(A) Doxycycline (doxy)-repressible Sp7-cre/loxP mouse model used to activate Ai9/TdTomato expression for lineage tracing experiments. In no doxy-fed mice, TdT is expressed in embryonic-derived …
Relates to FACS analysis in panels B, D, F, H.
. (A) Tumor growth in Osx-cre;TdT and WT;TdT male mice injected subcutaneously with B16-F10 tumor cells. (B) Body weight of the mice showed in (A) at the day of tumor injection (day 0) and at the …
(A) Experimental design for isolating TdTOSX+ cells from primary tumors isolated from doxy-fed Osx-cre;TdT mice inoculated with B16-F10. TdTOSX- cells comprise all TdT negative cells including the …
Relates to Real-Time PCR data.
(A) Model showing isolation of TdTOSX+ cells from B16-F10 primary tumors injected into doxy-fed Osx-cre;TdT mice and re-inoculation of TdTOSX+ cells together with B16-F10 tumor cells into WT …
(A) Fluorescence images of WT;TdT and Osx-cre;TdT mice inoculated with PyMT tumors in the MFP. Slides for fluorescence images were counterstained with DAPI (blue), magnification 200X. (B) …
Relates to FACS analysis.
(A–K) Quantitative Real-Time PCR for (A) Sp7 (Osterix), (B) Runx2, (C) Bglap (Osteocalcin), (D) Ibsp (Bone sialoprotein), (E) Alpl (Tnap, Tissue non-specific alkaline phosphatase), (F) S100a4 (Fsp1),…
Relates to Real-Time PCR.
(A–B) Representative FACS dot plots and quantification of TdTOSX+ cells in the peripheral blood of tumor-free and B16-F10 or PyMT tumor bearing mice. Significance was determined by student t-test …
Relates to FACS analysis in panels B, C, D, E, F, G, H.
(A) Schematic representation of TAM administration and tumor injection in Osx-creERT2;TdT mice. (B) Representative image of flow cytometry dot plots and quantification of TdTOSX+;CD45+ and TdTOSX+;CD…
Relates to FACS analysis.
(A) Immunofluorescence on paraffin-embedded B16-F10 tumors isolated from Osx-cre;TdT reporter mice. Staining was done using anti-RFP (red) to detect TdTomato and anti-CD45 (green) to stain immune …
(A) Multidimensional Principal Component Analysis of RNAseq data obtained from four tumor stroma subsets sorted according to TdTOSX and CD45 expression. (B) Venn diagram depicting uniquely and …
Real Time PCR performed on TdTOSX+;CD45+ and TdTOSX+;CD45- populations sorted from B16-F10 tumors in doxy-fed mice. Each sample is a pool of two tumors. Data show three genes (A) Cd163l1, (B) Wdr79 …
Relates to Real-Time PCR data.
(A–B) Quantification of FACS analysis showing the percentage of TdTOSX+;CD45+ and TdTOSX-;CD45+ populations in the tumor and bone marrow of Osx-cre;TdT mice injected subcutaneously with B16-F10 …
Relates to FACS analysis.
(A) Representative gate strategy analysis of total tumor mass stained for CD45, myeloid immune markers CD11b, F4/80 and Gr1. Analysis performed in both TdTOSX+ and TdTOSX- populations. Identical …
(A) Schematic of hematopoietic differentiation. (B–E) Flow cytometry quantification of TdTOSX+ cells in the bone marrow of (B–C) doxy-fed Osx-cre;TdT mice and (D–E) TAM-treated Osx-creERT2;TdT mice …
Relates to FACS analysis.
(A) Representative gate strategy analysis of bone marrow stained for HSC subsets. TdTOSX+ cells were quantified in each subset. Fluorescence minus one (FMO) controls were used to set the gates. WT;Td…
(A) Real Time PCR analysis comparing sorted hematopoietic stem cells (HSC), multipotent progenitors (MPP), common myeloid progenitors (CMP), common lymphoid progenitors (CLP) and whole bone marrow …
Relates to Real-Time PCR in panels A, B.
List of the 50 most highly expressed genes in the TdTOSX+;CD45- cells compared to the TdTOSX-;CD45- cells isolated from the TME.
external_gene_name | description | TdTOSX+CD45- logFC | TdTOSX+CD45- linearFC |
---|---|---|---|
Reg1 | regenerating islet-derived 1 | 7.675801 | 204.477899 |
Fbln7 | fibulin 7 | 7.184048 | 145.416614 |
Adcyap1r1 | adenylate cyclase activating polypeptide 1 receptor 1 | 6.864422 | 116.51905 |
Col24a1 | collagen, type XXIV, alpha 1 | 6.788179 | 110.521192 |
Moxd1 | monooxygenase, DBH-like 1 | 6.74623 | 107.35382 |
Fmod | fibromodulin | 6.739645 | 106.864989 |
4930562D21Rik | RIKEN cDNA 4930562D21 gene | 6.149924 | 71.008705 |
Angpt4 | angiopoietin 4 | 6.131627 | 70.113799 |
Tmem132c | transmembrane protein 132C | 5.933002 | 61.095844 |
Frem1 | Fras1 related extracellular matrix protein 1 | 5.906166 | 59.969876 |
Cxcl5 | chemokine (C-X-C motif) ligand 5 | 5.88881 | 59.252728 |
Ccl19 | chemokine (C-C motif) ligand 19 | 5.748355 | 53.756054 |
Rab15 | RAB15, member RAS oncogene family | 5.696057 | 51.842269 |
Prlr | prolactin receptor | 5.673651 | 51.04334 |
Msx1 | msh homeobox 1 | 5.657661 | 50.480738 |
Pabpc4l | poly(A) binding protein, cytoplasmic 4-like | 5.657301 | 50.46814 |
Reg3g | regenerating islet-derived 3 gamma | 5.651841 | 50.2775 |
Rflna | refilin A | 5.651765 | 50.274843 |
Adamts3 | a disintegrin-like and metallopeptidase (reprolysin type) with thrombospondin type 1 motif, 3 | 5.52646 | 46.092506 |
Lrp3 | low density lipoprotein receptor-related protein 3 | 5.465865 | 44.196651 |
Clstn2 | calsyntenin 2 | 5.41628 | 42.703438 |
Adam5 | a disintegrin and metallopeptidase domain 5 | 5.36495 | 41.210775 |
H2-M11 | histocompatibility 2, M region locus 11 | 5.335531 | 40.378937 |
Ptx3 | pentraxin related gene | 5.320565 | 39.962237 |
Rtl3 | retrotransposon Gag like 3 | 5.252916 | 38.131634 |
Gm26682 | predicted gene, 26682 | 5.252803 | 38.128648 |
Morc1 | microrchidia 1 | 5.251594 | 38.096689 |
Hoxc6 | homeobox C6 | 5.176329 | 36.160165 |
Mcpt8 | mast cell protease 8 | 5.146576 | 35.422062 |
Gabrb3 | gamma-aminobutyric acid (GABA) A receptor, subunit beta 3 | 5.110873 | 34.556218 |
Sbspon | somatomedin B and thrombospondin, type 1 domain containing | 5.085426 | 33.952042 |
Akap6 | A kinase (PRKA) anchor protein 6 | 5.073102 | 33.663235 |
Hmcn1 | hemicentin 1 | 5.060346 | 33.366905 |
Aqp2 | aquaporin 2 | 5.031264 | 32.701036 |
Gria3 | glutamate receptor, ionotropic, AMPA3 (alpha 3) | 5.011924 | 32.265589 |
AI464131 | expressed sequence AI464131 | 5.001414 | 32.03138 |
Muc13 | mucin 13, epithelial transmembrane | 4.997132 | 31.936452 |
Syt4 | synaptotagmin IV | 4.994666 | 31.881898 |
Spon1 | spondin 1, (f-spondin) extracellular matrix protein | 4.916033 | 30.190712 |
Caln1 | calneuron 1 | 4.85996 | 29.039801 |
Hoxc4 | homeobox C4 | 4.844155 | 28.723415 |
Tbx2 | T-box 2 | 4.842042 | 28.681376 |
Jph2 | junctophilin 2 | 4.816833 | 28.18456 |
Frzb | frizzled-related protein | 4.79214 | 27.706253 |
Galnt5 | polypeptide N-acetylgalactosaminyltransferase 5 | 4.77782 | 27.432606 |
Gm29100 | predicted gene 29100 | 4.77255 | 27.332593 |
Tspyl5 | testis-specific protein, Y-encoded-like 5 | 4.771509 | 27.312867 |
Spag17 | sperm associated antigen 17 | 4.770653 | 27.296671 |
Ldoc1 | regulator of NFKB signaling | 4.7479 | 26.86954 |
4833422C13Rik | RIKEN cDNA 4833422C13 gene | 4.730678 | 26.550693 |
List of the 50 most highly expressed genes in the TdTOSX+;CD45+ cells compared to the TdTOSX-;CD45+ cells isolated from the TME.
external_gene_ name | description | TdTOSX+CD45+ logFC | TdTOSX+CD45+ linearFC |
---|---|---|---|
Cd163l1 | CD163 molecule-like 1 | 6.482112 | 89.394369 |
Ltb | lymphotoxin B | 6.217037 | 74.389998 |
Klrg1 | killer cell lectin-like receptor subfamily G, member 1 | 5.915697 | 60.367363 |
Foxp3 | forkhead box P3 | 5.882423 | 58.991007 |
Trbv19 | T cell receptor beta, variable 19 | 5.83901 | 57.242322 |
Cd5 | CD5 antigen | 5.822965 | 56.609196 |
Gm4759 | predicted gene 4759 | 5.712238 | 52.426999 |
Tcrg-C1 | T cell receptor gamma, constant 1 | 5.709921 | 52.342868 |
Icos | inducible T cell co-stimulator | 5.588856 | 48.129715 |
Gm19585 | predicted gene, 19585 | 5.577582 | 47.755056 |
Fasl | Fas ligand (TNF superfamily, member 6) | 5.571314 | 47.548033 |
Rln3 | relaxin 3 | 5.518062 | 45.824955 |
Ubash3a | ubiquitin associated and SH3 domain containing, A | 5.432308 | 43.180511 |
Pcsk1 | proprotein convertase subtilisin/kexin type 1 | 5.376329 | 41.537117 |
Ccr8 | chemokine (C-C motif) receptor 8 | 5.32423 | 40.06386 |
Gimap3 | GTPase, IMAP family member 3 | 5.257508 | 38.253185 |
Slamf1 | signaling lymphocytic activation molecule family member 1 | 5.220641 | 37.288037 |
Klrb1f | killer cell lectin-like receptor subfamily B member 1F | 5.204067 | 36.86212 |
Ikzf3 | IKAROS family zinc finger 3 | 5.185332 | 36.386518 |
Trdv4 | T cell receptor delta variable | 5.179288 | 36.234394 |
Trat1 | T cell receptor associated transmembrane adaptor 1 | 5.144648 | 35.374755 |
Pdcd1 | programmed cell death 1 | 5.092017 | 34.107504 |
Izumo1r | IZUMO1 receptor, JUNO | 5.053635 | 33.212045 |
Cd3e | CD3 antigen, epsilon polypeptide | 5.042174 | 32.949246 |
Itk | IL2 inducible T cell kinase | 5.027182 | 32.608632 |
Ctla4 | cytotoxic T-lymphocyte-associated protein 4 | 4.978043 | 31.516668 |
Actn2 | actinin alpha 2 | 4.919967 | 30.27316 |
Trbv1 | T cell receptor beta, variable 1 | 4.891164 | 29.674756 |
Dkkl1 | dickkopf-like 1 | 4.820628 | 28.258804 |
Cd300e | CD300E molecule | 4.801355 | 27.883791 |
Pglyrp2 | peptidoglycan recognition protein 2 | 4.80032 | 27.863797 |
Cd226 | CD226 antigen | 4.766441 | 27.21709 |
Themis | thymocyte selection associated | 4.760082 | 27.097397 |
Dpep3 | dipeptidase 3 | 4.694473 | 25.892693 |
Cd209a | CD209a antigen | 4.650261 | 25.111233 |
Cd27 | CD27 antigen | 4.648725 | 25.084512 |
Gpr55 | G protein-coupled receptor 55 | 4.621113 | 24.608975 |
Ccr6 | chemokine (C-C motif) receptor 6 | 4.596004 | 24.18439 |
Gpr174 | G protein-coupled receptor 174 | 4.594683 | 24.16225 |
A630023P12Rik | RIKEN cDNA A630023P12 gene | 4.586499 | 24.025577 |
Cd28 | CD28 antigen | 4.564255 | 23.657981 |
Cd3g | CD3 antigen, gamma polypeptide | 4.547609 | 23.386577 |
Lta | lymphotoxin A | 4.534293 | 23.171712 |
Ccr3 | chemokine (C-C motif) receptor 3 | 4.526527 | 23.047318 |
Gzmb | granzyme B | 4.518658 | 22.921957 |
Lrrc66 | leucine rich repeat containing 66 | 4.47147 | 22.184347 |
Xirp2 | xin actin-binding repeat containing 2 | 4.462937 | 22.053513 |
St8sia1 | ST8 alpha-N-acetyl-neuraminide alpha-2,8-sialyltransferase 1 | 4.460924 | 22.022769 |
Sit1 | suppression inducing transmembrane adaptor 1 | 4.417363 | 21.367754 |
Tnfrsf4 | tumor necrosis factor receptor superfamily, member 4 | 4.381868 | 20.84844 |
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Gene (Mus musculus) | Sp7(Osx) | NCBI Gene | RRID:MGI:2153568 | NCBI ID:170574 |
Genetic reagent (Mus musculus) | Tg(Sp7-tTA,tetO-EGFP/cre)1Amc/J (Osx-cre) | The Jackson Laboratories | Cat#006361 RRID:MGI:3689350 | C57Bl/6 |
Genetic reagent (Mus musculus) | B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J (TdT) | The Jackson Laboratories | RRID:MGI3813511 | C57Bl/6 |
Genetic reagent (Mus musculus) | Sp7-creERT2/Ai9tdTomato (Osx-creERT2;TdT) | PMID:31768488 | Cat#007909 RRID:MGI:4829803 | Prof. Silva MJ (Washington University in St Louis) C57Bl/6 |
Cell line (Mus musculus, C57Bl/6) | B16-F10 melanoma cell line | ATCC | RRID:CRL-6475-LUC2 | C57Bl/6 |
Cell line (Mus musculus) | PyMT breast cancer cell line | PMID:27216180 | Prof. Weilbaecher KN (Washington University in St Louis) C57Bl/6 | |
Cell line (Mus musculus) | Immortalized Cancer Associated Fibroblast cell line | PMID:27264173 | Prof Longmore GD (Washington University in St Louis) | |
Antibody | Rat anti-mouse CD45-APCeFluor780(clone 30-F11) | eBioscience | Cat #47-0451-82 RRID:AB_1548781 | FACS (1:400) |
Antibody | Rat anti-mouse CD45-PE Cy7 (clone 30-F11) | eBioscience | Cat #25-0451-82 RRID:AB_2734986 | FACS (1:400) |
Antibody | Rat anti-mouse CD11b-AlexaFluor700 (clone M1/70) | eBioscience | Cat#56-0112-82 RRID:AB_657585 | FACS (1:400) |
Antibody | Rat anti-mouse Gr1(Ly6G)-FITC (clone RB6-8C5) | Miltenyi Biotec | Cat#130-102-837 RRID:AB_2659858 | FACS (1:100) |
Antibody | Rat anti-mouse F4/80 PerCP Cy5.5 (clone BM8) | Biolegend | Cat#123126 RRID:AB_10802654 | FACS (1:400) |
Antibody | Hamster anti-mouse CD3e-PE (clone 145–2 C11) | eBioscience | Cat#A14714 RRID:AB_2534230 | FACS (1:200) |
Antibody | Rat anti-mouse CD4-APC (clone RM4-5) | BD Biosciences | Cat#553051 RRID:AB_398528 | FACS (1:200) |
Antibody | Rat anti-mouse CD8a-FITC (clone 53–6.7) | BD Biosciences | Cat#561966 RRID:AB_10896291 | FACS (1:200) |
Antibody | Mouse anti-mouse NK1.1 (clone PK136) | Biolegend | Cat#108722 RRID:AB_2132712 | FACS (1:200) |
Antibody | Rat anti-mouse CD45R (B220)-Biotin (clone RA3-6B2) | eBioscience | Cat#13-0452-82 RRID:AB_466449 | FACS (0.4 ul/sample) |
Antibody | Hamster anti-mouse CD3e-Biotin (clone 145–2 C11) | eBioscience | Cat#13-0031-82 RRID:AB_466319 | FACS (0.4 ul/sample) |
Antibody | Rat anti-mouse Ter119 (clone TER119) | eBioscience | Cat# 13-5921-82 RRID:AB_466797 | FACS (0.4 ul/sample) |
Antibody | Rat anti-mouse Ly-6G/Ly-6C Biotin (clone RB6-8C5), | eBioscience | Cat#13-5931-82 RRID:AB_466800 | FACS (0.2 ul/sample) |
Antibody | Rat anti-mouse CD41Biotin (clone MWReg30) | eBioscience | Cat#13-0411-82 RRID:AB_763484 | FACS (1 ul/sample) |
Antibody | Streptavidin-BV510 | BD Biosciences | Cat#563261 | FACS (0.5 ul/sample) |
Antibody | Rat anti-mouse Sca-1 BV711 (clone D7) | BD Biosciences | Cat#563992 RRID:AB_2738529 | FACS (0.25 ul/sample) |
Antibody | Rat anti-mouse c-kit(CD117)-APCeFluor780 (clone ACK2) | eBioscience | Cat#47-1172-82 RRID:AB_1582226 | FACS (1 ul/sample) |
Antibody | Rat anti-mouse CD16/32-BUV395 (clone 2.4G2) | BD Biosciences | Cat#740217 RRID:AB_2739965 | FACS (0.5 ul/sample) |
Antibody | Rat anti-mouse CD34-FITC (clone RAM34) | eBioscience | Cat#11-0341-82 RRID:AB_465021 | FACS (4 ul/sample) |
Antibody | Rat anti-mouse CD150(SLAM)-BV421 (clone TC15-12F12.2) | Biolegend | Cat#115943 RRID:AB_2650881 | FACS (1 ul/sample) |
Antibody | Hamster anti-mouse CD48-PE Cy7 (clone HM48-1) | eBioscience | Cat#25-0481-80 RRID:AB_1724087 | FACS (0.15 ul/sample) |
Antibody | Rat anti-mouse CD135(Flt3)-APC (clone A2F10) | eBioscience | Cat#17-1351-82 RRID:AB_10717261 | FACS (2 ul/sample) |
Antibody | Rat anti-mouse CD45 (clone 30-F11) | Invitrogen | Cat#14-0451-82 RRID:AB_467251 | IF, IHC (1:200) |
Antibody | Rabbit anti-mouse RFP/TdT | Rockland | Cat#600-401-379 RRID:AB_2209751 | IF,IHC (1:500) |
Antibody | Goat-anti-Rat IgG-Biotin | Thermo Fisher | Cat#31830 RRID:AB_228355 | IF, IHC (1:500) |
Antibody | Streptavidin-HRP | Bio-Rad | Cat#STAR5B | IF, IHC (1:500) |
Antibody | Goat-anti-rabbit Alexa-fluor647 | Thermo Fisher | Cat#A21244 RRID:AB_2535812 | IF (4 ug/ml) |
Antibody | Rabbit anti-mouse Sp7/Osx | Abcam | Cat#ab227820 | IF (1:500) |
Antibody | Goat anti-rabbit AlexaFluor488 | Abcam | Cat#ab150077 RRID:AB_2630356 | IF (1:1000) |
Sequence-based reagent | Cyclophilin | This paper | PCR primers | 5’-AGC ATA CAG GTC CTG GCA TC-3’ and 5’-TTC ACC TTC CCA AAG ACC AC-3’ |
Sequence-based reagent | Sp7(Osx) | This paper | PCR primers | 5’-AAG GGT GGG TAG TCA TTT GCA-3’ and 5’-CCC TTC TCA AGC ACC AAT GG-3’ |
Sequence-based reagent | S100A4(Fsp-1) | This paper | PCR primers | 5’-TGA GCA ACT TGG ACA GCA ACA-3’ and 5’-TTC CGG GGT TCC TTA TCT GGG-3’ |
Sequence-based reagent | Acta2(α-SMA) | This paper | PCR primers | 5’-GTC CCA GAC ATC AGG GAG TAA-3’ and 5’-TCG GAT ACT TCA GCG TCA GGA-3’ |
Sequence-based reagent | Col1a1 | This paper | PCR primers | 5’-GGC CTT GGA GGA AAC TTT GC-3’ and 5’-GGG ACC CAT TGG ACC TGA AC-3’ |
Sequence-based reagent | Bglap(Ocn) | This paper | PCR primers | 5’-GGA CTG AGG CTC TGT GAG GT-3’ and 5’-CAG ACA CCA TGA GGA CCA TC-3’ |
Sequence-based reagent | Runx2 | This paper | PCR primers | 5’-GTT ATG AAA AAC CAA GTA GCC AGG-3’ and 5’-GTA ATC TGA CTC TGT CCT TGT GGA-3’ |
Sequence-based reagent | BSP | This paper | PCR primers | 5’-AGG ACT AGG GGT CAA ACA C-3’ and 5’-AGT AGC GTG GCC GGT ACT TA-3’ |
Sequence-based reagent | TNAP | This paper | PCR primers | 5’-GGG GAC ATG CAG TAT GAG TT-3’ and 5’-GGC CTG GTA GTT GTT GTG AG-3’ |
Commercial assay or kit | TSA FITC System | Perkin Elmer | Cat#NEL701A001KT | |
Commercial assay or kit | BOND Intense R detection kit | Leica Biosystem | Cat#DS9263 | |
Commercial assay or kit | BOND Polymer Refine Red detection kit | Leica Biosystem | Cat#DS9390 | |
Commercial assay or kit | Avidin/Biotin Blocking kit | Vector Labs | Cat#SP2001 RRID:AB_2336231 | |
Software, algorithm | STAR | STAR | RRID:SCR_015899 | Version 2.0.4b |
Software, algorithm | Subread | Subread | RRID:SCR_009803 | Version 1.4.5 |
Software, algorithm | R | R | RRID:SCR_001905 | Version 3.4.1 |
Software, algorithm | EdgeR | EdgeR | RRID:SCR_012802 | Version 3.20.2 |
Software, algorithm | LIMMA | LIMMA | RRID:SCR_010943 | Version 3.34.4 |
Software, algorithm | GAGE | GAGE | RRID:SCR_017067 | Version 2.28.0 |
Other | VECTASHIELD Mounting Medium with DAPI | Vector Labs | Cat#H1200 RRID:AB_2336790 | one drop |
Other | ProLong Gold with DAPI | Invitrogen | Cat#P10144 | one drop |