Cristian Coarfa

Supplementary Figures and Tables

Primary graft dysfunction (PGD) is a major cause of early morbidity and mortality after lung transplantation. During blood-based ex vivo lung perfusion (EVLP), hemolysis releases plasma-free hemoglobin (pfHb) and other erythrocyte-derived products that may promote endothelial injury and inflammation. We investigated whether EVLP-associated hemolysis is associated with cytokine release, endothelial dysfunction, and grade 3 PGD (PGD3). In this prospective, single-center observational study, end-EVLP perfusate from 57 double-lung transplant recipients supported with the Organ Care System Lung was analyzed for pfHb, 26 cytokines, blood counts, and associations with PGD3 within 72 h after transplantation. A mock EVLP blood-loop model was used to evaluate the effects of red blood cell storage duration and circuit run time on hemolysis. Generated hemolytic perfusates were applied to human lung microvascular endothelial cells to assess barrier injury and inflammatory activation. Among the 57 recipients, 32 (56.1%) developed PGD3 at any time point within 72 h after transplantation. Higher end-EVLP pfHb, interleukin-6, and interleukin-8 levels were associated with PGD3, and pfHb correlated positively with interleukin-6 and interleukin-8. Hemolysis increased with longer blood storage and EVLP duration, with storage duration emerging as the dominant contributor in the blood-loop model. Hemolytic perfusate disrupted endothelial junctions, increased permeability, and induced interleukin-6 and interleukin-8 release. EVLP-associated hemolysis is linked to endothelial barrier injury, inflammatory activation, and PGD3 after lung transplantation. Reducing hemolysis through improved blood-product selection and EVLP circuit management may represent a modifiable strategy to improve posttransplant outcomes.

Enteroaggregative Escherichia coli (EAEC) is a leading cause of diarrhea worldwide. Interestingly, EAEC disease outcomes vary dramatically among infected individuals, ranging from asymptomatic colonization to acute and chronic diarrhea. While previous research has focused on EAEC virulence factors, emerging evidence suggests that host genetic and cellular background may be equally important in determining disease susceptibility. To understand host-driven disease heterogeneity, we performed single-cell RNA sequencing on human colon organoids (HCOs) derived from three healthy adult donors following EAEC 042 infection. Surprisingly, despite comparable EAEC adherence across donors, we observed striking donor-specific responses to infection at multiple levels. A total of seven distinct colonic epithelial cell clusters were identified, with striking donor-specific baseline compositions. Following infection, the HCO donors exhibited three distinct response phenotypes evident in both cell type remodeling and gene regulation: hyperresponsive, intermediate, and non-responsive. We further observed extensive donor-specific cell-cell communication networks at baseline and after EAEC infection. This study suggests that the heterogeneity in host epithelial response to EAEC infection extends to the single-cell level. While the colonic regions from which the HCOs were derived represent a potential confounding factor, the magnitude of organoid-to-organoid variation exceeded previously reported regional differences, suggesting intrinsic host factors as a crucial part of response heterogeneity. These findings establish a paradigm for understanding infectious disease susceptibility through the human organoid model system. IMPORTANCE: Understanding why similar pathogen exposures produce vastly different clinical outcomes is critical for combating infectious diseases. By conducting single-cell RNA sequencing on human colon organoids derived from three different donors, we show that each donor possesses unique intrinsic cellular compositions and distinct transcriptomic responses to enteroaggregative Escherichia coli (EAEC) infection. These findings challenge the traditional pathogen-focused paradigm and highlight the critical role of host factors in infectious disease outcomes. This study also validates human organoids as a powerful platform for studying host heterogeneity, which may help predict disease susceptibility and optimize treatment for diverse patient populations in the future.

Purpose: SMARCB1-deficient renal medullary carcinoma (RMC) is an ultrarare, lethal malignancy refractory to therapies approved for other renal cell carcinomas, and its rarity makes randomized trials logistically unfeasible. We investigated whether wild-type (WT) epidermal growth factor receptor (EGFR) is a therapeutically actionable dependency in RMC and translated this insight into a deployable clinical regimen. Experimental Design: EGFR expression was assessed by Clinical Laboratory Improvement Amendments–certified immunohistochemistry in RMC tumors and by integrated RNA and chromatin immunoprecipitation sequencing of primary tumors and adjacent kidney. Panitumumab was compared with erlotinib in patient- and cell line–derived xenografts, and its mechanism defined by immunoblotting, confocal co-localization, cell-cycle and apoptosis assays, and natural killer (NK) cell coculture. Panitumumab-based therapy was then evaluated prospectively in 26 patients with RMC across 10 centers on two continents. Results: RMC showed uniformly high membranous EGFR (median H-score 300), with enhancer and promoter reprogramming at the EGFR locus and a ligand switch from EGF to epiregulin and amphiregulin. EGFR expression was uncoupled from SMARCB1 status in vitro. Panitumumab outperformed erlotinib in RMC xenografts, suppressed AKT and ERK1/2 signaling, and routed EGFR to LAMP1-positive lysosomes in vitro; its effect was predominantly cytostatic, with minimal NK cell–mediated cytotoxicity. Clinically, panitumumab-based therapy achieved a 53.9% objective response rate, including 15.4% complete responses, with median progression-free and overall survival rates of 5.8 and 9.5 months, respectively. Conclusions: WT EGFR is a foundational dependency in RMC that is effectively targeted by panitumumab-based therapy. These findings, derived from a nonrandomized registry, provide a biological and clinical rationale for further prospective validation.

Rationale: Preclinical familial pulmonary fibrosis (FPF) represents an early stage of fibrotic lung disease, yet the compartment- and cell-specific molecular programs preceding fibrosis remain poorly understood. Objective: To define spatially organized molecular signatures associated with preclinical FPF and identify tissue-informed circulating biomarkers linked to early fibrotic remodeling. Methods: We performed integrated multi-omic profiling of histologically preserved and remodeled lung regions from subjects with preclinical FPF, Idiopathic Pulmonary Fibrosis (IPF), and controls using spatial transcriptomics, single-nucleus RNA sequencing (snRNAseq), and blood proteomics. Differential expression and pathway enrichment analyses were performed across spatial compartments and epithelial cell states. Results: Histologically preserved lung regions in preclinical FPF demonstrated transcriptional abnormalities including stress-response, ciliary, and extracellular matrix-associated programs despite minimal architectural distortion. Spatial analyses identified alterations in alveolar niche molecular programs accompanied by increasing profibrotic signaling across preserved and tissue remodeled lung compartments. Compared with advanced IPF, preclinical FPF retained epithelial repair and surfactant-associated signatures. Integration with snRNAseq demonstrated enrichment of alveolar and airway epithelial cell dysregulated states associated with transitional phenotypes previously implicated in IPF. Compartment- and epithelial-associated transcriptional signatures identified in lung tissue were partially represented in the peripheral blood. Conclusion: Preclinical FPF is characterized by compartment- and cell-specific molecular programs that precede established fibrosis. We identified distinct alveolar, airway, and vascular molecular signatures and epithelial remodeling states represented in the peripheral blood. These findings provide an initial framework for molecular classification of early stages of pulmonary fibrosis and support future studies evaluating minimally invasive approaches for disease stratification and precision therapeutics. At a Glance Commentary: Scientific Knowledge on the Subject: The molecular events preceding a diagnosis of pulmonary fibrosis remain poorly understood. Most mechanistic studies in Idiopathic Pulmonary Fibrosis (IPF) have relied on end-stage explanted lungs, limiting insight into the compartment- and cell-specific molecular programs associated with early stages of pulmonary fibrosis. What this study adds to the field: Using integrated spatial transcriptomics, single-cell sequencing, and peripheral blood proteomic profiling, we demonstrate that preclinical familial pulmonary fibrosis (FPF) is characterized by compartment- and cell-specific molecular programs that precede clinically detectable fibrosis. Spatial analyses identified distinct alveolar, airway, and vascular molecular signatures, while single cell analysis confirmed the presence of epithelial dysregulated states. These signatures are partially represented in the peripheral blood. Our findings provide an initial framework for biologically informed classification of early stages of pulmonary fibrosis and future minimally invasive approaches for disease stratification.

Idiopathic pulmonary fibrosis (IPF) is a devastating chronic lung disorder with limited treatment options. Macropinocytosis is one of the key cellular processes involved in nutrient consumption from the extracellular environment under stress conditions. Here, we studied the role of macropinocytosis in experimental pulmonary fibrosis models. We found that macropinocytosis is increased in human lung fibroblasts (HLFs) derived from patients with IPF. The inhibition of macropinocytosis with 5-(n-ethyl-n-isopropyl)-amiloride (EIPA) inhibited profibrotic responses in IPF-derived and TGF-β1–stimulated HLFs and reduced pulmonary fibrosis in bleomycin-injured (Bleo-injured) mice. EIPA exerted its antifibrotic effects by regulating amino acid uptake, mammalian target of rapamycin complex 1 (mTORC1) activation and mesenchyme homeobox1 ( MEOX1 ) expression in activated HLFs. Fittingly, genetic inhibition of macropinocytosis also ameliorated lung fibroblast activation and pulmonary fibrosis in mice. Using IPF-derived precision cut lung slices (PCLSs), we observed robust repression of profibrotic gene expression programs in EIPA-treated PCLSs across different fibroblast subpopulations. Finally, we found that imipramine (Imi), a tricyclic antidepressant approved by the FDA, effectively inhibited macropinocytosis and ameliorated profibrotic responses in lung fibroblasts, Bleo-injured mice, and IPF-derived PCLSs. Taken together, our results suggest that macropinocytosis inhibition can be considered as a potential therapeutic strategy to treat pulmonary fibrosis.

GSEA results in ESR1m models of diferentially expressed siATR and siChk1 RSR defect signature GSEA normalized enrichment scores and significance values in ESR1m models. Gene signatures were developed utilizing differentially expressed genes from siATR and siChk1 knockdown models from McGrail et al as described in Materials and Methods.

Representative gating for flow cytometry analyzing percent BrdU uptake relative to PI in MCF-7 cells analyzed via flow cytometry All treatments were performed in charcoal stripped media after 48 hours of estrogen deprivation in charcoal stripped media followed by 36 hours of Lovastatin (10 µM) to synchronize cell cycles. Gating for BrdU is shown for both WT and Y537S cells untreated or treated with 0.5 mM Hydroxyurea (HU).

Proximity ligation assay of ER-PARP1 co-localization in Y537C cells Representative images of proximity ligation assay ER-PARP1 co-localization puncta in Y537C cells treated for 48 hours with Fulv (100 nM), Olap (5 µM), or Fulv+Olap compared to untreated control (Ctrl) following 5-day estrogen deprivation in charcoal stripped media.