Deprivation of the semi-essential amino acid arginine is well tolerated by the organism in vivo but appears to be detrimental for some tumors. It was shown both in vitro and in vivo that application of arginine-degrading enzymes harbors cytostatic effects and inhibits tumor growth. This phenomenon serves as a good rationale for the development of a metabolic enzymotherapeutic approaches to be combined with classical treatment regimes. It was previously demonstrated [Vynnytska et. al, Anticancer Drugs, 2011] that low concentrations of the arginine natural analogue canavanine accelerate and augment arginine deprivation-induced apoptosis when co-introduced with the arginine-degrading enzyme arginase. This effect was selective for tumor cells, not exclusive for arginine auxotrophic cells, and did not impact normal human cells.

We particularly aimed to evaluate the anticancer potential of the combined treatment of external irradiation and arginine starvation alone or together with canavanine using a spheroid-based test platform [Friedrich et al., Nature Protoc, 2009] and spheroid regrowth probability as an analytical endpoint with clinical relevance. This turned out to be a particularly relevant methodological approach because several cell types showed loss of sensitivity to arginine deficiency in the more physiological 3-D cellular context as compared to 2-D culture assays. With the set-up, we could reveal that low concentrations of canavanine do not affect radioresponse of colorectal adenocarcinoma HT29 spheroids when cultured in complete, arginine-containing medium. By contrast, pre-incubation of the spheroids in arginine-free culture medium or with the arginine-degrading enzyme human recombinant arginase resulted in a 1.5 time decrease in HT29 spheroid control dose 50 (SCD50 = 50% of spheroids do not regrowth and are "cured"). And, SCD50 even decreased by a factor of 3 when arginine-deprivation was combined with canavanine treatment before irradiation.

Our data strongly suggest that arginine deprivation by enzymotherapy alone or in combination with chemotherapeutical approaches (e.g., canavanine) is a promising strategy to sensitize (colon) cancer cells to irradiation. Extended pre-clinical studies are underway and options for translation into the clinic are under consideration. The challenges for such combination treatment will be discussed.