Human pancreatic cancer tumors are nutrient poor and tumor cells actively scavenge extracellular protein

Abstract

Glucose and amino acids are key nutrients supporting cell growth. Amino acids are imported as monomers, but an alternative route induced by oncogenic KRAS involves uptake of extracellular proteins via macropinocytosis and subsequent lysosomal degradation of these proteins as a source of amino acids. In this study, we examined the metabolism of pancreatic ductal adenocarcinoma (PDAC), a poorly vascularized lethal KRAS-driven malignancy. Metabolomic comparisons of human PDAC and benign adjacent tissue revealed that tumor tissue was low in glucose, upper glycolytic intermediates, creatine phosphate, and the amino acids glutamine and serine, two major metabolic substrates. Surprisingly, PDAC accumulated essential amino acids. Such accumulation could arise from extracellular proteins being degraded through macropinocytosis in quantities necessary to meet glutamine requirements, which in turn produces excess of most other amino acids. Consistent with this hypothesis, active macropinocytosis is observed in primary human PDAC specimens. Moreover, in the presence of physiologic albumin, we found that cultured murine PDAC cells grow indefinitely in media lacking single essential amino acids and replicate once in the absence of free amino acids. Growth under these conditions was characterized by simultaneous glutamine depletion and essential amino acid accumulation. Overall, our findings argue that the scavenging of extracellular proteins is an important mode of nutrient uptake in PDAC.

Figure 1. Metabolomic analysis of human PDAC compared to benign adjacent pancreatic tissue

(A) Schematic of experimental procedure: PDAC and benign adjacent tissue from n = 49 patients were collected during pancreatic resectomy, frozen in liquid nitrogen, ground, extracted, and subjected to metabolite analysis by LC-MS. (B) Metabolites with significantly different levels between tumor and paired benign adjacent tissues (false discovery rate < 0.05). Y-axis is the ratio of metabolite concentrations in the tumor to benign adjacent tissue. Vertical lines represent range, boxes interquartile range, and horizontal lines median across the 49 patients. (C) Schematic illustration of possible mechanism underlying essential amino acid accumulation: Protein catabolism yields amino acids in the ratio found in protein; those used extensively for purposes other than making new protein are depleted, while the others buildup.

Figure 2. Human pancreatic tumor cells display macropinocytosis

(A) Hematoxylin and eosin (H&E) staining of tissue from a representative human pancreatic tumor. Scale bar is 100 μm. (B and C) An ex vivo macropinocytosis uptake assay using TMR-dextran as a marker of macropinosomes (red) indicates that CK19-positive pancreatic tumor cells (green) display high levels of macropinocytosis. DAPI staining (blue) identifies nuclei. Cell boundaries (red outline) in (B) were delineated on the basis of CK19 staining. The images shown in (C) represent a higher magnification of the boxed areas in (B). For (B) and (C) the scale bars are 20 μm and 5 μm, respectively. Images shown are Z-stack projections and are representative of five independent tumors.

Figure 3. Ras-mutant pancreatic cells are capable of growth in medium lacking essential amino acids when supplemented with physiological levels of serum protein

(A, B) Images of KRPC cells cultured in leucine-free medium. The cells survive and proliferate in the presence of 50 g/L BSA. (B) Cell counts from (A). (C) Intracellular leucine concentrations in KRPC cells grown in leucine-rich and leucine-free media, as measured by LC-MS. (D, E) KRPC cells cultured in amino acid-free medium grow using serum protein as their sole source of amino acids. For B–D, data are means ± SE, n ≥ 3.

Figure 4. Stable isotope tracing reveals substantial contribution of protein scavenging to amino acid pools

(A) Schematic of isotope tracing experiment enabling separate quantification of amino acids derived from serum protein and amino acids initially taken up as monomers. (B, C) Fractional contribution of extracellular protein to intracellular (B) and extracellular (C) amino acid pools in KRPC cells. (D, E) Bafilomycin A1, an inhibitor of lysosomal acidification, reduces intracellular (D) and extracellular (E) serum protein-derived amino acid pools in a dose-dependent fashion. For B–E, data are means ± SE of n ≥ 3.

Figure 5. Cells relying on protein scavenging for amino acid assimilation accumulate strictly proteinogenic amino acids

KRPC cells were grown in amino acid-replete or amino acid-free medium and supplemented with physiological levels of albumin. (A) Fractional contribution of serum protein-derived carbon to intracellular amino acid pools. (B, C) Relative intracellular amino acid abundances. (D, E) Net uptake or excretion of amino acids into the medium. Data are means ± SE of n ≥ 3.