Periodic Reporting for period 1 - PNECtumor (Mechanisms regulating pulmonary neuroendocrine cell (PNEC) growth and tumorigenesis)
Reporting period: 2019-01-01 to 2020-12-31
Lung neuroendocrine neoplasms (LNENs) account for 20-25% of all lung cancers and can be subdivided into high-grade carcinomas and low grade neuroendocrine tumors, carcinoids. Because they exhibit features of neuroendocrine differentiation, all LNENs are thought to arise from a rare lung cell population, called pulmonary neuroendocrine cells (PNECs). Despite their involvement in LNENs and their implication in a number of other respiratory diseases, PNECs are an understudied cell type. Their normal function is not well defined and relatively little is known about their contribution to cancer development and progression. The experiments performed for this MSCA Action were focused on the functional characterization of PNECs and the elucidation of the pathways involved in the transformation of this cell type during LNEN formation.
One of the biggest barriers to studying PNECs has been the lack of tractable model systems that can be used to isolate and study this rare cell type. The objectives of the project were (1) to define PNEC heterogeneity and identify changes in gene expression associated with PNEC proliferation, (2) to define optimal conditions for PNEC expansion within a lung organoid culture system and (3) to use lung organoids to characterize key pathways involved in LNEN formation.
One of the biggest barriers to studying PNECs has been the lack of tractable model systems that can be used to isolate and study this rare cell type. The objectives of the project were (1) to define PNEC heterogeneity and identify changes in gene expression associated with PNEC proliferation, (2) to define optimal conditions for PNEC expansion within a lung organoid culture system and (3) to use lung organoids to characterize key pathways involved in LNEN formation.
Aim1: Define PNEC heterogeneity and identify changes in gene expression associated with PNEC proliferation
Through the work package for this aim, I generated a sizable single cell RNA sequencing (scRNA-seq) dataset comprising lineage labeled PNECs derived from mice either treated with an agent that induces acute lung injury, naphthalene, or vehicle. The acute lung injury induced by naphthalene ablates secretory Club cells in the lung, which, in turn induces PNEC proliferation. The PNECs included in the scRNA-seq dataset were collected at time points corresponding to maximum PNEC proliferation after injury. Our preliminary analysis of this dataset revealed several cellular clusters, suggesting that PNECs are indeed a heterogeneous population. We also found that several clusters in our analysis consisted almost entirely of PNECs derived from naphthalene injured mice and that cells in these clusters showed higher expression of several neuropeptides, including Calca, which has been implicated in other injury responses in the ling. These results highlight the previously unappreciated heterogeneity of the PNEC populations and uncover potential molecular mediators of the PNEC response to injury.
Aim 2: Define optimal conditions for PNEC expansion within a lung organoid culture system
The host laboratory, the lab of Dr. Hans Clevers, has derived organoid cultures from human adult lung tissue (Sachs et al. 2019). Organoids derived from human lung tissue, are representative of the human airways and have therefore been termed airway organoids (AOs). Although PNECs are located mostly in the airways and proximal lungs, standard adult AOs do not contain PNECs precluding the use of standard AOs to study this cell type. Because the proportion of PNECs present in the human lung is greatest at fetal time points and goes down in adulthood, to establish a new organoid model that contains PNECs, we tested different media recipes on AOs generated from fetal lung tissue. We identified 2 signaling molecules as key components of what we termed, PNEC expansion media. Fetal AOs isolated and grown in PNEC expansion media maintained high levels of expression of PNEC markers and contained multiple PNECs integrated into the epithelial structure of the AOs even at late passages. Next, we established a protocol for PNEC differentiation. Through these efforts we identified a small molecule modulator of cellular signaling that synergized with NOTCH inhibition to induce an up to 11 fold increase in the expression of PNEC marker genes and the number of PNECs in AOs. To resolve transcriptional changes during PNEC differentiation and to further define PNECs, we have performed single cell RNA-seq of fetal AOs during the course of induced PNEC differentiation, starting at day 1 of differentiation and ending with day 10. These analyses are ongoing.
Altogether, we have identified cellular signals that induce the differentiation of PNEC precursors, thereby allowing us to generate large numbers of these, normally very rare, differentiated cells in vitro. This model system will undoubtedly facilitate the study of PNECs and contribute to future studies on this cell type in my own work and in the work of the broader community. The results of these studies will be submitted for publication in the next year.
Sachs, Norman, Angelos Papaspyropoulos, Domenique D. Zomer van Ommen, Inha Heo, Lena Böttinger, Dymph Klay, Fleur Weeber, et al. 2019. “Long‐term Expanding Human Airway Organoids for Disease Modeling.” The EMBO Journal 38 (4): e100300–320.
Aim 3: Use lung organoids to characterize key pathways involved in LNET formation.
Lung NENs comprise both low grade, well-differentiated NETs and high grade, poorly differentiated NECs. In particular, lung NENs can be subdivided into the high-grade carcinomas, small cell lung cancer (SCLC) and large cell neuroendocrine carcinomas (LCNEC), and the low grade tumors, atypical carcinoids (AC), classified as intermediate grade, and typical carcinoids (TC), classified as low grade. One of the aims of this project was to generate an organoid model of AC and SCLC genesis. To achieve this, I propose to use CRISPR/Cas9-mediated genome editing in PNEC-differentiated human lung organoids to induce inactivating mutations in genes that are commonly altered in these tumor types. This strategy will allow me to determine how these alterations influence the behavior of PNECs and, ultimately, to decipher their mechanistic role in NEN tumorigenesis.
Thus far, we have generated the molecular tools needed for achieving this aim: lenti-viral vectors that express Cas9 and GFP driven from a PNEC-specific promoter, and transient expression vectors for the expression of sgRNAs against RB1, TP53, PTEN, and MEN1. To account for the potential influence of PNEC heterogeneity in response to specific oncogenic alterations, we cloned 3 lentiviral vectors, each in which expression of Cas9 is driven by one of three PNEC promoters: ASCL1, NEUROD1, or CHGA. We expect the first two promoters to drive expression of Cas9 in early PNECs. The third promoter, CHGA, is expected to drive expression of Cas9 in mature, fully differentiated PNECs. In the vectors we have generated, expression of Cas9 is linked to expression of GFP. Lentiviral preparations have been produced for each of the 3 different vectors and organoids will be infected in the coming months.
Through the work package for this aim, I generated a sizable single cell RNA sequencing (scRNA-seq) dataset comprising lineage labeled PNECs derived from mice either treated with an agent that induces acute lung injury, naphthalene, or vehicle. The acute lung injury induced by naphthalene ablates secretory Club cells in the lung, which, in turn induces PNEC proliferation. The PNECs included in the scRNA-seq dataset were collected at time points corresponding to maximum PNEC proliferation after injury. Our preliminary analysis of this dataset revealed several cellular clusters, suggesting that PNECs are indeed a heterogeneous population. We also found that several clusters in our analysis consisted almost entirely of PNECs derived from naphthalene injured mice and that cells in these clusters showed higher expression of several neuropeptides, including Calca, which has been implicated in other injury responses in the ling. These results highlight the previously unappreciated heterogeneity of the PNEC populations and uncover potential molecular mediators of the PNEC response to injury.
Aim 2: Define optimal conditions for PNEC expansion within a lung organoid culture system
The host laboratory, the lab of Dr. Hans Clevers, has derived organoid cultures from human adult lung tissue (Sachs et al. 2019). Organoids derived from human lung tissue, are representative of the human airways and have therefore been termed airway organoids (AOs). Although PNECs are located mostly in the airways and proximal lungs, standard adult AOs do not contain PNECs precluding the use of standard AOs to study this cell type. Because the proportion of PNECs present in the human lung is greatest at fetal time points and goes down in adulthood, to establish a new organoid model that contains PNECs, we tested different media recipes on AOs generated from fetal lung tissue. We identified 2 signaling molecules as key components of what we termed, PNEC expansion media. Fetal AOs isolated and grown in PNEC expansion media maintained high levels of expression of PNEC markers and contained multiple PNECs integrated into the epithelial structure of the AOs even at late passages. Next, we established a protocol for PNEC differentiation. Through these efforts we identified a small molecule modulator of cellular signaling that synergized with NOTCH inhibition to induce an up to 11 fold increase in the expression of PNEC marker genes and the number of PNECs in AOs. To resolve transcriptional changes during PNEC differentiation and to further define PNECs, we have performed single cell RNA-seq of fetal AOs during the course of induced PNEC differentiation, starting at day 1 of differentiation and ending with day 10. These analyses are ongoing.
Altogether, we have identified cellular signals that induce the differentiation of PNEC precursors, thereby allowing us to generate large numbers of these, normally very rare, differentiated cells in vitro. This model system will undoubtedly facilitate the study of PNECs and contribute to future studies on this cell type in my own work and in the work of the broader community. The results of these studies will be submitted for publication in the next year.
Sachs, Norman, Angelos Papaspyropoulos, Domenique D. Zomer van Ommen, Inha Heo, Lena Böttinger, Dymph Klay, Fleur Weeber, et al. 2019. “Long‐term Expanding Human Airway Organoids for Disease Modeling.” The EMBO Journal 38 (4): e100300–320.
Aim 3: Use lung organoids to characterize key pathways involved in LNET formation.
Lung NENs comprise both low grade, well-differentiated NETs and high grade, poorly differentiated NECs. In particular, lung NENs can be subdivided into the high-grade carcinomas, small cell lung cancer (SCLC) and large cell neuroendocrine carcinomas (LCNEC), and the low grade tumors, atypical carcinoids (AC), classified as intermediate grade, and typical carcinoids (TC), classified as low grade. One of the aims of this project was to generate an organoid model of AC and SCLC genesis. To achieve this, I propose to use CRISPR/Cas9-mediated genome editing in PNEC-differentiated human lung organoids to induce inactivating mutations in genes that are commonly altered in these tumor types. This strategy will allow me to determine how these alterations influence the behavior of PNECs and, ultimately, to decipher their mechanistic role in NEN tumorigenesis.
Thus far, we have generated the molecular tools needed for achieving this aim: lenti-viral vectors that express Cas9 and GFP driven from a PNEC-specific promoter, and transient expression vectors for the expression of sgRNAs against RB1, TP53, PTEN, and MEN1. To account for the potential influence of PNEC heterogeneity in response to specific oncogenic alterations, we cloned 3 lentiviral vectors, each in which expression of Cas9 is driven by one of three PNEC promoters: ASCL1, NEUROD1, or CHGA. We expect the first two promoters to drive expression of Cas9 in early PNECs. The third promoter, CHGA, is expected to drive expression of Cas9 in mature, fully differentiated PNECs. In the vectors we have generated, expression of Cas9 is linked to expression of GFP. Lentiviral preparations have been produced for each of the 3 different vectors and organoids will be infected in the coming months.
This project has resulted in a novel system for culturing human pulmonary neuroendocrine cells (PNECs) in airway organoids. In doing so, we have identified the signals that direct PNEC progenitor differentiation to the PNEC fate. I expect the model system that we have generated to be used by other biologists interested in studying PNECs and lung NENs. Moreover, the mechanistic studies that I am currently undertaking using this new model system, will elucidate aspects of lung NEN initiation and progression that are likely to impact patient stratification and may point to new therapeutic options for patients with lung NENs.