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. 2024 May 20;4(5):100760.
doi: 10.1016/j.crmeth.2024.100760. Epub 2024 Apr 26.

Systematic analysis of proteome turnover in an organoid model of pancreatic cancer by dSILO

Alison B Ross  1 , Darvesh Gorhe  1 , Jenny Kim Kim  1 , Stefanie Hodapp  1 , Lela DeVine  2 , Karina M Chan  3 , Iok In Christine Chio  4 , Marko Jovanovic  5 , Marina Ayres Pereira  6
Affiliations

Systematic analysis of proteome turnover in an organoid model of pancreatic cancer by dSILO

Alison B Ross et al. Cell Rep Methods. .

Abstract

The role of protein turnover in pancreatic ductal adenocarcinoma (PDA) metastasis has not been previously investigated. We introduce dynamic stable-isotope labeling of organoids (dSILO): a dynamic SILAC derivative that combines a pulse of isotopically labeled amino acids with isobaric tandem mass-tag (TMT) labeling to measure proteome-wide protein turnover rates in organoids. We applied it to a PDA model and discovered that metastatic organoids exhibit an accelerated global proteome turnover compared to primary tumor organoids. Globally, most turnover changes are not reflected at the level of protein abundance. Interestingly, the group of proteins that show the highest turnover increase in metastatic PDA compared to tumor is involved in mitochondrial respiration. This indicates that metastatic PDA may adopt alternative respiratory chain functionality that is controlled by the rate at which proteins are turned over. Collectively, our analysis of proteome turnover in PDA organoids offers insights into the mechanisms underlying PDA metastasis.

Keywords: CP: Biotechnology; CP: Cancer biology; PDA; SILAC; dSILO; metastases; protein half-life; protein turnover; respirasome.

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Conflict of interest statement

Declaration of interests The authors declare no competing interests.

Figures

None
Graphical abstract
Figure 1
Figure 1
Biological model and experimental design (A) Workflow for preparation of organoid lines for dSILO and global proteomics. Paired organoid cultures were established from the KrasG12D; p53R172H; PdxCre (KPC) mouse model of pancreatic cancer, comprising primary tumors (n = 4), diaphragm (n = 2), and liver metastases (n = 3). Organoid cultures were treated with Nutlin-3a for selection of cells with Trp53 LOH. The resulting panel of paired tumors (n = 4) and metastases (n = 5) was used for dSILO (protein turnover) and global proteomics (protein abundance) analyses. (B) Representative hematoxylin and eosin (H&E) staining of parental tissues (primary tumors and metastases, “Met”) used for the establishment of organoid lines. Bright-field (BF) pictures of tumor and metastasis organoids used for treatment with Nutlin-3a. Scale bars: 100 μm for H&E pictures and 2 mm for BF pictures. (C) Overview of primary and metastatic organoid pairs from each mouse in this study. (D) PCR-based genotyping of primary tumor (“T”) and metastasis (“M”) organoids, before and after Nutlin-3a treatment. Upon treatment with Nutlin-3a, tumor organoids with the loss of their wild-type (WT) allele copy of Trp53 were enriched.
Figure 2
Figure 2
Experimental workflow and validation of method to determine protein half-lives in PDA organoids (A) Experimental workflow for pulsing and quantification of protein half-lives in PDA organoids. (B–F) Heatmaps for protein heavy (H)/light (L) ratios (ln(H/L + 1)) across all biological replicates over the course of 36 h. (G) Coefficients of determination (R2) of the fit of the linear heavy/light label incorporation (in a semi-log plot; see STAR Methods for details) for each tumor and metastasis organoid line. Percentages represent the number of proteins with half-lives determined and R2 >0.7. Data are shown as mean ± SD.
Figure 3
Figure 3
Mitochondrial proteins turn over faster in metastases compared to tumors (A–E) Histograms of protein half-life frequency distributions for each metastasis vs. tumor pair. The “n” value in each graph corresponds to the number of proteins shared between each tumor and metastases pair. Differences between half-life distributions were assessed using the Wilcoxon test. (F) Volcano plot of differentially turned over proteins in metastases compared to primary tumors. Blue dots indicate proteins with significantly decreased half-lives in metastasis compared to tumor (n = 486, p < 0.05, log2FC < −0.32). Vertical red lines represent the bottom (left, log2FC = −0.32) and top (right, log2FC = 0.32) log2FC cutoffs applied. The horizontal dotted line represents the p value cutoff (p = 0.05). p value established by paired t test. (G) KEGG pathway enrichment analysis from DAVID showing pathways with shortened half-lives (i.e., faster turnover) in metastases compared to tumors. The x axis represents the −log10(p value). Only pathways with p <0.05 are shown. “n” represents the number of proteins associated with each pathway. (H) Keyword (KW) cellular compartment analysis from DAVID showing cellular compartments with shortened half-lives (i.e., faster turnover) in metastases compared to tumors. The x axis represents the −log10(p value). Only cellular compartments with p <0.05 are shown. “n” represents the number of proteins associated with each cellular compartment. Mitochondrial and inner mitochondrial membrane compartments are marked in green.
Figure 4
Figure 4
Mitochondrial respiratory chain complexes turn over faster in metastases than in primary tumors (A) Half-life cumulative distributions of proteins found to be in complexes and those without protein binding partners per CORUM. Proteins found within complexes did not display significant differences in half-lives compared with randomly grouped (shuffled) proteins within the dataset (Kolmogorov-Smirnov test, p > 0.05). (B) Variance cumulative distribution of half-lives of proteins found to be in complexes per CORUM compared to randomly shuffled groups of proteins from the dataset. Proteins found within complexes show less variance in their half-lives than randomly grouped (shuffled) proteins within the dataset (Kolmogorov-Smirnov test, p < 0.05). (C) Mean log2 fold-change differences in half-lives of the top five protein complexes with significantly different turnover rates in metastases compared to primary tumors after curating for redundant complex assignments (Kolmogorov-Smirnov test, p < 0.001). (D) Half-life comparison of differentially turned over respirasome proteins in tumors and metastases (two-sided paired t test, p < 0.05). (E) Log2 fold changes (FCs) of half-lives and protein abundances of overlapping proteins from the datasets of respiratory chain complexes I–V. Significance was assessed using a two-sided paired t test (p < 0.05). Data are shown as mean ± SD.
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