Isolation of epithelial, endothelial, and immune cells from lungs of transgenic mice with oncogene-induced lung adenocarcinomas.

AJRCMB Articles in Press. Published on 27-October-2014 as 10.1165/rcmb.2014-0312MA

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Isolation of Epithelial, Endothelial, and Immune Cells From Lungs of Transgenic Mice with Oncogene-Induced Lung Adenocarcinomas

Amlak Bantikassegn1, Xiaoling Song1 and Katerina Politi1, 2, 3, 4 1

Yale Cancer Center, Yale University School of Medicine, New Haven, Connecticut, 06510 Department of Pathology, Yale University School of Medicine, New Haven, Connecticut 06510 3 Department of Medicine (Section of Medical Oncology), Yale University School of Medicine, New Haven, Connecticut 06510 2

4

Address Correspondence: Katerina Politi, PhD Department of Pathology Yale Cancer Center Yale University School of Medicine 333 Cedar Street, SHM-I 234D New Haven, CT 06510 USA Office Tel: 203-737-5921 Lab Tel: 203-737-6215 Fax: 203-785-7531 Email: [email protected] Key Words: EpCAM, CD45, CD31, epithelial cell isolation, lung adenocarcinoma, transgenic mouse models Running Title: Cell Isolation From Tumor-bearing Mouse Lungs List ONE descriptor number: 8.24 (Pulmonary Laboratory Science: Methodology) Body word count: 3958 Abstract word count: 182 Author Contributions Conception and design: AB, XS, KP Performed experiments: AB Analysis and interpretation: AB, XS, KP Manuscript writing and editing: AB, XS, KP

Copyright © 2014 by the American Thoracic Society


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ABSTRACT Genetically engineered mouse models of lung adenocarcinoma have proven invaluable for understanding mechanisms of tumorigenesis, therapy response, and drug resistance. However, mechanistic studies focused on studying these processes in tumor-bearing mouse lungs are confounded by the fact that, in most cases, relevant signaling pathways are analyzed in whole lung preparations, which are composed of a heterogeneous mixture of cells. Given our increasing knowledge about the roles played by different subpopulations of cells in the development of lung adenocarcinoma, separating the major cellular compartments of the tumor microenvironment is recommended to allow for a precise analysis of relevant pathways in each isolated cell type. In this study, we optimized magnetic and fluorescence-based isolation protocols to segregate lung epithelial (CD326/EpCAM positive), endothelial (CD31 positive), and immune cells (CD45 positive), with high purity, from the lungs of transgenic mice with mutant EGFR-induced lung adenocarcinomas. This approach, which can potentially be extended to additional lung adenocarcinoma models, enables delineation of the molecular features of individual cell types that can be used to gain insight into their roles in lung adenocarcinoma initiation, progression, and response to therapy.

2 Copyright © 2014 by the American Thoracic Society

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INTRODUCTION The main compartments in the adult mouse lung are: trachea, bronchi, bronchioles, and alveoli. Several types of epithelial cells characterize these different compartments. Clara cells, for example, primarily found in the bronchioles, produce Clara cell secretory protein (CCSP/CC10) [1], which helps mitigate inflammation and oxidative stress in the lungs [2]. The alveolar sacs, in contrast, are composed of alveolar type I cells (ATI) and alveolar type II cells (ATII). ATI cells mediate gas exchange [3]; ATII cells maintain alveolar fluid balance, produce surfactant proteins, and differentiate into ATI cells [4]. Surfactant protein C (SP-C) in particular is a unique marker for ATII cells [5, 6]. Other types of epithelial cells-such as basal, goblet, and ciliated cells- make up the bronchi and larger airways of the lung. Lung adenocarcinoma, the most common histopathological subtype of lung cancer, is thought to arise from the atypical proliferation of cells located in the bronchioles and alveoli [712]. In genetically engineered mouse models (GEMMs) of lung adenocarcinoma, induced by frequently observed oncogenic drivers such as mutant Kras or EGFR, tumors are mainly composed of ATII cells (although both Clara and ATII cells have been implicated in Krasinduced models of lung adenocarcinoma) [10, 13, 14]. These mouse models are extensively used: to study mechanisms associated with Clara and/or ATII cell proliferation, to analyze signaling pathways important in tumorigenesis, and to identify possible molecular targets for therapies. In addition to epithelial cells, which spur the development of lung adenocarcinoma in the Kras and EGFR mutant mouse models, hematopoietic and endothelial cells are actively involved in shaping the tumor microenvironment in the lungs. For example, it has been shown that increased activity of the Stat3c pathway in ATII cells promotes inflammation and immune cell infiltration in murine lung adenocarcinomas [15]. It has also been shown that EGFR-mutant



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epithelial cells dampen the antitumor activity of immune cells through the PD-1 pathway, and that blocking this pathway impairs tumor growth [16]. Thus, immune cells and lung epithelial cells are intimately linked in mice with lung adenocarcinoma. Endothelial cells also play an active role in the development of progression of lung cancer. There is evidence, for example, that overexpression of HIF2alpha can lead to increased angiogenesis and reduced survival in mice expressing oncogenic Kras in the lungs [17]. Moreover, clinical data suggest that combined inhibition of EGFR and VEGFR (Vascular Endothelial Growth Factor Receptor) is beneficial [18]. Furthermore, there is evidence that a subset of immune (Gr+CD11b+) cells promote angiogenesis and endothelial cell proliferation; indicating that immune and endothelial cells are also linked in the development of tumors [19]. Thus, being able to isolate immune, endothelial, and epithelial cells from tumor-bearing mouse lungs is important to precisely elucidate the molecular framework of the tumor microenvironment in lung adenocarcinoma. Magnetic-activated cell sorting (MACS) is a technique in which cells can be depleted (or positively selected) by using micro-beads that target specific cell-surface antigens. Epithelial cells from normal mouse lungs have previously been isolated using MACS by first depleting CD45pos hematopoietic cells and then selecting for epithelial cell-adhesion molecule (EpCAM/CD326)-expressing cells [20]. EpCAM is expressed on Clara, ATII, and even potentially on tumor-initiating cells [20-23]. Moreover, EpCAM, which can be used to isolate mouse lung epithelial cells [20-22] is overexpressed in lung adenocarcinoma [24] and is being studied as a target for cancer therapy [25]. A significant fraction of EpCAM positive cells, however, are also positive for the endothelial cell marker CD31pos, possibly due to the very vascularized nature of the lung epithelium and the tight association of cells at the endothelialepithelial interface [26]. Thus, there is a need to effectively deplete CD31pos cells for optimal



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epithelial cell purity. This is even more crucial when isolating cells from tumors, which can have an increased level of angiogenesis and endothelial cell recruitment [27]. In this study, we optimized a protocol for the magnetic-based isolation of cells from the lungs of transgenic mice with lung adenocarcinomas, by depleting CD45pos cells and CD31pos cells prior to the positive selection of EpCAM expressing cells. Using this technique we isolated high purity fractions of immune, endothelial, and epithelial cells from the lungs of mice with lung adenocarcinoma. We compared this approach to fluorescence-activated cell sorting (FACS) of EpCAM, CD45, and CD31-expressing cells from lung single-cell suspensions. Implementation of these protocols will be helpful to shed light on the molecular signatures of the three major cellular compartments of the tumor microenvironment in lung adenocarcinoma; and thus, contribute to delineating mechanisms of tumorigenesis, therapy response, and drug resistance.

MATERIALS AND METHODS Isolation of cells from mouse models of EGFR-driven lung adenocarcinoma Previously described CCSP-rtTA (+) TetOEGFRL858R (+) bitransgenic mice that develop lung adenocarcinomas were used (Supplementary Fig. 1A) [14]. This model employs a construct in which a tetracycline-responsive element (TetO), activated by the reverse tetracycline transactivator (rtTA) in the presence of doxycycline, is upstream of a mutant EGFR cDNA harboring a lung adenocarcinoma-associated point mutation (EGFRL858R). The CCSP-rtTA transgenic strain was used to direct expression of rtTA to the lung epithelium; in this line, rtTA is mainly expressed in ATII cells [28, 29]. Expression of mutant EGFR protein in these epithelial cells,

following

induction

with

doxycycline,

induces

lung

adenocarcinomas

with



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bronchioloalveolar carcinoma features. Magnetic resonance imaging of bitransgenic CCSP-rtTA (+) TetOEGFRL858R (+) mice shows widespread tumorigenesis in the lungs (Supplementary Fig. 1C) compared to monotransgenic CCSP-rtTA (+) TetOEGFRL858R (-) mice, which do not develop tumors (Supplementary Fig. 1B). These transgenic mice are widely used in studies to understand the mechanistic basis of mutant EGFR-induced lung tumorigenesis, drug response, and resistance to therapies targeting EGFR [30-32]. CCSP-rtTA (+) TetOEGFRL858R (+) mice develop tumors approximately 60 days after doxycycline induction. For these studies, mice were euthanized with CO2 asphyxiation upon tumor development, the lungs were extracted from the chest cavity, and a lung single-cell suspension was made through proteolytic digestion of the lungs. MACS or FACS was used to isolate epithelial, endothelial, and immune cells from the lung single-cell suspension. We determined the purity of the isolated cells using western blot, flow cytometry, and/or immunocytochemistry (ICC). The viability and yield of the isolated cells were also assessed. See Supplementary Information for detailed protocols of all the techniques used (including the MACS and FACS protocols). All procedures regarding the handling of the mice were performed in accordance with the guidelines established by the Yale University Institutional Animal Care and Use Committee.

Reagents Reagents used for MACS and/or FACS were purchased from Miltenyi Biotec Inc. (Bergisch Gladbach, Germany) and include: Mouse Lung Dissociation Kit (cat: 130-095-927), CD31 MicroBeads (cat: 130-097-418), CD45 MicroBeads (cat: 130-052-301), EpCAM-PE (130102-265), Anti-PE MicroBeads (cat: 130-048-801), CD31-FITC (cat: 130-102-519), CD45-APC



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(cat: 130-102-544), CD45-PE (cat: 130-102-781), CD146-APC (cat: 130-102-846), and PEB buffer/autoMACS Running Buffer (cat: 130-091-221). Other reagents used include: PBS, DMEM, 1x Red Blood Cell Lysis Buffer (e-Biosciences, San Diego, CA, USA, cat: 00-4333), and SYTOX Red Dead Cell Stain (Life Technologies, Carlsbad, CA, USA, cat: S34859). Reagents used for western blot and/or immunocytochemistry include: anti-CD45 (Abcam, Cambridge, UK, cat: ab10558), anti-ProSurfactant protein C (Abcam, Cambridge, UK, cat: ab90716), anti-PECAM-1 (Santa Cruz Biotechnology Inc., Dallas, TX, USA, cat: sc-1506), anti-EGFRL858R (Cell Signaling Technology, Danvers, MA cat: 3197S), anti-Aquaporin 5 antibody (Abcam, Cambridge, UK, cat: ab76013), anti-TTF1 (Abcam, Cambridge, UK, cat: ab76013), anti-Actin (Santa Cruz Biotechnology Inc., Dallas, TX, USA, cat: sc-8432), Antirabbit IgG, HRP-linked antibody (Cell Signaling Technology, Danvers, MA, cat: 7074S), Alexa Fluor 594 Donkey anti-rabbit IgF (Life Technologies, Carlsbad, CA, USA, cat: A-21207), VECTASHIELD Mounting Medium with DAPI (Vector Laboratories, Burlingame, CA, USA cat: H-1200. The T1α serum was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at The University of Iowa, Department of Biology, Iowa City, IA 52242.

RESULTS Optimization of a MACS-based protocol for the isolation of cells from lungs of tumorbearing mice EGFRL858R mutant lung tumors grow diffusely through the lung [14], therefore, it is difficult to dissect distinct nodules of mutant epithelial cells for downstream analysis. To overcome this challenge, our initial efforts were centered on optimizing a method for the



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isolation of epithelial cells from whole lungs of CCSP-rtTA (+) TetOEGFRL858R (+) tumorbearing mice. To accomplish this, we first tested the purity of epithelial cells obtained using three different MACS protocols (Fig. 1A). Single-cell suspensions from tumor-bearing mouse lungs (using flow cytometry and gating on CD45neg cells) contained ~20% EpCAMpos epithelial cells and ~59% CD31pos cells (Fig. 1A. I). To enrich for epithelial cells, we first tested whether depleting CD45pos cells and performing a positive selection step for EpCAM (without CD31 depletion) increased epithelial cell purity, which it did by ~3.5x (Fig. 1A. II). We found that the EpCAM positive selection step was critical to achieve high epithelial cell purity (>70%) since doing CD31/CD45 depletion alone (without EpCAM positive selection) did not lead to similar levels of enrichment (Fig. 1A. III). Finally, to further enrich for epithelial cells, we tested CD31/CD45 depletion (sequential or simultaneous) followed by EpCAM positive selection. This protocol was optimal for enriching epithelial cells such that they composed >90% of the isolated fraction (Fig. 1A. IV). If the CD31 depletion step was excluded prior to EpCAM positive selection, the epithelial cell fraction contained ~20% endothelial cells (Fig. 1A. II). Depletion of CD31pos cells ensured that almost no endothelial cells were present in the epithelial cell fraction (Fig. 1A. III, IV). EpCAM positive selection alone therefore does not fully distinguish between endothelial and epithelial cells, and a CD31 depletion step is necessary (prior to EpCAM positive selection) for maximum epithelial cell purity. The antibodies and micro-beads used for the depletion and positive selection steps were titrated for optimal cell fraction purities (see Supplementary Information for details). To isolate immune, endothelial, and epithelial cells from individual mice using MACS, we had to determine the exact order in which each selection step should be performed to achieve a high purity of cells. We found that it is best to do the CD31 selection before and not after the


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EpCAM positive selection. This is because once the epithelial cells go through positive selection, they still have the micro-beads adhering to their surface, and subsequent depletion steps attempting to negatively select CD31 cells from the epithelial cells using an external magnetic system will be unsuccessful. For the same reason, since CD31 is expressed on some immune cells while endothelial cells do not express CD45, it is best to perform the CD45 selection before CD31 selection. In summary, we obtained the highest purity of epithelial cells (along with immune and endothelial cells) from tumor-bearing mice by sequentially selecting for CD45, CD31and EpCAM (see Fig. 1B, center panel).

Assessing the quality of MACS-based protocol To assess the purity of the sequential CD45, CD31 and EpCAM MACS-based isolation strategy performed on tumor-bearing mouse lungs, we examined the different isolated fractions using flow cytometry (Fig. 1B). EpCAMpos epithelial cells composed ~4% of the total lung cell population prior to separation (Fig. 1B. I), but they were significantly enriched in the EpCAMpos fraction, where they represented >90% of the cells (Fig. 1B. II). On average we observed a 24fold enrichment of EpCAMpos epithelial cells (Table 1) using our protocol. In the pre-separation fraction (Fig. 1B. I), there are two sub-populations of CD45pos cells, those that express EpCAM and those that do not. On average, ~75% of the cells were double positive for CD45 and EpCAM in EGFRL858R tumor-bearing mice before MACS was performed. This result is consistent with studies indicating that EpCAM is expressed on hematopoietic cells of the mouse lung [33, 34]. This emphasizes the need to perform the CD45 depletion before the EpCAM positive selection to avoid positively selecting EpCAMposCD45pos immune cells along with the EpCAMpos CD45neg epithelial cells (resulting in a low purity of epithelial cells).



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The pre-separation fraction was composed of ~84.0% CD45pos cells and ~6% CD31pos cells (Table 1 and Fig. 1B. III); on the other hand, the EpCAMpos fraction had 90% (Fig. 1B. IV, V). The purities (mean ± SEM) of the different fractions are summarized in Table 1. To further confirm the nature of the isolated cell fractions, we used immunoblotting with antibodies for cell-type specific markers. Western blot analysis showed that EGFRL858R and surfactant protein C (SP-C) were significantly enriched in the EpCAMpos fraction when compared to the pre-separation fraction (Fig. 2A). Furthermore, CD45 is only localized to the CD45pos fraction, and PECAM (endothelial cell marker) is only detected in the CD31pos fraction. We also observed TTF-1 expression predominantly in the EpCAMpos fraction. TTF-1 is a transcription factor that is clinically used as a marker for lung adenocarcinoma [35]. These results confirm that we successfully isolated epithelial, endothelial, and immune cells; and it validates the purity analysis obtained with flow cytometry. The lack of SP-C and EGFRL858R in the CD45pos fraction supports the idea that EpCAMposCD45pos cells (Fig. 1B. I) are immune cells that express EpCAM and not epithelial cells that express CD45. We also observed that a small fraction of epithelial cells (~5-6%) are found in the CD31pos fraction, which explains the low level of TTF1 expression in this fraction (Fig. 2A). To determine whether alveolar type I (AT I) cells are also enriched with this protocol, we probed for the ATI specific proteins (Aquaporin 5 and T1α), which were both enriched in the EpCAMpos fraction (compared to the pre-separation fraction)(Fig. 2C).


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In addition to the western blot data, immunocytochemical analysis of the different fractions further confirmed that epithelial cells expressing EGFRL858R are enriched when comparing the pre-separation and the EpCAMpos fractions (Fig. 2B), where approximately 77% of the epithelial cells were found to express EGFRL858R. To further assess the identity of the isolated fractions (Fig 2D), we stained the CD31pos fraction with antibodies to CD31 and CD146 (endothelial cell marker) conjugated to fluorochromes. Using flow cytometry, we found that ~95% of the CD31pos fraction is double positive for the two markers (D. I), confirming that this fraction predominantly contains endothelial cells. We also further evaluated the identity of cells in the CD45pos fraction (isolated from EGFRL858R tumor-bearing mice). We found that ~91% of the CD45pos cells were double positive for CD45 and F4/80 (a macrophage marker) (D. II), and ~16.1% of the population was double positive for CD45 and CD3 (T-cell marker) (D. III). Of note, cells that express both macrophage and lymphoid markers have been described which explains why we observed a small fraction of cells that are both F4/80pos and CD3pos[36]. To determine the viability of MACS-isolated cells, we labeled cells from the CD31pos, CD45pos, and EpCAMpos fractions with the SYTOX Red Dead Cell Stain (Fig. 2E). Cells that were positive for SYTOX Red were not viable. We found that 96.0% ± 0.4 of the CD45pos cells (Table 1, Fig 2E. I) and 90.2 ± 2.5 of the CD31pos cells (Table 1, Fig 2E. II) were viable. We observed that the shorter the total time of isolation, the higher the viability. The best viability for EpCAMpos cells was obtained when CD45pos and CD31pos cells were depleted simultaneously, which significantly decreases the total isolation time (by about 40 minutes) and avoids extra stress on the cells due to two sequential depletion steps before the EpCAM positive selection. We observed that this protocol noticeably increased the viability for EpCAMpos cells without



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significantly affecting the purity or yield. Using this protocol, the viability of EpCAMpos cells was 74.0% ± 2.4 (Table 1, Fig 2E. III), the disadvantage being that the CD31pos and CD45pos cells cannot be isolated separately using this approach. At the end of the isolation process, there is a fourth fraction characterized as CD45neg CD31neg EpCAMneg. We observed that this fraction is mainly composed of lingering red blood cells, fibroblasts, and possibly other stromal cells. Flow, western, and ICC analysis indicate that these cells do not express candidate proteins characteristic of epithelial, endothelial, or immune cells (data not shown). Comparison of MACS vs. FACS-based cell isolation An alternative approach to separate cell populations is that of using fluorescenceactivated cell sorting (FACS). To compare and contrast, MACS vs. FACS, we used CD31-FITC, CD45-APC, and EpCAM-PE to sort cells from the lungs of EGFRL858R tumor-bearing mice based on a gating scheme as illustrated in Figure 3A. Western blot analysis of the sorted fractions revealed that the EpCAMpos fraction is enriched for SP-C and EGFRL858R (Fig. 3B). We also observed that the CD31 fraction is enriched for PECAM, and the CD45 fraction is enriched for the CD45 protein (data not shown). Since all the markers are localized to their respective fractions with high specificity, we conclude that the FACS-based protocol is also successful in isolating endothelial, epithelial, and immune cells from mouse lungs. To define the similarities and differences between the two methods, we compared the purities, viabilities and yields from the sorted fractions as summarized in Table 1. The MACS protocol takes ~5-6 hours to complete per mouse, but it is possible to do isolations from more than one mouse concurrently provided that sufficient magnets are available. On the other hand, the FACS protocol takes ~3 hours per mouse, but samples from only one mouse at a time can be



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isolated. The FACS isolated EpCAMpos and CD31pos fractions (Table 1) have slightly higher purities and less debris compared to those isolated by MACS. On the other hand, we observed higher viabilities and yield using the MACS protocol compared to that of the FACS protocol for all the isolated fractions. Although other reasons inherent to the FACS protocol might account for the yield discrepancy, one explanation is that we use doublet discrimination gating to avoid sorting cells that adhere to one another (which otherwise might result in fractions with low purity). Quantitative and qualitative comparisons of the two protocols are summarized in Table 1 and Table 2 respectively.

DISCUSSION As our understanding of the complexity of the tumor microenvironment grows, interest in the molecular features of individual cell types that make up tumors is also increasing. Such studies require the isolation of different cell types for precise and detailed molecular and cellular investigations to elucidate mechanisms that underlie inflammation, tumor initiation, angiogenesis, and drug resistance. In this study, we optimized a MACS protocol to isolate the major cellular compartments of the lung from tumor-bearing transgenic mice with lung adenocarcinomas, the major histopathological subtype of lung cancer. As a proof-of-principle for our study, we used transgenic mice that develop lung adenocarcinomas upon expression of an EGFR mutant (EGFRL858R) found in human tumors [14]. Our approach is generalizable and can be expanded to other mouse models of lung cancer. We sequentially performed CD45 selection, CD31 selection, and EpCAM positive selection on tumor-bearing lungs to respectively isolate immune, endothelial, and epithelial cells from the same tumor-bearing lungs: a protocol that may help



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studies aimed at understanding the relationship between these cell types in tumor initiation and progression [19, 37]. Furthermore, we found that we could achieve high numbers of viable cells in all the isolated cell fractions using the MACS protocol, which is promising for any downstream studies that require the use of viable cells such as cell culture [37]. In addition to optimizing the MACS protocol, we used antibodies conjugated to fluorochromes that recognize EpCAM, CD45 and CD31 to perform FACS-based isolations analogous to those of the MACS protocol. We found that MACS protocol was generally superior in generating higher viabilities and yields compared to that of the FACS protocol (Tables 1 and 2). On the other hand, the overall purities obtained with FACS for EpCAMpos and CD31pos cells were better than when magnetic beads were used. There are drawbacks inherent in using FACS/MACS. First, the length of time that is required to process tissue and isolate cells means that gene expression and signaling pathway activation may change during the isolation process. When comparing experiments, therefore, at a minimum, it is important to take steps to make sure that the amount of time required for isolation is kept constant for each mouse studied. Moreover, analysis of signaling events using flow cytometry without cell isolation can be performed and compared to results obtained in isolated cell populations as a control. Second, we also isolated immune, endothelial, and epithelial cells from lungs of wild type EGFRL858R (-) mice, and found that the yields for all the cell types were considerably decreased compared to cells isolated from tumor-bearing lungs (Supplementary Figure 2) potentially due to increased epithelial cell proliferation, inflammation

and

angiogenesis in tumor-bearing lungs. However, Western blot analysis of the cells isolated from wild type mice showed a similar level of enrichment compared to cells isolated from tumor bearing lungs (data not shown).


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It is worth noting that there are discrepant findings regarding the proportion of epithelial cells, in particular ATII cells, in relation to the total lung cellularity in normal mice. Roper et al. used the SP-C promoter to drive the expression of Enhanced Green Fluorescent Protein (EGFP), and found that ATII cells make up less than 1% of total lung cells [38]. This finding is congruent with our study in which we identified ~1.1% EpCAM positive cells in normal mice (data not shown) and ~4% in EGFRL858R tumor bearing mice (Fig. 1. B. I.). A similar study that used the SP-C promoter to drive H2B-GFP expression indicated that ATII cells make up 6-8% of normal lung cells [39]. On the other hand, studies using pap staining to label ATII cells found that they make up 19.5% [40] and 40% [41] of the total lung-cell population in normal mice. The lungs of mice with EGFR-induced tumors exhibit a very high proportion of inflammatory cells, which might also contribute to the low percentage of epithelial cells identified in these lungs prior to cell separation. More studies are needed to accurately determine the exact proportion of epithelial cells in the lungs of normal and tumor-bearing mice. EpCAM, in addition to being expressed on Clara and ATII cells, is also expressed on terminally differentiated lung epithelial cells such as ATI and ciliated bronchiolar cells. By using this marker to isolate epithelial cells, we do not necessarily distinguish between tumor cells and normal epithelial cells (and indeed enrich for these cells aswell, Fig. 2C), although immunocytochemistry for EGFRL858R showed that most of these cells are positive for the oncogene. Another caveat of using EpCAM, is that cancer cells that undergo epithelial to mesenchymal transition may lose EpCAM expression; and thus, EpCAM-based enrichment methods lack the ability to capture these cells . Future experiments to refine strategies to capture these cells will be important.



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The ability to separate the major populations of cells from tumor-bearing mouse lungs is likely to contribute to studies aimed at determining mechanisms of carcinogenesis and drug resistance. Since in the tumor microenvironment many cell subpopulations have overlapping signaling mechanisms relevant to lung cancer, deconstructing the lung into its basic cellular components and analyzing characteristics of these cellular fractions separately is more advantageous than studying the tumor environment in its totality (which introduces confounding factors due to cellular heterogeneity). This approach can be applied to many lung cancer models and will allow for more accurate signaling, gene expression, and sequencing studies.

ACKNOWLEDGMENTS We thank Mary Ann Melnick, Deborah Ayeni, and Zimei Zhang for their assistance. We also extend our appreciation to the Dhodapkar lab for allowing us to use their FACSCalibur, Gouzel Tokmoulina for her technical expertise at the Yale Cell Sorter Core Facility, and Dr. Rakesh Verma for his insightful input. This work was supported by NIH/NCI grant (R01 120247), the Canary Foundation and the Labrecque Foundation to K.P. XS received support from the Yale Cancer Center and Uniting Against Lung Cancer.



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Table 1. Assessment of the purity, viability, and yield of cells isolated using a FACS or MACSbased protocol from mice with lung adenocarcinoma. (Mean ± SEM)

EpCAM

pos

MACS CD45pos

CD31

pos

EpCAM

pos

FACS CD45pos

CD31pos

Pre separation (% of total cells)

3.8% ± 0.5

83.9% ± 4.0

5.9% ± 0.8

3.2% ± 0.2

84.0% ± 0.9

7.5% ± 0.8

Post separation (% of total cells)

92.9% ± 1.2

93.7% ± 1.7

90.6% ± 0.6

97.4% ± 0.7

95.8% ± 1.1

98.7% ± 1.0

(p = 0.03)#

(p = 0.38)#

(p = 0.001)#

30x

1.1x

13.2x

57.2% ± 6.0

90.0%* ± 1.2

65.8% ± 5.7

(p = 0.06)#

(p = 0.01)#

(p = 0.02)#

Enrichment (post/pre) Viability

24.4x 74.0% ± 2.4

1.1x 96.0% ± 0.4

15x 90.2% ± 2.5

Average number of cells per 2.07± 6.23 ± 5.1 ± 0.85 5.6 ± 0.66 1.8 ± 1.5± 0.06 6 7 6 5 7 tumor-bearing 0.49 x10 1.36 x10 x10 x10 0.37x10 x106 ** lung (p = 0.06)# (p = 0.05)# (p = 0.02)# * Viability assay was performed using Trypan Blue staining ** The mice were on doxycycline ~ 60 days # p---value computed using a t---test comparing analogous fractions isolated using MACS and FACS.



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Table 2. Qualitative comparison of FACS and MACS-based isolations from lungs of mice with adenocarcinoma

MACS

FACS

Total time of Isolation/mouse

5-6 hrs. Possible to do simultaneous isolations from several mice

3 hrs. Allows for isolation from only one mouse at a time

Yield

Comparatively Higher yield

Lower Yield

Viability

Higher viability *

Lower viability

Purity

Lower overall purity

Higher Purity for EpCAMpos and CD31pos fractions

*Epithelial cell viability higher when doing CD45/CD31 simultaneous depletion


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FIGURE LEGENDS Figure 1. Optimization of the MACS protocol for isolating lung epithelial cells. (A) Flow cytometry analysis of single cell suspensions from the lungs of a tumor-bearing CCSP-rtTA (+) TetOEGFRL858R (+) mouse (A. I) prior to performing MACS-based separations. Analysis of the CD45neg cells is shown. The whole single cell suspension was then depleted of CD45pos cells followed either directly by EpCAM positive selection (A. II) or CD31 depletion (A. III, IV). EpCAM positive selection was then performed on a subset of the CD45negCD31neg cells (A. IV). The highest epithelial cell purity was achieved using the latter protocol. All the fractions analyzed were gated on CD45neg cells. (B). Flow-based analysis of the EpCAMpos, CD31pos, and CD45pos fractions (B. II, IV, V, VI) isolated using sequential CD45, CD31, and EpCAM selection (see center panel) compared to the pre-separation fraction (B. I, III). Flow analysis was performed on a FACSCalibur using CD31-FITC, CD45-APC, and EpCAM-PE antibodies.

Figure 2. Molecular and cellular characterization of the MACS-isolated cell factions. (A) Western blot analysis on MACS-isolated cells from an EGFRL858R tumor-bearing mouse for SPC, EpCAM, TTF-1, CD45, PECAM and EGFRL858R as indicated. (B) Immunocytochemistry for EGFRL858R in pre-separation cells, in EpCAMpos cells, and in CD31pos/CD45pos cells. Cells were incubated with an EGFR (L858R) primary antibody (red) followed by a secondary antibody (Alexa Fluor 594) and DAPI (blue) before being imaged (Scale bars: 50µm). (C) Lysates from cells isolated from an EGFRL858R tumor-bearing mouse probed with antibodies to Aquaporin 5 and T1α (ATI cell markers) are shown. Of note, the center lane shows lysates of cells following CD31/CD45 simultaneous depletion (not sequential). (D) Flow cytometry analysis of isolated non-epithelial cell fractions as indicated to confirm their identity. (E) Sytox red staining on



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MACS-isolated CD45, CD31, and EpCAM expressing cells from tumor-bearing (EGFRL858R) mice as indicated. The fractions were stained with Sytox Red to visualize the proportion of viable cells. Of note, the EpCAMpos fraction was isolated using CD31/CD45 simultaneous depletion (not sequential) before the EpCAM positive selection to maximize viability.

Figure 3. FACS based isolation of EpCAMpos, CD45pos, and CD31pos cells from tumorbearing (EGFRL858R) mouse lungs. (A) Cells from a single-cell suspension of lung tissue were labeled with EpCAM-PE, CD31-FITC and CD45-APC before being sorted using the BD FACSAria. Cells were gated as follows: CD45-APC expressing cells (Gate:1), EpCAM-PE expressing cells (Gate:2), and EpCAM-PE/CD45APC double negative cells (Gate: 3). To isolate the CD31-FITC expressing cells, we visualized the EpCAM-PE/CD45-APC double negative cells on a CD31-FITC histogram and identify cells positive for CD31-FITC (Gate:5) separately from the cells that are CD31-FITC, EpCAM-PE, and CD45-APC triple negative (Gate:4). Therefore, to isolate CD45pos, EpCAMpos, CD31pos, and triple negative cells; we sorted from gates 1, 2, 5, and 4 respectively. (B) Western blot analysis on FACS-isolated cells from an EGFRL858R tumor-bearing mouse for SP-C and EGFRL858R.



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REFERENCES 1.

2. 3.

4. 5.

6. 7.

8.

9.

10.

11. 12.

13.

14.

15.

Jojic V, Shay T, Sylvia K, Zuk O, Sun X, Kang J, Regev A, Koller D, Best AJ, Knell J, et al: Identification of transcriptional regulators in the mouse immune system. Nat Immunol 2013, 14:633-643. Lawrence T, Natoli G: Transcriptional regulation of macrophage polarization: enabling diversity with identity. Nat Rev Immunol 2011, 11:750-761. Thauvin-Robinet C, Munck A, Huet F, de Becdelievre A, Jimenez C, Lalau G, Gautier E, Rollet J, Flori J, Nove-Josserand R, et al: CFTR p.Arg117His associated with CBAVD and other CFTR-related disorders. J Med Genet 2013, 50:220-227. Fehrenbach H: Alveolar epithelial type II cell: defender of the alveolus revisited. Respir Res 2001, 2:33-46. Phelps DS, Floros J: Localization of pulmonary surfactant proteins using immunohistochemistry and tissue in situ hybridization. Exp Lung Res 1991, 17:985995. Kalina M, Mason RJ, Shannon JM: Surfactant protein C is expressed in alveolar type II cells but not in Clara cells of rat lung. Am J Respir Cell Mol Biol 1992, 6:594-600. Iselin LD, Wahl P, Studer P, Munro JT, Gautier E: Associated lesions in posterior wall acetabular fractures: not a valid predictor of failure. J Orthop Traumatol 2013, 14:179-184. Jakubzick C, Gautier EL, Gibbings SL, Sojka DK, Schlitzer A, Johnson TE, Ivanov S, Duan Q, Bala S, Condon T, et al: Minimal differentiation of classical monocytes as they survey steady-state tissues and transport antigen to lymph nodes. Immunity 2013, 39:599-610. Mingueneau M, Kreslavsky T, Gray D, Heng T, Cruse R, Ericson J, Bendall S, Spitzer MH, Nolan GP, Kobayashi K, et al: The transcriptional landscape of alphabeta T cell differentiation. Nat Immunol 2013, 14:619-632. Jackson EL, Willis N, Mercer K, Bronson RT, Crowley D, Montoya R, Jacks T, Tuveson DA: Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras. Genes Dev 2001, 15:3243-3248. Thaete LG, Malkinson AM: Cells of origin of primary pulmonary neoplasms in mice: morphologic and histochemical studies. Exp Lung Res 1991, 17:219-228. Gunning WT, Stoner GD, Goldblatt PJ: Glyceraldehyde-3-phosphate dehydrogenase and other enzymatic activity in normal mouse lung and in lung tumors. Exp Lung Res 1991, 17:255-261. Sutherland KD, Song JY, Kwon MC, Proost N, Zevenhoven J, Berns A: Multiple cellsof-origin of mutant K-Ras-induced mouse lung adenocarcinoma. Proceedings of the National Academy of Sciences of the United States of America 2014, 111:4952-4957. Politi K, Zakowski MF, Fan PD, Schonfeld EA, Pao W, Varmus HE: Lung adenocarcinomas induced in mice by mutant EGF receptors found in human lung cancers respond to a tyrosine kinase inhibitor or to down-regulation of the receptors. Genes Dev 2006, 20:1496-1510. Li Y, Du H, Qin Y, Roberts J, Cummings OW, Yan C: Activation of the signal transducers and activators of the transcription 3 pathway in alveolar epithelial cells induces inflammation and adenocarcinomas in mouse lung. Cancer Res 2007, 67:8494-8503.



16.

17.

18.

19.

20. 21.

22.

23. 24.

25.

26.

27.

28. 29.

Page 22 of 39

Akbay EA, Koyama S, Carretero J, Altabef A, Tchaicha JH, Christensen CL, Mikse OR, Cherniack AD, Beauchamp EM, Pugh TJ, et al: Activation of the PD-1 pathway contributes to immune escape in EGFR-driven lung tumors. Cancer Discov 2013, 3:1355-1363. Kim WY, Perera S, Zhou B, Carretero J, Yeh JJ, Heathcote SA, Jackson AL, Nikolinakos P, Ospina B, Naumov G, et al: HIF2alpha cooperates with RAS to promote lung tumorigenesis in mice. J Clin Invest 2009, 119:2160-2170. Kato T, Seto T, Goto K, Atagi S, Hosomi Y, Yamamoto N, Hida T, Maemondo M, Nakagawa K, Nagase S, et al: Eroltinib plus bevacizumab (EB) versus erlotinib alone (E) as first-line treatment for advanced EGFR mutation-positive nonsquamous nonsmall cell lung cancer (NSCLC): An open-label randomed trial. . In 2014 ASCO Aunnual Meeting. Chicago, Illinois: J. Clin Oncol, abstr 8005; 2014. Yang L, DeBusk LM, Fukuda K, Fingleton B, Green-Jarvis B, Shyr Y, Matrisian LM, Carbone DP, Lin PC: Expansion of myeloid immune suppressor Gr+CD11b+ cells in tumor-bearing host directly promotes tumor angiogenesis. Cancer Cell 2004, 6:409421. Messier EM, Mason RJ, Kosmider B: Efficient and rapid isolation and purification of mouse alveolar type II epithelial cells. Exp Lung Res 2012, 38:363-373. Podio NS, Baroni MV, Badini RG, Inga M, Ostera HA, Cagnoni M, Gautier EA, Garcia PP, Hoogewerff J, Wunderlin DA: Elemental and isotopic fingerprint of Argentinean wheat. Matching soil, water, and crop composition to differentiate provenance. J Agric Food Chem 2013, 61:3763-3773. Fujino N, Kubo H, Ota C, Suzuki T, Suzuki S, Yamada M, Takahashi T, He M, Suzuki T, Kondo T, Yamaya M: A novel method for isolating individual cellular components from the adult human distal lung. Am J Respir Cell Mol Biol 2012, 46:422-430. Visvader JE, Lindeman GJ: Cancer stem cells in solid tumours: accumulating evidence and unresolved questions. Nat Rev Cancer 2008, 8:755-768. Kim Y, Kim HS, Cui ZY, Lee HS, Ahn JS, Park CK, Park K, Ahn MJ: Clinicopathological implications of EpCAM expression in adenocarcinoma of the lung. Anticancer Res 2009, 29:1817-1822. Di Paolo C, Willuda J, Kubetzko S, Lauffer I, Tschudi D, Waibel R, Pluckthun A, Stahel RA, Zangemeister-Wittke U: A recombinant immunotoxin derived from a humanized epithelial cell adhesion molecule-specific single-chain antibody fragment has potent and selective antitumor activity. Clin Cancer Res 2003, 9:2837-2848. Yamamoto H, Yun EJ, Gerber HP, Ferrara N, Whitsett JA, Vu TH: Epithelial-vascular cross talk mediated by VEGF-A and HGF signaling directs primary septae formation during distal lung morphogenesis. Dev Biol 2007, 308:44-53. Moon WS, Park HS, Yu KH, Park MY, Kim KR, Jang KY, Kim JS, Cho BH: Expression of betacellulin and epidermal growth factor receptor in hepatocellular carcinoma: implications for angiogenesis. Hum Pathol 2006, 37:1324-1332. Tichelaar JW, Lu W, Whitsett JA: Conditional expression of fibroblast growth factor7 in the developing and mature lung. J Biol Chem 2000, 275:11858-11864. Fisher GH, Wellen SL, Klimstra D, Lenczowski JM, Tichelaar JW, Lizak MJ, Whitsett JA, Koretsky A, Varmus HE: Induction and apoptotic regression of lung adenocarcinomas by regulation of a K-Ras transgene in the presence and absence of tumor suppressor genes. Genes Dev 2001, 15:3249-3262.



Page 23 of 39

30.

31.

32.

33.

34.

35. 36.

37.

38.

39.

40. 41.

Politi K, Fan PD, Shen R, Zakowski M, Varmus H: Erlotinib resistance in mouse models of epidermal growth factor receptor-induced lung adenocarcinoma. Dis Model Mech 2010, 3:111-119. Regales L, Gong Y, Shen R, de Stanchina E, Vivanco I, Goel A, Koutcher JA, Spassova M, Ouerfelli O, Mellinghoff IK, et al: Dual targeting of EGFR can overcome a major drug resistance mutation in mouse models of EGFR mutant lung cancer. J Clin Invest 2009, 119:3000-3010. de Bruin EC, Cowell C, Warne PH, Jiang M, Saunders RE, Melnick MA, Gettinger S, Walther Z, Wurtz A, Heynen GJ, et al: Reduced NF1 expression confers resistance to EGFR inhibition in lung cancer. Cancer Discov 2014, 4:606-619. Nelson AJ, Dunn RJ, Peach R, Aruffo A, Farr AG: The murine homolog of human EpCAM, a homotypic adhesion molecule, is expressed by thymocytes and thymic epithelial cells. Eur J Immunol 1996, 26:401-408. Gautier EL, Shay T, Miller J, Greter M, Jakubzick C, Ivanov S, Helft J, Chow A, Elpek KG, Gordonov S, et al: Gene-expression profiles and transcriptional regulatory pathways that underlie the identity and diversity of mouse tissue macrophages. Nat Immunol 2012, 13:1118-1128. Stenhouse G, Fyfe N, King G, Chapman A, Kerr KM: Thyroid transcription factor 1 in pulmonary adenocarcinoma. J Clin Pathol 2004, 57:383-387. Baba T, Ishizu A, Iwasaki S, Suzuki A, Tomaru U, Ikeda H, Yoshiki T, Kasahara M: CD4+/CD8+ macrophages infiltrating at inflammatory sites: a population of monocytes/macrophages with a cytotoxic phenotype. Blood 2006, 107:2004-2012. Zhao T, Ding X, Du H, Yan C: Myeloid-derived suppressor cells are involved in lysosomal Acid lipase deficiency-induced endothelial cell dysfunctions. J Immunol 2014, 193:1942-1953. Roper JM, Staversky RJ, Finkelstein JN, Keng PC, O'Reilly MA: Identification and isolation of mouse type II cells on the basis of intrinsic expression of enhanced green fluorescent protein. Am J Physiol Lung Cell Mol Physiol 2003, 285:L691-700. Lee JH, Kim J, Gludish D, Roach RR, Saunders AH, Barrios J, Woo AJ, Chen H, Conner DA, Fujiwara Y, et al: Surfactant protein-C chromatin-bound green fluorescence protein reporter mice reveal heterogeneity of surfactant protein C-expressing lung cells. Am J Respir Cell Mol Biol 2013, 48:288-298. Harrison JH, Jr., Porretta CP, Leming K: Purification of murine pulmonary type II cells for flow cytometric cell cycle analysis. Exp Lung Res 1995, 21:407-421. Corti M, Brody AR, Harrison JH: Isolation and primary culture of murine alveolar type II cells. Am J Respir Cell Mol Biol 1996, 14:309-315.


Figure 1


Single-cell suspension

CD45 Depletion

CD31 Fibroblasts RBCs, etc… CD45

CD45neg CD31neg EpCAMpos

EpCAM Selection

CD45neg EpCAMpos Fibroblasts,

RBCs, etc…

EpCAM

CD31

CD45 Fib/ RBCs

CD45pos Fraction

CD31-FITC

EpCAM

CD31

EpCAM

CD31pos Fraction

(II) CD31-FITC

(I)

EpCAM Selection

Page 24 of 39

EpCAM Fib/ RBCs

(IV)

(III) CD31-FITC

Extraction of lungs and proteolytic digestion

CD45neg CD31neg

CD31 Depletion

CD31-FITC

(A)

EpCAM-PE

EpCAM-PE

CD45 Depletion EpCAM Positive Selection

CD45 Depletion CD31 Depletion

EpCAM-PE Pre Separation Fraction

EpCAM-PE CD45 Depletion CD31 Depletion EpCAM positive Selection

(B) (II)

CD45-APC

EpCAM Positive Selection

Fibroblasts/ RBCs

CD45 EpCAM

Pre Separation Fraction

CD31

CD45pos Fraction

CD31pos Fraction

(IV) CD45pos Fraction

CD31-FITC

CD31-FITC

(III) Pre-separation Fraction

CD45-APC

Fib RBCs

CD45

EpCAMpos Fraction

CD45neg CD31neg EpCAMneg

(V)

Copyright © 2014 by the American Thoracic Society CD45-APC

CD45-APC

EpCAM-PE

(VI) EpCAMpos Fraction

CD31pos Fraction

CD31-FITC

EpCAM-PE

EpCAM

CD31-FITC

CD31

CD31 Depletion

EpCAMpos Fraction CD45 Depletion

CD45-APC

(I) Pre-separation Fraction

CD45-APC

Figure 2


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B)

A) Pre-sep. fraction

CD45+ Fraction

Pre-sep. fraction

CD45/31+ Fraction

CD31+ EpCAMpos Fraction Fraction

Pre-sep. fraction

EGFRL858R

CD31pos/CD45pos Fraction

EpCAMpos Fraction

SP-C TTF1 PECAM CD45

Actin

C)

EpCAMpos Fraction

EGFRL858R T1 Aqua5 Actin

(I) CD31pos Fraction

F4/80-APC

CD146-APC (I) CD45pos Fraction

(II) CD31pos Fraction

Sytox Red

Sytox Red

CD45-PE

CD45-APC (III) EpCAMpos Fraction

Sytox Red

E)

(III) CD45pos Fraction CD3-PerCP -Cy5.5

CD45-PE

CD31-FITC

D)

(II) Fraction

CD45pos

CD31-FITC Copyright © 2014 by the American Thoracic Society

EpCAM-PE


Page 26 of Figure 339

(A) Sort From Gate:1

Gate:2

EpCAM-PE

EpCAM-PE

EpCAMpos Fraction

Pre-sorting Fraction CD45pos Fraction

EpCAM-PE

Sort From Gate:2

Gate:3

CD45-APC

Gate:1 CD45-APC

CD45-APC

CD45neg/CD31neg/EpCAMneg Fraction

CD31pos Fraction Gate:4

Gate:5 Sort From Gate:5

Sort From Gate:4 CD31-FITC

CD31-FITC

(B)

Pre-sep. CD45+ CD31+ EpCAMpos fraction Fraction Fraction Fraction EGFRL858R

SPC Actin


CD31-FITC


Page 27 of 39

SUPPLEMENTARY INFORMATION SUPPLEMENTARY MATERIALS AND METHODS Equipment The following equipment was acquired from Miltenyi Biotec Inc. (Bergisch Gladbach, Germany): gentleMACS Dissociator (cat: 130-093-235), QuadroMACS Magnet (cat: 130-091051), LS columns (cat: 130-042-401), Pre-separation 30µm Filters (cat: 130-041-407), and CTubes (cat: 130-093-237). Other instruments used include: FACSCalibur (BD Biosciences) with FlowJo, CytoSpin 4 Cytocentrifuge (Thermo Scientific), Bruker Mini 4T MRI, MoFlo XDP, ChemiDoc MP Imaging system (Bio-Rad) with Image Lab software, Microscope Axio Imager.A2 (Carl Zeiss AG) with AxioVision imaging software, X-Cite 120Q (Lumen Dynamics, Ontario, Canada), Countess Automated Cell Counter (with slides and Trypan Blue) (Life Technologies), mouse dissection scissors/forceps, 22G needle, 10cc syringe, 100µm strainers, 40µm strainers, 15ml/50ml falcon tubes, 5ml round bottom polystyrene tubes with a cell strainer cap (BD Biosciences).

Procedure for MACS-Based Isolation (A) Lung Extraction 1. Euthanize mouse using CO2 asphyxiation, and place carcass on a dissection board with ventral side facing up. 2. Open thoracic cavity and sever renal artery until a pool of blood surfaces. 3. Flush the heart with 10ml of PBS to remove blood from the lungs (using 10cc syringe and a 22G needle). Keep flushing lung until lobes are relatively clear and devoid of



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blood. This is an important step in order to remove as many erythrocytes as possible from the lung single-cell suspension. 4. Sever the trachea and use forceps to pull it towards the posterior end of the mouse until lungs/heart are extracted from the chest cavity. 5. Meticulously remove all tissues surrounding the lung such as: thymus, heart, efferent /afferent blood vessels, trachea, and connective tissues. 6. If the lung is still profusely permeated with blood (it is especially hard to remove blood from tumor-bearing lungs), flush individual lobes as best as possible with PBS. (B) Lung Dissociation kit Preparation (Miltenyi, cat: 130-095-927) 1. Prepare 1x Dissociation Buffer from the stock 20x Dissociation with distilled water. 2. Prepare Enzyme D by reconstituting the lyophilized powder with 3mL of 1x Dissociation Buffer (make appropriate aliquots, and store them in -20°C). 3. Prepare Enzyme F by reconstituting the lyophilized powder with 1mL 1x Dissociation Buffer (make appropriate aliquots, and store them in -20°C) (C) Proteolytic Digestion to make Lung Single-Cell Suspension [38] 1. Transfer the lobes into the gentleMACS C Tube (Miltenyi, cat: 130-093-237) containing an enzyme mix Note: Prepare enzyme mix by adding 2.4mL of 1x Dissociation Buffer, 100μL of Enzyme D and 15μL of Enzyme F into a gentleMACS C Tube. 2. Tightly close C Tube and attach it upside down onto the sleeve of the gentleMACS Dissociator (Miltenyi, cat: 130-093-235). 3. Run gentleMACS Dissociator protocol: mouse_lung_01.



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4. After termination of the program, detach C Tube from the Dissociator. The lung lobes will not be completely dissociated after this step. 5. Place sample in incubator/shaker for 30min at 37°C. 6. Attach C Tube upside down onto the sleeve of the gentleMACS Dissociator. 7. Run the gentleMACS Program m_lung_02. The lobes should disintegrate into a suspension after this step. 8. After termination of the program, detach C Tube from the gentleMACS Dissociator. 9. Perform a short centrifugation step to collect the sample material at the bottom. 10. Filter the cell suspension using a 100μm strainer placed on a 50mL falcon tube. Repeat this step using a 40μm strainer. 11. Rinse both strainers with 1ml PEB Buffer (Miltenyi, cat: 130-091-221) before discarding. 12. Centrifuge single-cell suspension (300g/10min/4°C). 13. Re-suspend pellet in 8-10ml of DMEM, and determine cell number using an automated cell counter (Life Technologies, cat: C10310)

Note: We found that using DMEM (as opposed to PEB buffer) during the isolation process significantly increases the viability of cells.

(D) CD45/CD31 Depletion There are two options to consider when doing negative selection of CD45 and CD31 cells from the lung single-cell suspension based on desired end result:



Option 1:

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Two separate depletion steps using two different columns: CD45 depletion

followed by CD31 depletion (sequential negative selection). This is best when separate endothelial and immune cell fractions are desired. Option 2: Depletion of both CD45 and CD31 using the same column (simultaneous negative selection). This is best for achieving a high viability of epithelial cells. The micro-beads used are 50nM in size and will label 20-40% of epitopes on the cell surface. All volumes are designed for 107 cells unless otherwise specified. If number of cells is less than 107 , use the same volumes, Scale up volumes if the number of cells is more than 107. 1. Centrifuge suspension (300g/10min/4°C). Pipette off supernatant completely (sucking off liquid using a vacuum is not recommended). 2. Re-suspend cell pellet in 90μL of DMEM per 107 total cells. 3. Add 10μL of CD45 MicroBeads per 107 cells (Miltenyi, cat: 130-052-301) and/or add 0.7μL of CD31 MicroBeads per 107 cells (Miltenyi, cat: 130-097-418). 4. Mix well and incubate for 15 minutes in 4°C. 5. Wash cells by adding 1-2 ml of buffer per 107 cells and centrifuge (300g/10min/4°C). 6. Pipette off supernatant, and re-suspend up to 108 cells in 500μl of DMEM. 7. Place an LS column (Miltenyi, cat: 130-042-401) in the QuadroMACS magnet (Miltenyi, cat: 130-091-051). 8. Wash column 3x with 1ml of DMEM, and collect flow-through with 15 ml flacon tubes. Make sure no bubbles form when adding DMEM to the column. Discard falcon tubes, and place a 30μm pre-separation filter (Miltenyi, cat: 130-041-407) over the column before applying the single-cell suspension labeled with microbeads.



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9. Collect the magnetically unlabeled cells (cells that do not express CD45 and/or CD31) that are part of the flow-through in a 15ml falcon tube. Wash column 3x with 1ml DMEM (without changing the falcon tube). Make sure to let each wash leave the column before starting the next wash. This cell fraction contains the epithelial cells. 10. Remove the LS column from the QuadroMACS magnet and place it on top of a new 15 ml flacon tube. Add 5ml DMEM to the column and apply a constant yet firm pressure with an LS column plunger until all the liquid is pushed out of the column and into a falcon tube. This fraction is characterized as CD45pos, CD31pos, or CD45posCD31pos, depending on if doing CD45 depletion, CD31 depletion, or CD45/CD31 double depletion respectively. 11. Determine cell yield for all fractions using an automated cell counter. (E) EpCAM Positive Selection Note: The EpCAM positive selection is performed on the fraction from step D9, the flowthrough cells after CD45 and CD31 depletions. 1) Centrifuge suspension (300g/10min/4°C), and pipette off supernatant. 2) Re-suspend cells in 50μl of PEB buffer per 106 cells (use the same dilution if cells are fewer than 106 , scale up accordingly if total cell count is greater than 106). Note: We found that DMEM interferes with a successful positive selection of epithelial cells using EpCAM-PE (data not shown); thus, we use PEB buffer for the majority of the steps in the positive selection process. 3) Add EpCAM-PE in a 1:10 dilution (Miltenyi, 130-102-265). For example: After re-suspending 2x107 cells in 1000μl of PEB buffer, add 100μl of EpCAM-PE.



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4) Mix well and incubate for 10min in 4°C in the dark. 5) Wash cells by adding 1-2mL of buffer per 107 cells and centrifuge (300g/10min/4°C). 6) Remove supernatant, re-suspend pellet in 80μl of PEB buffer per 107 cells, and add 20μl of anti-PE MicroBeads (per 107 total cells) (Miltenyi, cat: 130-048-801), 7) Mix well and incubate for 15min in 4°C in the dark. 8) Wash cells by adding 1-2ml of buffer per 107 cells and centrifuge (300g/10min/4°C). 9) Remove supernatant and re-suspend up to 108 cell in 500μl of PEB buffer. 10) Place a new LS column in the QuadroMACS magnet. 11) Wash column 3x with 1ml of PEB buffer, and collect flow-through with 15 ml flacon tubes. Make sure no bubbles form when adding PEB to the column. 12) Place a 30μm pre-separation filter over the column and apply cell suspension. 13) Collect the magnetically unlabeled cells (cells that do not express EpCAM) that are part of the flow-through in a 15ml falcon tube. Wash column 3x with 1ml PEB buffer (without changing the falcon tube). Make sure to let each wash leave the column before starting the next wash. This cell fraction mainly contains RBCs/fibroblasts and is characterized as CD45negCD31negEpcamneg. 14) Remove the LS column from the QuadroMACS magnet and place it on top of a new 15 ml flacon tube. Add 5ml DMEM to the column and apply pressure with an LS column plunger. This fraction is characterized as CD45negCD31negEpcampos, and contains the epithelial cells. 15) Determine cell yield for all the fractions using automated cell counter.

Procedure for FACS-Based Isolation



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1) Make a single-cell suspension from a mouse lung (follow the steps as highlighted in sections A-C in the MACS-based Isolation protocol). 2) Centrifuge suspension (300g/10min/4°C), and pipette off supernatant. 3) Re-suspend pellet in 1-2ml of Red Blood Cell Lysis Buffer (e-Biosciences, cat: 00-4333) and incubate for 1min in room temperature. 4) Wash cells by adding 1-2ml of PEB buffer per 107 cells, centrifuge suspension (300g/10min/4°C), discard supernatant, and re-suspend pellet in 5ml of PEB buffer. 5) Determine cell numbers using automated cell counter and adjust the concentration to 106 cells/ 50µl by centrifuging and re-suspending in PEB buffer. 6) Add CD45-APC (Miltenyi, cat: 130-102-544), EpCAM-PE, and CD31-FITC (Miltenyi, cat: 130-102-519) in a 1:10 dilution (incubate for 10min in 4°C in the dark). 7) Wash cells by adding 1-2ml of PEB buffer per 107 cells and centrifuge (300g/10min/4°C). 8) Discard supernatant and re-suspended cells in a maximum concentration of 6x107 cells/ml using PEB buffer. 9) Filter cells using a 5ml round bottom polystyrene tube with a cell strainer cap (BD Biosciences, cat: #352235) before sorting. 10) Isolate cells using a FACS machine with the appropriate lasers and filters (we used Beckman Coulter MoFlo). Use a 100µm nozzle and a low pressure setting for optimal viability. Make sure to discriminate debris and doublets from being sorted. Using the gating scheme as illustrated in Figure 3A, sort into polystyrene round bottom tubes with 500µl of DMEM already in them.



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Western blot 2x105 to 2x106 cells were lysed using RIPA lysis buffer (20µl-200µl) with Halt Protease and Phosphatase Inhibitor Cocktail (Thermo Scientific, Waltham, MA, USA, cat: #78440) mixed in a 100:1 dilution. The lysed cells were incubated on ice for 30min and spun for 10min at 300g. The supernatant was removed and quantified for protein concentrations using DC-Protein Assay (Bio-Rad Life Science, Hercules, CA, USA cat: 500-0112) and NanoDrop 1000 spectrophotometer (Thermo Scientific). Equal amounts of denatured protein were used in SDSPAGE, and then transferred to a nitrocellulose membrane. The membrane was washed with blocking buffer (60min), probed with a primary antibody (12hours in 4°C), washed with TBS buffer (3x, for 5min), probed with a secondary antibody (60min in 25°C), and washed with TBS buffer (3x, for 10min). The protein signals were detected using SuperSignal West Pico (Thermo Scientific, Waltham, MA, USA, cat: 34077) or Femto (Thermo Scientific, Waltham, MA, USA, cat: 34095) depending on the strength of the bands. The membranes were imaged and analyzed using ChemiDoc MP Imaging system with Image Lab software. All primary and secondary antibodies were used at concentrations recommended by the manufacturer.

Immunocytochemistry TPX Sample Chambers (Termo Scientific, cat: A78710018), Cytoclips (Termo Scientific, cat: 59910052), White Filter Cards (Termo Scientific, cat: 5991022), and microscope slides were assembled. 2x104 to 2x105 cells were spun on slides using the assembled chambers and a cytospin (5min at 400rpm). An Elite Mini PAP Pen (Diagnostic Biosystem, cat: K042) was used to circumscribe the cells with a water-repellent barrier before they were fixed, permeabilized, and stained using BD Cytofix/Cytoperm Solution Kit (BD Biosciences, cat: 555028). The cells



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are incubated with the desired primary antibody overnight in 4°C. Thereafter, the cells were washed with PBS (3x for 5min), and incubated with a fluorescent secondary antibody for 60min in 25°C in the dark. Finally, 200µl of DAPI-containing mounting media (Vector Laboratories, Burlingame, CA, USA cat: H-1200) was added before a coverslip was placed gently on top of the cells. The cells were imaged using Microscope Axio Imager.A2 with X-Cite 120Q, and images were analyzed using AxioVision software. All primary and secondary antibodies were used at concentrations recommended by the manufacturer.

Flow Cytometry 1x105 to 9x106 cells were suspended in 50µl PEB buffer per 106 cells. CD45-APC, EpCAM-PE, and CD31-FITC were added in a 1:10 dilution. The suspension was incubated for 10min at 4°C in the dark, washed with 1-2ml of PEB buffer (per 107 cells), and centrifuged (300g/10min/4°C). The supernatant was discarded and the cells were re-suspended in 100µl400µl PEB buffer. The suspension was filtered using a 5ml round bottom polystyrene tube with a cell strainer cap. We performed the flow cytometry on a BD FACSCalibur, and we analyzed the data using FlowJo.

Viability Assay Viability assays on MACS-isolated cells were performed by staining the isolated CD31pos, CD45pos, and EpCAMpos fractions with CD31-FITC, CD45-PE (Miltenyi, cat: 130-102781), and EpCAM-PE respectively along with SYTOX Red Dead Cell Stain (Life Technologies, cat: S34859) as illustrated in Figure 2D. Cells that were positive for SYTOX Red were not viable. The viability assay used for FACS-isolated cells is identical to that of the viability assay



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used for MACS-isolated cells for the CD31pos and EpCAMpos fractions, but not for the CD45pos fraction. Because the emission peak of APC (660nm) is almost identical to that of SYTOX Red (640nm), we cannot use these two fluorochromes simultaneously for assessing cell viability. Since we used CD45-APC for isolating immune cells using FACS, we can not use SYTOX Red co-staining as an indicator of viability for these cells. Instead, we used the Countess Automated Cell Counter/Trypan Blue staining to determine the proportion of viable cells in the FACSisolated CD45pos fraction.

Magnetic Resonance Imaging Mice were anesthetized using isoflurane prior to imaging. Images were collected with a 4T small-animal Bruker horizontal-bore spectrometer while gated on post-expiratory periods. The mice were supplied with O2 alongside isoflurane during MR imaging. All procedures regarding the handling of the mice were performed in accordance with the guidelines established by the Yale University Institutional Animal Care.



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SUPPLEMENTARY FIGURE LEGENDS Supplementary Figure 1: EGFRL858R mouse model of lung adenocarcinoma. (A) When doxycycline (dox) is administered to CCSP-rtTA (+) TetOEGFRL858R (+) mice, the dox binds to rtTA, a protein expressed under the control of the CCSP promoter specific to lung epithelial cells. The complex binds to the TetO promoter upstream of the EGFRL858R cDNA, and induces the transcription of the cDNA encoding the mutant receptor. Magnetic resonance and gross images of representative (B) control non-tumor-bearing CCSP-rtTA (+) TetOEGFRL858R (-) and (C) tumor-bearing CCSP-rtTA (+) TetOEGFRL858R(+) mouse lungs are shown.

Supplementary Figure 2: Quantification of cell numbers isolated from wild type EGFRL858R (-) mice and tumor bearing EGFRL858R (+) mice. For all the isolated fractions (CD45, CD31, and EpCAM), the yields from tumor-bearing mice were significantly higher compared to those of wild type mice (Mean ± SEM).



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Supplementary Figure 1 (A) Lung speciﬁc promoter

CCSP-‐rtTA (+) TetOEGFRL858R (+)

CCSP

Dox +

Doxycycline Diet (Day 1)

EGFR (L858R)

TetO

(B)

CCSP-‐rtTA (+) TetOEGFRL858R (-‐) H

CCSP-‐rtTA (+) TetOEGFRL858R (+)

rtTA

rtTA

rtTA

Doxycycline Diet (Day 60) Poly A tail

(C) CCSP-‐rtTA (+)

TetOEGFRL858R (+)

H

H

H

cm



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8×1077 7×107 6×107 5×10 1×107

Wild Type EGFR L858R (-) mice Tumor-Bearing EGFR L858R (+) mice

8×106 6×106 4×106 2×106 s

s Ep

C

A

M

po

po 31 D C

D

45

po

s

0

C

Cell Number Per Mouse

Supplementary Figure 2

Isolated Cell Fractions


Isolation and cultivation of microvascular endothelial cells from rat lungs: effects of gelatin substratum and serum.

Isolation and Characterization of Human Lung Lymphatic Endothelial Cells.

Conditionally induced RAGE expression by proximal airway epithelial cells in transgenic mice causes lung inflammation.

Copper Deficiency in the Lungs of TNF-α Transgenic Mice.

Type II epithelial cells from the lung of Syrian hamsters: isolation and metabolism.

Isolation and Cultivation of Primary Brain Endothelial Cells from Adult Mice.

Isolation and in vitro culture of vascular endothelial cells from mice.

J mice.

High frequency of mammary adenocarcinomas in metallothionein promoter-human growth hormone transgenic mice created from two different strains of mice.

Characterization of a transgenic mouse model exhibiting spontaneous lung adenocarcinomas with a metastatic phenotype.

Isolation of endothelial cells and pericytes from swine corpus luteum.

Isolation and culture of endothelial cells from the embryonic forebrain.

Isolation and characterization of endothelial progenitor cells from Rhesus monkeys.

Gli promotes epithelial-mesenchymal transition in human lung adenocarcinomas.

Isolation of Endothelial Progenitor Cells from Human Umbilical Cord Blood.

Transgenic mice as immune system models.

Isolation of Cancer Epithelial Cells from Mouse Mammary Tumors.

Isolation of microvascular endothelial cells from cadaveric corneal limbus.

Isolation and identification of epithelial and stromal stem cells from eutopic endometrium of women with endometriosis.

Isolation of amiloride-resistant clones from dog kidney epithelial cells.

Isolation and metabolism of granular pneumocytes from rat lungs.

Occlusive lung arterial lesions in endothelial-targeted, fas-induced apoptosis transgenic mice.

Lung oxygen consumption and mitochondria of alveolar epithelial and endothelial cells.

Utility of Genomic Analysis in Differentiating Synchronous and Metachronous Lung Adenocarcinomas from Primary Adenocarcinomas with Intrapulmonary Metastasis.