Extracellular Matrix Collagen I Differentially Regulates the Metabolic Plasticity of Pancreatic Ductal Adenocarcinoma Parenchymal Cell and Cancer Stem Cell

Abstract

Pancreatic ductal adenocarcinoma (PDAC) has a 5-year survival rate of less than 10 percent largely due to the intense fibrotic desmoplastic reaction, characterized by high levels of extracellular matrix (ECM) collagen I that constitutes a niche for a subset of cancer cells, the cancer stem cells (CSCs). Cancer cells undergo a complex metabolic adaptation characterized by changes in metabolic pathways and biosynthetic processes. The use of the 3D organotypic model in this study allowed us to manipulate the ECM constituents and mimic the progression of PDAC from an early tumor to an ever more advanced tumor stage. To understand the role of desmoplasia on the metabolism of PDAC parenchymal (CPC) and CSC populations, we studied their basic metabolic parameters in organotypic cultures of increasing collagen content to mimic in vivo conditions. We further measured the ability of the bioenergetic modulators (BMs), 2-deoxyglucose, dichloroacetate and phenformin, to modify their metabolic dependence and the therapeutic activity of paclitaxel albumin nanoparticles (NAB-PTX). While all the BMs decreased cell viability and increased cell death in all ECM types, a distinct, collagen I-dependent profile was observed in CSCs. As ECM collagen I content increased (e.g., more aggressive conditions), the CSCs switched from glucose to mostly glutamine metabolism. All three BMs synergistically potentiated the cytotoxicity of NAB-PTX in both cell lines, which, in CSCs, was collagen I-dependent and the strongest when treated with phenformin + NAB-PTX. Metabolic disruption in PDAC can be useful both as monotherapy or combined with conventional drugs to more efficiently block tumor growth.

Keywords: bioenergetic modulators; cancer stem cells; chemoresistance; collagen I; glutamine; pancreatic ductal adenocarcinoma; treatment; tumor microenvironment.

Figure 1

Metabolic profiles of PDAC cells in 2D and in 3D in different ECM compositions. (A) After 7 days of growth in the different ECMs, the growth medium was collected and basic metabolic parameters such as glucose consumption, lactate release and ATP production were measured. Results are presented as mean ± SEM in triplicate of at least three independent experiments. Significantly different between groups: * p < 0.05; ** p < 0.01; *** p < 0.001 compared to 2D condition of each cell line. # p < 0.05; ## p < 0.01; ### p < 0.001 and ns. p > 0.05 compared the 3D ECMs of each cell line. (B) Expression of the major transporters responsible for glucose consumption and lactate release. Upper panels display representative Western blots for the indicated proteins and lower panels (C) show a quantitative analysis of their relative expression as standardized to CPCs cells on 2D. Significantly different between groups: * p < 0.05; ** p < 0.01; *** p < 0.001 compared to 2D CPCs. ### p < 0.001 compared 2D CSCs; + p < 0.05; +++ p < 0.001 comparing CPCs to CSCs on the same ECM.

Figure 2

Amino acid quantification by HPLC in PDAC cell lines. The growth medium was collected after 7 days of growth in different substrates and the amino acids quantified by HPLC. Results are presented as mean ± SEM in triplicate of at least three independent experiments. Significance between groups: * p < 0.05; compared to 2D condition of each cells; # < 0.05 compared to 3Ds; + < 0.05 comparing CPCs to CSCs on the same ECMs.

Figure 3

Effect of bioenergetic modulators (BMs), 2-DG, DCA and phenformin on PDAC cell proliferation and cell death. After 7 days of treatment with BMs (2-DG: 5 mM, DCA: 20 mM and phenformin: 0.01 mM) in different substrates, (A) cell growth was measured by the resazurin assay. (B) Cell death was quantified using the ethidium homodimer assay where the integrity density of dead or dying cells (stained red) was measured by the ImageJ software. Untreated cells were used as control. Results are presented as the mean ± SEM of triplicates from at least three independent experiments. Significance between groups: * p < 0.05; ** p < 0.01; *** p < 0.001 compared to untreated cells; ns: not significant.

Figure 4

Effect of BM treatment significantly synergizes with NAB-PTX cytotoxicity in PDAC cell growth (A) and death (B) in different ECMs. Cells were exposed with a fixed concentration of BMs (2-DG (5 mM), DCA (20 mM) and phenformin (Phen. 0.01 mM)) and NAB-PTX (10 mM), during the respective incubation time. Results represent the mean ± SEM of triplicates from at least three independent experiments. Significance between groups: * p < 0.05; ** p < 0.01; *** p < 0.001 compared to untreated cells. # p < 0.05; ## p < 0.01; ### p < 0.001 compared to cells treated only with NAB-PTX 10 nM; ns: not significant.