Fig 2: ACE2 and Other Entry Factors Are Expressed in the Choroid Plexus(A) Dot plot showing average expression and percentage of cells expressing SARS-CoV-2 entry factors ACE2 and TMRPSS2 in the five main clusters identified by scRNA-seq of days 27–53 ChP and day 55 telencephalic organoids (Pellegrini et al., 2020).(B) Allen Brain Atlas expression data of ACE2 (probe names: ACE2 A_23 and ACE2 CUST_16267) in different adult human brain regions, with highest expression in the ChP. Regions with an average Z score across the two probes (black line) of greater than 1 are shown.(C) Uniform Manifold Approximation and Projection (UMAP) plot showing subclustering of all ChP cell types identified by scRNA-seq. Imm ChP, immature ChP; lipid prod ChP, lipoprotein-producing ChP; Mat ChP, maturing ChP; NC, neural crest.(D) Dot plot showing average expression and percentage of cells for key marker genes present in the subclusters identified by scRNA-seq. Lipoprotein-producing ChPs express SARS-CoV-2 entry genes ACE2, TMPRSS2, and TMPRSS4.(E) Feature plot showing all cells expressing any level of ACE2.(F) UMAP plot of mouse embryonic and adult ChP cells (left) and feature plot for ApoJ (clusterin).(G) Immunoblot for ACE2, APOJ, and the loading control β-actin of days 33–53 ChP organoid protein lysates.(H) Exponentially Modified Protein Abundance Index (emPAI) values for lipid-related proteins in a previously published dataset (Pellegrini et al., 2020) of organoid CSF samples until day 146.(I) Representative confocal images of day 40 ChP tissue immunostained for the ChP epithelial marker HTR2C (serotonin receptor 2C) in magenta and ACE2 in green. Nuclei in blue are stained with DAPI. Scale bar: 50 μm.(J) gProfileR (Reimand et al., 2011) analysis of genes co-expressed with ACE2 showing significant enrichment (p < 0.05) for GO categories cellular component (GO:CC), molecular function (GO:MF), and biological process (GO:BP).

Fig 3: NRF2 and the OSR are associated with the iCAF signature(A and B) Pearson correlation coefficients between stress and cell type scores calculated from the scRNA-seq data described in Figure 4: (A) iCAFs, (B) musclePer. Results are shown as mean ± SD. The p values were calculated using one-sample t test (μ = 0).(C and D) FFPE TMAs of breast (n = 114), pancreas (n = 125), ovary (n = 93), and prostate (n = 104) cancer patients were stained by MxIF and analyzed using QuPath software. CD45− CK− cells were defined as non-immune stromal cells. Representative images are shown (C). Scale bars, 40 μM. For each patient, Pearson correlation coefficients between the staining intensities of NRF2 and the different CAF markers CLU, ɑSMA, and MHC class II were calculated (D).Results are shown as mean ± SD. The p values were calculated using one-way ANOVA, followed by Tukey’s multiple comparisons test.

Fig 4: NRF2 and the OSR contribute to the transition of normal fibroblasts to iCAFs(A–E) Patient data from the TCGA breast cancer dataset were analyzed for cellular composition using the CIBERSORTx63 algorithm, and the results were ordered by the stress score. n = 1,187.(F–O) Trajectory analyses.(F and K) UMAP plots of scRNA-seq data of breast (E) or pancreas (J) fibroblasts with data from normal samples, re-analyzed from publicly available datasets.46,54(G and L) Clusters were annotated based on the stromal gene signatures we described in Figure S3.(H and M) Trajectory analyses of breast and pancreas tumors and normal samples using the Monocle3 R toolkit.(I and N) Projections of the OSR score we defined previously.(J and O) Patient-level quantification of OSR scores. The p values were calculated using one-way ANOVA, followed by Tukey’s multiple comparisons test.(P and Q) Kaplan-Meier analysis of overall survival for low-grade breast cancer patients from the METABRIC cohort.64 Patients were stratified based on their OSR signature and iCAF/myCAF (P) or musclePer/matriPer (Q) ratio, calculated by CIBERSORTx (median was used as cutoff). The p values were calculated from a log rank test, and paired comparisons were calculated using the Survdiff function in R with false discovery rate (FDR) correction.(R) Heatmap of the differentially expressed oxidative stress-related genes from bulk RNA-seq between cell culture models of iCAFs, myCAFs, and quiescent PSCs, re-analyzed from Öhlund et al.65 Differentially expressed genes were filtered by a logFC (log fold change) threshold of 1.5 and adjusted p value of 0.05.(S) Immortalized PSCs were seeded in Matrigel for 3 days with either DMEM or KPC organoid conditioned medium (orgCM), and the OSR genes Ho1 and Sod3 were measured using qPCR.(T) Immortalized PSCs were seeded in 2D culture and depleted of Nrf2 using small interfering RNA (siRNA). Cells were then seeded in Matrigel for 3 days with orgCM, and known iCAF markers in this system, Clu and C3, were measured using qPCR. Results are shown as mean ± SD. The p values were calculated using two samples t test. Results are from 2 to 4 independent experiments.

Fig 5: CLU alleviated inflammation and fibrosis in cardiomyocytes. The relative mRNA expression of (A) IL-6, (B) IL-1β, (C) Col5a1, and (D) Col5a3 in AC16 cells under 3-HIT stimulation with or without CLU overexpression (n = 3). (E) Representative confocal immunofluorescence images showing Col1a2 expression in AC16 cells. The relative mRNA expression of (F) NLRP3, (G) GSDMD, (H) Caspase-1 in AC16 cells treated with “3-HIT” and “3-HIT” plus CLU overexpression (n = 3). (I) Confocal fluorescence images of intracellular ROS detected using DCFH-DA. (J) Quantification of ROS fluorescence intensity (n = 3).