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FoxA1 and FoxA2 drive gastric differentiation and suppress squamous identity in NKX2-1-negative lung cancer

Research Article
Cite this article as: eLife 2018;7:e38579 doi: 10.7554/eLife.38579
7 figures, 1 table and 7 additional files

Figures

Figure 1 with 1 supplement
FoxA1 and FoxA2 are required for mucinous lung adenocarcinoma formation.

Photomicrographs of lung neoplasia arising 11 weeks after initiation with PGK-Cre lentivirus. All mice are KrasLSL-G12D/+; Nkx2-1F/F and harbor conditional alleles of Foxa1 and/or Foxa2 as indicated. (A) Hematoxylin and eosin (H and E) and immunohistochemistry (IHC) for NKX2-1, FoxA1 and FoxA2. Arrows indicate neoplasia lacking expression of all three proteins. Scale bar: 100 microns. (B) Alcian blue stain for mucin production. Scale bar: 50 microns. (C) IHC for markers of gastrointestinal differentiation HNF4α, PK-L and PDX1. Scale bar: 100 microns.

https://doi.org/10.7554/eLife.38579.003
Figure 1—figure supplement 1
FoxA1 and FoxA2 are required for mucinous lung adenocarcinoma formation.

(A) IHC for indicated proteins on primary human mucinous lung adenocarcinoma. Images are representative of six independent primary tumors. Scale bar: 100 microns. (B) FOXA1 and FOXA2 mRNA levels in KRAS-mutant human lung adenocarcinoma and adjacent normal lung. Clusters were defined in Skoulidis et al. (2015) using TCGA data. HNF4α-positive/NKX2-1-negative tumors are found predominantly in KC cluster. (C) IHC for indicated proteins on lung neoplasia arising 11 weeks after initiation with PGK-Cre lentivirus. All mice are KrasLSL-G12D/+; Nkx2-1F/F and harbor conditional alleles of Foxa1 and/or Foxa2. Scale bar: 100 microns.

https://doi.org/10.7554/eLife.38579.004
Figure 2 with 1 supplement
FoxA1 and FoxA2 are required for initiation and proliferation of NKX2-1-deficient lung adenocarcinoma.

(A) Quantitation of tumor burden 5 weeks after initiation with PGK-Cre lentivirus in KrasLSL-G12D/+; Nkx2-1F/F mice of indicated genotype: control (n = 15), Foxa1F/F (n = 13), Foxa2F/F (n = 9) and Foxa1F/F; Foxa2F/F (n = 12). **p < 0.0002 vs. each group, Mann-Whitney. *p=0.0425 vs. control. Graphs represent mean ±S.D. (B) Quantitation of BrdU incorporation in lung neoplasia 5 weeks after initiation with PGK-Cre lentivirus in KrasLSL-G12D/+; Nkx2-1F/F mice of indicated genotype: control (n = 7), Foxa1F/F (n = 4), Foxa2F/F (n = 4) and Foxa1F/F; Foxa2F/F (n = 7). *p < 0.005 vs. each control, Mann-Whitney. Graphs represent mean ±S.D. (C) Representative IHC for BrdU in KrasLSL-G12D/+; Nkx2-1F/F mice of indicated genotype quantitated in Figure 2B. Scale bar: 100 microns. (D) Long-term survival after tumor initiation with PGK-Cre lentivirus in KrasLSL-G12D/+; Nkx2-1F/F mice of indicated genotype: control (n = 9), Foxa1F/F (n = 10), Foxa2F/F (n = 12) and Foxa1F/F; Foxa2F/F (n = 9). p < 0.0001, KrasLSL-G12D/+; Nkx2-1F/F; Foxa1F/F; Foxa2F/F mice vs. each control, Log-rank test.

https://doi.org/10.7554/eLife.38579.005
Figure 2—figure supplement 1
FoxA1 and FoxA2 are required for initiation and proliferation of NKX2-1-deficient lung adenocarcinoma.

(A) Percentage of tumor area expressing HNF4α in KrasLSL-G12D/+; Nkx2-1F/F; Foxa1F/F; Foxa2F/F mice analyzed at 5-week (n = 11) and 11-week (n = 7) timepoints and in survival analysis (n = 9). Tumors were initiated by PGK-Cre lentivirus. *p < 0.001 vs. 11 weeks and survival group, Mann-Whitney. Graphs represent mean ±S.D. (B) IHC for HNF4α in KrasLSL-G12D/+; Nkx2-1F/F; Foxa1F/F; Foxa2F/F mouse analyzed at 11-week timepoint. Arrow points to HNF4α-negative complete recombinant (left). Brown nuclear stain marks HNF4α-positive incomplete recombinant. Scale bar: 500 microns. (C) Quantitation of average lesion area 5 weeks after initiation with PGK-Cre lentivirus in KrasLSL-G12D/+; Nkx2-1F/F mice of indicated genotype: control (n = 15), Foxa1F/F (n = 13), Foxa2F/F (n = 9) and Foxa1F/F; Foxa2F/F (n = 12). **p < 0.001 vs. each group, Mann-Whitney. *p = 0.0251 vs. control. Graphs represent mean ±S.D. (D) Quantitation of lesions/mm5 weeks after initiation with PGK-Cre lentivirus in KrasLSL-G12D/+; Nkx2-1F/F mice of indicated genotype: control (n = 15), Foxa1F/F (n = 13), Foxa2F/F (n = 9) and Foxa1F/F; Foxa2F/F (n = 12). **p < 0.003 vs. each group, Mann-Whitney. *p=0.0171 vs. control. Graphs represent mean ±S.D. (E) Quantitation of proliferation marker MCM2 in lung neoplasia 5 weeks after initiation with PGK-Cre lentivirus in KrasLSL-G12D/+; Nkx2-1F/F mice of indicated genotype: control (n = 7), Foxa1F/F (n = 5), Foxa2F/F (n = 4) and Foxa1F/F; Foxa2F/F (n = 7). *p < 0.01 vs. each control, Mann-Whitney. Graphs represent mean ±S.D. (F) Quantitation of proliferation marker KI67 in lung neoplasia 5 weeks after initiation with PGK-Cre lentivirus in KrasLSL-G12D/+; Nkx2-1F/F mice of indicated genotype: control (n = 7), Foxa1F/F (n = 5), Foxa2F/F (n = 4) and Foxa1F/F; Foxa2F/F (n = 7). *p < 0.01 vs. each control, Mann-Whitney. Graphs represent mean ±S.D. (G) Representative IHC for proliferation markers MCM2 and KI67 in KrasLSL-G12D/+; Nkx2-1F/F mice of indicated genotype quantitated in Figure 2—figure supplement 1E–F. Scale bar: 100 microns. (H) Representative IHC for CC3 (cleaved caspase-3) in tumors from mice of indicated genotype. Inset depicts thymus (positive control). Scale bar: 100 microns. (I) H and E demonstrating extracellular mucin secretion (E) in tumors from KrasLSL-G12D/+; Nkx2-1F/F; Foxa1F/F mice (survival analysis). Scale bar: 100 microns.

https://doi.org/10.7554/eLife.38579.006
Figure 3 with 1 supplement
Deletion Nkx2-1, Foxa1 and Foxa2 at initiation blocks gastric differentiation and induces expression of squamocolumnar junctional markers in lung neoplasia.

(A) tSNE plot of single-cell mRNA-Seq data derived from murine lung tumor cells (n = 134). Cells were sorted based on tdTomato expression from mice of the following genotypes: KrasLSL-G12D/+(K), n = 1 mouse), KrasLSL-G12D/+; Nkx2-1F/F (KN), n = 1 mouse), KrasLSL-G12D/+; Nkx2-1F/F; Foxa1F/F; Foxa2F/F (KNF1F2, n = 2 mice). Color indicates cancer cell cluster. Shape indicates genotype of mouse from which cell was isolated. (B) tSNE plot of three cancer cell clusters (C1–C3) and a panel of normal murine tissue. ‘Glandular’ indicates glandular stomach. (C) IHC for indicated proteins in lung neoplasia 5 weeks after initiation with PGK-Cre lentivirus in KrasLSL-G12D/+; Nkx2-1F/F; Foxa1F/F; Foxa2F/F mice and NKX2-1-negative controls. SCJ: normal squamocolumnar junction (forestomach on left, glandular stomach on right). Scale bar: 100 microns.

https://doi.org/10.7554/eLife.38579.007
Figure 3—figure supplement 1
Deletion of Nkx2-1Foxa1 and Foxa2 at initiation blocks gastric differentiation and induces expression of squamocolumnar junctional markers in lung neoplasia.

(A) Correlation between differentially expressed genes identified in single-cell analysis (C1 vs. C2, X axis) and population analysis (KrasLSL-G12D/+(K) vs. KrasLSL-G12D/+; Nkx2-1F/F (KN), Y axis). (B) tSNE plot of single high-quality tumor cells (this study) and E18.5 type two pneumocytes (AT2) from murine lung (Treutlein et al.). Color indicates cluster. Shape indicates source of cell. (C) Principal component analysis (PCA) of cancer cell clusters (C1–C3) based on genes comprising invasive mucinous adenocarcinoma signature (Guo et al). (D) Hierarchical clustering on principal components (HCPC) of the three cancer cell clusters (C1–C3) and normal murine tissue. The first two dimensions of a principal component analysis (PCA) is overlayed with the hierarchical clustering of the first five PCA dimensions. Dot color indicates cluster to which sample was assigned in K-means clustering. Gray ovals highlight multiple samples from the same tissue. ‘Glandular’ indicates glandular stomach. (E) IHC for indicated proteins in lung neoplasia 5 weeks after initiation with PGK-Cre lentivirus in KrasLSL-G12D/+; Nkx2-1F/F; Foxa1F/F; Foxa2F/F mice and controls. Scale bar: 100 microns. (F) IHC for FoxA1 and FoxA2 at the squamocolumnar junction (SCJ) of glandular stomach and forestomach (left panels) and in proximal forestomach (right panels). Arrows point to SCJ. Scale bars: 100 microns.

https://doi.org/10.7554/eLife.38579.008
Figure 4 with 1 supplement
FoxA1 and FoxA2 are downregulated in the squamous component of murine and human adenosquamous lung carcinoma.

(A) H and E of AdSCC arising in KrasLSL-G12D/+; Nkx2-1F/F; Foxa2F/F mouse. Scale bar: 1000 microns. (B) H and E and IHC of adenocarcinoma (left) and squamous (right) components of AdSCC arising in KrasLSL-G12D/+; Nkx2-1F/F; Foxa2F/F mouse. Scale bar: 100 microns. (C) Dual IHC for ΔNp63 (brown) and FoxA1/2 (purple) in AdSCC arising in KrasLSL-G12D/+; Nkx2-1F/F; Foxa2F/F mouse. Scale bar: 1000 microns. (D) Percent of human AdSCC cases (n = 12) exhibiting downregulation of FoxA1 and/or FoxA2 expression in the SCC component as assessed by IHC. (E) Representative IHC for FoxA1 and FoxA2 in a human AdSCC exhibiting downregulation of both proteins in the SCC component. Scale bar: 100 microns.

https://doi.org/10.7554/eLife.38579.009
Figure 4—figure supplement 1
FoxA1 and FoxA2 are downregulated in the squamous component of murine and human adenosquamous lung carcinoma.

(A) Percent of KrasLSL-G12D/+; Nkx2-1F/F mice of indicated genotype with adenosquamous carcinoma (AdSCC) 20 weeks after initiation with PGK-Cre lentivirus. Control (n = 14), Foxa1F/F (n = 10), Foxa2F/F (n = 17) and Foxa1F/F; Foxa2F/F (n = 12). (B) IHC of adenocarcinoma (left) and squamous (right) components of AdSCC arising in KrasLSL-G12D/+; Nkx2-1F/F; Foxa1F/F mouse. Scale bar: 100 microns. (C) IHC for indicated proteins in adenocarcinoma (left) and squamous (right) components of AdSCC arising in KrasLSL-G12D/+; Nkx2-1F/F; Foxa1F/F; Foxa2F/F mouse. Scale bar: 100 microns.

https://doi.org/10.7554/eLife.38579.010
Figure 5 with 1 supplement
Uncoupling KRASG12D activation from lineage specifier deletion promotes squamous cell carcinoma formation in the lung.

(A) Schematic of experimental design. (B) H and E and IHC for indicated proteins in tumors from mice harboring the conditional alleles KrasFSF-G12D/+; RosaFSF-CreERT2; Nkx2-1F/F (controls) alone and in combination with either Foxa2F/F or Foxa1F/F; Foxa2F/F. ‘K’ indicates acellular keratin. All mice were given tamoxifen 1 week after tumor initiation. Tamoxifen administration consisted of six intraperitoneal doses over nine days, followed by tamoxifen-containing chow until the end of the experiment. Scale bar: 50 microns.

https://doi.org/10.7554/eLife.38579.011
Figure 5—figure supplement 1
Uncoupling KRASG12Dactivation from lineage specifier deletion promotes squamous cell carcinoma formation in the lung.

(A) IHC for FoxA2 in tumors from mice harboring the conditional alleles KrasFSF-G12D/+; RosaFSF-CreERT2; Nkx2-1F/F (controls) alone and in combination with either Foxa2F/F or Foxa1F/F; Foxa2F/F. Scale bar: 50 microns. (B) Low power H and E photomicrograph of keratinizing squamous cell carcinoma arising in KrasFSF-G12D/+; RosaFSF-CreERT2; Nkx2-1F/F; Foxa1F/F; Foxa2F/F mouse. Scale bar: 100 microns. ‘K’ indicates acellular keratin. (C) Percent of KrasFSF-G12D/+; RosaFSF-CreERT2; Nkx2-1F/F (Control, n = 19), KrasFSF-G12D/+; RosaFSF-CreERT2; Nkx2-1F/F; Foxa2F/F (n = 21), KrasFSF-G12D/+; RosaFSF-CreERT2; Nkx2-1F/F; Foxa1F/F; Foxa2F/F (n = 8) and KrasLSL-G12D/+; Nkx2-1F/F; Foxa1F/F; Foxa2F/F mice (n = 6) harboring keratinizing squamous cell carcinoma 5 weeks after initiation with Ad5-CMV-FlpO followed by tamoxifen (KrasFSF-G12D/+ mice) or Ad5-CMV-Cre (KrasLSL-G12D/+ mice). *p < 0.001, Chi-square test.

https://doi.org/10.7554/eLife.38579.012
Figure 6 with 1 supplement
SPC-positive cells give rise to squamous cell carcinoma when KRASG12D activation is uncoupled from lineage specifier deletion.

H and E and IHC for indicated proteins in neoplasia 5 weeks post initiation. In mice harboring the conditional alleles KrasFSF-G12D/+; RosaFSF-CreERT2; Nkx2-1F/F (controls) alone and in combination with Foxa1F/F; Foxa2F/F, lung tumors were in initiated with Ad5-SPC-FlpO adenovirus. In mice harboring the conditional alleles KrasLSL-G12D/+; RosaLSL-tdTomato; Nkx2-1F/F; Foxa1F/F; Foxa2F/F, tumors were initiated with Ad5-SPC-Cre (right column). All mice were given tamoxifen 1 week after tumor initiation. Tamoxifen administration consisted of four intraperitoneal doses over 5 days, followed by tamoxifen-containing chow until the end of the experiment. Scale bar: 100 microns.

https://doi.org/10.7554/eLife.38579.013
Figure 6—figure supplement 1
SPC-positive cells give rise to squamous cell carcinoma when KRASG12Dactivation is uncoupled from lineage specifier deletion.

(A) IHC for HA (purple) in lungs from KrasFSF-G12D/+; RosaFSF-CreERT2; CAG-LSL-HA-UPRT mice infected with Ad5-CMV-FlpO (top) or Ad5-SPC-FlpO (bottom). One week post-infection, mice were given four intraperitoneal injections of tamoxifen over 5 days, followed by tamoxifen chow. Lungs were analyzed 3 weeks post-infection. Scale bar: 100 microns. (B) Percent of mice harboring keratinizing squamous cell carcinoma (SCC) 5 weeks post tumor initiation. In mice harboring the conditional alleles KrasFSF-G12D/+; RosaFSF-CreERT2; Nkx2-1F/F (controls, n = 5)) alone and in combination with Foxa1F/F; Foxa2F/F (n = 8) lung tumors were initiated with Ad5-SPC-FlpO adenovirus. In mice harboring the conditional alleles KrasLSL-G12D/+; RosaLSL-tdTomato; Nkx2-1F/F; Foxa1F/F; Foxa2F/F (n = 5) tumors were initiated with Ad5-SPC-Cre (right column). All mice were given tamoxifen 1 week after tumor initiation. Tamoxifen administration consisted of four intraperitoneal doses over 5 days, followed by tamoxifen-containing chow until the end of the experiment. *p < 0.03 vs. each control, Chi-square test.

https://doi.org/10.7554/eLife.38579.014
Model of context-specific regulation of lung cancer identity by NKX2-1, FoxA1 and FoxA2.

SCJ: squamocolumnar junction of GI tract.

https://doi.org/10.7554/eLife.38579.015

Tables

Key resources table
Reagent type
(species)
or resource
DesignationSource or
reference
IdentifiersAdditional
information
Genetic reagent
(Mus musculus)
KrasLSL-G12DPMID:
11751630
Dr. Tyler Jacks
(Massachusetts
Institute of
Technology,
Cambridge,
Massachusetts)
Genetic reagent
(M. musculus)
KrasFSF-G12DPMID:
21512139
RRID:MGI:5007794Dr. Tyler Jacks
(Massachusetts
Institute of
Technology
, Cambridge,
Massachusetts)
Genetic reagent
(M. musculus)
Rosa26LSL-tdTomatoPMID:
20023653
RRID:MGI:4436847Jackson
Laboratories
(Bar Harbor, Maine)
Genetic reagent
(M. musculus)
Rosa26FSF-CreERT2PMID:
25326799
Dr. Dieter Saur
(Technische
Universität
München,
München, Germany)
Genetic reagent
(M. musculus)
Nkx2-1F/FPMID:
16601074
RRID:MGI:3653706Dr. Shioko Kimura
(National Cancer
Institute (NCI),
National Institutes
of Health, Bethesda,
Maryland)
Genetic reagent
(M. musculus)
Foxa1F/FPMID:
19141476
RRID:MGI:3831163Dr. Klaus H.
Kaestner (University
of Pennsylvania
School of Medicine,
Philadelphia,
Pennsylvania,
USA)
Genetic reagent
(M. musculus)
Foxa2F/FPMID:
10866673
RRID:MGI:2177357Dr. Klaus H.
Kaestner (University
of Pennsylvania
School of Medicine,
Philadelphia,
Pennsylvania, USA)
Genetic reagent
(M. musculus)
CAG-LSL-HA-UPRTPMID: 23307870Jackson
Laboratories
(Bar Harbor,
Maine)
Cell line293TPMID:
19561589
AntibodyRat monoclonal
anti-BrdU
AbcamCat. #:
ab6326, RRID:
AB_305426
IHC (1:100)
AntibodyRabbit monoclonal
anti-Cadherin 13
AbcamCat. #:
ab167407
IHC (1:250)
AntibodyRabbit polyclonal
anti-Cathepsin E
Lifespan
Biosciences
Cat. #:
LS-B523, RRID:AB_2087236
IHC (1:12000)
AntibodyRabbit monoclonal
anti-Caveolin 1
AbcamCat. #: ab192869IHC (1:4000)
AntibodyRabbit monoclonal
anti-CHIL3/4
AbcamCat. #: ab192029IHC (1:20000)
AntibodyRabbit monoclonal
anti-Cleaved-
caspase 3
Cell Signaling
Technology
Cat. #: 9664IHC (1:800)
AntibodyRabbit monoclonal anti-Cytokeratin-5AbcamCat #:
ab52635
(EP1691Y)
IHC (1:400)
AntibodyRabbit monoclonal
anti-Cytokeratin-7
AbcamCat #:
ab181598
(EP17078)
IHC (1:20,000)
AntibodyRat monoclonal
anti-Cytokeratin-8
Developmental
Studies Hybridoma
Bank
Cat. #:
TROMA-I, RRID:AB_531826
IHC (1:100)
AntibodyRabbit monoclonal
anti-Cytokeratin-
14
AbcamCat. #:
ab181595
(EPR17350)
IHC (1:4000)
AntibodyRabbit monoclonal
anti-FoxA1
AbcamCat. #:
ab173287
IHC (1:4000)
AntibodyRabbit monoclonal
anti-FoxA2
AbcamCat. #:
ab108422,
RRID:AB_11157157
IHC (1:1200)
AntibodyGoat polyclonal
anti-Galectin 4
R and D SystemsCat. #:
AF2128,
RRID:AB_
2297050
IHC (1:400)
AntibodyMouse monoclonal
anti-Gastrokine 1
AbnovaCat. #:
H00056287-M01, RRID:AB_1505437
IHC (1:50)
AntibodyRabbit monoclonal
anti-GDA
AbcamCat. #:
ab210606
IHC (1:5000)
Antibody Rabbit monoclonal
anti-HNF4α
Cell Signaling
Technology
Cat. #:
3113S, RRID:AB_2295208
IHC (1:500)
AntibodyRabbit monoclonal
anti-Ki67
AbcamCat. #:
ab16667, RRID:AB_302459
IHC (1:100)
AntibodyRabbit polyclonal
anti-MCM2
AbcamCat. #:
ab31159, RRID:AB_881276
IHC (1:800)
AntibodyPolyclonal goat
anti-MMP7
R and D
Systems
Cat. #:
AF2967, RRID:AB_664120
IHC (1:400)
AntibodyMouse monoclonal
anti-Muc5AC
AbnovaCat. #:
MAB13117
IHC (1:100)
AntibodyRabbit monoclonal
anti-NKX2-1
AbcamCat. #:
ab76013, RRID:
AB_1310784
IHC (1:2000)
AntibodyMouse monoclonal
anti-p40 (∆Np63)
Biocare MedicalCat. #: ACI 3066 CIHC (1:100)
AntibodyMouse monoclonal
anti-PDX1
Developmental
Studies
Hybridoma Bank
Cat. #:
F109-D12,
RRID:AB_1
157903
IHC (1:10)
AntibodyRat monoclonal
anti-PIGR
AbcamCat. #:
ab170321
IHC (1:400)
AntibodyRabbit monoclonal
anti-PK-LR
AbcamCat. #:
ab171744
IHC (1:500)
AntibodyRabbit polyclonal
anti-proSPC
MilliporeCat. #:
AB3786, RRID:
AB_91588
IHC (1:4000)
AntibodyRabbit polyclonal
anti-RFP
RocklandCat. #:
600-401-379
IHC (1:400)
AntibodyRabbit monoclonal
anti-SOX2
Cell Signaling
Technology
Cat. #:
3728, RRID:
AB_2194037
IHC (1:250)
AntibodyRabbit monoclonal
anti-VCAM1
AbcamCat. #:
ab134047,
RRID:AB_2721053
IHC (1:1000)
Recombinant
DNA reagent
Ad5-CMVCreGene Transfer
Vector Core,
University of
Iowa, IA
VVC-U of
Iowa-5-HT
Recombinant
DNA reagent
Ad5-CMVFlpoGene Transfer
Vector Core,
University of
Iowa, IA
VVC-U of
Iowa-530HT
Recombinant
DNA reagent
Ad5-SPC-CreGene Transfer
Vector Core,
University of
Iowa, IA
VVC-Berns-
1168
Recombinant
DNA reagent
Ad5-SPC-FlpOGene Transfer
Vector
Core, University of
Iowa, IA
VVC-Snyder-
6695
Recombinant
DNA reagent
PGK-CrePMID: 19561589
Recombinant
DNA reagent
VSVgPMID: 19561589
Recombinant
DNA reagent
Δ8.9PMID: 19561589
Recombinant
DNA reagent
SPC-FlpO shuttle
plasmid
this paper
Chemical
compound, drug
TamoxifenSigma-AldrichT5648-5G
Chemical
compound, drug
Tamoxifen
supplemented
chow
EnvigoTD.130858500 mg/kg of diet
Chemical
compound, drug
1X PBSThermoFisher
Scientific
20012050
Chemical
compound, drug
TrizolThermoFisher
Scientific
15596026
Commercial
assay, kit
BloxallVector
Laboratories
SP-6000
Commercial
assay, kit
Horse serumVector
Laboratories
S-2012
 commercial
assay, kit
Rodent
Block M
Biocare
Medical
RBM961
Commercial
assay, kit
ImmPRESS
anti-rabbit HRP
Vector
Laboratories
MP-7401
Commercial
assay, kit
ImmPRESS
anti-rat HRP
Vector
Laboratories
MP-7444
Commercial
assay, kit
ImmPRESS
anti-goat HRP
Vector
Laboratories
MP-7405
Commercial
assay, kit
Anti-mouse secondaryBiocare
Medical
MM620
Commercial
assay, kit
ImmPACT DAB
Peroxidase (HRP)
Substrate
Vector
Laboratories
SK-4105
Commercial
assay, kit
ImmPACT VIP
Peroxidase (HRP)
Substrate
Vector
Laboratories
SK-4605
Commercial
assay, kit
HematoxylinFisher
Scientific
6765003
Commercial
assay, kit
Collagena
se type I
ThermoFisher
Scientific
17100017
Commercial
assay, kit
ElastaseWorthington
Biochemical
Corporation
LS002280
Commercial
assay, kit
DispaseCorning354235
Commercial
assay, kit
Deoxyribonuclease ISigma-AldrichDN25
Commercial
assay, kit
Red Blood
Cell Lysis Buffer
eBioscience00-4333-57
Commercial
assay, kit
PureLink RNA
Mini kit
ThermoFisher
Scientific
12183018A

Additional files

Supplementary file 1

List of all single cells used in analysis with quality control metrics, genotype of mouse and cluster each cell was assigned.

In cells that failed quality control, cluster is not assigned and failed QC metric(s) are highlighted.

https://doi.org/10.7554/eLife.38579.016
Supplementary file 2

Table of gene expression levels in all high quality cells used in downstream analysis.

Cells are organized by cluster (C1: gray, C2: yellow, C3: green).

https://doi.org/10.7554/eLife.38579.017
Supplementary file 3

Marker genes from each cluster (generated with S3).

Genes differentially expressed between each cluster (pairwise comparisons, SCDE).

https://doi.org/10.7554/eLife.38579.018
Supplementary file 4

Genes differentially expressed between tumor cells sorted from KrasLSL-G12D/+ mice (K, n = 3 mice) and KrasLSL-G12D/+; Nkx2-1F/F mice (KN, n = 3 mice)

https://doi.org/10.7554/eLife.38579.019
Supplementary file 5

List of normal murine tissues used and their source.

https://doi.org/10.7554/eLife.38579.020
Supplementary file 6

Cosine similarity table quantitating similarity between single cell clusters and each normal tissue evaluated.

https://doi.org/10.7554/eLife.38579.021
Transparent reporting form
https://doi.org/10.7554/eLife.38579.022

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