Cancer-associated fibroblasts (CAFs) are a heterogeneous population of mesenchymal cells supporting tumor progression, whose origin remains to be fully elucidated. Osterix (Osx) is a marker of osteogenic differentiation, expressed in skeletal progenitor stem cells and bone-forming osteoblasts. We repor

Belle, Jad I

Celik, Hamza

Civitelli, Roberto

Faccio, Roberta

Fontana, Francesca

Ricci, Biancamaria

Tycksen, Eric

English

Digital Commons@Becker

Figures and figure supplementsOsterix-Cre marks distinct subsets of CD45- and CD45+ stromal populations inextra-skeletal tumors with pro-tumorigenic characteristicsBiancamaria Ricci et alRicci et al. eLife 2020;9:e54659. DOI: https://doi.org/10.7554/eLife.54659 1 of 19RESEARCH ARTICLEFigure 1. Embryonic and adult-derived Osx+ cells are present in primary tumors at extra-skeletal sites. (A)Doxycycline (doxy)-repressible Sp7-cre/loxP mouse model used to activate Ai9/TdTomato expression for lineagetracing experiments. In no doxy-fed mice, TdT is expressed in embryonic-derived osteolineage cells (left), while inmice fed a doxy diet until weaning, TdT is expressed in adult-derived osteolineage cells. (B–I) Flow cytometryanalysis and fluorescence images of primary tumors showing presence of TdTOSX+ cells in no-doxy fed Osx-cre;TdT mice or WT;TdT controls inoculated with B16-F10 melanoma subcutaneously (B–C), or with PyMT breastcancer cells in the mammary fat pad (MFP) (D–E), and in doxy-fed mice injected with B16-F10 subcutaneously (F–G), or with PyMT in the MFP (H–I). Slides for fluorescence images were counterstained with DAPI (blue),magnification 200X. Inserts are further magnified 4.5 folds. Data representative of a single experiment,experiments were repeated between 2 and 5 times. p values represent Student t-test statistical analysis.Ricci et al. eLife 2020;9:e54659. DOI: https://doi.org/10.7554/eLife.54659 2 of 19Research article Cancer Biology Immunology and InflammationFigure 1—figure supplement 1. No difference in tumor growth and body weight between sex and age matchedcre positive (Osx-cre;TdT) and negative (WT;TdT) mice. . (A) Tumor growth in Osx-cre;TdT and WT;TdT male miceinjected subcutaneously with B16-F10 tumor cells. (B) Body weight of the mice showed in (A) at the day of tumorinjection (day 0) and at the end point (day 14 post-injection). (C) Tumor growth in Osx-cre;TdT and WT;TdT femalemice injected in the MFP with PyMT tumor cells. (D) Body weight of the mice showed in (C) on day 0 and day 14post-injection.Ricci et al. eLife 2020;9:e54659. DOI: https://doi.org/10.7554/eLife.54659 3 of 19Research article Cancer Biology Immunology and InflammationFigure 2. TdTOSX+ cells from primary tumors express mesenchymal markers. (A) Experimental design for isolatingTdTOSX+ 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 tumor cells. Immortalized CAFs (iCAFs) and crushed long boneswere used as reference controls. (B–L) Quantitative Real-Time PCR for (B) Sp7(Osterix), (C) Runx2, (D) Bglap(Osteocalcin), (E) Ibsp (Bone sialoprotein), (F) Alpl (Tnap, Tissue non-specific alkaline phosphatase), (G) S100a4(Fsp1), (H) Acta2 (a-SMA) (I) Collagen1a1 (J) Collagen1a2, (K) Mmp2 (Metalloproteinase2) and (L) Mmp9(Metalloproteinase9) genes in TdTOSX+, TdTOSX- cells, iCAFs and bone. Each data point includes cells isolatedfrom 3 to 4 mice and from two independent experiments. (M–N) Real-Time PCR of Sp7(Osterix) in B16-F10 orPyMT cell lines, CAFs isolated from primary B16-F10 or PyMT tumors or from the remaining cells comprising thetumor minus the CAFs (susp) (n = 3–4 mice). (O) Sp7(Osx) expression based on the Finak Breast Cancer Stromagene set in the Oncomine cancer microarray database. Significance was determined by one-way ANOVA withTukey post-hoc test, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.Ricci et al. eLife 2020;9:e54659. DOI: https://doi.org/10.7554/eLife.54659 4 of 19Research article Cancer Biology Immunology and InflammationFigure 3. Tumor resident but not bone marrow resident TdTOSX+ cells increase tumor growth. (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 recipientmice at the ratio 5:1. Mice injected with B16-F10 alone were used as controls. (B) Tumor growth of experimental model described in (A) determined bycaliper measurement. n = 3/group, experiment repeated twice. Significance was determined by one-way ANOVA with Tukey post-hoc test. (C)Fluorescence images of bone sections showing presence of TdTOSX+ within the bone and bone marrow of naı̈ve Osx-cre;TdT, Osx-cre;TdT miceinoculated subcutaneously with B16-F10 tumors or WT;TdT mice used as negative control. Sections were counterstained with DAPI (blue), magnification200X. (D–E) Quantification of TdTOSX+ cells in the bone marrow of tumor-free and B16-F10 tumor bearing (D) Osx-cre;TdT mice (doxy-fed) and (E) Osx-creERT2;TdT (TAM-treated) determined by FACS. Experiment repeated twice. Significance was determined by student t-test statistical analysis. (F)Tumor growth of WT mice inoculated with bone marrow-derived TdTOSX+ cells from B16-F10 bearing doxy-fed Osx-cre;TdT mice together with B16-F10 tumor cells (ratio 5:1). Mice injected with B16-F10 alone were used as controls. n = 3/group. Significance was determined by two-way ANOVAfollowed by Tukey post-hoc test. ***p<0.001, ****p<0.0001.Ricci et al. eLife 2020;9:e54659. DOI: https://doi.org/10.7554/eLife.54659 5 of 19Research article Cancer Biology Immunology and InflammationFigure 3—figure supplement 1. TdTOSX+ cells increase in the bone marrow of PyMT tumor bearing mice. (A)Fluorescence images of WT;TdT and Osx-cre;TdT mice inoculated with PyMT tumors in the MFP. Slides forfluorescence images were counterstained with DAPI (blue), magnification 200X. (B) Quantification of TdTOSX+ cellsin the bone marrow of tumor-free and PyMT bearing mice. Experiment repeated twice. p value represents Studentt-test statistical analysis.Ricci et al. eLife 2020;9:e54659. DOI: https://doi.org/10.7554/eLife.54659 6 of 19Research article Cancer Biology Immunology and InflammationFigure 3—figure supplement 2. Differential expression of mesenchymal markers in TdTOSX+ cells isolated from tumor site or bone marrow. (A–K)Quantitative Real-Time PCR for (A) Sp7 (Osterix), (B) Runx2, (C) Bglap (Osteocalcin), (D) Ibsp (Bone sialoprotein), (E) Alpl (Tnap, Tissue non-specificalkaline phosphatase), (F) S100a4 (Fsp1), (G) Acta2 (a-SMA), (H) Collagen 1a1, (I) Collagen 1a2, (J) Mmp2 (Metalloproteinase2) and (K) Mmp9(Metalloproteinase9) genes in TdTOSX+ isolated from the B16-F10 melanoma primary tumors, bone marrow of B16-F10 bearing mice and bone marrowof naı̈ve tumor-free mice.Ricci et al. eLife 2020;9:e54659. DOI: https://doi.org/10.7554/eLife.54659 7 of 19Research article Cancer Biology Immunology and InflammationFigure 4. TdTOSX+ cells express the immune surface marker CD45. (A–B) Representative FACS dot plots and quantification of TdTOSX+ cells in theperipheral blood of tumor-free and B16-F10 or PyMT tumor bearing mice. Significance was determined by student t-test statistical analysis for eachFigure 4 continued on next pageRicci et al. eLife 2020;9:e54659. DOI: https://doi.org/10.7554/eLife.54659 8 of 19Research article Cancer Biology Immunology and InflammationFigure 4 continuedtumor model relative to no tumor controls. (C–D) Representative FACS dot plots and quantification of TdTOSX+;CD45+ and TdTOSX+;CD45-populations in the blood of doxy-fed Osx-cre;TdT mice injected with B16-F10 or PyMT cells. (E–H) Representative FACS dot plots and quantification ofTdTOSX+;CD45+ and TdTOSX+;CD45- populations in the bone marrow (E–F) or tumor site (G–H) of doxy-fed Osx-cre;TdT mice injected with B16-F10 orPyMT cells, respectively. Experiments were repeated at least twice. (I) Immunohistochemistry staining of paraffin-embedded B16-F10 tumors inoculatedinto Osx-cre;TdT reporter mice, pseudo colored with red representing TdTOSX+ cells (RFP stained), green representing CD45+ cells (DAB stained) andblue representing nuclei (hematoxylin), magnification 200X.Ricci et al. eLife 2020;9:e54659. DOI: https://doi.org/10.7554/eLife.54659 9 of 19Research article Cancer Biology Immunology and InflammationFigure 4—figure supplement 1. Presence of TdTOSX+ cells in bone marrow (BM) of TAM-pulsed Osx-creERT2;TdT reporter mice. (A) Schematicrepresentation of TAM administration and tumor injection in Osx-creERT2;TdT mice. (B) Representative image of flow cytometry dot plots andquantification of TdTOSX+;CD45+ and TdTOSX+;CD45- populations in the bone marrow of B16-F10 bearing mice. A cre negative mouse was used ascontrol to set gates. Experiment was repeated three times.Ricci et al. eLife 2020;9:e54659. DOI: https://doi.org/10.7554/eLife.54659 10 of 19Research article Cancer Biology Immunology and InflammationFigure 4—figure supplement 2. Co-expression of Osx-driven TdTomato and the immune marker CD45. (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 populations. Slides were counterstained with DAPI (blue), magnification 200X. (B) Staining for CD45 on a single cell suspension frombone marrow of Osx-cre;TdT reporter mice, magnification 200X. Yellow arrows point to TdTOSX+ single cells expressing CD45, while the white arrowpoints to a TdTOSX+ cell negative for CD45.Ricci et al. eLife 2020;9:e54659. DOI: https://doi.org/10.7554/eLife.54659 11 of 19Research article Cancer Biology Immunology and InflammationFigure 5. TdTOSX+;CD45- and TdTOSX+;CD45+ are two functionally distinct populations in the tumormicroenvironment. (A) Multidimensional Principal Component Analysis of RNAseq data obtained from four tumorstroma subsets sorted according to TdTOSX and CD45 expression. (B) Venn diagram depicting uniquely andcommonly expressed genes among the four groups. (C–D) First ten GO pathways obtained from the GO analysisof the log two fold-changes for (C) CD45 negative subsets and (D) CD45 positive subsets.Ricci et al. eLife 2020;9:e54659. DOI: https://doi.org/10.7554/eLife.54659 12 of 19Research article Cancer Biology Immunology and InflammationFigure 5—figure supplement 1. Validation of RNAseq data. Real Time PCR performed on TdTOSX+;CD45+ andTdTOSX+;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 and (C) Spock2 to be upregulated in the TdTOSX+;CD45+compared to TdTOSX-;CD45+ cells.Ricci et al. eLife 2020;9:e54659. DOI: https://doi.org/10.7554/eLife.54659 13 of 19Research article Cancer Biology Immunology and InflammationFigure 6. Subsets of myeloid and lymphoid cells in the tumor microenvironment and the bone marrow are derivedfrom TdTOSX+ cells. (A–B) Quantification of FACS analysis showing the percentage of TdTOSX+;CD45+ andTdTOSX-;CD45+ populations in the tumor and bone marrow of Osx-cre;TdT mice injected subcutaneously withB16-F10 tumor cells. Data showed as % of total CD45+ cells. (C–D) Quantification of FACS analysis showing thepercentage of tumor infiltrating myeloid and lymphoid populations within the TdTOSX+;CD45+ or TdTOSX-;CD45+subsets, each considered as 100%. (E) Lymphoid over myeloid ratio within the tumor infiltrating TdTOSX+;CD45+or the TdTOSX-;CD45+ subsets. Statistical analysis was performed by student t-test. (F–G) Quantification of FACSanalysis showing the percentage of the bone marrow resident myeloid and lymphoid populations within theTdTOSX+;CD45+ or TdTOSX-;CD45+ subsets, each considered as 100%. n = 3/group. (H) Tumor growth in miceinjected with B16-F10 tumor cells alone or together with tumor-derived TdTOSX+;CD45+ or TdTOSX-;CD45+ cellsat the ratio 1:5. n = 3–6/group.Ricci et al. eLife 2020;9:e54659. DOI: https://doi.org/10.7554/eLife.54659 14 of 19Research article Cancer Biology Immunology and InflammationFigure 6—figure supplement 1. Gate strategy for immune staining of tumor and bone marrow. (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 analysis was performed onBM samples. (B) Representative gate strategy analysis of BM stained for lymphoid immune markers CD3, CD8,CD4 and NK1.1. Analysis performed in both TdTOSX+ and TdTOSX- populations. Identical analysis was performedon tumor samples. Fluorescence minus one (FMO) controls were used to set the gates. WT;TdT mice were used toset the gate for TdT+ cells.Ricci et al. eLife 2020;9:e54659. DOI: https://doi.org/10.7554/eLife.54659 15 of 19Research article Cancer Biology Immunology and InflammationFigure 7. TdTOSX+;CD45+ cells derive from TdTOSX+ HSCs. (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 injected subcutaneously at 7 weeks of age with B16-F10 tumors. In (B and D)LSK and LK subsets are shown, while in (C and E) the LSK population is further divided into HSCs and MPP1-4subsets. Red numbers represent the average of TdTOSX+ cells in each specific subset. n = 2–6/group.Ricci et al. eLife 2020;9:e54659. DOI: https://doi.org/10.7554/eLife.54659 16 of 19Research article Cancer Biology Immunology and InflammationFigure 7—figure supplement 1. Gate strategy for HSC immunostaining of bone marrow. (A) Representative gatestrategy 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;TdT mice were used to set the gate forTdT+ cells.Ricci et al. eLife 2020;9:e54659. DOI: https://doi.org/10.7554/eLife.54659 17 of 19Research article Cancer Biology Immunology and InflammationFigure 8. Sp7 transcripts and Osx protein are expressed in a subset of HSCs isolated from bone marrow of naı̈ve mice. (A) Real Time PCR analysiscomparing sorted hematopoietic stem cells (HSC), multipotent progenitors (MPP), common myeloid progenitors (CMP), common lymphoid progenitorsFigure 8 continued on next pageRicci et al. eLife 2020;9:e54659. DOI: https://doi.org/10.7554/eLife.54659 18 of 19Research article Cancer Biology Immunology and InflammationFigure 8 continued(CLP) and whole bone marrow (WBM) from 7 to 9 week old mice. FACS sorted TdTOSX- cells from the tumor of Osx-cre;TdT mice were used as negativecontrol (n = 3–8/group). Statistical analysis was determined by two-way ANOVA followed by Tukey post-hoc test. *p<0.05, **p<0.01, ***p<0.001,****p<0.0001. (B) Real Time PCR analysis of isolated mature CD3+ T cells, conventional dendritic cells (cDC) and bone marrow-derivedmacrophages (mj) from WT naı̈ve mice. (C–E) Immunofluorescence for Osx in (C) primary osteoblasts differentiated from BMSC cultured for 4 days inosteogenic media, (D) TdTOSX+ HSCs sorted from Osx-cre;TdT reporter mice and (E) whole bone marrow cells from WT mice. DAPI is used for nuclearstaining. Magnification 200X.Ricci et al. eLife 2020;9:e54659. DOI: https://doi.org/10.7554/eLife.54659 19 of 19Research article Cancer Biology Immunology and Inflammation

Osterix-cre marks distinct subsets of CD45- and CD45+ stromal populations in extra-skeletal tumors with pro-tumorigenic characteristics

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Osterix-cre marks distinct subsets of CD45- and CD45+ stromal populations in extra-skeletal tumors with pro-tumorigenic characteristics

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