Journal: bioRxiv

Article Title: Phenotypic plasticity of ER+ breast cancer in the bone microenvironment

doi: 10.1101/2020.11.14.383000

Figure Lengend Snippet: Osteogenic cells promote loss of ER expression and reduction of ER activities during early stages of bone colonization A. Representative confocal image of bone metastasis showing a negative association between ER expression in cancer cells and alkaline phosphatase (ALP) expression in osteogenic cells. ER, ALP and nuclei (DAPI) are shown as yellow, red, and blue, respectively. Scale bars : 50µM B. Dot plot showing quantification of ER expression in MCF7 and two PDX (HCI011 and WHIM9) models of IIA-induced bone metastasis. Alkaline phosphatase (ALP) was used to identify osteogenic cells (red). Bone metastases were classified as Low or High according to ALP expression in surrounding osteogenic cells as depicted in Figure 2A. Each dot represents a cell, and 3-5 animals were combined. C. Graph representing the paired analysis of ER expression according to alkaline phosphatase (ALP) enrichment in the tumor microenvironment as shown in A. Mean expression of ER was obtained by averaging single cell data from 3-5 different mice. P-values derive from two-tailed paired Student’s t -test. D. Representative IF images of HCI011-derived primary cells and MCF7 cells in 3D monoculture and co-culture with human fetal osteoblast cell line (FOB) and mesenchymal stem cell line (MSC). ER, keratin 8 (K8) and nuclei (DAPI) are represented in red, grey, and blue, respectively. Scale bars: 100µm. E. Heatmap showing the mean intensity of ER in primary cells (HCI011) and breast cancer cell lines (MDA-MB-361, MCF7, ZR75-1, T47D, ZR75-30) in 3D monoculture (control) or co-culture with osteoclast precursors (U937), bone marrow stromal cells (Hs5), mouse pre-osteoblasts (MC3T3), human mesenchymal stem cells (MSC) and human pre-osteoblast (FOB). All co-cultures were performed in triplicate and images were captured with a 40x oil objective lens. F. Graph comparing ER expression in monoculture versus co-culture of multiple cell lines with FOB. P-value results from a two-tailed paired Student’s t -test. 3 separated experiments were used. Error bars: mean +/- standard deviation. G. Relative mRNA expression of ESR1 in 3D monoculture or co-culture of MCF7 with FOB. Data result from MCF7 cells only (FACS sorted). H. Dot plot representing ER transcriptional activity in MCF7 cells expressing pGL2 ERE-luciferase reporter. MCF7 cells were cultured in 3D with or without osteogenic cells (FOB and MSC) for 7 days. Luciferase activity was assessed using IVIS Lumina II in vivo system; n=10 technical replicates. Error bars: mean +/- standard deviation. I. Representative confocal images showing ER expression (red) in MCF7 single cell-derived populations (SCPs) in 3D monoculture or co-culture with FOB cells. Vimentin (VIM), Keratin 8 (CK8), and DAPI were used to identify osteoblasts (Green), cancer cells (grey) and cell nuclei (blue), respectively. Scale bars: 50µm. J. Quantified ER expression from confocal images of single cell-derived populations (SCP1-SCP4). A two-tailed paired Student’s t -test analysis reveals a strong negative correlation (r) between monoculture and FOB co-cultures (right panel n=5). Error bars: +/- standard error of the mean. Each dot represents a cell from 3 different images. K. Diagram showing the experimental design for positron emission tomography–computed tomography (PET-CT) imaging of MCF7 cells transplanted orthotopically or to bone via IIA injection. Two rounds of imaging were performed at week 1 and week 5 post transplantation using 18F-Fluoroestradiol (18F-FES) and 18F-Fluorodeoxyglucose (18F-FDG) with 2 days apart. L. Representative PET/CT scans showing the maximum intensity projection (MIP) visualization of radiolabeled 18F-Fluorodeoxyglucose (18F-FDG) and 18F-Fluoroestradiol (18F-FES) in bone. Early time point (Week 1) and late time point (Week 7) were used to depict the micro-metastasis stage (small) and the macrometastasis stage (large). MCF7 bone metastases were generated using Intra-iliac artery injection. Red arrows indicate tumor location (Joint area). A smaller scale (0.2-0.5 SUV-bw) was used for week 1 images to allow detection of small lesions while a scale of 100-200 SUV-bw was used for the macrometastasis stage. M. Axial view of representative PET/CT scans depicting the uptake of radiolabeled fluorodeoxyglucose (18F-FDG) and fluoroestradiol (18F-FES) in small and large lesions of MCF7 orthotopic tumors. Early time point (Week 1) and late time point (Week 7) were used to depict non palpable orthotopic tumor stage (small < 2mm) and the palpable tumor stage. Red arrows indicate expected tumor location (mammary gland). Color scales for early lesions (Week 1): 0.2-0.5 SUV-bw; Color scales for large lesions (Week 7): 100-200 SUV-bw. N. Relative quantification of radiolabeled 18F-FES uptake in small and large lesions of orthotopic and bone metastases. Each dot represents the mean standard uptake values (mean SUV-bw) of 18F-FES normalized to the mean SUV of 18F-FDG. Mann Whitney U -test is used for statistical analysis. n=5 mice per group.

Article Snippet: Human estrogen receptor positive (ER+) breast cancer cell lines MCF7, T47D, MDA-MB-361 and ZR75-30, pre-osteoblast cells hFOB-1.19, mesenchymal stem cells MSC, pre-osteoclast U937 and the mouse pre-osteoblast MC3T3-E1 were purchased from American Type Culture Collection (ATCC).

Techniques: Expressing, Two Tailed Test, Derivative Assay, Co-Culture Assay, Control, Standard Deviation, Activity Assay, Luciferase, Cell Culture, In Vivo, Positron Emission Tomography, Computed Tomography, Positron Emission Tomography-Computed Tomography, Imaging, Injection, Transplantation Assay, Generated, Quantitative Proteomics, MANN-WHITNEY

Journal: bioRxiv

Article Title: Phenotypic plasticity of ER+ breast cancer in the bone microenvironment

doi: 10.1101/2020.11.14.383000

Figure Lengend Snippet: Osteogenic cell-secreted FGFs and PDGFs promote endocrine resistance and bone addiction. A. Network depicting functional protein association between FGFR1, PDGFRB and ER using the STRING database. K means clustering ( k =3) was used to represent 3 major centroids (depicted as red, green, and cyan spheres) and their most closely associated proteins based on unsupervised data mining. B. Graph showing the gene expression of human FGFs in osteogenic cells (FOB). The data derives from RNA sequencing data of osteogenic cells (FOB). n= 3 technical replicates C. Representative IF images showing decreased ER expression (yellow) in tumors established in FGF2 (green) enriched bone microenvironments. Keratin 8 (red) is used to identify breast cancer cells. The scatter dot plot represents ER quantification from tumors according to FGF2 enrichment (Low and High) in adjacent stromal cells (n=3-4 samples). Mean expression is represented in blue. D. Graph depicting the inhibitory effect of recombinant FGF2 (20ng/ml) on ER expression in MCF7, ZR75-1 and T47D after 24h treatment. Data represent normalized ER over β-actin protein expression from 3 separate immunoblotting experiments. E. Bone-In-Culture-Array (BICA) assay showing synergistic effects between 2.5µM FGF2 inhibitor (BGJ398) and 20nM fulvestrant in MCF7, ZR75-1 and T47D models. F. Graph depicts the inhibitory effect of indicated PDGF recombinants on ER expression in MCF7 and ZR75-1 after 24h treatment. Data represent normalized ER over β-actin protein expression from Immunoblot quantification. G. Violin plot showing the effect of 20ng/ml PDGF recombinants (PDGF-BB, PDGF-CC, PDGF-DD) on bone-entrained MCF7 (MCF7-Bo) and SCP2 (SCP2-Bo) response to fulvestrant. BLI was assessed after 72h of treatment. H. Dot plot showing the effect of the pan FGFR inhibitor (BGJ398), and the PDGFRβ inhibitor (sunitinib) on osteoblast (FOB)- mediated MCF7 and ZR75-1 cell growth in 3D. I. Scatter plot showing negative Pearson correlation (r) between FGFR1, FGFR2, PDGFRA, PDGFRB, and ESR1 expression in clinical specimens of bone metastasis. n= 11 bone metastasis samples. P values: two tailed Paired Sample t Test.

Article Snippet: Human estrogen receptor positive (ER+) breast cancer cell lines MCF7, T47D, MDA-MB-361 and ZR75-30, pre-osteoblast cells hFOB-1.19, mesenchymal stem cells MSC, pre-osteoclast U937 and the mouse pre-osteoblast MC3T3-E1 were purchased from American Type Culture Collection (ATCC).

Techniques: Functional Assay, Gene Expression, RNA Sequencing, Expressing, Recombinant, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: Phenotypic plasticity of ER+ breast cancer in the bone microenvironment

doi: 10.1101/2020.11.14.383000

Figure Lengend Snippet: The bone microenvironment induces transient loss of ER expression in ER+ breast cancer cells. A. Representative H&E staining of spontaneous metastases of HCI011 and WHM9 tumors to spine and hind limb, respectively. Scale bar: 100μm. B. Human-specific ER IHC staining are shown for spontaneous metastasis of HCI011 and WHIM9, respectively. Bone matrix is annotated. Scale bar: 50μm. C. Confocal images showing immunofluorescence (IF) staining of ER (green), keratin 8 [k8] (red), and DAPI (blue) in orthotopic (mammary) and IIA-induced bone metastasis models of ER+ PDXs (HCI011 and WHIM9). Scale bars: 100µM. Representative images were captured with a 40x oil objective lens D. Dot plot depicting quantification of nuclear ER intensity from HCI011 primary tumor and bone metastasis specimens as illustrated in Figure 1A. Bone lesions were classified into “small” and “big” groups based on cell numbers captured by a same field with the cutoffs being < median – 0.5xS.D. (small) or > median + 0.5xS.D. (big). Each dot represents the fluorescence intensity of ER of a single cells. Cells from 3-5 different animals are plotted. E. Dot plot depicting quantification of nuclear ER intensity from WHIM9 primary tumor and bone metastasis specimens as illustrated in Figure 1A. Bone lesions were classified into “small” and “big” groups as defined in (B). Each dot represents the fluorescence intensity of ER of a single cells. Cells from 3-5 different animals are plotted. F. Representative IF images of MCF7 cells following orthotopic and bone transplantation in nude mice. Changes in ER expression are illustrated in primary tumor and bone metastasis at different stages of tumor progression. Early, intermediate and late phases are depicted from left to right. Green, red and blue represent IF staining of estrogen receptor (ER), cytokeratin (K8) and nucleus (DAPI). Scale bars, 50μm. G. Dot plot depicting quantification of nuclear ER intensity from MCF7 primary tumor and bone metastasis specimens as illustrated in (D). Bone lesions were classified into “small” and “big” as defined in (B). Each dot represents the fluorescence intensity of ER of a single cells. Cells from 6 different animals are plotted. H. Dot plot depicting quantification of nuclear ER intensity from MCF7 single cell-derived population 2 (SCP2) primary tumor and bone metastasis specimens. Bone lesions were classified into “small” and “big” groups as defined in (B). Each dot represents the fluorescence intensity of ER of a single cells. Cells from 4 different animals are plotted. I. Dot plot showing the mean-normalized ER intensity of all cancer models using from Figure 1A to 1F. P-values derive from a two-tailed paired Student’s t -test. J. Boxplot showing changes in ESR1 early signature in matched bone metastases and primary specimens from breast cancer patients (( https://github.com/npriedig/ ). All images were captured with Leica TCS SP5 confocal microscope. A 40x or 63x oil objective lens were used to capture all images (Immersion oil refractive index n=1.51). All quantifications were performed using ImageJ (Fiji). All statistical analyses represent a two-tailed unpaired Student’s t -test except when specified otherwise.

Article Snippet: Human estrogen receptor positive (ER+) breast cancer cell lines MCF7, T47D, MDA-MB-361 and ZR75-30, pre-osteoblast cells hFOB-1.19, mesenchymal stem cells MSC, pre-osteoclast U937 and the mouse pre-osteoblast MC3T3-E1 were purchased from American Type Culture Collection (ATCC).

Techniques: Expressing, Staining, Immunohistochemistry, Immunofluorescence, Fluorescence, Transplantation Assay, Derivative Assay, Two Tailed Test, Microscopy, Refractive Index

Journal: bioRxiv

Article Title: Phenotypic plasticity of ER+ breast cancer in the bone microenvironment

doi: 10.1101/2020.11.14.383000

Figure Lengend Snippet: A. ER IHC staining are shown for spontaneous metastasis of WHIM9, respectively. Representative pictures of metastatic lesions of different sizes are shown. Scale bar: 50μm. B. ER IHC staining of a macroscopic spontaneous metastasis is shown with bone matrix annotated. Scale bar, 100μm. C. Diagram representing key procedures for intra-iliac artery injection (to generate bone metastasis) from freshly harvested orthotopic PDX models. D. Scatter plots showing Pearson correlation (r) between bone metastasis sizes (cell count) and ER IF intensity. Images were acquired with 40x oil objective lens (Leica TCS SP5 confocal microscope). Each dot represents an image (n=22 for HCI011; n=21 for WHIM9; n=34 for MCF7; n=12 for SCP2) E. Scatter plots showing Pearson correlations (r) between orthotopic tumor sizes (cell count) and ER IF intensity. Images were acquired with 40x oil objective lens (Leica TCS SP5 confocal microscope). Each dot represents an image (n=8 for HCI011; n=6 for WHIM9; n= 7 For MCF7; n=9 for SCP2). P values: two tailed Paired Sample t Test. F. Representative images of orthotopic and IIA-induces bone metastasis from MCF7 and MCF7 single cell-derived population (SCP1-4). From the ventral view, right tumors show orthotopic tumors while left tumors are bone metastases.

Article Snippet: Human estrogen receptor positive (ER+) breast cancer cell lines MCF7, T47D, MDA-MB-361 and ZR75-30, pre-osteoblast cells hFOB-1.19, mesenchymal stem cells MSC, pre-osteoclast U937 and the mouse pre-osteoblast MC3T3-E1 were purchased from American Type Culture Collection (ATCC).

Techniques: Immunohistochemistry, Injection, Cell Counting, Microscopy, Two Tailed Test, Derivative Assay

Journal: bioRxiv

Article Title: Phenotypic plasticity of ER+ breast cancer in the bone microenvironment

doi: 10.1101/2020.11.14.383000

Figure Lengend Snippet: Osteogenic cells promote loss of ER expression and reduction of ER activities during early stages of bone colonization A. Representative confocal image of bone metastasis showing a negative association between ER expression in cancer cells and alkaline phosphatase (ALP) expression in osteogenic cells. ER, ALP and nuclei (DAPI) are shown as yellow, red, and blue, respectively. Scale bars : 50µM B. Dot plot showing quantification of ER expression in MCF7 and two PDX (HCI011 and WHIM9) models of IIA-induced bone metastasis. Alkaline phosphatase (ALP) was used to identify osteogenic cells (red). Bone metastases were classified as Low or High according to ALP expression in surrounding osteogenic cells as depicted in Figure 2A. Each dot represents a cell, and 3-5 animals were combined. C. Graph representing the paired analysis of ER expression according to alkaline phosphatase (ALP) enrichment in the tumor microenvironment as shown in A. Mean expression of ER was obtained by averaging single cell data from 3-5 different mice. P-values derive from two-tailed paired Student’s t -test. D. Representative IF images of HCI011-derived primary cells and MCF7 cells in 3D monoculture and co-culture with human fetal osteoblast cell line (FOB) and mesenchymal stem cell line (MSC). ER, keratin 8 (K8) and nuclei (DAPI) are represented in red, grey, and blue, respectively. Scale bars: 100µm. E. Heatmap showing the mean intensity of ER in primary cells (HCI011) and breast cancer cell lines (MDA-MB-361, MCF7, ZR75-1, T47D, ZR75-30) in 3D monoculture (control) or co-culture with osteoclast precursors (U937), bone marrow stromal cells (Hs5), mouse pre-osteoblasts (MC3T3), human mesenchymal stem cells (MSC) and human pre-osteoblast (FOB). All co-cultures were performed in triplicate and images were captured with a 40x oil objective lens. F. Graph comparing ER expression in monoculture versus co-culture of multiple cell lines with FOB. P-value results from a two-tailed paired Student’s t -test. 3 separated experiments were used. Error bars: mean +/- standard deviation. G. Relative mRNA expression of ESR1 in 3D monoculture or co-culture of MCF7 with FOB. Data result from MCF7 cells only (FACS sorted). H. Dot plot representing ER transcriptional activity in MCF7 cells expressing pGL2 ERE-luciferase reporter. MCF7 cells were cultured in 3D with or without osteogenic cells (FOB and MSC) for 7 days. Luciferase activity was assessed using IVIS Lumina II in vivo system; n=10 technical replicates. Error bars: mean +/- standard deviation. I. Representative confocal images showing ER expression (red) in MCF7 single cell-derived populations (SCPs) in 3D monoculture or co-culture with FOB cells. Vimentin (VIM), Keratin 8 (CK8), and DAPI were used to identify osteoblasts (Green), cancer cells (grey) and cell nuclei (blue), respectively. Scale bars: 50µm. J. Quantified ER expression from confocal images of single cell-derived populations (SCP1-SCP4). A two-tailed paired Student’s t -test analysis reveals a strong negative correlation (r) between monoculture and FOB co-cultures (right panel n=5). Error bars: +/- standard error of the mean. Each dot represents a cell from 3 different images. K. Diagram showing the experimental design for positron emission tomography–computed tomography (PET-CT) imaging of MCF7 cells transplanted orthotopically or to bone via IIA injection. Two rounds of imaging were performed at week 1 and week 5 post transplantation using 18F-Fluoroestradiol (18F-FES) and 18F-Fluorodeoxyglucose (18F-FDG) with 2 days apart. L. Representative PET/CT scans showing the maximum intensity projection (MIP) visualization of radiolabeled 18F-Fluorodeoxyglucose (18F-FDG) and 18F-Fluoroestradiol (18F-FES) in bone. Early time point (Week 1) and late time point (Week 7) were used to depict the micro-metastasis stage (small) and the macrometastasis stage (large). MCF7 bone metastases were generated using Intra-iliac artery injection. Red arrows indicate tumor location (Joint area). A smaller scale (0.2-0.5 SUV-bw) was used for week 1 images to allow detection of small lesions while a scale of 100-200 SUV-bw was used for the macrometastasis stage. M. Axial view of representative PET/CT scans depicting the uptake of radiolabeled fluorodeoxyglucose (18F-FDG) and fluoroestradiol (18F-FES) in small and large lesions of MCF7 orthotopic tumors. Early time point (Week 1) and late time point (Week 7) were used to depict non palpable orthotopic tumor stage (small < 2mm) and the palpable tumor stage. Red arrows indicate expected tumor location (mammary gland). Color scales for early lesions (Week 1): 0.2-0.5 SUV-bw; Color scales for large lesions (Week 7): 100-200 SUV-bw. N. Relative quantification of radiolabeled 18F-FES uptake in small and large lesions of orthotopic and bone metastases. Each dot represents the mean standard uptake values (mean SUV-bw) of 18F-FES normalized to the mean SUV of 18F-FDG. Mann Whitney U -test is used for statistical analysis. n=5 mice per group.

Article Snippet: Human estrogen receptor positive (ER+) breast cancer cell lines MCF7, T47D, MDA-MB-361 and ZR75-30, pre-osteoblast cells hFOB-1.19, mesenchymal stem cells MSC, pre-osteoclast U937 and the mouse pre-osteoblast MC3T3-E1 were purchased from American Type Culture Collection (ATCC).

Techniques: Expressing, Two Tailed Test, Derivative Assay, Co-Culture Assay, Control, Standard Deviation, Activity Assay, Luciferase, Cell Culture, In Vivo, Positron Emission Tomography, Computed Tomography, Positron Emission Tomography-Computed Tomography, Imaging, Injection, Transplantation Assay, Generated, Quantitative Proteomics, MANN-WHITNEY

Journal: bioRxiv

Article Title: Phenotypic plasticity of ER+ breast cancer in the bone microenvironment

doi: 10.1101/2020.11.14.383000

Figure Lengend Snippet: A. Growth curves of MCF7 and single cell-derived populations (SCP1-4) in 3D monoculture and co-culture with osteoprogenitor cells (MSC) in complete growth medium (10% FBS). Real-time images were acquired hourly using Incucyte. Values represent epifluorescence normalized to the earliest time point of co-culture. Error bars: +/- standard error of the mean (SEM). B. Table summarizing metastatic characteristics of MCF7 (Parental) and SCPs in vivo and in 3D co-culture C. Coronal, sagittal, and axial view or representative PET/CT images depicting the uptake of radiolabeled fluorodeoxyglucose (18F-FDG) and fluoroestradiol (18F-FES) in small and large lesions of MCF7 orthotopic and bone metastasis. Small lesions (Week 1) and larger lesions (Week 7) were used to depict early and late stage of tumor formation. Red arrows indicate expected tumor location in bone and mammary gland. Color scales for early lesions (Week 1): 0.2-0.5 SUV-bw; Color scales for large lesions (Week 7): 100-200 SUV-bw.

Article Snippet: Human estrogen receptor positive (ER+) breast cancer cell lines MCF7, T47D, MDA-MB-361 and ZR75-30, pre-osteoblast cells hFOB-1.19, mesenchymal stem cells MSC, pre-osteoclast U937 and the mouse pre-osteoblast MC3T3-E1 were purchased from American Type Culture Collection (ATCC).

Techniques: Derivative Assay, Co-Culture Assay, In Vivo, Positron Emission Tomography-Computed Tomography

Journal: bioRxiv

Article Title: Phenotypic plasticity of ER+ breast cancer in the bone microenvironment

doi: 10.1101/2020.11.14.383000

Figure Lengend Snippet: Osteogenic cells confer endocrine resistance A. Violin plot showing the response of luciferase-labelled MCF7 and ZR75-1 cells to 100nM of tamoxifen (4-Hydroxytamoxifen) and 20nM of fulvestrant in 3D monoculture or co-culture with osteogenic cells (FOB). Bioluminescence was acquired 72 hours post treatment using the IVIS Lumina II. Two-tailed unpaired Student’s t -test was used for statistical analysis (n=12 for MCF7; n=10 for ZR75-1). B. Heatmap depicting ER intensity in 3D monocultures and co-cultures of MCF7 cells with FOB following 24 hours treatment with 10nM 17β-estradiol (E2), 20nM fulvestrant (Fulv) and 100nM tamoxifen (4-OHT). Fulvestrant and tamoxifen were used in presence or not of 10nM of E2. Data represent the average of 5 different images. Each row shows the average of 5 random single cells from different images. C. Dot plots showing normalized bioluminescence intensity (BLI) of mammary tumor and IIA-induced bone lesions in non-ovariectomized (wild-type) and ovariectomized mice. Ovariectomy was performed 1 month after tumor transplant. Bioluminescence intensity (BLI) was obtained 2 weeks after ovariectomy and normalized to pre-ovariectomy BLI (n= 9 for control groups; n=12 for ovariectomized mammary group; n=11 for ovariectomized bone lesion group). D. Bioluminescent images of IIA-induced bone metastases in non-ovariectomized (wild-type) and ovariectomized mice (OV-mice). Different from the experiment described in C, here ovariectomy was performed before IIA-injection of MCF7 cells to mice. 8 weeks after injection, tumor bearing bones were harvested and BLI was assessed ex vivo. E. Graphical representation of estrogen replacement via drink water (E2-in-water) for bone metastasis formation in OV-mice. Bone metastasis progression was measured using tumor BLI before and after withdrawal of estrogen from drink water. n=5 mice. P-values ≤ 0.05 and 0.005 (relative to Week 0) are represented as (*) and (**), respectively. F. Graphs representing the proliferation of MCF7 (Par) and single cell-derived populations (SCP1-4) in monoculture and MSC co-culture following 1 week of treatment with 20nM fulvestrant and 100nM tamoxifen. n= 5 different cell lines. Two-tailed paired Student’s t -test was used for statistical analysis. G. Growth kinetics of naïve cells (Parental and SCP1-4) and bone-educated cells (MCF7- Bo, SCP1-Bo, SCP2-Bo, SCP3-Bo, SCP4-Bo) in complete media. n=10 technical replicates. Real-time images were obtained using Incucyte S3 system. H. Dot plot representing the BL intensity of bone-entrained (MCF7-Bo), mammary gland-entrained (MCF7-Ma) and naïve MCF7 cells (parental) after 10 days of 3D monoculture (blue) and FOB co-culture (red). Grey area highlight bone-derived (bone-entrained) MCF7 cells. Error bars: mean +/- standard deviation. I. Histogram showing the relative growth of non-entrained and bone-entrained MCF7 cells following 20nM fulvestrant treatment. Epifluorescence was measured 72h post treatment. n=12 for bone-entrained, n=4 for non-entrained cells. Two-tailed unpaired Student’s t -test was used for all statistical analysis. J. Violin plot showing the time course assessment of endocrine resistance phenotype in naïve and bone-entrained MCF7 and SCP2 cells over 8 passages (∼2 months). Median is shown in red. Cells were treated with vehicle or 20nM fulvestrant in estrogen-free medium. n=5-6 technical replicates per group. Two-tailed unpaired Student’s t -test was used for all statistical analysis.

Article Snippet: Human estrogen receptor positive (ER+) breast cancer cell lines MCF7, T47D, MDA-MB-361 and ZR75-30, pre-osteoblast cells hFOB-1.19, mesenchymal stem cells MSC, pre-osteoclast U937 and the mouse pre-osteoblast MC3T3-E1 were purchased from American Type Culture Collection (ATCC).

Techniques: Luciferase, Co-Culture Assay, Two Tailed Test, Control, Injection, Ex Vivo, Derivative Assay, Standard Deviation

Journal: bioRxiv

Article Title: Phenotypic plasticity of ER+ breast cancer in the bone microenvironment

doi: 10.1101/2020.11.14.383000

Figure Lengend Snippet: A. Heatmap showing estrogen independent promoting effect of osteogenic cells (FOB) on cancer cells (MCF7) in 3D co-culture. Real-time epifluorescence images were acquired with Incucyte live imaging for 6 days. n=3-5 replicates per condition. P values were calculated using ordinary two-way ANOVA (p<0.0001 for ZR75-1 and MCF7). B. Dot plot showing the effect of ovary removal (OV) on mouse weight in comparison to wild-type mice (Left), and the effect of OV on the rate of bone metastasis formation (right). For wild-type group, n= 8 mice; for OV group n= 6 mice). P values: two tailed Paired Sample t Test (p-value <0.05 is significant). C. Graph showing growth differences in MCF7 cells derived from different tissues. The diagram represents the work flow used to generate bone-entrained cells (MCF7-Bo). D. Representative luciferase images showing stimulatory effect of FOB on non-entrained (MCF7), bone-entrained (MCF7-Bo), and mammary gland-entrained (MCF7-Ma) cells. Images are acquired at day 10 post co-coculture.

Article Snippet: Human estrogen receptor positive (ER+) breast cancer cell lines MCF7, T47D, MDA-MB-361 and ZR75-30, pre-osteoblast cells hFOB-1.19, mesenchymal stem cells MSC, pre-osteoclast U937 and the mouse pre-osteoblast MC3T3-E1 were purchased from American Type Culture Collection (ATCC).

Techniques: Co-Culture Assay, Imaging, Comparison, Two Tailed Test, Derivative Assay, Luciferase

Journal: bioRxiv

Article Title: Phenotypic plasticity of ER+ breast cancer in the bone microenvironment

doi: 10.1101/2020.11.14.383000

Figure Lengend Snippet: Gap junctions and calcium signaling partially contribute to ER downregulation, and the bone microenvironment drives a global phenotypic shift involving multiple other pathways. A. Immunoblotting showing ER expression in ZR75-1 and MCF7 in monoculture or FOB-co-culture after 24h treatment with 1uM CX43 inhibitor (GAP19) and 1uM calcium signaling inhibitor (FK506). Keratin 19 and RFP were used for loading control specific to cancer cells. B. Immunoblotting showing the effect of 2mM Calcium (CaCl2) on ER expression in ZR75-1 and MCF7 cells. C. Violin plots depicting the inhibitory effect of calcium signaling disruptors (1µM FK506 and 10µM Carbenoxolone-CBX) on osteoblast-induced breast cancer cell growth (ZR75-1 and MCF7). Cancer cells were cultured in 3D with (grey) or without (orange) osteogenic cells (FOB). Bioluminescence intensity (BLI) was acquired 72h post treatment. Data results from 3 different experiments with 4-6 technical replicates. Two-tailed unpaired Student’s t -test was used for statistical analysis. D. Violin plots indicating synergism between fulvestrant (anti-ER) and FK506 (Calcium signaling inhibitor) on MCF7 and ZR75-1 cells grown in bone using BICA (Bone-In-Culture-Array). Cells were injected to bone using intra-iliac artery (IIA) injection. Hind limbs were harvested, and bone pieces were cultures ex vivo. Bioluminescence intensity (BLI) was assessed using IVIS Lumina II in vivo system. n= 6-18 bone pieces for each treatment group. Two-tailed unpaired Student’s t -test was used for statistical analysis. E. Diagram summarizing strategies used to evaluate molecular changes occurring in cancer cells when exposed to the bone microenvironment. Translating Ribosome Affinity Purification (TRAP) was used to generate and sequence breast cancer cell specific transcriptomes without alteration of cell-cell interaction in 3D co-culture of cancer cells (MCF7) and osteogenic cells (FOB). Reverse Phase Protein Arrays (RPPA) was used to assess protein alterations between naive cells (MCF7 and SCP2) and bone-entrained cells (MCF7-Bo and SCP2-Bo). F. Box plot depicting gene signature alternations in MCF7 monoculture (MSC-) and co-cultures (MSC) from TRAP sequencing. Specific colors represent different treatment conditions as indicated. G. Waterfall plot showing the gene ontology analysis of TRAP sequencing data PANTHER classification system. Signaling pathways were organized based on their false discovery rate (FDR). H. Heatmap depicting expression changes in luminal and stemness-related markers from RPPA data. Parental cells (MCF7 and SCP2), and bone-entrained breast cancer cells (MCF7-Bo and SCP2-Bo) are compared. 4 biological replicates and 3 technical replicates were used for each cell line (See Supplementary Table 1). I. Heatmap depicting expression changes in EMT/MET markers from RPPA data as describes in H. J. Heatmap depicting expression changes in receptor tyrosine kinases from RPPA data as described in H.

Article Snippet: Human estrogen receptor positive (ER+) breast cancer cell lines MCF7, T47D, MDA-MB-361 and ZR75-30, pre-osteoblast cells hFOB-1.19, mesenchymal stem cells MSC, pre-osteoclast U937 and the mouse pre-osteoblast MC3T3-E1 were purchased from American Type Culture Collection (ATCC).

Techniques: Western Blot, Expressing, Co-Culture Assay, Control, Cell Culture, Two Tailed Test, Injection, Ex Vivo, In Vivo, Affinity Purification, Sequencing, Protein-Protein interactions

Journal: bioRxiv

Article Title: Phenotypic plasticity of ER+ breast cancer in the bone microenvironment

doi: 10.1101/2020.11.14.383000

Figure Lengend Snippet: A. Heatmap depicting molecular pathways (PANTHERS) altered in MCF7 when cultured alone or in 3D with MSC and treated with vehicle (control), 10nM 17B-estradiol (E2), 10nM fulvestrant (ICI) or 100nM tamoxifen (Tam) for 24h. Data results from Translating Ribosome Affinity Purification (TRAP) sequencing analysis. B. Boxplot showing the promoting effect of osteogenic cells (MSC) on CX43 gene (GJA1) expression in 3D co-cultures of MCF7 (left). Scatter plot showing Pearson (r) correlation between GJA1 and ESR1 gene (right). All values result from Translating Ribosome Affinity Purification (TRAP) sequencing data. Colors are specific to treatment conditions. C. Effect of FOB conditioned media on ESR1 expression (left) and endocrine response. n=3 different cell models. For left panel (mRNA expression), P values: two tailed Paired Sample t Test (p-value <0.05 is significant). For cell growth measured by BLI (right panel), P values were calculated using ordinary two-way ANOVA. D. Table depicting signaling pathways involved in osteogenic cell-mediated breast cancer cell reprogramming based on MCF7 Translating Ribosome Affinity Purification (TRAP) sequencing analyzed.

Article Snippet: Human estrogen receptor positive (ER+) breast cancer cell lines MCF7, T47D, MDA-MB-361 and ZR75-30, pre-osteoblast cells hFOB-1.19, mesenchymal stem cells MSC, pre-osteoclast U937 and the mouse pre-osteoblast MC3T3-E1 were purchased from American Type Culture Collection (ATCC).

Techniques: Cell Culture, Control, Affinity Purification, Sequencing, Expressing, Two Tailed Test, Protein-Protein interactions

Journal: bioRxiv

Article Title: Phenotypic plasticity of ER+ breast cancer in the bone microenvironment

doi: 10.1101/2020.11.14.383000

Figure Lengend Snippet: A. Heatmap showing differentially expressed proteins from RPPA data. Parental cells (MCF7) were compared to bone-entrained cells (MCF7-Bo). B. Similar to A with single cell-derived cell population 2 (SCP2) used as cells. Parental cells (SCP2) were compared to bone-entrained cells (SCP2-Bo). C. Volcano plot indicating protein distribution in MCF7-Bo and SCP2-Bo cells relatively to parental cells (MCF7 and SCP2) based on expression fold change (Log2) and p-value (Log10). D. Immunoblotting showing the expression of PDGFRB, ER, and PR in parental (MCF7 and SCP2) and bone-entrained cells (MCF7-Bo and SCP2-Bo). E. Quantitative PCR showing changes in PDGFRB expression between parental and bone-educated MCF7 and SCP2 cells. (n= 3 technical replicates)

Article Snippet: Human estrogen receptor positive (ER+) breast cancer cell lines MCF7, T47D, MDA-MB-361 and ZR75-30, pre-osteoblast cells hFOB-1.19, mesenchymal stem cells MSC, pre-osteoclast U937 and the mouse pre-osteoblast MC3T3-E1 were purchased from American Type Culture Collection (ATCC).

Techniques: Derivative Assay, Expressing, Western Blot, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Phenotypic plasticity of ER+ breast cancer in the bone microenvironment

doi: 10.1101/2020.11.14.383000

Figure Lengend Snippet: Osteogenic cell-secreted FGFs and PDGFs promote endocrine resistance and bone addiction. A. Network depicting functional protein association between FGFR1, PDGFRB and ER using the STRING database. K means clustering ( k =3) was used to represent 3 major centroids (depicted as red, green, and cyan spheres) and their most closely associated proteins based on unsupervised data mining. B. Graph showing the gene expression of human FGFs in osteogenic cells (FOB). The data derives from RNA sequencing data of osteogenic cells (FOB). n= 3 technical replicates C. Representative IF images showing decreased ER expression (yellow) in tumors established in FGF2 (green) enriched bone microenvironments. Keratin 8 (red) is used to identify breast cancer cells. The scatter dot plot represents ER quantification from tumors according to FGF2 enrichment (Low and High) in adjacent stromal cells (n=3-4 samples). Mean expression is represented in blue. D. Graph depicting the inhibitory effect of recombinant FGF2 (20ng/ml) on ER expression in MCF7, ZR75-1 and T47D after 24h treatment. Data represent normalized ER over β-actin protein expression from 3 separate immunoblotting experiments. E. Bone-In-Culture-Array (BICA) assay showing synergistic effects between 2.5µM FGF2 inhibitor (BGJ398) and 20nM fulvestrant in MCF7, ZR75-1 and T47D models. F. Graph depicts the inhibitory effect of indicated PDGF recombinants on ER expression in MCF7 and ZR75-1 after 24h treatment. Data represent normalized ER over β-actin protein expression from Immunoblot quantification. G. Violin plot showing the effect of 20ng/ml PDGF recombinants (PDGF-BB, PDGF-CC, PDGF-DD) on bone-entrained MCF7 (MCF7-Bo) and SCP2 (SCP2-Bo) response to fulvestrant. BLI was assessed after 72h of treatment. H. Dot plot showing the effect of the pan FGFR inhibitor (BGJ398), and the PDGFRβ inhibitor (sunitinib) on osteoblast (FOB)- mediated MCF7 and ZR75-1 cell growth in 3D. I. Scatter plot showing negative Pearson correlation (r) between FGFR1, FGFR2, PDGFRA, PDGFRB, and ESR1 expression in clinical specimens of bone metastasis. n= 11 bone metastasis samples. P values: two tailed Paired Sample t Test.

Article Snippet: Human estrogen receptor positive (ER+) breast cancer cell lines MCF7, T47D, MDA-MB-361 and ZR75-30, pre-osteoblast cells hFOB-1.19, mesenchymal stem cells MSC, pre-osteoclast U937 and the mouse pre-osteoblast MC3T3-E1 were purchased from American Type Culture Collection (ATCC).

Techniques: Functional Assay, Gene Expression, RNA Sequencing, Expressing, Recombinant, Western Blot, Two Tailed Test

Journal: bioRxiv

Article Title: Phenotypic plasticity of ER+ breast cancer in the bone microenvironment

doi: 10.1101/2020.11.14.383000

Figure Lengend Snippet: EZH2 integrates multiple signals from the bone microenvironment and drives the phenotypic shift of ER+ breast cancer cells. A. Graphs showing the association of histone modifications with FGF2 gene signatures using the Enrichr platform ( https://amp.pharm.mssm.edu/Enrichr/ ). Processed ChIP-sequencing data was obtained from epigenomic roadmap project (Roadmap Epigenomics Consortium et al., 2015). Signatures are sorted based on p-value ranking. Only p-value < 0.05 and <0.01 were shown for the top and bottom panel, respectively. B. Immunoblotting showing changes in H3K27me3 and EZH2 expression in nuclear extracts of SCP2 cells following 24h treatment with 20nM FGF2 recombinant (FGF2r). Total histone 3 (H3) and actin-β were used as loading controls for H3k27me3 and EZH2, respectively. C. Primary cells generated from HCI011 (ER+ PDX) were cultured in 3D and treated with 1µM pan FGFR inhibitor (BGJ398) or vehicle for 24h. Representative images depict EZH2 expression in indicated conditions. D. Box plot representing the gene set variation score (GSVA) of EZH2 target genes (Lu et al.) in MCF7 monoculture (MSC-) and co-culture with MSCs (MSC+) from TRAP-sequencing. Each color represents a specific treatment as indicated. Cells were cultured (estrogen-free medium) in 3D and treated with 10nM estradiol and fulvestrant, and 100nM tamoxifen for 24h. E. Graph shows reductions in EZH2 target gene expression in MCF7 cells following 3D monoculture (-MSC) and co-culture (+MSC) and TRAP sequencing. EZH2 signature genes were selected from previous studies . F. Quantitative PCR of stemness-related genes in MCF7 cells from 3D monoculture and co-culture with FOB (osteoblast), human bone marrow (marrow), and U937 (osteoclast) cells. All conditions were FACS-sorted for RFP-labeled MCF7cells before mRNA extraction and qPCR. G. IF quantification of EZH2 expression in multiple metastases and primary tumor originating from the same mouse. 2×10 5 MCF7 cells were transplanted orthotopically and via IIA injection (bone) to nude mice, which led to tumor formation at multiples sites (Lung, ovary, bone and Mammary gland). Metastases derived from same animal were harvested for immunofluorescence studies. H. Representative confocal images showing co-expression of ER (purple) and EZH2 (Green) in 3D models of MCF7 and HCI011 primary cells. Keratin 8 (red) and DAPI (Blue) were used to identify epithelial cells and cell nuclei. I. IF quantification of ER (purple) and EZH2 (green) in MCF7 and HCI011 3D monocultures and co-cultures with osteogenic cells (FOB). Keratin 8 -K8 (red) and DAPI (blue) were used to identify epithelial cells and cell nuclei. J. IF quantification showing changes in EZH2 and ER according to bone metastasis size. Micrometastases (micro) represent early stages while macrometastases (overt) represent late stages of bone metastasis. K. Quantitative PCR showing the effect of siRNA downregulation of EZH2 on ESR1 expression. n= 3 technical replicates. L. Quantitative PCR showing the effect of EZH2 inhibitor EPZ011989 on ESR1 expression after 24 hours of treatment. n=4 cell lines. P value shows two-tailed paired Student’s t test.

Article Snippet: Human estrogen receptor positive (ER+) breast cancer cell lines MCF7, T47D, MDA-MB-361 and ZR75-30, pre-osteoblast cells hFOB-1.19, mesenchymal stem cells MSC, pre-osteoclast U937 and the mouse pre-osteoblast MC3T3-E1 were purchased from American Type Culture Collection (ATCC).

Techniques: ChIP-sequencing, Western Blot, Expressing, Recombinant, Generated, Cell Culture, Co-Culture Assay, Sequencing, Targeted Gene Expression, Real-time Polymerase Chain Reaction, Labeling, Extraction, Injection, Derivative Assay, Immunofluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: Phenotypic plasticity of ER+ breast cancer in the bone microenvironment

doi: 10.1101/2020.11.14.383000

Figure Lengend Snippet: A. Immunoblotting showing the effect of FGF2 and PDGF-DD recombinant on histone modifications in SCP2. Cells were treated with 20ng/ml recombinant for 24h. B. Quantitative PCR showing the promoting effect of Calcium on EZH2 expression in MCF7 cells (n= 3 independent experiments). C. Quantitative PCR showing knockdown effect of 2 different siRNAs (s4916 and s4918) on EZH2 after 72h. MCF7 cells and HCI011 primary cells were used (n= 3 replicates). D. EZH2 target genes correlate with distant metastasis-free survival in patients with ER-positive, but not in ER-negative, breast cancer. Survival curves were generated from GOBO database ( http://co.bmc.lu.se/gobo/gsa.pl ) after inputting EZH2 target gene set. E. Scatter plots showing negative correlations between EZH2 and luminal markers in breast cancer patients. Gene expression was extracted from METABRIC dataset ( http://www.cbioportal.org ./ ). All correlations are significant (n=1866 and p-values < 10 -5 ).

Article Snippet: Human estrogen receptor positive (ER+) breast cancer cell lines MCF7, T47D, MDA-MB-361 and ZR75-30, pre-osteoblast cells hFOB-1.19, mesenchymal stem cells MSC, pre-osteoclast U937 and the mouse pre-osteoblast MC3T3-E1 were purchased from American Type Culture Collection (ATCC).

Techniques: Western Blot, Recombinant, Real-time Polymerase Chain Reaction, Expressing, Knockdown, Generated, Gene Expression

Journal: bioRxiv

Article Title: Phenotypic plasticity of ER+ breast cancer in the bone microenvironment

doi: 10.1101/2020.11.14.383000

Figure Lengend Snippet: Short-term treatment of an EZH2 inhibitor restores endocrine sensitivity of breast cancer bone metastasis. A. LOEWE analysis of fulvestrant and EZH2 inhibitor (EPZ011989) combination in 3D co-culture of mammary gland-entrained (MCF7-Ma) and bone-entrained (MCF7-Bo) MCF7 cells. Graphs were generated using the Combenefit interactive platform. B. Representative images showing beneficial effects of fulvestrant and EPZ011989 combination treatment on IIA-induced bone metastases. 4 arms were formed: (i) control group (Ctrl/ctrl) received vehicle treatment; (ii) EPZ011989 group (EPZ011989) received EZH2 inhibitor followed by vehicle treatment; (iii) fulvestrant group (Fulv) received vehicle treatment followed by fulvestrant treatment; (iv) combination treatment group (EPZ/Fulv) received both EPZ011989 and Fulvestrant treatment. EPZ011989 was used as a neoadjuvant for 3 weeks (125mg/kg; oral gavage; twice a day) before fulvestrant treatment (250mg/kg; subcutaneous injection, once per week for 2 weeks). Tumor progression free survival (PFS) curve was generated to represent the effect of indicated treatments. Log-rank (Mantel-Cox) test was used for statistical analysis. P values <0.05 are represented as (*). C. Graph showing the bioluminescence intensity of IIA-induced bone metastases normalized to day 1 of treatment. Each dot represents an image. D. Ex vivo quantification of tumor bioluminescence intensity in bone. E. Representative PET-CT images showing 18F-FDG uptake in hind limb bones of fulvestrant treated and EPZ011989/fulvestrant treated metastases. 2×10 5 dissociated tumors cells from freshly harvested PDXs (HCI011) were injected to mammary gland of nude mice. A week after primary tumors were removed, EPZ011989 treatment (125mg/kg; oral gavage; twice a day) started for 3 weeks, followed by fulvestrant treatment (250mg/kg; subcutaneous injection, once per week) for 2 weeks. Residual tumors were challenged with estrogen supplementation in drink water before 18F-FDG PET-CT imaging. F. Quantification of 18F-FDG uptake (mean SUV-bw) in hind limbs to evaluate spontaneous metastasis from HCI011 PDXs. A 90% thresholding of the maximum standard uptake value (SUV-bw) was used to remove background signals. Two-tailed unpaired Student’s t -test was used for statistical analysis. G. Proposed model summarizing the mechanisms involved in breast cancer bone metastasis and endocrine resistance. A subset of ER+ breast cancers express FGFR1 which may confer survival advantages during metastasis. FGFs are important for stem cell maintenance and migration. Here, we found that FGF2 secreted from osteoblasts can activate FGFR signaling in a paracrine manner, leading to increased EZH2 expression, chromatin alteration, and subsequent downregulation of estrogen receptor (ER) in the osteogenic niche. This promotes estrogen independent growth of micrometastases. The proximity of cancer cells to osteoblasts is gradually lost with macrometastasis formation resulting in ER re-expression in advanced tumors. These changes are also associated with acquired PDGFRB expression which further contributes in maintaining stemness in overt lesions of bone metastases. As PDGFRB expression persists outside of the bone microenvironment, it could be a potential marker for bone metastasis.

Article Snippet: Human estrogen receptor positive (ER+) breast cancer cell lines MCF7, T47D, MDA-MB-361 and ZR75-30, pre-osteoblast cells hFOB-1.19, mesenchymal stem cells MSC, pre-osteoclast U937 and the mouse pre-osteoblast MC3T3-E1 were purchased from American Type Culture Collection (ATCC).

Techniques: Co-Culture Assay, Generated, Control, Injection, Ex Vivo, Positron Emission Tomography-Computed Tomography, Imaging, Two Tailed Test, Migration, Expressing, Marker

Journal: bioRxiv

Article Title: Phenotypic plasticity of ER+ breast cancer in the bone microenvironment

doi: 10.1101/2020.11.14.383000

Figure Lengend Snippet: A. Diagram showing experimental procedures and treatment conditions used to assess therapeutic effects of EZH2 inhibitor (EPZ011989) in combination with fulvestrant on MCF7 bone metastasis. Tumor burden was acquired by bioluminescence (BLI). Dot plot show similar weight between vehicle and EZP011989 group after 3 weeks of treatment (n= 19 mice per group). n.s: non-significant (P value <0.05) B. Diagram showing experimental procedures and treatment conditions used to assess therapeutic effects of EZH2 inhibitor (EPZ011989) on spontaneous metastases of HCI011 PDXs when used in combination with fulvestrant (Fulv). Mouse weight was assessed between both treatment groups. n= 4 mice per group. n.s: non-significant (P value <0.05) . C. Representative 18F-NAF PET/CT scans showing spontaneous metastasis sites (hot spot) in bone following fulvestrant monotherapy or combination (mice pre-treated for 3 weeks with EPZ011989). Hot spots (red arrows) show enrichment of radiolabeled 18F-Sodium Fluoride (18F-NAF).

Article Snippet: Human estrogen receptor positive (ER+) breast cancer cell lines MCF7, T47D, MDA-MB-361 and ZR75-30, pre-osteoblast cells hFOB-1.19, mesenchymal stem cells MSC, pre-osteoclast U937 and the mouse pre-osteoblast MC3T3-E1 were purchased from American Type Culture Collection (ATCC).

Techniques: Positron Emission Tomography-Computed Tomography