Journal: Nature

Article Title: Cooperative nutrient scavenging is an evolutionary advantage in cancer

doi: 10.1038/s41586-025-08588-w

Figure Lengend Snippet: a , Inhibitors used to identify proteases responsible for cooperation. Cila, cilastatin; Dox, doxycycline; iAN, aminopeptidase N inhibitor; JPM, JPM-OEt; Lina, linagliptin; SC, Sigma C-, S-, T- and D-peptidase inhibitor cocktail. b , Growth curves (bottom) and representative images (top) of A375 cells growing on Ala-Gln and treated with bestatin or with bestatin and Gln. Scale bars, 100 µm. c , We synthesized PEGylated analogues of bestatin to reduce its cell permeability. All tested analogues inhibited growth on Ala-Gln with no effects on cells growing on Gln. d , The strategy used to identify CNDP2 as the key extracellular aminopeptidase for cooperative peptide cleavage. Proteomic analysis of conditioned media revealed the presence of 16 secreted aminopeptidases (SAPs). APs, aminopeptidases; PSM, peptide-spectrum match. e , Growth curves of lung KP Keap1 -KO ( Cndp2 -wild-type (WT)) and isogenic Cndp2 -KO cells, and Cndp2 -KO cells expressing CNDP2 from a CMV promoter. Plots show relative cell numbers and population viability over time. f , Cell lysates and conditioned media from Cndp2 -WT and Cndp2 -KO cells were immunoblotted for CNDP2. Representative data from three independent experiments. For gel source data, see Supplementary Fig. . g , Recombinant CNDP2 (rCNDP2) was used to estimate the levels of endogenous CNDP2 in CM. Right, quantification of the enhanced chemiluminescence (ECL) from blots on the left. Data representative of three independent experiments. For gel source data, see Supplementary Fig. . a.u., arbitrary units. h , Growth of lung KP Cndp2 -KO cells on Ala-Gln is rescued by extracellular rCNDP2. Data are mean ± s.d. of three replicates. Student’s t -test. i , Conditioned medium from Cndp2 -KO cells does not stimulate growth in cultures of Cndp2 -WT cells. Conditioned medium was collected from Cndp2 -WT or Cndp2 -KO cells grown in Ala-Gln for 24 h. Data points depict averages from four visual fields. Two-tailed t -tests.

Article Snippet: Cells were treated with bestatin (Cayman Chemicals 70520 or Sigma-Aldrich B8385), GPNA ( l -γ-glutamyl- p -nitroanilide; Selleckchem S6670), BenSer (Sigma-Aldrich 13900), DAPT (Tocris 2634), Doxycycline (Tocris 4090), Linagliptin (Selleckchem S3031), anti-aminopeptidase N antibody (Millipore MABF2147), Cilastatin (SCBT sc-201312), JPM-OEt (Fisher, 502029515), Protease Inhibitor Cocktail (Sigma-Aldrich P8340), vacuolin-1 (Cayman Chemicals 20425), 5-( N -ethyl- N -isopropyl)amiloride (EIPA; Sigma-Aldrich A3085) and 3-methyladenine (3-MA; Selleckchem S2767).

Techniques: Synthesized, Analogues, Permeability, Expressing, Recombinant, Two Tailed Test