Three-dimensional environment renders cancer cells profoundly less susceptible to a single amino acid starvation

Abstract

Increased amino acid requirement of malignant cells is exploited in metabolic antitumor therapy, e.g., enzymotherapies based on arginine or methionine deprivation. However, studies on animal models and clinical trials revealed that solid tumors are much less susceptible to single amino acid starvation than could be expected from the in vitro data. We conducted a comparative analysis of the response of several tumor cell lines to single amino acid starvation in 2-D monolayer versus 3-D spheroid culture. We revealed for the first time that in comparison with monolayer culture tumor cells, spheroids are much less susceptible to the deprivation of individual amino acids (i.e., arginine, leucine, lysine or methionine). Accordingly, even after prolonged (up to 10 days) starvation, spheroid cells could readily resume proliferation when appropriate amino acid was resupplemented. In the case of arginine deprivation, similar apoptosis induction was detected both in 2-D and 3-D culture, suggesting that this process does not determine the level of tumor cell sensitivity to this kind of treatment. It was also observed that spheroids much better mimic the in vivo ability of tumor cells to utilize citrulline as arginine precursor for growth in amino acid deficient environment. We conclude that 3-D spheroid culture better reflects in vivo tumor cell response to single amino acid starvation than 2-D monolayer culture and should be used as an integral model in the studies of this type of antitumor metabolic targeting.

Proliferation and motility of hepatocellular, pancreatic and gastric cancer cells grown in a medium without glucose and arginine, but with galactose and ornithine

“…One example is the use of arginase, which degrades arginine and induces apoptosis in cancer cells (30). A previous study indicated that cancer cells are resistant to arginine deprivation in a three-dimensional environment (31). The current study used HSM, which does not contain glucose or arginine, and the results revealed that that deprivation of glucose and arginine may synergistically suppress cell proliferation and induce apoptosis.…”

Arginine enzymatic deprivation and diet restriction for cancer treatment

“…One such limitation arises from the upregulation of ASS expression in many tumors in response to arginine starvation, leading to the appearance of the ASS-positive tumor relapse insensitive to the therapy (Vynnytska-Myronovska et al, 2012). Also, results showed that tumor cells become profoundly more resistant to arginine withdrawal in in vitro 3D spheroid models relative to respective monolayer cultures (Vynnytska-Myronovska et al, 2013). This phenomenon is consistent with the results of animal studies and ongoing clinical trials which showed that arginine deprivation is effective in inhibiting tumor growth but not in inducing tumor regression.…”

Co-application of canavanine and irradiation uncouples anticancer potential of arginine deprivation from citrulline availability

“…Consequently, CRC was excluded from the list of tumors defined as the responders to arginine deprivation therapy [4, 7]. However, our recent in vitro data indicate that cancer cells might be radiosensitized in the absence of arginine even if they express citrulline-to-arginine converting enzymes [18, 19]. Therefore, it is reasonable to speculate that arginine deprivation therapy could prime CRC and other ASS1-positive malignancies to both standard-of-care and novel combinational therapies.…”

“…The present study was thus designed to 1) prove that, despite the inducible mode of ASS1 expression, arginine metabolism can still be considered as a promising target in CRC treatment and 2) gain an insight into the presumed adverse compensatory mechanism of citrulline conversion to arginine. Human CRC cell lines were grown both in conventional two-dimensional (2-D) monolayer cultures, as well as in 3-D spheroid cultures, which were proposed as a more reliable tool for evaluating metabolic anticancer therapies before turning into whole animal studies [19]. …”