Diverse metabolic response of cancer cells treated with a 213Bi-anti-EGFR-immunoconjugate

Abstract

Evaluation of treatment response is among the major challenges in modern oncology. We herein used a monoclonal antibody targeting the EGF receptor (EGFR) labelled with the alpha emitter ²¹³Bi (²¹³Bi-anti-EGFR-MAb). EJ28Luc (bladder) and LN18 (glioma) cancer cells, both overexpressing EGFR, were incubated for 3 h with the radioimmunoconjugate. To assess the responses in the core carbon metabolism upon this treatment, these cancer cell lines were subsequently cultivated for 18 h in the presence of [U-¹³C₆]glucose. ¹³C-enrichment and isotopologue profiles of key amino acids were monitored by gas chromatography-mass spectrometry (GC/MS), in order to monitor the impacts of the radionuclide-treatment upon glucose metabolism. In comparison to untreated controls, treatment of EJ28Luc cells with ²¹³Bi-anti-EGFR-MAb resulted in a significantly decreased incorporation of ¹³C from [U-¹³C₆]glucose into alanine, aspartate, glutamate, glycine, proline and serine. In sharp contrast, the same amino acids did not display less ¹³C-enrichments during treatment of the LN18 cells. The data indicate early treatment response of the bladder cancer cells, but not of the glioma cells though cell lines were killed following ²¹³Bi-anti-EGFR-MAb treatment. The pilot study shows that the ¹³C-labelling approach is a valid tool to assess the responsiveness of cancer cells upon radionuclide-treatment in considerable metabolic detail.

Figure 1

Cellular metabolism of glucose. The figure shows relevant steps in metabolization of glucose following uptake from the extracellular milieu. Red circles indicate the analyzed amino acids, i.e. alanine (Ala), aspartate (Asp), glutamate (Glu), proline (Pro), serine (Ser), and glycine (Gly), with regard to enrichment of ¹³C in mock-treated controls and cells incubated with ²¹³Bi-anti-EGFR-MAb. Results of ¹³C-enrichment in EJ28Luc bladder cancer cells. Red arrows indicate significantly decreased enrichment of ¹³C in ²¹³Bi-anti-EGFR-MAb treated cells compared to controls. ETC electron transport chain; (adapted from).

Figure 2

Overall enrichment of ¹³C in analyzed amino acids of untreated control cells and ²¹³Bi-anti-EGFR-MAb treated cells. Results are shown for EJ28-Luc bladder cancer cells (A) and LN18 glioma cells (B). ²¹³Bi-anti-EGFR-MAb treatment of EJ28-Luc cells resulted in a significant decrease of ¹³C-enrichment in alanine, aspartate, glutamate, and serine (***p < 0.0001 compared to controls). Moreover, we observed a slight (nonsignificant = n.s.) decrease in proline. Enrichment of ¹³C varied from 1.6% in proline to 4.5% in alanine (A). In contrast, no significant changes were observed for LN18 cells.

Figure 3

Isotopologue profiling with regard to ¹³C in alanine, aspartate, glutamate, glycine, proline, and serine following incubation of EJ28Luc cells with ²¹³Bi-anti-EGFR-MAb compared to untreated controls. Just as in Fig. 2 showing overall enrichment of ¹³C, a significantly lower enrichment of ¹³C in the various C-atoms following ²¹³Bi-anti-EGFR-MAb treatment was only observed for alanine, aspartate, glutamate, and serine but not for glycine and proline. SHMT serine hydroxymethyl transferase, ALT alanine aminotransferase, PC pyruvate carboxylase, PDH pyruvate dehydrogenase.

Figure 4

Isotopologue profiling with regard to ¹³C in alanine, aspartate, glutamate, glycine, proline, and serine following incubation of LN18 cells with ²¹³Bi-anti-EGFR-MAb compared to untreated controls. As expected from the results shown in Fig. 2, isotopologue profiling did not reveal significant differences between ²¹³Bi-anti-EGFR-MAb treated and untreated LN18 cells, except for the serine isotopologue containing three ¹³C-atoms (M + 3). However, the frequency of the different ¹³C isotopologues was identical for all amino acids in both cell lines analyzed (EJ28Luc and LN18). For example, in proline consisting of five C-atoms, the most frequent isotopologues contained two ¹³C-atoms and isotopologues with four or five ¹³C atoms could not be detected in both cell lines. SHMT serine hydroxymethyl transferase, ALT alanine aminotransferase, PC pyruvate carboxylase, PDH pyruvate dehydrogenase.