S100 family proteins are linked to organoid morphology and EMT in pancreatic cancer

Abstract

Epithelial-mesenchymal transition (EMT) is a continuum that includes epithelial, partial EMT, and mesenchymal states, each of which is associated with cancer progression, invasive capabilities, and ultimately, metastasis. We used a lineage-traced sporadic model of pancreatic cancer to generate a murine organoid biobank from primary and secondary tumors, including sublines that underwent partial EMT and complete EMT. Using an unbiased proteomics approach, we found that organoid morphology predicts the EMT state, and the solid organoids are associated with a partial EMT signature. We also observed that exogenous TGFβ1 induces solid organoid morphology that is associated with changes in the S100 family, complete EMT, and the formation of high-grade tumors. S100A4 may be a useful biomarker for predicting EMT state, disease progression, and outcome in patients with pancreatic cancer.

Fig. 1. Generation of a murine organoid biobank with different morphological features.

A Schematic representation of the workflow to generate organoids from primary and metastatic secondary tumors in the KPC model. YFP + cells FACS isolated from Pdx-Cre; Rosa^YFP (CY) mice were used to generate normal pancreatic organoids, while YFP + cells FACS isolated from either primary tumors or secondary tumors (liver, lung, and diaphragm) from Pdx-Cre; Kras^G12V; p53^R172H; Rosa^YFP (CKPY) mice were used to generate tumor organoids. B Representative brightfield (top) and H&E (bottom) images of normal pancreatic, primary tumor and metastatic tumor organoids. Representative glandular and solid subtype organoids are shown. Scale bar = 100 μm. C Representative FACS plots for isolation of YFP + EpCAM+ and YFP + EpCAM- cells from normal pancreas (N = 5), low-grade tumor (N = 5) and high-grade tumor (N = 4) for the generation of organoids. D Percentage of live YFP + EpCAM+ cells isolated from the normal pancreas from CY mice (N = 5), and either low-grade tumors (N = 5) or high-grade (N = 4) pancreatic tumors from CKPY mice. Each dot represents an individual mouse. Data are presented as mean + /− SEM. *p < 0.05, Student’s t-test. E Representative H&E images of the primary tumor and corresponding YFP + (left), YFP + EpCAM + (middle) and YFP + EpCAM- (right) organoids. Scale bar = 100 μm. F Quantification of the organoid morphology for each MO line, represented as glandular (green) or solid (red).

Fig. 2. Murine organoid morphology correlates with partial EMT state.

A Schematic representation of organoid lines used for label-free based quantitative proteomics analysis. Organoids with different morphologies were subjected to cell lysis followed by on-bead enzymatic digestion and subsequent mass spectrometry analysis. B Principal component analysis plot of 3546 of the most variable proteins across organoid lines, including YFP + EpCAM+ normal (N = 5), YFP + EpCAM+ tumor (N = 9), YFP + EpCAM- tumor (N = 9) and YFP + secondary tumors (Met; N = 8) organoids. The plot shows the separation of samples based on different principal components (PCs). Glandular (G) and solid (S) organoid lines are indicated. C The organoid signature score of the six previously described EMT subtypes [22] including YFP + EpCAM + (E+; N = 9), YFP + EpCAM- (E-; N = 9) and YFP + secondary (Met; N = 8) tumor organoids. Glandular (G) and solid (S) organoid lines are indicated. D Volcano plot illustrating the log₂ protein ratios in whole cell lysates of organoids, comparing glandular YFP + EpCAM+ with solid YFP + EpCAM- tumor organoids. Proteins were deemed differentially regulated in the log2 fold change in protein expression was ≥ 1-fold and exhibited an adjusted p ≤ 0.05. E mRNA expression of S100a4 and S100a14 in YFP + (Y), YFP + EpCAM + (E + ) and YFP + EpCAM- (E-) organoids. Each dot represents an individual organoid line. Glandular (open circle) and solid (closed circle) organoid lines are indicated. Data is relative to Gapdh, presented as mean + /− SEM. *p < 0.05; **p < 0.01; ***p < 0.001, Mann–Whitney test.

Fig. 3. TGF-β1 treatment induces a mesenchymal signature in tumor organoids.

A Schematic representation of the treatment of murine tumor organoids seeded as single cells with recombinant TGFβ1 and timing of quantification of the morphology and gene expression signatures. B Representative brightfield images of YFP + EpCAM + (N = 6), YFP + EpCAM- (N = 6) and YFP + secondary (Met, N = 6) tumor organoids on day 3 post TGFβ1 treatment. The pre-treatment (PRE) organoid morphology is indicated. Representative images of organoids post-treatment (POST) are shown. Arrows indicate a branching phenotype. Images are representative of 3 biological replicates. Scale bar = 300 μm. C Quantification of the tumor organoid morphology 3 days post TGFβ1 treatment. YFP + EpCAM + (N = 6), YFP + EpCAM- (N = 6) and YFP + secondary (Met; N = 6) tumor organoids. Organoids are grouped as glandular (G) and solid (S) morphologies pre-treatment (PRE). Quantification of glandular (green), solid (red) and branching (blue) post cytokine treatment (POST). Data includes 3 biological replicates and is presented + /− SEM. ****p < 0.0001, Chi-square test. D, E mRNA expression levels of mesenchymal markers, Vim, Cdh2, Fn1, Snai1, Snai2, Zeb1 (D); and S100 family members, S100a4, S100a14 (E), in YFP + EpCAM + (E+), YFP + EpCAM- (E-) primary tumor organoids and YFP + secondary tumor organoids following the addition of TGFβ1 (blue). Each dot represents an individual organoid line. Organoids are grouped as glandular (G) and solid (S) morphologies. Data includes three biological replicates and is presented as log10 fold change relative to the vehicle (white) control, mean + /− SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, paired t-test.

Fig. 4. TGFβ1 induces the expression of IL-6 family cytokines.

A Schematic representation of the treatment of murine tumor organoids seeded as single cells with recombinant TGFβ1 and the timing of quantification of gene expression signatures. B–E mRNA expression levels of IL-6 family cytokines Il6, Lif, and Il11 (B); IL-6 family cytokine receptors il6st (C); Il6r, Lifr, and Il11r (D), and the IL-6 cytokine family target gene, Socs3 (E), 3 days post treatment with TGFβ1. Each dot represents an individual organoid. Organoids are grouped into glandular (G) and solid (S) types. Data includes three biological replicates and is presented as log10 fold change relative to the vehicle (white) control (mean ± SEM). *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, paired t-test.

Fig. 5. IL-6 or LIF do not alter tumor organoid morphology or induce EMT.

A Schematic representation of the treatment of murine organoids seeded as single cells with recombinant IL-6 or LIF and timing of quantification of the morphology and gene expression signatures. B Representative brightfield images of YFP + EpCAM + (E+; N = 6), YFP + EpCAM- (E-) and YFP + secondary (Met; N = 12) tumor organoids on day 3 post treatment (POST) with the indicated cytokine. Organoids are grouped as glandular (G) and solid (S) morphologies pre-treatment (PRE). Scale bar = 300 μm. C Quantification of the tumor organoid morphology 3 days post treatment with the indicated cytokine. YFP + EpCAM + (E + , N = 6), YFP + EpCAM- (E-, N = 12 primary and secondary). Glandular (green), solid (red) and branching (blue). Data is presented + /− SEM. Results are not significant, Chi-square test. D mRNA expression levels of S100 family proteins, S100a4 and S100a14, in YFP + EpCAM + (E+), YFP + EpCAM- (E-) primary tumor organoids and YFP + secondary tumor organoids following the addition of the indicated cytokine (IL-6, brown; LIF, red). Each dot represents an individual organoid. Organoids are grouped as glandular (G) and solid (S) morphologies. Data includes 3 biological replicates and is presented as log10 fold change relative to the vehicle (white) control, mean + /− SEM. paired t-test.

Fig. 6. Organoid-derived allografts resemble original tumors and exhibit an EMT phenotype.

A Schematic representation of the subcutaneous transplant of organoids into gender matched wild-type C57BL/6 mice to generate allograft lines. B Quantification of the number of low-grade (orange) and high-grade (blue) allograft tumors, generated from YFP + EpCAM + (E+), YFP + EpCAM- (E-) primary tumor organoids and YFP + secondary tumor organoids. Organoids are grouped as glandular (G) and solid (S) morphologies. C Representative H&E images of the tumor organoid, and corresponding allograft generated from YFP + EpCAM + (E + ), YFP + EpCAM- (E-) primary tumor organoids and YFP + secondary tumor organoids. Organoids are grouped as glandular (G) and solid (S) morphologies. D, E mRNA expression of Tgfb1, Tgfbr2 (D), and the S100 family proteins, S100a4, S100a14 (E), in low-grade (N = 4 tumors) and high-grade (N = 4 tumors) allograft tumors derived from YFP + EpCAM+ MO, YFP + EpCAM- MO, and YFP + secondary tumor MO. Each dot represents an individual tumor. Data is relative to Gapdh, presented as mean + /− SEM. *p < 0.05, **p < 0.01, Mann–Whitney test. F–G Kaplan–Meier plots of overall survival for PDAC patients based on high (top 33%) and low mRNA expression (bottom 33%) of S100A4 and S100A14 obtained from the TCGA-PAAD dataset. Plots and p-value were determined using the OncoLnc web tool [64].