Project title: A Human Pluripotent Stem Cell-based Model for Lung Carcinoid

Description

Chen is working to develop a novel human cell-based disease model for lung carcinoid, converting normal pulmonary neuroendocrine cells derived from human pluripotent stem cells (hPSCs) into NETs. If successful, the lung carcinoid model using human cells derived from hPSCs may be a valuable research tool for further studies of these forms of cancer.

What question will the researchers try to answer?

Because lung carcinoid is a rare form of cancer, it is difficult to obtain clinical tumor tissues to conduct in-depth research. In addition, few cell lines or disease models have been established for robust and reliable mechanistic studies. Chen’s team will establish an innovative tumor model of human cell origin that recapitulates the clinical features of lung carcinoids that will allow for more extensive studies of these rare cancers.

Why is this important?

Primary tissues, disease models, and cell line resources to study the biology of pulmonary carcinoid are currently scarce, and the existing cell models cannot fully recapitulate the disease features. As a result, no definitive molecular markers or genetic drivers have been identified and little is known regarding potential targets for therapy. New models and tools are urgently needed to understand the tumor’s biology in order to develop novel treatment approaches for these rare tumors.

What will researchers do?

Lung carcinoid is composed of cells with properties of pulmonary neuroendocrine cells (PNECs). Chen’s team established the first method to produce large numbers of PNECs, which are believed to be the precursors of pulmonary neuroendocrine tumors, through the differentiation of human pluripotent stem cells (hPSCs). By manipulating the genetic factors or signaling pathways recurrent in lung carcinoids, they will convert the hPSC-derived normal PNECs into cancerous cells in culture and in other disease models. The team will further define the similarities between the genetic and physiological features of the lung carcinoids from hPSCs and the ones in patient samples. Through these studies, they aim to create novel disease models as needed research resources for advancing the understanding of the biology of lung carcinoids and the development of new treatments.

How might this improve the treatment of NETs?

Dr. Chen and his team propose to use these novel approaches to pursue important questions, including whether PNECs serve as the cell of origin for pulmonary carcinoids and explore the high-frequency mutations that can initiate and promote pulmonary tumorigenesis. Moreover, their team will work to establish cancerous cell lines from the hPSC-derived pulmonary carcinoid tumor tissues. If successful, these studies will generate a foundation to identify cancer genes and pathways that may serve as potential therapeutic targets. They expect the models and cell lines created in this project to serve as an innovative research platform for testing new targeted agents and for mechanistic studies of these rare diseases.

What is the next step?

This study will determine the feasibility of modeling lung carcinoids with human cells derived from hPSCs. If successful, this methodology will pave a novel avenue for developing human cell-based models for neuroendocrine tumors in other organs. Moreover, the models and cell lines created in this project will be exploited for studying disease mechanisms and for high-throughput drug or genetic screening.

Outcomes:

Pulmonary carcinoids are rare neuroendocrine tumors (NETs) with their incidence notably increased compared to NETs in other sites in recent decades. Current research of pulmonary carcinoids has been hindered by the low frequency of the disease and scarcity of disease models and research resources for in-depth mechanistic or functional studies. This project aims to create novel disease models as needed research resources for advancing understanding of the biology and developing new treatments for pulmonary carcinoids. Pulmonary carcinoids are believed to originate from pulmonary neuroendocrine cells (PNECs). In our previously published research, we have established, to our knowledge, the first method to produce high proportions of functioning PNEC by differentiation of human pluripotent stem cells (hPSCs). The availability of hPSC-derived PNECs paves a new avenue for modeling and studying lung NE tumors including lung carcinoids. We hypothesize that the hPSC-derived PNEC can transform to pulmonary carcinoids by the common mutations specifically implicated in these tumors.

With the support by the Neuroendocrine Tumor Research Foundation (NETRF), we have successfully generated six hPSC lines with inducible over-expression or deletion of the candidate genes that are commonly mutated in pulmonary carcinoids. These include the familial mutation MEN1, and sporadic mutations IGF1R, ARID1, EGFR. We used two main methods to insert the genetic mutation, inducible mutations which could be induced specifically in PNEC differentiation stage or mutation expression controlled by a PNEC marker. After validation of the gene expression at protein level, these hPSC cell lines are differentiated into lung lineage including PNECs. The PNECs can be purified by a yellow fluorescent protein (YFP) marker and characterized for their capacity to transform to pulmonary carcinoids. A series of experiments like counting cell numbers, calculation of mitotic index and colony formation were then performed to evaluate the proliferation, apoptosis, and oncogenic transformation of PNECs in culture. In addition, we set up a PNEC culture with genetic mutations commonly found in small cell lung cancer (SCLC), another type of lung neuroendocrine tumor, as controls. In particular, we used the newly developed cell models combined with gene targeting tools and functional studies (as CRISPR gene screening) to test the high-frequency mutations for their capacity to transform the PNECs to pulmonary carcinoid. We also tested the role of the recurrent genetic mutations in pulmonary carcinoids with the aim to identify key driver genes or signaling pathways during disease initiation and progression.

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