons (0.5-12 Gy). Following photon irradiation survival and viability of normoxic cells were significantly lower than those of hypoxic cells at all doses analysed. In contrast, cell death induced by (213)Bi-anti-EGFR-MAb turned out to be independent of cellular oxygenation. These results demonstrate that α-particle emitting (213)Bi-immunoconjugates eradicate hypoxic tumor cells as effective as normoxic cells. Therefore, (213)Bi-radioimmunotherapy seems to be an appropriate strategy for treatment of hypoxic tumors.

Figure 1. Photon irradiation of CAL33 cells in the aluminium hypoxic chamber.

Culture flasks were arranged in three levels, separated by lead plates (thickness 1.31 cm; distance 6.5 cm). Thus, irradiation doses were reduced by half (D₁, D_1/2, D_1/4), respectively, as confirmed by dosimetric measurements. Cells were irradiated with three different doses in one irradiation experiment. Hypoxia was established by repeated aspiration of air and influx of 95% N₂, 5% CO₂.

Figure 2. Western blot analysis of HIF-1α expression in CAL33 cells under normoxia and hypoxia.

A) HIF-1α expression at different times (0–24 h) after setup of hypoxia; 3 h+3 h: incubation of cells done twice for 3 hours under hypoxia, interrupted by a 10 min phase under normoxic conditions. B) HIF-1α expression at different times (0–180 min) after release of CAL33 from hypoxia and incubation under normoxic conditions; control: normoxic cells; CoCl₂: cells treated with CoCl₂ used as positive control; 160 K, 105 K, 75 K molecular weight markers (RPN800, GE Healthcare). Unspecific staining of protein bands other than HIF-1α was used as sample loading control.

Figure 3. Clonogenic survival and viability of normoxic and hypoxic cells after irradiation with photons or ²¹³Bi-anti-EGFR-MAb.

Results of single experiments are shown as pale coloured graphs, results of means as bold coloured graphs. A) Number of clones (clonogenic assay) and B) absorbance (WST viability assay) as a function of activity concentration (²¹³Bi-anti-EGFR-MAb) or photon dose, respectively.

Figure 4. Relative risk for cell survival/viability after irradiation with photons/²¹³Bi-anti-EGFR-MAb under normoxia/hypoxia.

Relative risk (RR) (▪) denotes RR-fold higher survival of CAL33 cells irradiated under hypoxia compared to normoxia. RR was calculated according to the Generalized Linear Mixed Model (GLMM). Overall cell survival/viability is significantly (RR-fold) higher under hypoxia, if the 95%- confidence interval (•) doesn't include the value 1 (red line).

Figure 5. Relative risk for cell survival (A)/viability (B) at different photon doses/²¹³Bi-anti-EGFR-MAb activity concentrations under normoxia/hypoxia.

Relative risk (RR) (▪) denotes RR-fold higher survival of CAL33 cells irradiated under hypoxia compared to normoxia. RR was calculated according to the Generalized Linear Mixed Model (GLMM). Overall cell survival/viability is significantly (RR-fold) higher under hypoxia, if the 95%- confidence interval (•) doesn't include the value 1 (red line).