Fulvestrant-Mediated Attenuation of the Innate

Published OnlineFirst August 27, 2020; DOI: 10.1158/0008-5472.CAN-20-1705

CANCER RESEARCH | TUMOR BIOLOGY AND IMMUNOLOGY

Fulvestrant-Mediated Attenuation of the Innate Immune Response Decreases ERþ Breast Cancer Growth In Vivo More Effectively than Tamoxifen A C Annelie Abrahamsson, Gabriela Vazquez Rodriguez, and Charlotta Dabrosin

ABSTRACT ◥ Although blocking estrogen-dependent signaling is a corner- released into the cancer microenvironment by the cancer cells stone of adjuvant treatment for breast cancer, 25% of patients sampled via microdialysis in vivo revealed that 38 proteins were experience recurrent disease. Stroma events including innate altered following both treatments; 25 of these proteins were asso- immune responses are key in cancer progression. How different ciated with immune response and were altered by fulvestrant only. estrogen receptor (ER)–targeting therapies, including the partial Compared with tamoxifen, fulvestrant significantly affected inflam- agonist tamoxifen and the pure antagonist fulvestrant, affect the matory proteins released by murine stroma cells. Importantly, þ tumor stroma has not yet been elucidated. Fulvestrant is used in only in vivo microdialysis of human ER breast cancer revealed that postmenopausal patients, and its effects in the presence of estradiol the majority of affected proteins in murine models were upregulated remain undetermined. Here we observe that fulvestrant decreases in patients. Together, these results suggest that fulvestrant targets þ þ ER breast cancer growth compared with tamoxifen in the presence ER breast cancer more effectively than tamoxifen even in the of physiologic levels of estradiol in human breast cancer in nude presence of estradiol, mainly by attenuation of the innate immune mice and in murine breast cancer in immune-competent mice. response. Fulvestrant significantly inhibited macrophage and neutrophil infiltration in both models. These effects were corroborated in a Significance: These findings demonstrate novel effects of the þ zebrafish model where fulvestrant inhibited neutrophil- and mac- pure antiestrogen fulvestrant in ER breast cancer and evaluate rophage-dependent cancer cell dissemination more effectively than its effects under physiologic levels of estradiol, representative of tamoxifen. A comprehensive analysis of 234 human proteins premenopausal patients.

Introduction Despite the success of these therapies, approximately 25% of patients will experience recurrent disease (7). The SERD fulvestrant Exposure to sex steroids such as estrogens plays an important is not used in the adjuvant setting, but is used as a second-line role in the development and progression of breast cancer. Up to þ treatment for postmenopausal patients with metastatic breast can- 80% of all breast cancers are estrogen receptor positive (ER ;1). cer who develop resistance to other antiestrogen therapies, includ- Blocking ER signaling is a cornerstone in both adjuvant and ing tamoxifen (8). Although ER breast cancer is more common in metastatic treatment of breast cancer (2, 3). Different strategies premenopausal women than in postmenopausal women, the major- are used to target ER-dependent signaling, including aromatase ity of premenopausal breast cancers express the ER (9). Further inhibitors (AI) that reduce estrogen synthesis, selective estrogen studies of the mechanisms underlying these different therapeutic receptor modulators (SERM), and selective estrogen receptor down- approaches are needed to potentiate antiestrogen therapy for pre- regulators (SERD). Whereas SERMs may elicit both agonistic and þ menopausal women with ER breast cancer. antagonistic effects in different organs depending on their affinity to Events in the stroma, such as immune responses and angiogen- the receptor or on the ratio of ERa and ERb in tissues, SERDs elicit esis, are hallmarks of cancer and play major roles in cancer no agonistic actions as ligand binding enhances the ER to be progression (10). Several studies suggest that the effects of anties- destroyed (4, 5). The standard-of-care adjuvant treatment is AIs trogen therapies as well as chemotherapy are dependent on stroma- for postmenopausal women and tamoxifen, a SERM, for premen- related signatures (11, 12). Tamoxifen may induce a microenvi- opausal women (6). Treatment with an AI or tamoxifen for five to ronment suppressive to breast cancer cells, which in turn may ten years is associated with an increase in overall survival (6). potentiate the effects of the drug (13). We have recently shown that estradiol enhances the innate immune response by increasing þ the infiltration of macrophages into ER breast cancer and attract- Department of Oncology and Department of Biomedical and Clinical Sciences, € € ing neutrophils to the invasive margin (14, 15). Additionally, Linkoping University, Linkoping, Sweden. þ low metastatic ER cells may become highly metastatic in the Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). presence of neutrophils and macrophages, an effect that is poten- tiated during estradiol exposure (14, 15). Several chemoattractants Corresponding Author: Charlotta Dabrosin, Professor of Oncology, Linkoping€ € and angiogenesis stimulators also are increased during exposure to University, University Hospital, Division of Oncology, SE-581 85 Linkoping, – Sweden. Phone: 46-10-1038595; E-mail: [email protected] estradiol (14 18). The epithelial-to-mesenchymal transition (EMT) of breast cancer Cancer Res 2020;80:1–13 cells can contribute to increased tumor progression and metastases, doi: 10.1158/0008-5472.CAN-20-1705 whereas a reversal of EMT to a mesenchymal-to-epithelial transition 2020 American Association for Cancer Research. reduces stemness and metastatic capacity of cancer cells (19, 20). An

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EMT can be stimulated by several signaling pathways and via cross- procedure generates tumor cells with maintained expression of ER at talks with different cell types in the tumor microenvironment (21, 22). significant levels as early carcinoma stages still express the receptor, In addition, it has been determined that myeloid-derived immune cells whereas ER expression decreases at later stages of tumor progression as such as macrophages and neutrophils support EMT in several cancer previously described (25, 27, 28). Mice were treated with fulvestrant forms, including breast cancer (23, 24). (5 mg/mouse twice per week, s.c.) or tamoxifen (1 mg/mouse every Whether different antiestrogen therapeutic approaches can atten- second day, s.c.) in addition to the estradiol exposure. Tumor areas uate stromal events, including the innate immune response and EMT, were calculated using the formula length/2 width/2 p. is unknown. Here, we demonstrate that the SERD fulvestrant, com- þ pared with tamoxifen, enhanced tumor regression in ER experimen- Microdialysis in mice tal breast cancer, in murine immunocompetent and immunodeficient Prior to the microdialysis experiment, tumor-bearing mice with breast cancer models, in the presence of physiologic levels of estradiol. size-matched tumors were anesthetized with i.p. injections of keta- Fulvestrant significantly reduced the number of tumor-infiltrating mine/xylazine. An anesthetic state was maintained as needed by macrophages and neutrophils on both models and reduced EMT repeated s.c. injections of ketamine/xylazine. Body temperatures were markers more effectively than tamoxifen. In the zebrafish, fulvestrant, maintained using a heat lamp. Microdialysis probes with 4-mm compared with tamoxifen, significantly inhibited estradiol induced membranes (CMA 20, 100-kDa cutoff; CMA Microdialysis AB) were þ ER cancer cell dissemination in the presence of neutrophils and inserted into tumor tissue and connected to a microdialysis pump monocytes. In a comprehensive analysis in vivo of 234 proteins (CMA 102; CMA Microdialysis AB) perfused at 0.6 mL/min with released into the cancer microenvironment, 38 were significantly 154 mmol/L NaCl and 60 g/L hydroxyethyl starch (Voluven; Fresenius changed after antiestrogen therapy, and 25 were significantly altered Kabi), as previously described (16, 17). After a 60-minute equilibrium by fulvestrant only. The majority of the proteins were associated with period, outgoing perfusates (i.e., microdialysates) were collected and immune function. Of the 38 proteins found to be altered in experi- stored at 80C for subsequent analysis. þ mental breast cancer, 36 were detectable in human ER breast cancer in vivo. Furthermore, 31 of these proteins were significantly upregu- Zebrafish tumor xenograft model lated, supporting the clinical relevance of these findings. Together, our MCF7 cells were treated with E2 1 nmol/L 48 hours before experi- data suggest that the SERD fulvestrant, compared with tamoxifen, ments and labeled with Fast DiI oil red dye (Thermo Fisher Scientific þ more effectively targets ER breast cancer both by direct effects on the cat. #D3899), 4 mg/mL in PBS, 24 hours before injections. cancer cells and by alterations in the tumor stroma. Human neutrophils and monocytes were isolated from venous blood from a healthy female donor. Peripheral blood mononuclear cells were obtained by gradient separation with Ficoll-Paque (GE Materials and Methods Healthcare cat. #17-1440-02) and monocytes were separated by neg- Cell line ative isolation by using the Dynabeads Untouched Human Monocytes þ The ER cell lines MCF7 (ATCC cat. #HTB-22, RRID:CVCL_ kit (Thermo Fisher Scientific cat. #11350D) by following the provider's 0031) reauthenticated using the short tandem repeat profiling at instructions. Neutrophils were isolated as described previously the Uppsala Genome Center were maintained in DMEM (Gibco cat. (PMID:30105032). # 11880-028) with 10% FBS (Gibco cat. #26140-079), 2 mmol/L The animal ethics committee at Linkoping€ University approved all glutamine (Gibco cat. # 25030-024) and 50 IU/mL/50 mg/mL zebrafish experiments. Transgenic Tg(fli1:EGFP)y1 zebrafish embryos penicillin-G/streptomycin (Gibco cat. #15070-063). were collected and maintained in E3 embryo medium with 0.2 mmol/L 1-phenyl-2-thiourea (PTU) at 28C. Dil-labeled MCF7 cells were Breast cancer models injected with 50% neutrophils or with 10% monocytes into the The Institutional Animal Ethics Committee at Linkoping€ Univer- perivitelline space of 2-day-old zebrafish embryos. Correctly injected sity approved this study, which conformed to regulatory standards embryos were selected under fluorescence and incubated in E3/PTU þ of animal care. Oophorectomized athymic mice and FVB/N mice E2 1 nmol/L tamoxifen 1 mmol/L fulvestrant 1 mmol/L at 28C (Balb/C-nu/nu, 6–8 weeks old, Scanbur) were housed at Linkoping€ during 1 or 3 days where indicated. After incubation, anesthetized University in ventilated cages with a light/dark cycle of 12/12 hours zebrafish embryos were assessed for cancer cell dissemination in the with rodent chow and water available ad libitum. Mice were anesthe- tail region under fluorescence. Images of disseminated cancer cells tized via intraperitoneal (i.p.) injection of ketamine/xylazine and were acquired with the Olympus CellSens Imaging software version implanted with a subcutaneous (s.c.) 3-mm pellet containing either 1.16 (Olympus CellSens Software, RRID:SCR_016238) by using an 17b-estradiol (0.18 mg/60-day release; Innovative Research of Amer- Olympus BX43 light/fluorescence microscope (10/0.30 magnifica- ica) or placebo. The active pellet releases serum concentrations of tion) with excitation filters BP460-495 and BP530-550, and Olympus 150 to 250 pmol/L estradiol (25). DP72 CCD camera. Seven days after surgery, 5 106 MCF7 cells or 1 106 or tumor cells derived from a transgenic mouse strain expressing polyoma Immunohistochemistry middle T (PyMT) antigen under the control of the mouse mammary Formalin-fixed tumors were paraffin-embedded and cut in 4-mm tumor virus long terminal repeat (26) were injected into the dorsal sections, deparaffinized, and exposed to rat anti-mouse F4/80 mammary fat pads in 200 mL PBS. Because MCF7 cells require estrogen (Abcam cat. #ab6640, RRID:AB_1140040), rat anti-mouse Ly6G (BD for tumor formation and growth, a nonestrogen control group was not Biosciences cat. #551459, RRID:AB_394206), rat on mouse HRP possible in this in vivo model. The PyMT mice develop spontaneous Polymer Kit (BioCare Medical cat. #RT517), rabbit anti-human von adenocarcinomas of all mammary epithelium by 8 to 10 weeks of age. Willebrand factor (Agilent cat. #A0082, RRID:AB_2315602), mouse These tumors were excised, dissociated in a collagenase/dispase solu- anti-human Ki67 (Agilent cat. #M7240, RRID:AB_2142367), rabbit tion (100 mL PBS with 25 mg collagenase/250 mg dispase; Roche) to anti-human N-cadherin (clone EPR19654, Abcam cat. #ab207608), generate a single-cell suspension and cultured until confluence. This mouse anti-human E-cadherin (Novus Biologicals cat. #NBP2-47827),

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mouse anti-human CD68 (Agilent cat. #GA60961-2), rabbit anti- Fluidigm Corporation). The generated fluorescent signal directly human Slug (Abcam cat. #ab27568, RRID:AB_777968), rabbit anti- correlated with protein abundance. The output from the Proseek human Snail (Novus Biologicals cat. #NBP2–27293), rabbit anti- Multiplex protocol was correlated in quantitation cycles (Cq) pro- mouse Fibroblast activation protein (FAP; Abcam cat. #ab218164), duced by the BioMark Real-Time PCR Software. To minimize vari- mouse anti-human estrogen receptor a (Agilent cat. #M7047, RRID: ation within and between runs, the data were normalized using both an AB_2101946), rabbit anti-human vimentin (Abcam cat. #ab16700, internal control (extension control) and an interplate control, and RRID:AB_443435), Dako EnVision þ System-HRP Labeled Polymer transformed using a predetermined correction factor. The prepro- anti-rabbit (Dako cat. #K4002), and anti-mouse (Dako cat. #K4000). cessed data were provided in the arbitrary unit, normalized protein Sections were counterstained with Mayer's hematoxylin. Negative expression (NPX), on a log2 scale, which then were linearized using the controls did not show staining. Images of 10 areas of each tumor formula 2NPX. A high NPX value corresponded to a high protein section from 3 to 4 mice in each treatment group, and three tumor concentration. Values represented a relative quantification, which sections and three normal breast tissue sections from each patient were means that no comparison of absolute levels between different proteins acquired on an Olympus BX43 microscope ( 40/0.75 magnification) could be made. and digitally analyzed and quantified using ImageJ software version 1.52n (ImageJ, RRID:SCR_003070). Statistical analysis For immunofluorescence, sections were exposed to rat anti-mouse The Wilcoxon signed-rank test was used to compare paired obser- Ly6G, incubated with conjugated donkey anti-rat antibody (Alexa vations. The Student t test was used for quantitative data. For all tests, flour 594, Thermo Fisher Scientific cat. #A-21209, RRID:AB_2535795) P < 0.05 was considered significant. The proteomics data were analyzed and mounted using SlowFade Gold containing 40,6-diamidino-2- using the Student t test followed by the Benjamini, Krieger, and phenylindole (DAPI; Invitrogen cat. #S36938). Samples were visual- Yekutieli procedure to assess the false discovery rate (FDR), which ized with an Olympus BX43 light/fluorescence microscope ( 40/0.75 was set to 5%. Only proteins above the limit of detection in ≥50% of the magnification) with excitation filters BP360-370 and BP530-550 using samples were included in the analyses. Data were expressed as the an Olympus DP72 CCD camera, analyzed by CellSens Imaging mean standard deviation (SD). All analyses were performed using software, and converted to RGB images with the same threshold using Prism 7.0 (GraphPad Software). ImageJ.

Human study Results þ The Regional Ethical Review Board of Linkoping,€ Sweden, Fulvestrant inhibited the growth of experimental ER breast approved the study, which was carried out in accordance with the cancer in vivo more effectively than tamoxifen þ Declaration of Helsinki. All subjects gave written informed consent. ER breast cancers were established orthotopically in the mam- þ Ten women with ongoing early ER breast cancer underwent mary fat pad in oophorectomized nude mice. Physiologic levels of microdialysis the day before their scheduled surgery. During micro- estradiol were maintained in all animals. At similar tumor sizes, dialysis, one catheter was inserted into the cancer tissue and a treatment with either tamoxifen or fulvestrant was initiated in the second catheter was inserted into normal adjacent breast tissue. The presence of estradiol and continued for 24 days. As demonstrated microdialysis catheters (M Dialysis AB) consisted of a 10-mm-long in Fig. 1A, fulvestrant treatment resulted in significantly decreased tubular dialysis membrane (diameter 0.52 mm, 100,000 atomic tumor growth compared with tamoxifen as measured both during mass cutoff) glued to the end of a double-lumen tube. Catheters tumor growth in vivo by tumor area and at the end of treatment by were inserted via a splitable introducer (M Dialysis AB), connected tumor weight. Both antiestrogen treatments decreased cell prolif- to a microinfusion pump (M Dialysis AB), and perfused with 154 eration as compared with untreated tumors, and fulvestrant exhib- mmol/L NaCl and 60 g/L hydroxyethyl starch (Voluven; Fresenius ited significantly decreased proliferation as compared with tamox- Kabi) at 0.5 mL/min. Prior to the insertion of each catheter, each ifen (Fig. 1B). As expected, fulvestrant induced downregulation of area was treated with 0.5 mL lidocaine (10 mg/mL) intracutane- ER in the tumor cells (Fig. 1C). ously. After a 60-minute equilibration period, the outgoing perfus- ate was stored at 80Cforsubsequentanalysis. Molecular characterization of the extracellular þ Human monocytes, isolated as described above, were cultured microenvironment in vivo in experimental ER breast cancer 24 hours in 96-well plates in DMEM with 2 mmol/L glutamine and during antiestrogen therapy 10% heat-inactivated FBS, containing E2 1 nmol/L 1 mmol/L Next, we wanted to examine whether the treatments differentially tamoxifen or 1 mmol/L fulvestrant. Culture media were analyzing affected the release of proteins into the extracellular space, thereby for ENA78, FGF basic, G-CSF, GM-CSF, INFg, IL1a, IL1b, IL2, IL4, altering the intercellular cross-talk. Tumors from the different treat- IL5, IL6, IL8, IL10, IL17, CCL2, CCL3, CCL4, CCL5, and TNFa ment groups were subjected to microdialysis to sample the extracel- using Magnetic Luminex High Performance Assay Kit (R&D Systems lular proteins in vivo. In total, 234 individual human proteins (listed in cat. # LUHM000) analyzed with a multiplex bead reader FlexMap Supplementary Table 1) were detected in the microdialysates of the 3D (Luminex Corporation). breast cancer tumors from the mice. After FDR correction, we þ observed significant alterations in 38 proteins from the ER tumors Olink proteomics of mice treated with fulvestrant; of these, 4 were significantly upre- þ Samples were submitted on 96-well PCR plates to Olink Proteomics gulated and 34 were significantly downregulated (Fig. 2A). In ER for analyses. In brief, 1 mL of undiluted sample was incubated with tumors from tamoxifen-treated animals, 12 proteins were significantly proximity antibody pairs tagged with DNA reporter molecules. After downregulated and 1 protein was significantly upregulated (Fig. 2A). binding of the antibodies to their corresponding antigens, the respec- All 13 proteins that were significantly changed in the tamoxifen- tive DNA tails formed an amplicon by proximity extension, which was treated group were also significantly altered in the fulvestrant-treated quantified by high-throughput real-time PCR (BioMark HD System; group (Fig. 2B). Thus, 25 proteins were significantly changed in the

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Figure 1. Fulvestrant inhibited the growth of experimental ERþ breast cancer more effectively than tamoxifen in vivo. Oophorectomized athymic mice supplemented with physiologic levels of estradiol (E2) were injected with MCF7 into the dorsal mammary fat pads. At similar tumor sizes, mice either continued with E2 or were additionally treated with tamoxifen (E2 þ Tam; 1 mg/mouse every second day, s.c.) or fulvestrant (E2 þ Fulv; 5 mg/mouse every 3 days, s.c.). A, Tumor growth. , P < 0.01; , P < 0.001 vs. control (E2) and ##, P < 0.01 vs. E2 þ Tam. Tumor weight at the end of the experiment. , P < 0.01; , P < 0.001. Data, mean SD. B, Tumor sections from each treatment group were stained for proliferation (Ki67) and quantiﬁed as the percentage of area with positive staining in the different treatment groups. , P < 0.05; , P < 0.01; , P < 0.001. Data, mean SD. C, Tumor sections from each treatment group were stained for ERa and quantiﬁed as the percentage of cells with positive staining in the different treatment groups. , P < 0.001. Data, mean SD.

fulvestrant-treated group that were not changed in the tamoxifen- cantly increased the levels of murine IL6 and murine IL10 and treated group, whereas no proteins were changed only in the tamoxifen decreased the levels of murine CCL2, whereas murine CCL5 was group (Fig. 2B). unaffected. No differences were detected in murine TGFa, CCL3, The majority of proteins released from human cancer cells were CCL20, CXCL1, or CXCL9. associated with immune function. Therefore, we next determined To determine whether the hormone treatments could affect the whether each treatment affected inﬂammatory proteins released by secretome of macrophages per se, we set up cultures of freshly stroma cells of murine origin. A total of nine proteins could be isolated human monocytes and exposed them to estradiol alone and detected in all samples. As shown in Fig. 2C, fulvestrant signiﬁ- in combination with tamoxifen or fulvestrant. Indeed, we found

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that fulvestrant, in the presence of estradiol, signiﬁcantly decreased Fulvestrant decreased the inﬁltration of innate immune cells the levels of ENA78, ILb, IL8, CCL2, CCL3, CCL4, and TNFa and angiogenesis more potently than tamoxifen in þ (Fig. 2D). Tamoxifen added to estradiol did not change the levels of experimental ER breast cancer any of the detected cytokines. FGFbasic,G-CSF,GM-CSF,INFg, Several of the proteins that were selectively changed in the fulves- IL1a,IL2,IL4,IL5,IL6,IL10,IL17werebelowthedetectionlimitof trant group were associated with immune function and angiogenesis. the assay. Therefore, we investigated whether there were any differences between

Figure 2. Molecular characterization of the extracellular microenvironment in vivo in experimental ERþ breast cancer during antiestrogen therapy. Oophorecto- mized athymic mice supplemented with physiologic levels of estradiol (E2) were injected with MCF7 into the dorsal mammary fat pads. At similar tumor sizes, mice either continued with E2 or were additionally treated with tamoxifen (E2 þ Tam; 1 mg/mouse every second day, s.c.) or fulvestrant (E2 þ Fulv; 5 mg/mouse every 3 days, s.c.). Size- matched tumors from the different treatment groups underwent microdialysis for sampling of extracellular proteins in vivo. A, Volcano plots illustrating the log10 statistical significance (FDR- adjusted P value) in relation to the log2-fold change of 234 human proteins released by the cancer cells treated with E2 þ Tam or E2 þ Fulv vs. control (E2). Proteins that passed the FDR-adjusted P < 0.05 significance threshold and were downregulated (log2-fold change <1.0) are marked in blue. Proteins that passed the FDR-adjusted P < 0.05 significance threshold and were upregulated (log2-fold change >1.0) are marked in red. B, A Venn diagram illustrating significantly altered proteins after FDR correction for E2 þ Tam or E2 þ Fulv vs. control (E2). Proteins that were downregulated are marked in blue and proteins that were upregulated are marked in red. C, Murine immune-regulating proteins released by the stroma. , P < 0.05; , P < 0.01; , P < 0.001. Data, mean SD. D, Freshly isolated human monocytes were cultured for 24 hours in the presence of estradiol (E2), E2 þ tamoxifen (Tam) or E2 þ fulvestrant (Fulv) and secreted cytokines were quantified as described in Materials and Methods. , P < 0.05; , P < 0.01. Data, mean SD.

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the treatment groups in the number of infiltrating innate immune cells Angiogenesis was measured as the percent vessel area. Both treat- into the tumors or vessel area. Neutrophils were predominantly found in ments resulted in decreased angiogenesis compared with the control the invasive margin of the tumors, but almost no neutrophils were group (Fig. 3C). In addition, fulvestrant was more efficient in decreas- detected within the tumor tissue, as previously described (15). Therefore, ing angiogenesis compared with tamoxifen (Fig. 3C). No differences we quantified the number of neutrophils in the invasive margin. Both were found in the amount of tumor-associated fibroblasts between the treatments resulted in a decreased number of neutrophils compared different treatments (Fig. 3D). with the control group (Fig. 3A). In addition, fulvestrant significantly reduced the number of neutrophils compared with tamoxifen (Fig. 3A). Changes in EMT markers after fulvestrant and tamoxifen þ In contrast to neutrophils, macrophages were detected within the treatment of experimental ER breast cancer tumor stroma. Therefore, we quantified the percentage of area that The cross-talk between cancer cells and immune cells has been was positively stained for the macrophage marker F4/80. Both treat- shown to affect the EMT of cancer cells (29). We investigated ments resulted in a significantly decreased area of infiltrating macro- whether the reduced number of innate immune cells in the tumors phages compared with the control group (Fig. 3B). Fulvestrant also of tamoxifen- or fulvestrant-treated mice resulted in changes in significantly decreased the area of infiltrating macrophages compared EMT markers. During the EMT, E-cadherin is downregulated and with tamoxifen (Fig. 3B). N-cadherin is upregulated. E-cadherin was expressed in all tumors

Figure 3. Fulvestrant reduced innate immune cells in experimental ERþ breast cancer more effectively than tamoxifen in vivo. Oophorectomized athymic mice supplemented with physiologic levels of estradiol (E2) were injected with MCF7 into the dorsal mammary fat pads. At similar tumor sizes, mice either continued with E2 or were additionally treated for 24 days with tamoxifen (E2 þ Tam; 1 mg/mouse every second day, s.c.) or fulvestrant (E2 þ Fulv; 5 mg/mouse every 3 days, s.c.). A, Ten hotspot areas of each tumor were selected to quantify the number of neutrophils (Ly6G; red) infiltrating the invasive margin. Scale bar, 20 mm. , P < 0.05; , P < 0.01; , P < 0.001. Data, mean SD. B, Tumor sections stained for infiltrating macrophages were quantified as the percentage of area with positive staining for the macrophage marker F4/80. Scale bar, 20 mm. , P < 0.001. Data, mean SD. C, Ten hotspot areas of each tumor were selected for quantification of the vessel area stained with von Willebrand factor. Scale bar, 100 mm. , P < 0.05; , P < 0.001. Data, mean SD. D, Ten hotspot areas of each tumor were selected for quantification of tumor-associated fibroblast stained with FAP. Scale bar, 20 mm. Data, mean SD.

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with no detectable changes between the three groups (Fig. 4A). A Fulvestrant decreased the infiltration of innate immune cells þ stronger expression of N-cadherin was detected across all groups, more potently than tamoxifen in experimental ER breast and both tamoxifen and fulvestrant induced a significant down- cancer in immunocompetent mice regulation of N-cadherin compared with tumors in the control The advantages of using the nude mouse model of cancer growth is group (Fig. 4B). In addition, fulvestrant significantly decreased N- that human cancer cells can be investigated, and as the stroma is of cadherin compared with tamoxifen (Fig. 4B). The transcription murine origin, it is possible to distinguish events originating from the factor Snail, which promotes mesenchymal transition, was signif- cancer cells (human proteins) or from the stroma (murine proteins). icantly downregulated by both treatments compared with control By using the nude Balb/C-nu/nu mouse, investigations of the innate tumors (Fig. 4C). The transcription factor Slug was also down- immune system are possible as these mice have an intact innate regulated by both treatments and significantly decreased in the immunity, including B cells and NK cells, but lack T cells. fulvestrant-treated tumors compared with the tamoxifen group However, to further understand the effects on innate immune þ (Fig. 4D). The mesenchymal protein vimentin was not detectable cells in an immunocompetent host, we next set up mice with ER in any tumors. breast cancer from the PyMT transgenic mouse model. Fulvestrant

Figure 4. EMT expression in experimental ERþ breast cancer treated with fulvestrant or tamoxifen. Oophorectomized athymic mice supplemented with physiologic levels of estradiol (E2) were injected with MCF7 into the dorsal mammary fat pads. At similar tumor sizes, mice either continued with E2 or were additionally treated for 24 days with tamoxifen (E2 þ Tam; 1 mg/mouse every second day, s.c.) or fulvestrant (E2 þ Fulv; 5 mg/mouse every 3 days, s.c.). A, Tumor sections stained for E-cadherin were quantified as the percentage of area with positive staining. B, Tumor sections stained for the EMT marker N-cadherin were quantified as the percentage of area with positive staining. C, Tumor sections stained for the EMT marker Snail were quantified as the percentage of cells with intense staining. D, Tumor sections stained for the EMT marker Slug were quantified as the percentage of cells with intense staining. Scale bar, 20 mm. , P < 0.05; , P < 0.001. Data, mean SD.

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þ reduced tumor growth significantly compared with tamoxifen and proliferation of ER breast cancer tumors in mice, fulvestrant (Fig. 5A). At the end of the experiment, tumor weights were: induced profound effects in the tumor stroma by significantly 508 mg 43 (SD) in the E2-alone group, 460 112 in the E2 reducing the infiltration of macrophages and neutrophils as com- þ Tam group, and 310 42 in the E2 þ Fulv group, leading to E2 pared with tamoxifen in both immunodeficient and immunocom- versus E2 þ Tam, not significant, E þ Tam versus E2 þ Fulv, P < petent mice. Additionally, in the presence of estradiol, fulvestrant 0.01, and E2 versus E2 þ Fulv, P < 0.001 n ¼ 7ineachgroup. was more effective than tamoxifen in inhibiting cancer cell dissem- Additionally, similar to the results in nude mice, fulvestrant sig- ination mediated by neutrophils and monocytes. In vivo sampling of nificantly decreased the number of neutrophils in the invasive 234 extracellular proteins identified 38 proteins related to inflam- margin of the tumors and infiltration of macrophages into the mation that were significantly altered after fulvestrant and tamox- tumor tissue (Fig. 5B and C). ifen therapy; 25 of these proteins were significantly changed in fulvestrant-treated animals only.Ofthese38proteins,36were þ þ Fulvestrant decreased ER cancer cell dissemination more detected in ER human breast cancer in vivo and 31 of these were potently than tamoxifen upregulated. Thus, the proteins that were targeted by fulvestrant in As metastasis is the major cause of death in breast cancer patients, the animal model were also upregulated in humans. This suggests we next investigated if there were any differences between the treat- clinical relevance of the identified immune-mediated mechanism of ments of the capacity of cancer cells to disseminate. In the zebrafish action by antiestrogen therapy. model of cancer cell metastases, fulvestrant inhibited estradiol- The intercellular cross-talk between cancer cells and stroma cells in þ dependent dissemination of ER breast cancer cells in the presence the tissue microenvironment is a key determinant for carcinogenesis of neutrophils and monocytes more effectively than tamoxifen and the efficacy of cancer therapy (12, 13). Innate immune cells such as (Fig. 5D and E). macrophages and neutrophils constitute a major component of the stroma in human breast cancer (30, 31). All immune cells express ERs, þ Upregulation of proteins in human ER breast cancer in vivo and estrogen governs several signaling pathways within these cells (32). To elucidate whether human breast cancer exhibit increased infil- Whether estrogen exposure results in a pro- or anti-inflammatory state tration of innate immune cells and if the affected proteins in exper- is dependent on hormone levels. At normal physiologic levels, as þ imental ER breast cancer were present—and therefore druggable—in observed during the menstrual cycle, estradiol promotes the produc- human breast cancer, we stained human breast cancers for CD68 and tion of proinflammatory cytokines. In contrast, at very high levels (e.g., þ performed microdialysis in women with ER breast cancer prior to as observed during pregnancy), estradiol promotes an anti- their surgery. Human breast cancers exhibited increased numbers of inflammatory response (32). In the present study, we administered innate immune cells compared with normal adjacent breast tissue menstrual cycle-related physiologic levels of estradiol, thus reflecting (Fig. 6A). Of the 38 proteins that were significantly up- or down- premenopausal breast cancer conditions. Fulvestrant has previously regulated in experimental breast cancer samples, 36 were detectable in been shown to be more effective than tamoxifen after estrogen the human samples; only UMOD (uromodulin) and IL20A were not withdrawal (33). Our data suggest that fulvestrant also exerts increased detectable. Of the 36 detected proteins, 31 were significantly upregu- efficacy compared with tamoxifen in the presence of estradiol, sug- þ lated in ER breast cancers compared with normal adjacent breast gesting that fulvestrant would also be effective in premenopausal tissue (Figs. 6B and 7A–C). women. Based on the types of extracellular proteins released by the cancer cells as identified during microdialysis, the major difference between fulvestrant and tamoxifen therapy may be in the mediation of Discussion the inflammatory response and angiogenesis. Several inflammatory þ Today, approximately 25% of patients with ER breast cancer will proteins were significantly affected by the two treatments. Whereas have a recurrence of their disease despite adjuvant antiestrogen bothtreatmentsincreasedIL1RA, whichisantitumorigenic (16,34,35), therapies. Thus, there is a need to potentiate ER-targeting therapies fulvestrant but not tamoxifen significantly decreased IL2RA levels. As against breast cancer. Previous studies have demonstrated that an inhibitor of IL2, IL2RA has been associated with cancer progres- events in the stroma are determinants for the efficiency of cancer sion (36). The immune regulatory proteins ITGAM (Integrin Alpha therapies. How different ER targeting therapies may affect the tumor M), LILBRs (leukocyte immunoglobulin-like receptor subfamily B), stroma has not yet been elucidated. In our current study, the ER TIMD4 (T-cell immunoglobulin and mucin domain containing 4), antagonist fulvestrant was identified as a more efficient therapy and CCL18, which were more effectively decreased by fulvestrant þ against ER breast cancer than the SERM tamoxifen in the presence compared with tamoxifen, have been shown to be upregulated in of physiologic levels of estradiol. In addition to decreased growth cancer and associated with enhanced tumor growth and poor

Figure 5. Fulvestrant reduced innate immune cell infiltration in ERþ breast cancer in immune-competent mice and decreased ERþ breast cancer cell dissemination in zebrafish. Oophorectomized FVB/N mice were supplemented with a physiologic level of estradiol (E2) and injected with PyMT tumor cells in the mammary fat pad and treated for 11 days with tamoxifen (E2 þ Tam; 1 mg/mouse every second day, s.c.) or fulvestrant (E2 þ Fulv; 5 mg/mouse every 3 days, s.c.) or left untreated. A, Tumor growth. , P < 0.05; , P < 0.01; ###, P < 0.001 vs. E2. Data, mean SD. B, Ten hotspot areas of each tumor were selected to quantify the number of neutrophils (Ly6G; red) infiltrating the invasive margin. Scale bar, 20 mm. , P < 0.01; , P < 0.001. Data, mean SD. C, Tumor sections stained for infiltrating macrophages were quantified as the percentage of area with positive staining for the macrophage marker F4/80. Scale bar, 20 mm. , P < 0.05; , P < 0.001. Data, mean SD. D, Zebrafish embryos, with green blood vessels, were coinjected with MCF7 þ 50% human neutrophils (Neu). All embryos were exposed to estradiol (E2) and treated with tamoxifen (Tam) or fulvestrant (Fulv), as described in Materials and Methods. The number of disseminated cells was counted 24 hours after injection. Scale bar, 100 mm. n ¼ 20–22 in each group. , P < 0.05; , P < 0.01. Data, mean SD. E, Zebrafish embryos were coinjected with MCF7 þ 10% human monocytes (MQ). All embryos were exposed to estradiol (E2) and treated with tamoxifen (Tam) or fulvestrant (Fulv), as described in Materials and Methods. The number of disseminated cells was counted 3 days after injection. Scale bar, 100 mm. n ¼ 20–22 in each group. , P < 0.05; , P < 0.01. Data, mean SD. BV, blood vessels. Arrowheads, disseminated cancer cells.

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þ survival (37–40). These inflammatory proteins may affect macro- nonmetastatic ER breast cancer cells into metastatic cells (14, 15). phage and neutrophil infiltration into tumors. Reducing the number Additionally, innate immune cells may be activated by various cues þ of infiltrating innate immune cells into ER breast cancer may be in the tumor microenvironment into tumor-promoting or antitu- critical, as it has been shown previously that these cells may turn morigenicphenotypesdependingonthetypeandamountsof

Figure 6. Extracellular levels of proteins with immune modulating functions in human ERþ breast cancer in vivo. A, Sections from breast cancers and normal adjacent breast tissue from patients were stained with CD68, a marker for innate immune cells, and percentage of area was quantiﬁed as described in Materials and Methods section (n ¼ 30 in each group; cancer and normal breast tissue, respectively). Representative sections are depicted. Data, mean SD. Scale bar, 20 mm. B, Patients with breast cancer underwent microdialysis 1 day prior to their surgery. One catheter was inserted into the breast cancer (red bars) and another catheter was inserted into normal adjacent breast tissue (white bars) for in vivo collection of extracellular proteins. The multiplex proximity extension assay was used for protein quantiﬁcation. The data represent protein abundance in linear values (2NPX as described in Materials and Methods). , P < 0.05; , P < 0.01. Data, mean SD.

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secreted factors. Our data show that while fulvestrant decreased the These results were corroborated in vitro where fulvestrant affected number of innate immune cells, the treatment also increased the secreted levels of several cytokines without affecting the number stroma-derived inflammatory cytokines. This may be explained by of cells. Inflammation and angiogenesis are intertwined processes, phenotypic changes of the immune cells by fulvestrant as the and several proteins regulate both of these processes. Two of the different treatments had no effect on tumor-associated fibroblasts. most potent proteins in these events are VEGF and IL8, which were

Figure 7. Extracellular levels of angiogenic factors, growth factors, and metabolic proteins in human ERþ breast cancer in vivo. Patients with breast cancer underwent microdialysis 1 day prior to their surgery. One catheter was inserted into the breast cancer (red bars) and another catheter was inserted into normal adjacent breast tissue (white bars) for in vivo collection of extracellular proteins. The multiplex proximity extension assay was used for protein quantiﬁcation. The data represent protein abundance in linear values (2NPX as described in Materials and Methods). A, Extracellular levels of angiogenic factors. B, Extracellular levels of growth factors. C, Extracellular levels of metabolic proteins. , P < 0.05; , P < 0.01. Data, mean SD.

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affected by both treatments in line with previous published important roles (50). Thus, therapeutic effects directly on tumor cells data (17, 18, 41, 42). Fulvestrant but not tamoxifen also affected as well as indirect effects via infiltrated immune cells may result in additional potent proangiogenic proteins; ANGPTL3 (angiopoie- changes of the EMT program. þ tin-related protein 3) and AP-N (aminopeptidase N), both of which Targeting the ER in ER breast cancer is the current gold standard have been implicated in breast cancer progression, were signifi- for both adjuvant and metastatic treatment. We have shown here that cantly decreased (43, 44). In line with the decreased levels of direct targeting of the ER with the pure antiestrogen fulvestrant is a þ proangiogenic proteins, fulvestrant decreased tumoral angiogenesis more efficient approach in treating ER breast cancer than direct more potently than tamoxifen. The clinical relevance of our exper- targeting with the partial agonist tamoxifen in the presence of estradiol. imental findings was confirmed in our clinical human samples from Our findings suggest that the effectiveness of fulvestrant is due to both þ ER breast cancer, where the majority of the affected proteins were direct effects on the cancer cells and profound effects on the tumor also upregulated. stroma. Fulvestrant also exerted increased effects in the tumor stroma as Currently, fulvestrant is the only approved SERD in clinical murine IL6 and murine IL10, which have been linked with a good practice. Due to its low bioavailability, the only administrative prognosis of breast cancer, were significantly increased compared with route for therapy is intramuscular injections, which limits its tamoxifen, whereas murine CCL2 was similarly decreased by both clinical use. Several early-phase studies are currently ongoing to fulvestrant and tamoxifen (45). These results support that stromal cells test oral SERDs compared with fulvestrant in patients with meta- expressing ERs respond to antiestrogen therapy and are consistent static cancer (https://clinicaltrials.gov). If oral SERDs are as efficient with prior studies (14, 15, 46). as fulvestrant, then more patients may be able to benefitfromthis In addition to the inflammatory mediators, a number of therapy. Moreover, as fulvestrant had major effects on intercellular growth factors and proteins involved in metabolism were also affected cross-talk and immune function, combinations of fulvestrant by the different treatments. In particular, some growth factors and andimmune-mediatedtherapiesmay be interesting to explore. proteins responded to fulvestrant only including the growth factors Finally, although fulvestrant is used currently only in postmeno- TNXB (tenacin-X), PLTP (phospholipid transfer protein), SERPINA7 pausal patients, our data support that fulvestrant may be effective in (thyroxine-binding globulin) and the tumor suppressor proteins premenopausal patients. AXIN1 and Dkk-1 (Dickkopf-related protein 1) corroborating previous data showing that the downregulation of AXIN1 by estrogen plays Disclosure of Potential Conflicts of Interest þ an important role in ER breast cancer (47). No potential conflicts of interest were disclosed. Metastatic disease is the major cause of breast cancer–associated þ mortality, including ER breast cancer. The majority of ER-expressing Authors’ Contributions primary breast cancers maintain the ER at the metastatic site and A. Abrahamsson: Validation, investigation, methodology, writing-original draft, – almost a third of initially ER-negative primary tumors gain ER writing review, and editing. G. Vazquez Rodriguez: Investigation, methodology, writing–review, and editing. C. Dabrosin: Conceptualization, resources, formal expression in the metastatic lesion (48). Thus, investigating metastatic þ analysis, supervision, funding acquisition, validation, investigation, visualization, mechanisms of ER breast cancer is key for improved treatments. – þ methodology, writing-original draft, project administration, writing review, and Hitherto, there are no experimental breast cancer models in which ER editing. primary breast cancer spontaneously metastasizes with maintained ER expression. However, the zebrafish metastasis model allows for such Acknowledgments investigations (49). Our present data clearly showed that fulvestrant This work was supported by grants to C. Dabrosin from the Swedish Cancer þ inhibited ER primary tumor growth as well as cancer cell dissem- Society (2018/464), the Swedish Research Council (2018-02584), LiU-Cancer, and € ination more efficiently than tamoxifen. Fulvestrant also affected EMT ALF of Linkoping University Hospital. features of the cancer cells by downregulation of N-cadherin expres- The costs of publication of this article were defrayed in part by the payment of page sion, which empower cells from primary tumors to become metastatic. charges. This article must therefore be hereby marked advertisement in accordance Interestingly, similar results have previously been shown in estrogen- with 18 U.S.C. Section 1734 solely to indicate this fact. dependent lung cancer growth (46). The EMT program is activated by several pathways, among which, signaling between cancer cells and the Received May 20, 2020; revised July 7, 2020; accepted August 18, 2020; microenvironment in general and immune cells in particular play published first August 27, 2020.

References 1. Grann VR, Troxel AB, Zojwalla NJ, Jacobson JS, Hershman D, Neugut AI. 6. Burstein HJ, Curigliano G, Loibl S, Dubsky P, Gnant M, Poortmans P, et al. Hormone receptor status and survival in a population-based cohort of patients Estimating the beneﬁts of therapy for early-stage breast cancer: the St. Gallen with breast carcinoma. Cancer 2005;103:2241–51. International Consensus Guidelines for the primary therapy of early breast 2. Goldhirsch A, Ingle JN, Gelber RD, Coates AS, Thurlimann B, Senn HJ. cancer 2019. Ann Oncol 2019;30:1541–57. Thresholds for therapies: highlights of the St Gallen International Expert 7. Ruhstaller T, Giobbie-Hurder A, Colleoni M, Jensen MB, Ejlertsen B, de Consensus on the primary therapy of early breast cancer 2009. Ann Oncol Azambuja E, et al. Adjuvant letrozole and tamoxifen alone or sequentially for 2009;20:1319–29. postmenopausal women with hormone receptor-positive breast cancer: long- 3. Lewis JS, Jordan VC. Selective estrogen receptor modulators (SERMs): mechan- term follow-Up of the BIG 1-98 trial. J Clin Oncol 2019;37:105–14. isms of anticarcinogenesis and drug resistance. Mutat Res 2005;591:247–63. 8. Robertson JF, Come SE, Jones SE, Beex L, Kaufmann M, Makris A, et al. 4. Jordan VC. Antiestrogens and selective estrogen receptor modulators as mul- Endocrine treatment options for advanced breast cancer–the role of fulvestrant. tifunctional medicines. 2. Clinical considerations and new agents. J Med Chem Eur J Cancer 2005;41:346–56. 2003;46:1081–111. 9. Clarke CA, Keegan TH, Yang J, Press DJ, Kurian AW, Patel AH, et al. Age- 5. Jordan VC.. Antiestrogens and selective estrogen receptor modulators as mul- speciﬁc incidence of breast cancer subtypes: understanding the black-white tifunctional medicines. 1. Receptor interactions. J Med Chem 2003;46:883–908. crossover. J Natl Cancer Inst 2012;104:1094–101.

OF12 Cancer Res; 80(20) October 15, 2020 CANCER RESEARCH

Comparison of Antiestrogen Therapies in Breast Cancer

10. Hanahan D, Weinberg RA. Hallmarks of cancer: the next generation. Cell 2011; 29. Marcucci F, Bellone M, Caserta CA, Corti A. Pushing tumor cells towards a 144:646–74. malignant phenotype: stimuli from the microenvironment, intercellular com- 11. Busch S, Ryden L, Stal O, Jirstrom K, Landberg G. Low ERK phosphorylation in munications and alternative roads. Int J Cancer 2014;135:1265–76. cancer-associated fibroblasts is associated with tamoxifen resistance in pre- 30. Mantovani A, Allavena P, Sica A, Balkwill F. Cancer-related inflammation. menopausal breast cancer. PLoS One 2012;7:e45669. Nature 2008;454:436–44. 12. Farmer P, Bonnefoi H, Anderle P, Cameron D, Wirapati P, Becette V, et al. A 31. Whiteside TL. The tumor microenvironment and its role in promoting tumor stroma-related gene signature predicts resistance to neoadjuvant chemotherapy growth. Oncogene 2008;27:5904–12. in breast cancer. Nat Med 2009;15:68–74. 32. Kovats S. Estrogen receptors regulate innate immune cells and signaling path- 13. Hattar R, Maller O, McDaniel S, Hansen KC, Hedman KJ, Lyons TR, et al. ways. Cell Immunol 2015;294:63–9. Tamoxifen induces pleiotrophic changes in mammary stroma resulting in 33. Osborne CK, Coronado-Heinsohn EB, Hilsenbeck SG, McCue BL, Wakeling AE, extracellular matrix that suppresses transformed phenotypes. Breast Cancer Res McClelland RA, et al. Comparison of the effects of a pure steroidal antiestrogen 2009;11:R5. with those of tamoxifen in a model of human breast cancer. J Natl Cancer Inst 14. Svensson S, Abrahamsson A, Rodriguez GV, Olsson AK, Jensen L, Cao Y, et al. 1995;87:746–50. CCL2 and CCL5 are novel therapeutic targets for estrogen-dependent breast 34. Bar D, Apte RN, Voronov E, Dinarello CA, Cohen S. A continuous delivery cancer. Clin Cancer Res 2015;21:3794–805. system of IL-1 receptor antagonist reduces angiogenesis and inhibits tumor 15. Vazquez Rodriguez G, Abrahamsson A, Jensen LD, Dabrosin C. Estradiol development. Faseb J 2004;18:161–3. promotes breast cancer cell migration via recruitment and activation of neu- 35. Elaraj DM, Weinreich DM, Varghese S, Puhlmann M, Hewitt SM, Carroll NM, trophils. Cancer Immunol Res 2017;5:234–47. et al. The role of interleukin 1 in growth and metastasis of human cancer 16. Lindahl G, Saarinen N, Abrahamsson A, Dabrosin C. Tamoxifen, flaxseed, and xenografts. Clin Cancer Res 2006;12:1088–96. the lignan enterolactone increase stroma- and cancer cell-derived IL-1Ra and 36. Jiang T, Zhou C, Ren S. Role of IL-2 in cancer immunotherapy. Oncoimmunol- decrease tumor angiogenesis in estrogen-dependent breast cancer. Cancer Res ogy 2016;5:e1163462. 2011;71:51–60. 37. Kang X, Kim J, Deng M, John S, Chen H, Wu G, et al. Inhibitory leukocyte 17. Garvin S, Dabrosin C. Tamoxifen inhibits secretion of vascular endothelial immunoglobulin-like receptors: immune checkpoint proteins and tumor sus- growth factor in breast cancer in vivo. Cancer Res 2003;63:8742–8. taining factors. Cell Cycle 2016;15:25–40. 18. Aberg UW, Saarinen N, Abrahamsson A, Nurmi T, Engblom S, Dabrosin C. 38. Gabrilovich DI, Nagaraj S. Myeloid-derived suppressor cells as regulators of the Tamoxifen and flaxseed alter angiogenesis regulators in normal human breast immune system. Nat Rev Immunol 2009;9:162–74. tissue in vivo. PLoS One 2011;6:e25720. 39. Tan X, Zhang Z, Yao H, Shen L. Tim-4 promotes the growth of colorectal cancer 19. Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY, et al. The epithelial- by activating angiogenesis and recruiting tumor-associated macrophages via the mesenchymal transition generates cells with properties of stem cells. Cell 2008; PI3K/AKT/mTOR signaling pathway. Cancer Lett 2018;436:119–28. 133:704–15. 40. Su S, Liu Q, Chen J, Chen J, Chen F, He C, et al. A positive feedback loop between 20. Polyak K, Weinberg RA. Transitions between epithelial and mesenchymal mesenchymal-like cancer cells and macrophages is essential to breast cancer states: acquisition of malignant and stem cell traits. Nat Rev Cancer 2009;9: metastasis. Cancer Cell 2014;25:605–20. 265–73. 41. Bendrik C, Dabrosin C. Estradiol increases IL-8 secretion of normal human 21. Ricciardi M, Zanotto M, Malpeli G, Bassi G, Perbellini O, Chilosi M, et al. breast tissue and breast cancer in vivo. J Immunol 2009;182:371–8. Epithelial-to-mesenchymal transition (EMT) induced by inflammatory priming 42. Dabrosin C.Variability of vascular endothelial growth factor in normal human elicits mesenchymal stromal cell-like immune-modulatory properties in cancer breast tissue in vivo during the menstrual cycle. J Clin Endocrinol Metab 2003;88: cells. Br J Cancer 2015;112:1067–75. 2695–8. 22. Yang J, Weinberg RA. Epithelial-mesenchymal transition: at the crossroads of 43. Carbone C, Piro G, Merz V, Simionato F, Santoro R, Zecchetto C, et al. development and tumor metastasis. Dev Cell 2008;14:818–29. Angiopoietin-like proteins in angiogenesis, inflammation and cancer. Int J Mol 23.TohB,WangX,KeebleJ,SimWJ,KhooK,WongWC,etal.Mesen- Sci 2018;19:431. chymal transition and dissemination of cancer cells is driven by myeloid- 44. Wickstrom M, Larsson R, Nygren P, Gullbo J. Aminopeptidase N (CD13) as a derived suppressor cells infiltrating the primary tumor. PLoS Biol 2011;9: target for cancer chemotherapy. Cancer Sci 2011;102:501–8. e1001162. 45. Ahmad N, Ammar A, Storr SJ, Green AR, Rakha E, Ellis IO, et al. IL-6 and IL-10 24. Sangaletti S, Tripodo C, Santangelo A, Castioni N, Portararo P, Gulino A, are associated with good prognosis in early stage invasive breast cancer patients. et al. Mesenchymal transition of high-grade breast carcinomas depends on Cancer Immunol Immunother 2018;67:537–49. extracellular matrix control of myeloid suppressor cell activity. Cell Rep 46. Hamilton DH, Griner LM, Keller JM, Hu X, Southall N, Marugan J, et al. 2016;17:233–48. Targeting estrogen receptor signaling with fulvestrant enhances immune and 25. Dabrosin C, Margetts PJ, Gauldie J. Estradiol increases extracellular levels of chemotherapy-mediated cytotoxicity of human lung cancer. Clin Cancer Res vascular endothelial growth factor in vivo in murine mammary cancer. Int J 2016;22:6204–16. Cancer 2003;107:535–40. 47. Chimge NO, Little GH, Baniwal SK, Adisetiyo H, Xie Y, Zhang T, et al. RUNX1 26. Guy CT, Cardiff RD, Muller WJ. Induction of mammary tumors by expression of prevents oestrogen-mediated AXIN1 suppression and beta-catenin activation in polyomavirus middle T oncogene: a transgenic mouse model for metastatic ER-positive breast cancer. Nat Commun 2016;7:10751. disease. Mol Cell Biol 1992;12:954–61. 48. Lindstrom LS, Karlsson E, Wilking UM, Johansson U, Hartman J, Lidbrink EK, 27. Lin EY, Jones JG, Li P, Zhu L, Whitney KD, Muller WJ, et al. Progression et al. Clinically used breast cancer markers such as estrogen receptor, proges- to malignancy in the polyoma middle T oncoprotein mouse breast cancer terone receptor, and human epidermal growth factor receptor 2 are unstable model provides a reliable model for human diseases. Am J Pathol 2003; throughout tumor progression. J Clin Oncol 2012;30:2601–8. 163:2113–26. 49. Rouhi P, Jensen LD, Cao Z, Hosaka K, Lanne T, Wahlberg E, et al. Hypoxia- 28. Dabrosin C, Palmer K, Muller WJ, Gauldie J. Estradiol promotes growth and induced metastasis model in embryonic zebrafish. Nat Protoc 2010;5:1911–8. angiogenesis in polyoma middle T transgenic mouse mammary tumor explants. 50. Chaffer CL, Weinberg RA. A perspective on cancer cell metastasis. Science 2011; Breast Cancer Res Treat 2003;78:1–6. 331:1559–64.

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Fulvestrant-Mediated Attenuation of the Innate Immune Response Decreases ER + Breast Cancer Growth In Vivo More Effectively than Tamoxifen

Annelie Abrahamsson, Gabriela Vazquez Rodriguez and Charlotta Dabrosin

Cancer Res Published OnlineFirst August 27, 2020.

Updated version Access the most recent version of this article at: doi:10.1158/0008-5472.CAN-20-1705

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