Development of a small cell lung cancer organoid model to study cellular interactions and survival after chemotherapy.

Introduction: Small-cell-lung-cancer (SCLC) has the worst prognosis of all lung cancers because of a high incidence of relapse after therapy. While lung cancer is the second most common malignancy in the US, only about 10% of cases of lung cancer are SCLC, therefore, it is categorized as a rare and recalcitrant disease. Therapeutic discovery for SCLC has been challenging and the existing pre-clinical models often fail to recapitulate actual tumor pathophysiology. To address this, we developed a bioengineered 3-dimensional (3D) SCLC co-culture organoid model as a phenotypic tool to study SCLC tumor kinetics and SCLC-fibroblast interactions after chemotherapy. Method: We used functionalized alginate microbeads as a scaffold to mimic lung alveolar architecture and co-cultured SCLC cell lines with primary adult lung fibroblasts (ALF). We found that SCLCs in the model proliferated extensively, invaded the microbead scaffold and formed tumors within just 7 days. We compared the bioengineered tumors with patient tumors and found them to recapitulate the pathology and immunophenotyping of the patient tumors. When treated with standard chemotherapy drugs, etoposide and cisplatin, we observed that some of the cells survived the chemotherapy and reformed the tumor in the organoid model. Result and Discussion: Co-culture of the SCLC cells with ALFs revealed that the fibroblasts play a key role in inducing faster and more robust SCLC cell regrowth in the model. This is likely due to a paracrine effect, as conditioned media from the same fibroblasts could also support this accelerated regrowth. This model can be used to study cell-cell interactions and the response to chemotherapy in SCLC and is also scalable and amenable to high throughput phenotypic or targeted drug screening to find new therapeutics for SCLC.

Small cell lung cancer co-culture organoids provide insights into cancer cell survival after chemotherapy.

Small-cell-lung-cancer (SCLC) has the worst prognosis of all lung cancers because of a high incidence of relapse after therapy. We developed a bioengineered 3-dimensional (3D) SCLC co-culture organoid as a phenotypic tool to study SCLC tumor kinetics and SCLC-fibroblast interactions during relapse. We used functionalized alginate microbeads as a scaffold to mimic lung alveolar architecture and co-cultured SCLC cell lines with primary adult lung fibroblasts (ALF). We found that SCLCs in the model proliferated extensively, invaded the microbead scaffold and formed tumors within just 7 days. We compared the bioengineered tumors with patient tumors and found them to recapitulate the pathology and immunophenotyping of the patient tumors better than the PDX model developed from the same SCLC cell line. When treated with standard chemotherapy drugs, etoposide and cisplatin, the organoid recapitulated relapse after chemotherapy. Co-culture of the SCLC cells with ALFs revealed that the fibroblasts play a key role in inducing faster and more robust SCLC cell regrowth in the model. This was a paracrine effect as conditioned medium from the same fibroblasts was responsible for this accelerated cell regrowth. This model is also amenable to high throughput phenotypic or targeted drug screening to find new therapeutics for SCLC.

Targeting PEA3 transcription factors to mitigate small cell lung cancer progression.

Small cell lung cancer (SCLC) remains a lethal disease with a dismal overall survival rate of 6% despite promising responses to upfront combination chemotherapy. The key drivers of such rapid mortality include early metastatic dissemination in the natural course of the disease and the near guaranteed emergence of chemoresistant disease. Here, we found that we could model the regression and relapse seen in clinical SCLC in vitro. We utilized time-course resolved RNA-sequencing to globally profile transcriptome changes as SCLC cells responded to a combination of cisplatin and etoposide-the standard-of-care in SCLC. Comparisons across time points demonstrated a distinct transient transcriptional state resembling embryonic diapause. Differential gene expression analysis revealed that expression of the PEA3 transcription factors ETV4 and ETV5 were transiently upregulated in the surviving fraction of cells which we determined to be necessary for efficient clonogenic expansion following chemotherapy. The FGFR-PEA3 signaling axis guided the identification of a pan-FGFR inhibitor demonstrating in vitro and in vivo efficacy in delaying progression following combination chemotherapy, observed inhibition of phosphorylation of the FGFR adaptor FRS2 and corresponding downstream MAPK and PI3K-Akt signaling pathways. Taken together, these data nominate PEA3 transcription factors as key mediators of relapse progression in SCLC and identify a clinically actionable small molecule candidate for delaying relapse of SCLC.

Transcriptional analysis of cystic fibrosis airways at single-cell resolution reveals altered epithelial cell states and composition.

Cystic fibrosis (CF) is a lethal autosomal recessive disorder that afflicts more than 70,000 people. People with CF experience multi-organ dysfunction resulting from aberrant electrolyte transport across polarized epithelia due to mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CF-related lung disease is by far the most important determinant of morbidity and mortality. Here we report results from a multi-institute consortium in which single-cell transcriptomics were applied to define disease-related changes by comparing the proximal airway of CF donors (n = 19) undergoing transplantation for end-stage lung disease with that of previously healthy lung donors (n = 19). Disease-dependent differences observed include an overabundance of epithelial cells transitioning to specialized ciliated and secretory cell subsets coupled with an unexpected decrease in cycling basal cells. Our study yields a molecular atlas of the proximal airway epithelium that will provide insights for the development of new targeted therapies for CF airway disease.

Direct Exposure to SARS-CoV-2 and Cigarette Smoke Increases Infection Severity and Alters the Stem Cell-Derived Airway Repair Response.

Current smoking is associated with increased risk of severe COVID-19, but it is not clear how cigarette smoke (CS) exposure affects SARS-CoV-2 airway cell infection. We directly exposed air-liquid interface (ALI) cultures derived from primary human nonsmoker airway basal stem cells (ABSCs) to short term CS and then infected them with SARS-CoV-2. We found an increase in the number of infected airway cells after CS exposure with a lack of ABSC proliferation. Single-cell profiling of the cultures showed that the normal interferon response was reduced after CS exposure with infection. Treatment of CS-exposed ALI cultures with interferon ß-1 abrogated the viral infection, suggesting one potential mechanism for more severe viral infection. Our data show that acute CS exposure allows for more severe airway epithelial disease from SARS-CoV-2 by reducing the innate immune response and ABSC proliferation and has implications for disease spread and severity in people exposed to CS.

Direct exposure to SARS-CoV-2 and cigarette smoke increases infection severity and alters the stem cell-derived airway repair response.

Most demographic studies are now associating current smoking status with increased risk of severe COVID-19 and mortality from the disease but there remain many questions about how direct cigarette smoke exposure affects SARS-CoV-2 airway cell infection. We directly exposed mucociliary air-liquid interface (ALI) cultures derived from primary human nonsmoker airway basal stem cells (ABSCs) to short term cigarette smoke and infected them with live SARS-CoV-2. We found an increase in the number of infected airway cells after cigarette smoke exposure as well as an increased number of apoptotic cells. Cigarette smoke exposure alone caused airway injury that resulted in an increased number of ABSCs, which proliferate to repair the airway. But we found that acute SARS-CoV-2 infection or the combination of exposure to cigarette smoke and SARS-CoV-2 did not induce ABSC proliferation. We set out to examine the underlying mechanism governing the increased susceptibility of cigarette smoke exposed ALI to SARS-CoV-2 infection. Single cell profiling of the cultures showed that infected airway cells displayed a global reduction in gene expression across all airway cell types. Interestingly, interferon response genes were induced in SARS-CoV-2 infected airway epithelial cells in the ALI cultures but smoking exposure together with SARS-CoV-2 infection reduced the interferon response. Treatment of cigarette smoke-exposed ALI cultures with Interferon ß-1 abrogated the viral infection, suggesting that the lack of interferon response in the cigarette smoke-exposed ALI cultures allows for more severe viral infection and cell death. In summary, our data show that acute smoke exposure allows for more severe proximal airway epithelial disease from SARS-CoV-2 by reducing the mucosal innate immune response and ABSC proliferation and has implications for disease spread and severity in people exposed to cigarette smoke.

Distinct Spatiotemporally Dynamic Wnt-Secreting Niches Regulate Proximal Airway Regeneration and Aging.

Our understanding of dynamic interactions between airway basal stem cells (ABSCs) and their signaling niches in homeostasis, injury, and aging remains elusive. Using transgenic mice and pharmacologic studies, we found that Wnt/ß-catenin within ABSCs was essential for proliferation post-injury in vivo. ABSC-derived Wnt ligand production was dispensable for epithelial proliferation. Instead, the PDGFRα+ lineage in the intercartilaginous zone (ICZ) niche transiently secreted Wnt ligand necessary for ABSC proliferation. Strikingly, ABSC-derived Wnt ligand later drove early progenitor differentiation to ciliated cells. We discovered additional changes in aging, as glandular-like epithelial invaginations (GLEIs) derived from ABSCs emerged exclusively in the ICZ of aged mice and contributed to airway homeostasis and repair. Further, ABSC Wnt ligand secretion was necessary for GLEI formation, and constitutive activation of ß-catenin in young mice induced their formation in vivo. Collectively, these data underscore multiple spatiotemporally dynamic Wnt-secreting niches that regulate functionally distinct phases of airway regeneration and aging.

High-Throughput Drug Screening Identifies a Potent Wnt Inhibitor that Promotes Airway Basal Stem Cell Homeostasis.

Mechanisms underpinning airway epithelial homeostatic maintenance and ways to prevent its dysregulation remain elusive. Herein, we identify that ß-catenin phosphorylated at Y489 (p-ß-cateninY489) emerges during human squamous lung cancer progression. This led us to develop a model of airway basal stem cell (ABSC) hyperproliferation by driving Wnt/ß-catenin signaling, resulting in a morphology that resembles premalignant lesions and loss of ciliated cell differentiation. To identify small molecules that could reverse this process, we performed a high-throughput drug screen for inhibitors of Wnt/ß-catenin signaling. Our studies unveil Wnt inhibitor compound 1 (WIC1), which suppresses T-cell factor/lymphoid enhancer-binding factor (TCF/LEF) activity, reduces ABSC proliferation, induces ciliated cell differentiation, and decreases nuclear p-ß-cateninY489. Collectively, our work elucidates a dysregulated Wnt/p-ß-cateninY489 axis in lung premalignancy that can be modeled in vitro and identifies a Wnt/ß-catenin inhibitor that promotes airway homeostasis. WIC1 may therefore serve as a tool compound in regenerative medicine studies with implications for restoring normal airway homeostasis after injury.

Modeling Progressive Fibrosis with Pluripotent Stem Cells Identifies an Anti-fibrotic Small Molecule.

Progressive organ fibrosis accounts for one-third of all deaths worldwide, yet preclinical models that mimic the complex, progressive nature of the disease are lacking, and hence, there are no curative therapies. Progressive fibrosis across organs shares common cellular and molecular pathways involving chronic injury, inflammation, and aberrant repair resulting in deposition of extracellular matrix, organ remodeling, and ultimately organ failure. We describe the generation and characterization of an in vitro progressive fibrosis model that uses cell types derived from induced pluripotent stem cells. Our model produces endogenous activated transforming growth factor ß (TGF-ß) and contains activated fibroblastic aggregates that progressively increase in size and stiffness with activation of known fibrotic molecular and cellular changes. We used this model as a phenotypic drug discovery platform for modulators of fibrosis. We validated this platform by identifying a compound that promotes resolution of fibrosis in in vivo and ex vivo models of ocular and lung fibrosis.