Lung fibrogenic microenvironment in mouse reconstitutes human alveolar structure and lung tumor

Bankert, R.B., Hess, S.D., and Egilmez, N.K.

(2002). SCID mouse models to study human

cancer pathogenesis and approaches to therapy:

potential, limitations, and future directions. Front.

Biosci. 7. c44–62.

Barkauskas, C.E., Cronce, M.J., Rackley, C.R.,

Bowie, E.J., Keene, D.R., Stripp, B.R., Randell,

S.H., Noble, P.W., and Hogan, B.L. (2013). Type 2

alveolar cells are stem cells in adult lung.

J. Clinical Investig. 123, 3025–3036. https://doi.

org/10.1172/jci68782.

Berman, J.S., Serlin, D., Li, X., Whitley, G., Hayes,

J., Rishikof, D.C., Ricupero, D.A., Liaw, L.,

Goetschkes, M., and O’Regan, A.W. (2004).

Altered bleomycin-induced lung fibrosis in

osteopontin-deficient mice. Am. J. Physiol. Lung

Cell Mol. Physiol. 286, L1311–L1318. https://doi.

org/10.1152/ajplung.00394.

Bibby, M.C. (2004). Orthotopic models of cancer

for preclinical drug evaluation: advantages and

disadvantages. Eur. J. Cancer 40, 852–857.

https://doi.org/10.1016/j.ejca.2003.11.021.

Borok, Z., Gillissen, A., Buhl, R., Hoyt, R.F.,

Hubbard, R.C., Ozaki, T., Rennard, S.I., and

Crystal, R.G. (1991). Augmentation of functional

prostaglandin E levels on the respiratory

epithelial surface by aerosol administration of

prostaglandin E. Am. Rev. Resp. Dis. 144, 1080–

1084. https://doi.org/10.1164/ajrccm/144.5.1080.

Carey, W.A., Taylor, G.D., Dean, W.B., and

Bristow, J.D. (2010). Tenascin-C deficiency

attenuates TGF-ss-mediated fibrosis following

murine lung injury. Am. J. Physiol. 299, L785–L793.

https://doi.org/10.1152/ajplung.00385.

Chen, L., Acciani, T., Le Cras, T., Lutzko, C., and

Perl, A.K. (2012). Dynamic regulation of plateletderived growth factor receptor alpha expression

in alveolar fibroblasts during realveolarization.

Am. J. Resp. Cell Mol. Biol. 47, 517–527. https://

doi.org/10.1165/rcmb.2012-0030OC.

Chijiwa, T., Kawai, K., Noguchi, A., Sato, H.,

Hayashi, A., Cho, H., Shiozawa, M., Kishida, T.,

Morinaga, S., Yokose, T., et al. (2015).

Establishment of patient-derived cancer

xenografts in immunodeficient NOG mice. Int. J.

Oncol. 47, 61–70. https://doi.org/10.3892/ijo.

2015.2997.

Choi, J., Park, J.E., Tsagkogeorga, G., Yanagita,

M., Koo, B.K., Han, N., and Lee, J.H. (2020).

Inflammatory signals induce AT2 cell-derived

damage-associated transient progenitors that

mediate alveolar regeneration. Cell Stem Cell 27,

366–382.e7. https://doi.org/10.1016/j.stem.2020.

06.020.

Cross, D.A., Ashton, S.E., Ghiorghiu, S., Eberlein,

C., Nebhan, C.A., Spitzler, P.J., Orme, J.P., Finlay,

M.R., Ward, R.A., Mellor, M.J., et al. (2014).

AZD9291, an irreversible EGFR TKI, overcomes

T790M-mediated resistance to EGFR inhibitors in

lung cancer. Cancer Discov. 4, 1046–1061. https://

doi.org/10.1158/2159-8290.CD-14-0337.

Fehrenbach, H. (2001). Alveolar epithelial type II

cell: defender of the alveolus revisited. Resp. Res.

2, 33–46.

Garcia-Prieto, E., Gonzalez-Lopez, A., Cabrera,

S., Astudillo, A., Gutierrez-Fernandez, A., FanjulFernandez, M., Batalla-Solis, E., Puente, X.S.,

Fueyo, A., Lopez-Otin, C., and Albaiceta, G.M.

(2010). Resistance to bleomycin-induced lung

fibrosis in MMP-8 deficient mice is mediated by

interleukin-10. PLoS One 5, e13242. https://doi.

org/10.1371/journal.pone.0013242.

Garcia, O., Hiatt, M.J., Lundin, A., Lee, J., Reddy,

R., Navarro, S., Kikuchi, A., and Driscoll, B. (2016).

Targeted type 2 alveolar cell depletion. A

dynamic functional model for lung injury repair.

Am. J. Resp. Cell Mol. Biol. 54, 319–330. https://

doi.org/10.1165/rcmb.2014-0246OC.

Hagimoto, N., Kuwano, K., Nomoto, Y., Kunitake,

R., and Hara, N. (1997). Apoptosis and expression

of Fas/Fas ligand mRNA in bleomycin-induced

pulmonary fibrosis in mice. Am. J. Resp. Cell Mol.

Biol. 16, 91–101. https://doi.org/10.1165/ajrcmb.

16.1.8998084.

Hasegawa, K., Sato, A., Tanimura, K., Uemasu, K.,

Hamakawa, Y., Fuseya, Y., Sato, S., Muro, S., and

Hirai, T. (2017). Fraction of MHCII and EpCAM

expression characterizes distal lung epithelial

cells for alveolar type 2 cell isolation. Resp. Res.

18, 150. https://doi.org/10.1186/s12931-0170635-5.

Hay, J., Shahzeidi, S., and Laurent, G. (1991).

Mechanisms of bleomycin-induced lung damage.

Arch. Toxicol. 65, 81–94.

Helene, M., Lake-Bullock, V., Zhu, J., Hao, H.,

Cohen, D.A., and Kaplan, A.M. (1999). T cell

independence of bleomycin-induced pulmonary

fibrosis. J. Leukoc. Biol. 65, 187–195.

Herzog, E.L., Brody, A.R., Colby, T.V., Mason, R.,

and Williams, M.C. (2008). Knowns and unknowns

of the alveolus. Proc. Am. Thorac. Soc. 5, 778–782.

https://doi.org/10.1513/pats.200803-028HR.

Karampitsakos, T., Tzilas, V., Tringidou, R.,

Steiropoulos, P., Aidinis, V., Papiris, S.A., Bouros,

D., and Tzouvelekis, A. (2017). Lung cancer in

patients with idiopathic pulmonary fibrosis. Pulm.

Pharmacol. Therapeut. 45, 1–10. https://doi.org/

10.1016/j.pupt.2017.03.016.

King, B.A., and Kingma, P.S. (2011). Surfactant

protein D deficiency increases lung injury during

endotoxemia. Am. J. Resp. Cell Mol. Biol. 44,

709–715. https://doi.org/10.1165/rcmb.20090436OC.

Lama, V., Moore, B.B., Christensen, P., Toews,

G.B., and Peters-Golden, M. (2002).

Prostaglandin E2 synthesis and suppression of

fibroblast proliferation by alveolar epithelial cells

is cyclooxygenase-2-dependent. Am. J. Resp.

Cell Mol. Biol. 27, 752–758. https://doi.org/10.

1165/rcmb.4857.

Mediavilla-Varela, M., Boateng, K., Noyes, D.,

and Antonia, S.J. (2016). The anti-fibrotic agent

pirfenidone synergizes with cisplatin in killing

tumor cells and cancer-associated fibroblasts.

BMC Cancer 16, 176. https://doi.org/10.1186/

s12885-016-2162-z.

Moeller, A., Ask, K., Warburton, D., Gauldie, J.,

and Kolb, M. (2008). The bleomycin animal

model: a useful tool to investigate treatment

options for idiopathic pulmonary fibrosis? Int. J.

Biochem. Cell Biol. 40, 362–382. https://doi.org/

10.1016/j.biocel.2007.08.011.

Mulugeta, S., Nureki, S.I., and Beers, M.F. (2015).

Lost after translation: insights from surfactant for

understanding the role of alveolar epithelial

dysfunction and cell quality control in fibrotic lung

disease. Am. J. Physiol. Lung Cell. Mol. Physiol.

https://doi.org/10.1152/ajplung.00139.2015.

Nureki, S.I., Tomer, Y., Venosa, A., Katzen, J.,

Russo, S.J., Jamil, S., Barrett, M., Nguyen, V.,

Kopp, M., Mulugeta, S., and Beers, M.F. (2018).

Expression of mutant Sftpc in murine alveolar

epithelia drives spontaneous lung fibrosis. The

Journal of Clin. Investig. 128, 4008–4024. https://

doi.org/10.1172/jci99287.

Panos, R.J., Bak, P.M., Simonet, W.S., Rubin, J.S.,

and Smith, L.J. (1995). Intratracheal instillation of

keratinocyte growth factor decreases hyperoxiainduced mortality in rats. J. Clin. Investig. 96,

2026–2033. https://doi.org/10.1172/jci118250.

Povedano, J.M., Martinez, P., Flores, J.M.,

Mulero, F., and Blasco, M.A. (2015). Mice with

pulmonary fibrosis driven by telomere

dysfunction. Cell Rep. 12, 286–299. https://doi.

org/10.1016/j.celrep.2015.06.028.

Rock, J.R., Barkauskas, C.E., Cronce, M.J., Xue, Y.,

Harris, J.R., Liang, J., Noble, P.W., and Hogan,

B.L. (2011). Multiple stromal populations

contribute to pulmonary fibrosis without

evidence for epithelial to mesenchymal

transition. Prod. Natl. Acad. of Sci. USA 108,

E1475–E1483. https://doi.org/10.1073/pnas.

1117988108.

Rogliani, P., Calzetta, L., Cavalli, F., Matera, M.G.,

and Cazzola, M. (2016). Pirfenidone, nintedanib

and N-acetylcysteine for the treatment of idiopathic pulmonary fibrosis: a systematic review

and meta-analysis. Pulm. Pharmacol. Therapeut.

40, 95–103. https://doi.org/10.1016/j.pupt.2016.

07.009.

Sahai, E., Astsaturov, I., Cukierman, E., DeNardo,

D.G., Egeblad, M., Evans, R.M., Fearon, D.,

Greten, F.R., Hingorani, S.R., Hunter, T., et al.

(2020). A framework for advancing our

understanding of cancer-associated fibroblasts.

Nat. Rev. Cancer 20, 174–186. https://doi.org/10.

1038/s41568-019-0238-1.

iScience 25, 104912, September 16, 2022

17

iScience

ll

Article

OPEN ACCESS

Sgalla, G., Iovene, B., Calvello, M., Ori, M.,

Varone, F., and Richeldi, L. (2018). Idiopathic

pulmonary fibrosis: pathogenesis and

management. Resp. Res. 19, 32. https://doi.org/

10.1186/s12931-018-0730-2.

Sisson, T.H., Mendez, M., Choi, K., Subbotina, N.,

Courey, A., Cunningham, A., Dave, A.,

Engelhardt, J.F., Liu, X., White, E.S., et al. (2010).

Targeted injury of type II alveolar epithelial cells

induces pulmonary fibrosis. Am. J. Respir. Crit.

Care Med. 181, 254–263. https://doi.org/10.1164/

rccm.200810-1615OC.

Suzuki, H., Nagai, K., Yamaki, H., Tanaka, N., and

Umezawa, H. (1969). On the mechanism of action

of bleomycin: scission of DNA strands in vitro and

in vivo. J. Antibiotics 22, 446–448.

Taguchi, F., Koh, Y., Koizumi, F., Tamura, T., Saijo,

N., and Nishio, K. (2004). Anticancer effects of

ZD6474, a VEGF receptor tyrosine kinase

inhibitor, in gefitinib ("Iressa")-sensitive and

resistant xenograft models. Cancer Sci. 95,

984–989.

Tang, N., Zhao, Y., Feng, R., Liu, Y., Wang, S., Wei,

W., Ding, Q., An, M.S., Wen, J., and Li, L. (2014).

Lysophosphatidic acid accelerates lung fibrosis

by inducing differentiation of mesenchymal stem

18

iScience 25, 104912, September 16, 2022

cells into myofibroblasts. J. Cell Mol. Med. 18,

156–169. https://doi.org/10.1111/jcmm.12178.

190, 1402–1412. https://doi.org/10.1164/rccm.

201404-0744OC.

Tsukui, T., Ueha, S., Abe, J., Hashimoto, S.,

Shichino, S., Shimaoka, T., Shand, F.H., Arakawa,

Y., Oshima, K., Hattori, M., et al. (2013).

Qualitative rather than quantitative changes are

hallmarks of fibroblasts in bleomycin-induced

pulmonary fibrosis. Am. J. Pathol. 183, 758–773.

https://doi.org/10.1016/j.ajpath.2013.06.005.

Yi, E.S., Williams, S.T., Lee, H., Malicki, D.M., Chin,

E.M., Yin, S., Tarpley, J., and Ulich, T.R. (1996).

Keratinocyte growth factor ameliorates radiationand bleomycin-induced lung injury and mortality.

Am. J. Pathol. 149, 1963–1970.

Tsukui, T., Ueha, S., Shichino, S., Inagaki, Y., and

Matsushima, K. (2015). Intratracheal cell transfer

demonstrates the profibrotic potential of

resident fibroblasts in pulmonary fibrosis. Am. J.

Pathol. 185, 2939–2948. https://doi.org/10.1016/j.

ajpath.2015.07.022.

Wang, R., Ibarra-Sunga, O., Verlinski, L., Pick, R.,

and Uhal, B.D. (2000). Abrogation of bleomycininduced epithelial apoptosis and lung fibrosis by

captopril or by a caspase inhibitor. Am. J. Physiol.

279, L143–L151.

Weng, T., Poth, J.M., Karmouty-Quintana, H.,

Garcia-Morales, L.J., Melicoff, E., Luo, F., Chen,

N.Y., Evans, C.M., Bunge, R.R., Bruckner, B.A.,

et al. (2014). Hypoxia-induced deoxycytidine

kinase contributes to epithelial proliferation in

pulmonary fibrosis. Am. J. Respir. Crit. Care Med.

Zacharias, W.J., Frank, D.B., Zepp, J.A., Morley,

M.P., Alkhaleel, F.A., Kong, J., Zhou, S., Cantu, E.,

and Morrisey, E.E. (2018). Regeneration of the

lung alveolus by an evolutionarily conserved

epithelial progenitor. Nature 555, 251–255.

https://doi.org/10.1038/nature25786.

Zepp, J.A., Zacharias, W.J., Frank, D.B.,

Cavanaugh, C.A., Zhou, S., Morley, M.P., and

Morrisey, E.E. (2017). Distinct mesenchymal

lineages and niches promote epithelial selfrenewal and myofibrogenesis in the lung. Cell

170, 1134–1148.e10. https://doi.org/10.1016/j.

cell.2017.07.034.

Zheng, D., Limmon, G.V., Yin, L., Leung, N.H., Yu,

H., Chow, V.T., and Chen, J. (2012). Regeneration

of alveolar type I and II cells from Scgb1a1expressing cells following severe pulmonary

damage induced by bleomycin and influenza.

PLoS One 7, e48451. https://doi.org/10.1371/

journal.pone.0048451.

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STAR+METHODS

KEY RESOURCES TABLE

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Antibodies

Rat CD45-PE-Cy7 (Clone: 30-F11)

eBioscience

Cat# 25-0451-82

Rat CD31-PE-Cy7 (Clone: 390)

BD Biosciences

Cat# 561410

Rat EpCAM-APC (Clone: G8.8)

eBioscience

Cat# 17-5791-82

Rat EpCAM-FITC (Clone: G8.8)

eBioscience

Cat# 11-5791-82

Rat MHCII-eFluor450 (Clone: I-A/I-E)

eBioscience

Cat# 48-5321-82

Mouse HLA-DR-eFluor450 (Clone: LN3)

Thermo Fisher

Cat# 48-9956-42

Mouse EpCAM-eFluor660 (Clone: 1B7)

Thermo Fisher

Cat# 50-9326-42

Mouse CD45-PE-Cy7 (Clone: 2D1)

Thermo Fisher

Cat# 25-9459-41

Mouse CD31-PE-Cy7 (Clone: WM059)

Thermo Fisher

Cat# 25-0319-41

Mouse Rabbit proSP-C

Millipore

Cat# AB3796

Rat Ki-67-FITC (Clone: SolA15)

eBioscience

Cat# 11-5698-80

Mouse anti-proSP-C

Abcam

Cat# ab40879

Mouse anti-STEM121

TaKaRa

Cat# Y40410

Mouse anti-Human Nucleoli

Abcam

Cat# ab190710

Rabbit anti-Ki-67

Millipore

Cat# AB9260

Hamster anti-podoplanin (T1a)

Abcam

Cat# ab11936

Rabbit anti-aSMA

Abcam

Cat# ab7817

Mouse anti-desmin

Abcam

Cat# ab6322

Rabbit anti-GFP

Abcam

Cat# ab183734

Rabbit anti-CD31

Abcam

Cat# ab28364

Dr Hisataka Sabe

RRID: Addgene_32751

Bacterial and virus strains

pEGFP-N1 vector

Chemicals, peptides, and recombinant proteins

Anti-mouse IgG1 Alexa Fluor 488

Thermo Fisher

Cat# A11001

Anti-rabbit IgG Alexa Fluor 594

Thermo Fisher

Cat# A11012

Anti-mouse IgG2a Alexa Fluor 647

Thermo Fisher

Cat# A28181

Lipofectamine 2000

Invitrogen

Cat# 11668019

Doxycycline-containing feed (600 ppm)

Oriental Yeast

Cat# D11072802

Tamoxifen

Toronto Research Chemicals

Cat# T006000

Bleomycin

Nihon Kayaku

CAS# 9041-93-4

Gefitinib

Selleck

Cat# S5098

Collagenasetype I

Gibco

Cat# 17100017

Dispase

Corning

Cat# 354235

DAPI

Life Technologies

Cat# 62248

Matrigel

Corning

Cat# 356234

GeneChip WT PLUS Reagent Kit

Affymetrix

Cat# 902280

GeneChip Mouse Gene 2.0 ST Array

Applied Biosystems

Cat# 902500

Clariom S Assay, Mouse

Applied Biosystems

Cat# 902931

Critical commercial assays

(Continued on next page)

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OPEN ACCESS

Continued

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Deposited data

Microarray data (GeneChip Mouse Gene 2.0 ST Array)

This paper

GSE208444

Microarray data (Clariome S, Mouse)

This paper

GSE208373

A549

ATCC

CCL-185

NCI-H441

ATCC

HTB-174

NCI-H1975

ATCC

CRL-5908

NCI-H226

ATCC

CRL-5826

NCI-H460

ATCC

HTB-177

NCI-H1299

ATCC

CRL-5803

PC-9

IBL cell bank

RRID: CVCL_B260

Lewis lung carcinoma

Taiho Pharmaceutical

RRID: CVCL_5653

Mouse: C57/BL6J

The Jackson Laboratory

Strain code: 632

Mouse: CB17.Cg-PrkdcscidLystbg-J/CrlCrlj

The Jackson Laboratory

Strain code: 250

Mouse: C57BL/6-Tg (CAG-EGFP)

Dr Masataka Asagiri

Strain code: 329

Mouse: B6.Cg-Tg(Scgb1a1-rtTA)1Jaw/J

Dr Machiko Ikegami and Jeffrey A. Whitsett

Strain code: 6232

Mouse: B6;C3-Tg(ACTA1-rtTA,

Dr Machiko Ikegami and Jeffrey A. Whitsett

Strain code: 12433

Mouse: ROSAmT/mG

The Jackson Laboratory

Strain code: 37456

Mouse: Sftpc-CreERT2

The Jackson Laboratory

Strain code: 28054

Mouse: Scgb1a1-CreERT2

The Jackson Laboratory

http://www.informatics.jax.org/

Experimental models: Cell lines

Experimental models: Organisms/strains

tetO-cre)102Monk/J

allele/MGI:5660121

Software and algorithms

GeneSpring software version 13.1

Agilent Technologies

Transcriptome Analysis Console Software version 4.0.1

Applied Biosystems

Database for Annotation, Visualization,

Laboratory of Human Retrovirology

and Integrated Discovery (DAVID)

and Immunoinformatics

SYNAPSE VINCENT software version 5

Fujifilm

JMP ver. 10

SAS Institute

https://www.chem-agilent.com/

contents.php?id=27881

https://tools.thermofisher.com/content/sfs/

brochures/tac_software_datasheet.pdf

https://david.ncifcrf.gov/

https://www.fujifilm.com/jp/ja/healthcare/

healthcare-it/it-3d/vincent

RRID:SCR_014242

RESOURCE AVAILABILITY

Lead contact

Further information and requests for resources and reagents should be directed to and will be fulfilled by

the lead contact, Dr. Atsuyasu Sato (atsuyasu@kuhp.kyoto-u.ac.jp).

Materials availability

The study did not generate new unique reagents.

Data and code availability

Microarray datasets have been deposited at GEO and are publicly available as of the date of publication.

Accession numbers are listed in the key resources table.

The paper does not report original code.

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OPEN ACCESS

Any additional information required to reanalyze the data reported in this paper is available from the

lead contact on request.

EXPERIMENTAL MODEL AND SUBJECT DETAILS

The study protocols were approved by the Animal Research Committee of Kyoto University (ID: MedKyo

13533). Written informed consent for the use of lung parenchyma or cancer tissue and for subsequent

use in the in vivo study was obtained according to a protocol approved by the Kyoto University Hospital

Institutional Review Board (approved numbers: R1280 and R1486).

METHOD DETAILS

Cell lines and reagents

A549 (CCL-185), NCI-H441 (H441; HTB-174), NCI-H1975 (H1975; CRL-5908), NCI-H226 (H226; CRL-5826),

NCI-H460 (H460; HTB-177), and NCI-H1299 (H1299; CRL-5803) cells were obtained from the American

Type Culture Collection (Rockville, MD). PC-9 cells were obtained from the IBL cell bank (Gunma, Japan).

Lewis lung carcinoma (LLC) cells were kindly supplied by Taiho Pharmaceutical (Tokyo, Japan). LLC-GFP

was established by lipofection. The EGFP-N1 vector was kindly supplied by Dr Hisataka Sabe of Hokkaido

University, Japan, and was transfected into LLC cells using Lipofectamine 2000 (Invitrogen, Cat. 11668019,

Carlsbad, CA) according to the manufacturer’s instructions. For the in vivo lung cancer treatment

assay, mice were treated orally with a single bolus dose of either vehicle or gefitinib (Selleck Chemicals,

Houston, TX).

Animals

Eight-week-old C57BL/6J mice (the Jackson Laboratory) and CB17.Cg-PrkdcscidLystbg-J/CrlCrlj mice (SCIDBeige mice) (the Jackson Laboratory) were purchased for use in this study. C57BL/6-Tg(CAG-EGFP) mice

(GFP mice) were generously provided by Dr. Masataka Asagiri. Double-transgenic Scgb1a1-rtTA (Line

1)/(tetO)7CMV-Cre mice (a gift from Dr Machiko Ikegami and Jeffrey A. Whitsett) were bred with

ROSAmT/mG mice (the Jackson Laboratory) to generate triple-transgenic Scgb1a1-rtTA/(tetO)7CMV-Cre/

ROSAmT/mG mice. Doxycycline (600 ppm) was added to the chow starting from 5 weeks old to 8 weeks

old to activate Cre-mediated recombination in triple-transgenic mice. Sftpc-CreERT2 mice (the Jackson

Laboratory) or Scgb1a1-CreERT2 mice (the Jackson Laboratory) were bred with ROSAmT/mG mice to

generate Sftpc-CreERT2/ROSAmT/mG mice or Scgb1a1-CreERT2/ROSAmT/mG mice. Tamoxifen (Toronto

Research Chemicals, North York, Canada) was dissolved in corn oil (20 mg/ml) and injected intraperitoneally (200 mg/kg) for 4 consecutive days from 6 weeks old as previously reported (Barkauskas et al., 2013) to

activate Cre-mediated recombination. The mice were used for experiments 2 weeks after tamoxifen

injection.

Bleomycin (BLM)-induced lung injury

Mice were anaesthetized with isoflurane and hung upright at a 45-degree angle. BLM (Nihon Kayaku, Tokyo, Japan) (from 0.25 mg/kg to 2 mg/kg body weight in 100 mL of PBS) or PBS (control) was administered as

reported previously (King and Kingma, 2011).

The preparation of single-cell suspensions of murine lung

For AT2 cell isolation, single lung cells were obtained as we previously reported (Hasegawa et al., 2017). For

lung fibroblast isolation and analysis and cell sorting in the BLM model, protease solution [collagenase type

I (450 U/ml; Gibco, Grand Island, NY) and dispase (5 U/ml; Corning, Corning, NY) in HBSS] was used, and

whole lungs were incubated for 30 min at 37 C. The subsequent process was performed as we previously

reported (Hasegawa et al., 2017).

The preparation of single-cell suspensions of human lung parenchyma or cancer tissue

Human lung parenchyma or cancer tissue was obtained from patients undergoing lung resection at Kyoto

University Hospital because of lung cancer. All lung parenchyma or cancer samples were minced, transferred to the same protease mix as in the process of lung tissues in the murine BLM model, and incubated

at 37 C for 60 min. The subsequent steps were the same as in the preparation of a single-cell suspension of

murine lung (Hasegawa et al., 2017). Pathological evaluation for the establishment of xenografts was reviewed for consistency by a single pathologist (A.Y.).

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Flow cytometric analysis and sorting of lung cells

Antibodies used for flow cytometry are listed in Key resources table. We sorted live, single cells using a

FACSAria III Cell Sorter with FACSDiva ver. 8.0.1 (BD Biosciences). Murine AT2 cells were identified and

sorted as we previously reported, with a purity >98% (Hasegawa et al., 2017). Human AT2 cells were identified as CD45-CD31-EpCAM+HLA-DR+ cells using the same gating strategy as murine AT2 cells (Hasegawa

et al., 2017). Murine lung mesenchymal cells were identified as CD45-CD31-EpCAM- cells as described

previously (Zepp et al., 2017). Sorted cells were collected in DMEM containing 10% FBS, antibiotics, antimycotic solution, and 25 mM HEPES (Life Technologies, Gaithersburg, MD) for further experiments. For

intracellular staining, the cells were incubated with the fixable viability dye eFluor780 (eBioscience) and surface antigens, fixed, and permeabilized with fixation and permeabilization buffer (eBioscience) according

to the manufacturer’s instructions. Permeabilized cells were incubated with anti-Ki-67 antibody and antiproSP-C antibody. Anti-mouse proSP-C antibody was labelled with PE-antirabbit IgG antibody. Fixed cells

were analyzed using a BD LSR Fortessa (BD Biosciences). Appropriate isotype control samples were utilized

for all FACS analyses. The data were analyzed using FlowJo software (ver. 7.6.5, Tree Star, San Carlos, CA).

Immunohistological analysis

The antibodies used for immunohistochemistry are listed in Key resources table. Murine lungs were inflated, fixed at 25 cm H2O with 10% neutral buffered formalin and embedded in paraffin. Tissue sections

(4-mm thick) were immunostained with primary antibodies at 4 C overnight followed by incubation with

horseradish peroxidase (HRP)-conjugated secondary antibodies. Immunoreactivity was visualized by incubation with 3,3’-diaminobenzidine (DAB).

Immunofluorescence analysis

Antibodies used for immunofluorescence are listed in Key resources table. Murine lungs were inflated,

fixed at 25 cm H2O with 10% neutral buffered formalin and embedded in paraffin. Tissue sections (4-mm

thick) were immunostained with primary antibodies. Alexa 488-, Alexa 594-, and Alexa 647-conjugated secondary antibodies (1:200; Life Technologies) were used for immunofluorescence analysis. Tissue slides

were mounted with anti-fade solution containing DAPI (Life Technologies). Fluorescence images were

obtained using an LSM 710 confocal microscope (Carl Zeiss, Thornwood, NY), an SP-8 confocal microscope

(Leica Microsystems, Wetzlar, Germany), or a BIOREVO BZ-9000/BZ-X810 fluorescence microscope (Keyence, Osaka, Japan).

Hydroxyproline assay

Mice were sacrificed, and the right lungs or whole lungs were harvested for the assay. The lungs were transferred to gentleMACS M tubes (Miltenyi Biotech) containing 1.5 mL (for right lungs) or 3 ml (for whole lungs)

of saline and homogenized using the gentleMACS Dissociator program RNA-01. Then, 300 ml of lung homogenate was mixed with 300 mL of 12 N HCl and heated overnight at 107 C. Next, 20 mL of acid hydrolysates was mixed with 20 mL of citrate-acetate buffer and 400 mL of chloramine T solution and incubated for

20 min at room temperature. Then, 400 mL of aldehyde/perchloric acid solution was added to the tubes,

vortexed and incubated for 15 min at 65 C before measurement of the absorbance (optical density OD560).

AT2 fibroblast coculture

AT2 fibroblast coculture was performed according to a previous study (Barkauskas et al., 2013) with a slight

modification. Briefly, sorted AT2 cells (53103) and lung mesenchymal cells (13105) were resuspended in

50 mL of the medium and mixed with 50 mL of growth factor-reduced Matrigel (Corning). Then, 100 mL of

mixed cell suspension was placed in a 24-well 0.4-mm Transwell insert (Falcon), and 500 mL of the medium

was placed in the lower chamber. Cells were incubated at 37 C in a 5% CO2 environment, and the medium

was changed every other day. Y-27632 (10 mM) was added to the medium for the first 2 days of culture.

Immunofluorescence of AT2 colonies was performed as whole mount staining. Matrigel disks were fixed

with 4% paraformaldehyde in PBS for 15minat RT and then washed with PBS prior to blocking and immunostaining, as was done for the immunofluorescence staining of tissue sections.

AT2 cell transplantation

On day 10 after BLM treatment, mice were administered 4.03105 freshly isolated AT2 cells suspended in

100 mL of PBS following the same procedure as for BLM administration. The control group received

100 mL of PBS.

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RNA purification

Total RNA was purified using an RNeasy mini plus kit (Qiagen, Chatsworth, CA) according to the manufacturer’s protocol. The integrity of total RNA was assessed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA), and samples with an RNA integrity number (RIN) above 8.0 were used for further

analyses.

Microarray analysis

Total RNA samples (100 ng) were processed with a GeneChip WT PLUS Reagent Kit (Affymetrix) and

hybridized to a GeneChip Mouse Gene 2.0 ST Array for mesenchymal cell analysis or to a Clariom S Assay

for cancer cell analysis according to the manufacturer’s protocol. Raw data obtained with Affymetrix

GeneChip Operating Software were normalized and analyzed by GeneSpring software (version 13.1, Agilent Technologies, Santa Clara, CA, USA) for mesenchymal cell analysis or Transcriptome Analysis Console

Software (version 4.0.1, Applied Biosystems, CA, USA) for cancer cell analysis.

Real-time (RT) PCR

cDNA was synthesized from purified total RNA using an iScript cDNA synthesis kit (Bio-Rad Laboratories,

Hercules, CA). Quantitative RT-PCR was performed using TaqMan Gene Expression Assays and a StepOne

Plus Real-Time PCR ystem (Life Technologies).

Gene ontology analysis

The differentially expressed genes were subjected to gene ontology (GO) analysis using the Database for

Annotation, Visualization, and Integrated Discovery (DAVID) to find overrepresentations of GO terms in the

biological process (BP) category (GOTERM_BP_FAT, GOTERM_CC_FAT, and GOTERM_MF_FAT) (https://

david.ncifcrf.gov/. Accessed on November 27th, 2018). As background, the Mus musculus (mouse) whole

genome was used (Mm9). Statistical enrichment was determined using the default settings in DAVID.

Micro-CT imaging

Mice were anaesthetized with a continuous flow of 4% isoflurane/air mixture (2.0 L/min) and placed in the

chamber of the micro-CT system (LaTheta LCT-200, Aloka, Tokyo, Japan). The processing of the CT data

was performed using ImageJ software (ver.1.52a, National Institutes of Health, Bethesda, MD, USA). The

tumor volume was semi-automatically obtained using SYNAPSE VINCENT software (Fujifilm Co. Tokyo,

Japan).

QUANTIFICATION AND STATISTICAL ANALYSIS

Statistical analysis

The values are expressed as the means G SEM if not otherwise specified. Statistical analyses were performed using JMP (ver.10, SAS Institute, Cary, NC). Comparisons between two groups were performed

using the Wilcoxon rank sum test. The differences between more than two groups were analyzed with analysis of variance, and post hoc analysis was performed using the Tukey–Kramer test. GeneSpring software

(ver.13.1, Agilent Technologies, Santa Clara, USA) and Transcriptome Analysis Console Software (ver.4.0.1,

Applied Biosystems, California, USA) were used for microarray analysis. The processing of the CT data was

performed using ImageJ software (ver.1.52a, National Institutes of Health, Bethesda, MD, USA). SYNAPSE

VINCENT software (ver.5, Fujifilm Co. Tokyo, Japan) was used for the 3-D reconstruction of CT images and

calculation of tumor volume. The exact number of replicates in each group and the statistical tests used

were specified in Figure legends.

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