GPI/AMF inhibition blocks the development of the metastatic phenotype of mature multi-cellular tumor spheroids

Abstract

Epithelial-mesenchymal transition (EMT) and cellular invasiveness are two pivotal processes for the development of metastatic tumor phenotypes. The metastatic profile of non-metastatic MCF-7 cells growing as multi-cellular tumor microspheroids (MCTSs) was analyzed by determining the contents of the EMT, invasive and migratory proteins, as well as their migration and invasiveness potential and capacity to secrete active cytokines such as the glucose phosphate isomerase/AMF (GPI/AMF). As for the control, the same analysis was also performed in MCF-7 and MDA-MB-231 (highly metastatic, MDA) monolayer cells, and in stage IIIB and IV human metastatic breast biopsies. The proliferative cell layers (PRL) of mature MCF-7 MCTSs, MDA monolayer cells and metastatic biopsies exhibited increased cellular contents (2-15 times) of EMT (β-catenin, SNAIL), migratory (vimentin, cytokeratin, and fibronectin) and invasive (MMP-1, VEGF) proteins versus MCF-7 monolayer cells, quiescent cell layers of mature MCF-7 MCTS and non-metastatic breast biopsies. The increase in metastatic proteins correlated with substantially elevated cellular abilities for migration (18-times) and invasiveness (13-times) and with the higher level (6-times) of the cytokine GPI/AMF in the extracellular medium of PRL, as compared to MCF-7 monolayer cells. Interestingly, the addition of the GPI/AMF inhibitors erythrose-4-phosphate or 6-phosphogluconate at micromolar doses significantly decreased its extracellular activity (>80%), with a concomitant diminution in the metastatic protein content and migratory tumor cell capacity, and with no inhibitory effect on tumor lactate production or toxicity on 3T3 mouse fibroblasts. The present findings provide new insights into the discovery of metabolic inhibitors to be used as complementary therapy against metastatic and aggressive tumors.

Hypoglycemia Enhances Epithelial‐Mesenchymal Transition and Invasiveness, and Restrains the Warburg Phenotype, in Hypoxic HeLa Cell Cultures and Microspheroids

“…S5). Interestingly, only in the HH condition, cancer cells were able to secrete HPI/AMF (1.9–4 times vs. NN), an important cytokine linked to the migration onset (Gallardo‐Pérez et al, ). In fact, HPI/AMF release correlated with increased invasiveness under moderate HH (Fig.…”

“…Calcein fluorescence was detected at λ excitation of 485 nm and λ emission of 520 nm in a microplate reader (Nunclon™, Roskilde, Denmark). The breast triple negative highly metastatic MDA‐MB‐231 cell line was used as positive control of migration (Gallardo‐Pérez et al, ).…”

“…After 24 h exposure to NN or HH, 5 ml of DMEM (i.e., extracellular medium) was removed from the HeLa cell cultures (5 × 10 6 cells/ml) and incubated with 10% trichloroacetic acid (TCA) overnight at 4°C (Gallardo‐Pérez et al, ). The mixture was centrifuged at 8,900g for 30 min, and the pellet was recovered and re‐suspended in 100 μl loading buffer (glycerol 10%, SDS 2%, bromophenol blue 0.02%, Tris–HCl 62.5 mM, β‐mercaptoethanol 5%).…”

Co-culture of 3D tumor spheroids with fibroblasts as a model for epithelial–mesenchymal transition in vitro

“…Data obtained using these in vitro models may explain the involvement of interaction between cancer cells and the extracellular matrix (ECM) or between cancer cells and stromal cells in the induction of EMT. In contrast to these conventional 2-dimensional cell culture systems, 3-dimensional (3D) tumor spheroids (TSs) may be a more reliable model reflecting the characteristics of in vivo tumors more closely [12][13][14]. To incorporate tumor microenvironmental factors into TSs representing the architecture of in vivo tumors, a directmixed co-culture of TSs with fibroblasts using the hanging-drop method has been reported [15].…”

Changes in Protein Expression in Two Cholangiocarcinoma Cell Lines Undergoing Formation of Multicellular Tumor Spheroids In Vitro

“…Although the changes in the patterns of cellular aggregation can be triggered also by adding up cytokines to culture media, even the simple hypoxic and mechanical stimulations promote MCTS formation in HepG2 hepatocellular carcinoma [14] and breast cancer MCF-7 cultured cells [15] undergoing EMT as proved by the upregulation of vimentin and loss of E-cadherin expression [13]. …”