Hydrogel-embedded precision-cut lung slices support ex vivo culture of in vivo -induced premalignant lung lesions.

Lung cancer is the leading cause of global cancer death and prevention strategies are key to reducing mortality. Medical prevention may have a larger impact than treatment on mortality by targeting high-risk populations and reducing their lung cancer risk. Premalignant lesions (PMLs) that can be intercepted by prevention agents are difficult to study in humans but easily accessible in murine preclinical carcinogenesis studies. Precision-cut lung slices (PCLS) are underutilized as an ex vivo model for lung cancer studies due to limited culture time. Embedding PCLS within bioengineered hydrogels extends PCLS viability and functionality for up to six weeks. Here, we embedded PCLS generated from urethane-induced murine PMLs in cell-degradable and non-degradable hydrogels to study viability and activity of the tissues over six weeks. PMLs in hydrogel-embedded PCLS maintained viability, gene expression, and proliferation. Treatment of hydrogel-embedded PCLS containing urethane-induced PMLs with iloprost, a known lung cancer prevention agent, recapitulated in vivo gene expression and activity. These studies also showed that iloprost reduced proliferation and PML size in hydrogel-embedded PCLS, with some differences based on hydrogel formulation and suggested that hydrogel-embedded PCLS models may support long-term culture of in vivo generated PMLs to improve preclinical studies of lung cancer and prevention agents.

Targeting Tryptophan Catabolism in Ovarian Cancer to Attenuate Macrophage Infiltration and PD-L1 Expression.

High-grade serous carcinoma (HGSC) of the fallopian tube, ovary, and peritoneum is the most common type of ovarian cancer and is predicted to be immunogenic because the presence of tumor-infiltrating lymphocytes conveys a better prognosis. However, the efficacy of immunotherapies has been limited because of the immune-suppressed tumor microenvironment (TME). Tumor metabolism and immune-suppressive metabolites directly affect immune cell function through the depletion of nutrients and activation of immune-suppressive transcriptional programs. Tryptophan (TRP) catabolism is a contributor to HGSC disease progression. Two structurally distinct rate-limiting TRP catabolizing enzymes, indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase 2 (TDO2), evolved separately to catabolize TRP. IDO1/TDO2 are aberrantly expressed in carcinomas and metabolize TRP into the immune-suppressive metabolite kynurenine (KYN), which can engage the aryl hydrocarbon receptor to drive immunosuppressive transcriptional programs. To date, IDO inhibitors tested in clinical trials have had limited efficacy, but those inhibitors did not target TDO2, and we find that HGSC cell lines and clinical outcomes are more dependent on TDO2 than IDO1. To identify inflammatory HGSC cancers with poor prognosis, we stratified patient ascites samples by IL6 status, which correlates with poor prognosis. Metabolomics revealed that IL6-high patient samples had enriched KYN. TDO2 knockdown significantly inhibited HGSC growth and TRP catabolism. The orally available dual IDO1/TDO2 inhibitor, AT-0174, significantly inhibited tumor progression, reduced tumor-associated macrophages, and reduced expression of immune-suppressive proteins on immune and tumor cells. These studies demonstrate the importance of TDO2 and the therapeutic potential of AT-0174 to overcome an immune-suppressed TME. SIGNIFICANCE: Developing strategies to improve response to chemotherapy is essential to extending disease-free intervals for patients with HGSC of the fallopian tube, ovary, and peritoneum. In this article, we demonstrate that targeting TRP catabolism, particularly with dual inhibition of TDO2 and IDO1, attenuates the immune-suppressive microenvironment and, when combined with chemotherapy, extends survival compared with chemotherapy alone.

Hydrogel-Embedded Precision-Cut Lung Slices Model Lung Cancer Premalignancy Ex Vivo.

Lung cancer is the leading global cause of cancer-related deaths. Although smoking cessation is the best prevention, 50% of lung cancer diagnoses occur in people who have quit smoking. Research into treatment options for high-risk patients is constrained to rodent models, which are time-consuming, expensive, and require large cohorts. Embedding precision-cut lung slices (PCLS) within an engineered hydrogel and exposing this tissue to vinyl carbamate, a carcinogen from cigarette smoke, creates an in vitro model of lung cancer premalignancy. Hydrogel formulations are selected to promote early lung cancer cellular phenotypes and extend PCLS viability to six weeks. Hydrogel-embedded PCLS are exposed to vinyl carbamate, which induces adenocarcinoma in mice. Analysis of proliferation, gene expression, histology, tissue stiffness, and cellular content after six weeks reveals that vinyl carbamate induces premalignant lesions with a mixed adenoma/squamous phenotype. Putative chemoprevention agents diffuse through the hydrogel and induce tissue-level changes. The design parameters selected using murine tissue are validated with hydrogel-embedded human PCLS and results show increased proliferation and premalignant lesion gene expression patterns. This tissue-engineered model of human lung cancer premalignancy is the foundation for more sophisticated ex vivo models that enable the study of carcinogenesis and chemoprevention strategies.

Tissue-engineered models of lung cancer premalignancy.

Lung cancer is the leading global cause of cancer-related deaths. Although smoking cessation is the best preventive action, nearly 50% of all lung cancer diagnoses occur in people who have already quit smoking. Research into treatment options for these high-risk patients has been constrained to rodent models of chemical carcinogenesis, which are time-consuming, expensive, and require large numbers of animals. Here we show that embedding precision-cut lung slices within an engineered hydrogel and exposing this tissue to a carcinogen from cigarette smoke creates an in vitro model of lung cancer premalignancy. Hydrogel formulations were selected to promote early lung cancer cellular phenotypes and extend PCLS viability up to six weeks. In this study, hydrogel-embedded lung slices were exposed to the cigarette smoke derived carcinogen vinyl carbamate, which induces adenocarcinoma in mice. At six weeks, analysis of proliferation, gene expression, histology, tissue stiffness, and cellular content revealed that vinyl carbamate induced the formation of premalignant lesions with a mixed adenoma/squamous phenotype. Two putative chemoprevention agents were able to freely diffuse through the hydrogel and induce tissue-level changes. The design parameters selected using murine tissue were validated with hydrogel-embedded human PCLS and results showed increased proliferation and premalignant lesion gene expression patterns. This tissue-engineered model of human lung cancer premalignancy is the starting point for more sophisticated ex vivo models and a foundation for the study of carcinogenesis and chemoprevention strategies.

Precision Cut Lung Slices as a Preclinical Model for Non-Small Cell Lung Cancer Chemoprevention.

Lung cancer chemoprevention is critical to addressing cancer burden in high-risk populations. Chemoprevention clinical trials rely on data from preclinical models; however, in vivo studies have high financial, technical, and staffing requirements. Precision cut lung slices (PCLS) provide an ex vivo model that maintains the structure and function of native tissues. This model can be used for mechanistic investigations and drug screenings and reduces the number of animals and time required to test hypotheses compared with in vivo studies. We tested the use of PCLS for chemoprevention studies, demonstrating recapitulation of in vivo models. Treatment of PCLS with the PPARγ agonizing chemoprevention agent iloprost produced similar effects on gene expression and downstream signaling as in vivo models. This occurred in both wild-type tissue and Frizzled 9 knockout tissue, a transmembrane receptor required for iloprost's preventive activity. We explored new areas of iloprost mechanisms by measuring immune and inflammation markers in PCLS tissue and media, and immune cell presence with immunofluorescence. To demonstrate the potential for drug screening, we treated PCLS with additional lung cancer chemoprevention agents and confirmed activity markers in culture. PCLS offers an intermediate step for chemoprevention research between in vitro and in vivo models that can facilitate drug screening prior to in vivo studies and support mechanistic studies with more relevant tissue environments and functions than in vitro models. PREVENTION RELEVANCE: PCLS could be a new model for premalignancy and chemoprevention research, and this work evaluates the model with tissue from prevention-relevant genetic and carcinogen exposed in vivo mouse models, in addition to evaluating chemoprevention agents.

Loss of Frizzled 9 in Lung Cells Alters Epithelial Phenotype and Promotes Premalignant Lesion Development.

The transmembrane receptor Frizzled 9 (FZD9) is important for fetal neurologic and bone development through both canonical and non-canonical WNT/FZD signaling. In the adult lung, however, Fzd9 helps to maintain a normal epithelium by signaling through peroxisome proliferator activated receptor γ (PPARγ). The effect of FZD9 loss on normal lung epithelial cells and regulators of its expression in the lung are unknown. We knocked down FZD9 in human bronchial epithelial cell (HBEC) lines and found that downstream EMT targets and PPARγ activity are altered. We used a FZD9-/- mouse in the urethane lung adenocarcinoma model and found FZD9-/- adenomas had more proliferation, increased EMT signaling, decreased activation of PPARγ, increased expression of lung cancer associated genes, increased transformed growth, and increased potential for invasive behavior. We identified PPARγ as a transcriptional regulator of FZD9. We also demonstrated that extended cigarette smoke exposure in HBEC leads to decreased FZD9 expression, decreased activation of PPARγ, and increased transformed growth, and found that higher exposure to cigarette smoke in human lungs leads to decreased FZD9 expression. These results provide evidence for the role of FZD9 in lung epithelial maintenance and in smoking related malignant transformation. We identified the first transcriptional regulator of FZD9 in the lung and found FZD9 negative lesions are more dangerous. Loss of FZD9 creates a permissive environment for development of premalignant lung lesions, making it a potential target for intervention.

PPARgamma agonism inhibits progression of premalignant lesions in a murine lung squamous cell carcinoma model.

The N-nitroso-trischloroethylurea (NTCU)-induced mouse model of squamous lung carcinoma recapitulates human disease from premalignant dysplasia through invasive tumors, making it suitable for preclinical chemoprevention drug testing. Pioglitazone is a peroxisome proliferator-activated receptor γ (PPARγ) agonist shown to prevent lung tumors in preclinical models. We investigated pioglitazone's effect on lesion development and markers of potential preventive mechanisms in the NTCU model. Female FVB/N mice were exposed to vehicle, NTCU or NTCU + oral pioglitazone for 32 weeks. NTCU induces the appearance of basal cells in murine airways while decreasing/changing their epithelial cell makeup, resulting in development of bronchial dysplasia. H&E and keratin 5 (KRT5) staining were used to detect and grade squamous lesions in formalin fixed lungs. mRNA expression of epithelial to mesenchymal transition (EMT) markers and basal cell markers were measured by qPCR. Dysplasia persistence markers desmoglein 3 and polo like kinase 1 were measured by immunohistochemistry. Basal cell markers KRT14 and p63, club cell specific protein and ciliated cell marker acetylated tubulin were measured by immunofluorescence. Pioglitazone treatment significantly reduced squamous lesions and the presence of airway basal cells, along with increasing normal epithelial cells in the airways of NTCU-exposed mice. Pioglitazone also significantly influenced EMT gene expression to promote a more epithelial, and less mesenchymal, phenotype. Pioglitazone reduced the presence of squamous dysplasia and maintained normal airway cell composition. This work increases the knowledge of mechanistic pathways in PPARγ agonism for lung cancer interception and provides a basis for further investigation to advance this chemoprevention strategy.

Iloprost requires the Frizzled-9 receptor to prevent lung cancer.

Prevention of premalignant lesion progression is a promising approach to reducing lung cancer burden in high-risk populations. Substantial preclinical and clinical evidence has demonstrated efficacy of the prostacyclin analogue iloprost for lung cancer chemoprevention. Iloprost activates peroxisome proliferator-activated receptor gamma (PPARG) to initiate chemopreventive signaling and in vitro, which requires the transmembrane receptor Frizzled9 (FZD9). We hypothesized a Fzd 9 -/- mouse would not be protected by iloprost in a lung cancer model. Fzd 9 -/- mice were treated with inhaled iloprost in a urethane model of lung adenoma. We found that Fzd 9 -/- mice treated with iloprost were not protected from adenoma development compared to wild-type mice nor did they demonstrate increased activation of iloprost signaling pathways. Our results established that iloprost requires FZD9 in vivo for lung cancer chemoprevention. This work represents a critical advancement in defining iloprost's chemopreventive mechanisms and identifies a potential response marker for future clinical trials.

Non-Coding RNA and Frizzled Receptors in Cancer.

Frizzled receptors have been long recognized for their role in Wnt/ß-catenin signaling, a pathway known for its tumorigenic effects. More recent studies of frizzled receptors include efforts to understand non-coding RNA (ncRNA) regulation of these receptors in cancer. It has become increasingly clear that ncRNA molecules are important for regulating the expression of both oncogenic and tumor-suppressive proteins. The three most commonly described ncRNA molecules are microRNAs (miRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs). Here, we review ncRNA molecules that directly or indirectly affect frizzled protein expression and downstream signaling. Exploring these interactions highlights the potential of incorporating ncRNA molecules into cancer prevention and therapy strategies that target frizzled receptors. Previous investigations of frizzled receptors and ncRNA have established strong promise for a role in cancer progression, but additional studies are needed to provide the substantial pre-clinical evidence required to translate findings to clinical applications.