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. 2023 Jul:7:100080.
doi: 10.1016/j.adcanc.2022.100080. Epub 2022 Dec 7.

Benzophenone-3 exposure alters composition of tumor infiltrating immune cells and increases lung seeding of 4T1 breast cancer cells

Stephanie M Morin  1   2 , Kelly J Gregory  1 , Brenda Medeiros  3 , Tigist Terefe  1 , Reyhane Hoshyar  4 , Ahmed Alhusseiny  5 , Shiuan Chen  6 , Richard C Schwartz  4 , D Joseph Jerry  2 , Laura N Vandenberg  3 , Sallie S Schneider  1   2   7
Affiliations

Benzophenone-3 exposure alters composition of tumor infiltrating immune cells and increases lung seeding of 4T1 breast cancer cells

Stephanie M Morin et al. Adv Cancer Biol Metastasis. 2023 Jul.

Abstract

Environmental chemicals are a persistent and pervasive part of everyday life. A subset of environmental chemicals are xenoestrogens, compounds that bind to the estrogen receptor (ER) and drive estrogen-related processes. One such chemical, benzophenone-3 (BP3), is a common chemical in sunscreen. It is a potent UV protectant but also is quickly absorbed through the skin. While it has been approved by the FDA, there is a renewed interest in the safety of BP3, particularly in relation to breast cancer. The focus of this study was to examine the impact that BP3 has on triple negative breast cancer (TNBC) through alterations to cells in the immune microenvironment. In this study, we exposed female mice to one of two doses of BP3 before injecting them with a TNBC cell line. Several immune endpoints were examined both in the primary tissues and from in vitro studies of T cell behavior. Our studies revealed that in the lung tumor microenvironment, exposure to BP3 not only increased the number of metastases, but also the total area of tumor coverage. We also found that BP3 caused alterations in immune populations in a tissue-dependent manner, particularly in T cells. Taken together, our data suggest that while BP3 may not directly affect the proliferation of TNBC, growth and metastasis of TNBC-derived tumors can be altered by BP3 exposures via the alterations in the immune populations of the tumor microenvironment.

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Conflict of interest statement

Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Fig. 1.
Fig. 1.. BP3 induces 4T1 proliferation in vivo.
4T1s were treated directly with 30 μM BP3 and A. proliferation of 4T1 cancer cells was measured via MTS assay. Migration of the 4T1s was measured through B. scratch wound assay and C. transwell migration assay. D. Schematic of Balb/c mouse experiment. E. Tumor volume in mice injected with 4T1 in the mammary fat pad starting on day 7 post injection using the formula V = (W (2) * L)/2 where V is volume, W is tumor width, and L is length. F. Mice were weighed throughout the experiment, and the change in weight was calculated starting at time of injection. G. Tumor foci in IV lung sections from IV mice were counted upon staining with H&E. H. In the IV lung H&E sections, the percent tumor coverage was calculated via measurement of total tumor size divided by total lung area. I. IV lung sections were stained with Ki67, and the total number of positive cells in the metastasis were counted. J. Representative images of Ki67 staining in IV lung tumor metastases. K. IV lungs were stained with cleaved caspase 3 and total number of positive cells were counted in the metastases.
Fig. 2.
Fig. 2.. BP3 alters immune populations in a tissue specific manner.
A. Immunohistochemistry was performed on lungs from IV mice that were paraffine embedded, to observe immune populations. From left to right, Vital New Red for eosinophils, Nimp-14 for neutrophils, CD45r for B cells. Positively staining cells were counted in the metastases of the lung. B. Lungs from IV mice were stained with T cell markers (left to right) T helper cells, cytotoxic T cells, and Tregs. C. Representative images from the lung metastasis of the IV group staining with Foxp3. D. Tumor from IM mice were paraffin embedded, sectioned and stained for T cell markers.
Fig. 3.
Fig. 3.. BP3 alters cytokine expression
A. RNA was harvested from sections of whole tissue from both IM and IV mice exposed to BP3. mRNA levels of the various cytokines were analyzed by qRT-PCR. All qRT-PCR results were performed with n = 5 and results were normalized to ATCB. B, C. Cells were exposed to 30 μM BP3 in vitro or exposed to conditioned media from cells that were exposed to BP3. Supernatant was collected and protein levels of CCL2 or CCL3 were measured via ELISA.
Fig. 4.
Fig. 4.. BP3 alters T cell expansion and cytokine expression in vitro
T cells were harvested from spleens of female 5-week Balb/c mice and activated via the presence of plate-bound CD3, CD28, and IL-2 in the media. A. Cell number, % viability, and cell diameter were quantified using the ChemoMetec NucleoCounter Nc-202. B. Gene expression of T cell activation genes was measured via qRT-PCR. All qRT = PCR reactions were run in biological triplicate and technical duplicate. C. T cell related cytokines were measured from the media of activated T cells exposed to 30 μM BP3 through the use of MSD. D. T cell activation, senescence, and exhaustion gene expression markers were interrogated through the use of qRT-PCR.
Fig. 5.
Fig. 5.. Exposure to BP3 reduces the normal activity of T regulatory cells
T cells were activated and then polarized or not to the Tregs through the addition of TGFb and retinoic acid. A. Cell expression of the surface markers, CD4 and CD25, and the transcription factor, Foxp3 were measured by flow cytometry. B. Supernatant from the Tregs in A. was taken to measure secreted levels of Il-10 protein. C. Activated T cells or Tregs±BP3 were counted using the ChemoMetec NucleoCounter Nc-202. D, E. Supernatant from activated T cells or Tregs±BP3 was taken to measure secreted protein levels. F. RNA was isolated from activated T cells or Tregs±BP3 and gene expression of Treg associated genes was measured through qRT-PCR, all reactions were measured in biological triplicate, technical duplicate.
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References

    1. MacOn MB, Fenton SE, Endocrine disruptors and the breast: early life effects and later life disease, J. Mammary Gland Biol. Neoplasia 18 (2013) 43–61, 10.1007/s10911-013-9275-7. Preprint at. - DOI - PMC - PubMed
    1. Yager JD, Davidson NE, Estrogen carcinogenesis in breast cancer, N. Engl. J. Med. 354 (2006) 270–282. - PubMed
    1. Zhang HZ, Bennett JM, Smith KT, Sunil N, Haslam SZ, Estrogen mediates mammary epithelial cell proliferation in serum-free culture indirectly via mammary stroma-derived hepatocyte growth factor, Endocrinology 143 (2002) 3427–3434. - PubMed
    1. Feng Y, Manka D, Wagner KU, Khan SA, Estrogen receptor-α expression in the mammary epithelium is required for ductal and alveolar morphogenesis in mice, Proc. Natl. Acad. Sci. U. S. A. 104 (2007), 14718. - PMC - PubMed
    1. Knower KC, To SQ, Leung YK, Ho SM, Clyne CD, Endocrine disruption of the epigenome: a breast cancer link, Endocr. Relat. Cancer 21 (2014), 10.1530/ERC-13-0513. T33 Preprint at. - DOI - PMC - PubMed

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