Protocol for generation of lung adenocarcinoma organoids from clinical samples.
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ABSTRACT:
SUBMITTER: Li Z
PROVIDER: S-EPMC7779824 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:PurposePatient-derived organoids (PDO) of lung cancer has been recently introduced, reflecting the genomic landscape of lung cancer. However, clinical relevance of advanced lung adenocarcinoma organoids remains unknown. Here, we examined the ability of PDOs to predict clinical responses to targeted therapies in individual patients and to identify effective anticancer therapies for novel molecular targets.Experimental designEighty-four organoids were established from patients with advanced lung adenocarcinoma. Formalin-fixed, paraffin-embedded tumor specimens from corresponding patients were analyzed by whole-exome sequencing (n = 12). Organoids were analyzed by whole-exome sequencing (n = 61) and RNA sequencing (n = 55). Responses to mono or combination targeted therapies were examined in organoids and organoid-derived xenografts.ResultsPDOs largely retained somatic alterations including driver mutations of matching patient tumors. PDOs were able to recapitulate progression-free survival and objective responses of patients with non-small cell lung cancer receiving clinically approved tyrosine kinase inhibitors. PDOs recapitulated activity of therapeutic strategies under clinical investigation. YUO-071 harboring an EGFR exon 19 deletion and a BRAF G464A mutation and the matching patient responded to dabrafenib/trametinib combination therapy. YUO-004 and YUO-050 harboring an EGFR L747P mutation was sensitive to afatinib, consistent with the response in the matching patient of YUO-050. Furthermore, we utilized organoids to identify effective therapies for novel molecular targets by demonstrating the efficacy of poziotinib against ERBB2 exon 20 insertions and pralsetinib against RET fusions.ConclusionsWe demonstrated translational relevance of PDOs in advanced lung adenocarcinoma. PDOs are an important diagnostic tool, which can assist clinical decision making and accelerate development of therapeutic strategies.
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Project description:The lung is comprised of more than 40 distinct cell types that support a complex 3-dimensional (3D) architecture that is required for efficient lung function. Loss of this proper architecture can accommodate and promote lung disease, highlighting researchers' growing need to analyze lung structures in detail. Additionally, in vivo cellular and molecular response to chemical and physical signals, along with the recapitulation of gene-expression patterns, can be lost during the transition from complex 3D tissues to 2D cell culture systems. Therefore, technologies that allow for the investigation of lung function under normal and disease states utilizing the entirety of the lung architecture are required to generate a complete understanding of these processes. Airway cell-derived organoids that can recapitulate lung structure and function ex vivo while being amenable to experimental manipulation, have provided a new and exciting model system to investigate lung biology. In this perspective, we discuss emerging technologies for culturing lung-derived organoids, techniques to visualize organoids using high-resolution microscopy and the resulting information extracted from organoids supporting research focused on lung function and diseases.
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Project description:Next-generation sequencing (NGS) has recently been rapidly adopted in the molecular diagnosis of cancer, but it still faces some obstacles. In this study, 665 lung adenocarcinoma samples (558 TKI-naive and 107 TKI-relapsed samples) were interrogated using NGS, and the challenges and possible solutions of subjecting appropriate tissue samples to NGS testing were explored. The results showed that lower frequencies of HER2/BRAF/PIK3CA and acquired EGFR T790M mutations were observed in biopsy samples with <20% tumor cellularity than in those with ?20%, but there were no significant differences in the frequencies of EGFR or KRAS mutations. Moreover, tumor heterogeneity was assessed by heterogeneity score (HS), which was calculated through multiplying by 2 the mutant allele frequency (MAF) of tumor cells. In TKI-naive samples, intratumor heterogeneity could occur in EGFR, KRAS, HER2, BRAF, and PIK3CA mutant tumors, but the degree was variable. Higher EGFR, but lower BRAF and PIK3CA HS values were observed compared with KRAS HS. In TKI-relapsed samples, analysis of concomitant sensitizing EGFR and T790M MAFs showed that intratumor heterogeneity was common in acquired EGFR T790M mutant tumors. The mutational status between primary and metastatic tumors was usually concordant, but KRAS, HER2, and PIK3CA HS were significantly higher in metastatic tumors than in primary tumors. Additionally, the discordance rate of mutational status in multifocal lung adenocarcinomas diagnosed as equivocal or multiple primary tumors was high. Together, our findings demonstrate that a comprehensive quality assessment is necessary during tissue process to mitigate the challenges of poor tumor cellularity, tumor heterogeneity, and multifocal clonally independent tumors.
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Project description:The lung epithelium is derived from the endodermal germ layer, which undergoes a complex series of endoderm-mesoderm-mediated signaling events to generate the final arborized network of conducting airways (bronchi, bronchioles) and gas-exchanging units (alveoli). These stages include endoderm induction, anterior-posterior and dorsal-ventral patterning, lung specification, lung budding, branching morphogenesis, and, finally, maturation. Here we describe a protocol that recapitulates several of these milestones in order to differentiate human pluripotent stem cells (hPSCs) into ventral-anterior foregut spheroids and further into two distinct types of organoids: human lung organoids and bud tip progenitor organoids. The resulting human lung organoids possess cell types and structures that resemble the bronchi/bronchioles of the developing human airway surrounded by lung mesenchyme and cells expressing alveolar-cell markers. The bud tip progenitor organoids possess a population of highly proliferative multipotent cells with in vitro multilineage differentiation potential and in vivo engraftment potential. Human lung organoids can be generated from hPSCs in 50-85 d, and bud tip progenitor organoids can be generated in 22 d. The two hPSC-derived models presented here have been benchmarked with human fetal tissue and found to be representative of human fetal-like tissue. The bud tip progenitor organoids are thus ideal for exploring epithelial fate decisions, while the human lung organoids can be used to model epithelial-mesenchymal cross-talk during human lung development. In addition to their applications in developmental biology, human lung organoids and bud tip progenitor organoids may be implemented in regenerative medicine, tissue engineering, and pharmaceutical safety and efficacy testing.
| S-EPMC6531049 | biostudies-literature
Project description:Recent breakthroughs in 3-dimensional (3D) organoid cultures for many organ systems have led to new physiologically complex in vitro models to study human development and disease. Here, we report the step-wise differentiation of human pluripotent stem cells (hPSCs) (embryonic and induced) into lung organoids. By manipulating developmental signaling pathways hPSCs generate ventral-anterior foregut spheroids, which are then expanded into human lung organoids (HLOs). HLOs consist of epithelial and mesenchymal compartments of the lung, organized with structural features similar to the native lung. HLOs possess upper airway-like epithelium with basal cells and immature ciliated cells surrounded by smooth muscle and myofibroblasts as well as an alveolar-like domain with appropriate cell types. Using RNA-sequencing, we show that HLOs are remarkably similar to human fetal lung based on global transcriptional profiles, suggesting that HLOs are an excellent model to study human lung development, maturation and disease.
| S-EPMC4370217 | biostudies-literature
Project description:Lung cancer organoid (LCO) is a novel model of lung cancer that facilitates drug screening. However, the success rate of LCOs varies from 7% to 87%, and the culture medium compositions are markedly different. Airway organoid media can be used for LCO cultures, but this promotes the overgrowth of normal cell organoids especially in LCOs from intrapulmonary lesions. Several modified media are specifically utilized for promoting the cancer cell's growth. For culturing high-purity LCOs, cancer cells from metastatic lesions and malignant effusions are used. Recently, single-cell RNA sequencing has identified previously unknown cell populations in the lungs and lung cancer. This sequencing technology can be used to validate whether the LCO recapitulates the heterogeneity and functional hierarchy of the primary tumor. Several groups have attempted to culture LCOs with mesenchymal cells and immune cells to recapitulate the tumor microenvironment. Disease modeling using LCO provides novel insight into the pathophysiology of lung cancer and enables high-throughput screening for drug discovery and prognosis prediction. An LCO model would help to identify new concepts as a basis for lung cancer targeting by discovering innovative therapeutic targets.
| S-EPMC8616085 | biostudies-literature
Project description:Lung cancer is the most lethal malignancy in the world, and each year thousands of people die from this disease. Early detection has proven to increase the 5-year survival for this cancer in general, independent of the origination site in the lung. To address this challenge, we have used cell-based SELEX (Systematic Evolution of Ligands by Exponential Enrichment) to select a panel of aptamers capable of distinguishing lung adenocarcinoma cells from normal lung epithelial cells. These aptamers bind at physiological and formalin-fixed conditions and display affinity for their targets with apparent K(d')s in the nanomolar range. Our findings suggest that the selected aptamers have the potential to be used in clinical settings, as well as to improve classification of nonsurgical specimens, another current challenge in lung cancer.
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Project description:Identification of targetable molecular changes is essential for selecting appropriate treatment in patients with advanced lung adenocarcinoma. Methods: In this study, a Sanger sequencing plus Fluorescence In Situ Hybridization (FISH) sequential approach was compared with a Next-Generation Sequencing (NGS)-based approach for the detection of actionable genomic mutations in an experimental cohort (EC) of 117 patients with advanced lung adenocarcinoma. Its applicability was assessed in small biopsies and cytology specimens previously tested for epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK) mutational status, comparing the molecular changes identified and the impact on clinical outcomes. Subsequently, an NGS-based approach was applied and tested in an implementation cohort (IC) in clinical practice. Using Sanger and FISH, patients were classified as EGFR-mutated (n = 22, 18.8%), ALK-mutated (n = 9, 7.7%), and unclassifiable (UC) (n = 86, 73.5%). Retesting the EC with NGS led to the identification of at least one gene variant in 56 (47.9%) patients, totaling 68 variants among all samples. Still, in the EC, combining NGS plus FISH for ALK, patients were classified as 23 (19.7%) EGFR; 20 (17.1%) KRAS; five (4.3%) B-Raf proto-oncogene (BRAF); one (0.9%) Erb-B2 Receptor Tyrosine Kinase 2 (ERBB2); one (0.9%) STK11; one (0.9%) TP53, and nine (7.7%) ALK mutated. Only 57 (48.7%) remained genomically UC, reducing the UC rate by 24.8%. Fourteen (12.0%) patients presented synchronous alterations. Concordance between NGS and Sanger for EGFR status was very high (? = 0.972; 99.1%). In the IC, a combined DNA and RNA NGS panel was used in 123 patients. Genomic variants were found in 79 (64.2%). In addition, eight (6.3%) EML4-ALK, four (3.1%), KIF5B-RET, four (3.1%) CD74-ROS1, one (0.8%) TPM3-NTRK translocations and three (2.4%) exon 14 skipping MET Proto-Oncogene (MET) mutations were detected, and 36% were treatable alterations. Conclusions: This study supports the use of NGS as the first-line test for genomic profiling of patients with advanced lung adenocarcinoma.
| S-EPMC6770536 | biostudies-literature
Project description:AimsThe growing number of genomically targeted therapies has made genomic testing an important part of the care for patients with non-small cell lung cancer. However, limited tissue availability, cost and long turnaround times can create barriers to efficient genomic testing and subsequent treatment. Effective approaches to reduce these barriers are needed.Methods302 advanced lung adenocarcinomas from consecutive patients seen at University Hospitals Cleveland Medical Center (UHCMC) were tested inhouse using a hybrid DNA/RNA next-generation sequencing (NGS) panel. Sample testing was reflexed from pathology for all stage III or IV tumours. Genomic alterations were tiered according to their clinical relevance and reported with guideline-recommended therapies. Clinical implications of genomic testing results were assessed by manual chart review.ResultsWith a sample cohort consisting of 64% biopsies, 16% excisions/resections and 20% fine needle aspirations, the assay was reliable with a 95% success rate. The average turnaround time from receipt of unstained formalin-fixed paraffin embedded slides to reporting was 4.8±2.1 days, half of the recommended 10 days and similar to single-gene testing. Alterations with Food and Drug Administration-approved or the National Cancer Center Network guideline-recommended targeted therapies were found in 18% of cases. Within this group, 60% of patients went on genomically driven therapies.ConclusionsWe found our reflexed inhouse NGS assay to be reliable, cost-effective and efficient. Incorporation of reflex testing with our NGS assay led to an expansion of successful genomic profiling for all guideline-recommended alterations, and by including an expanded number of alterations within our panel we obtained clinically useful information outside the guidelines without changing cost or efficiency. This approach has enabled UHCMC clinicians to efficiently initiate genomically driven therapies for patients with lung adenocarcinoma.
| S-EPMC6900927 | biostudies-literature