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Triple-Negative Breast Cancer Cells Recruit Neutrophils by Secreting TGF-β and CXCR2 Ligands.

ABSTRACT: Tumor associated neutrophils (TANs) are frequently detected in triple-negative breast cancer (TNBC). Recent studies also reveal the importance of neutrophils in promoting tumor progression and metastasis during breast cancer. However, the mechanisms regulating neutrophil trafficking to breast tumors are less clear. We sought to determine whether neutrophil trafficking to breast tumors is determined directly by the malignant potential of cancer cells. We found that tumor conditioned media (TCM) harvested from highly aggressive, metastatic TNBC cells induced a polarized morphology and robust neutrophil migration, while TCM derived from poorly aggressive estrogen receptor positive (ER+) breast cancer cells had no activity. In a three-dimensional (3D) type-I collagen matrix, neutrophils migrated toward TCM from aggressive breast cancer cells with increased velocity and directionality. Moreover, in a neutrophil-tumor spheroid co-culture system, neutrophils migrated with increased directionality towards spheroids generated from TNBC cells compared to ER+ cells. Based on these findings, we next sought to characterize the active factors secreted by TNBC cell lines. We found that TCM-induced neutrophil migration is dependent on tumor-derived chemokines, and screening TCM elution fractions based on their ability to induce polarized neutrophil morphology revealed the molecular weight of the active factors to be around 12 kDa. TCM from TNBC cell lines contained copious amounts of GRO (CXCL1/2/3) chemokines and TGF-β cytokines compared to ER+ cell-derived TCM. TCM activity was inhibited by simultaneously blocking receptors specific to GRO chemokines and TGF-β, while the activity remained intact in the presence of either single receptor inhibitor. Together, our findings establish a direct link between the malignant potential of breast cancer cells and their ability to induce neutrophil migration. Our study also uncovers a novel coordinated function of TGF-β and GRO chemokines responsible for guiding neutrophil trafficking to the breast tumor.

SUBMITTER: SenGupta S

PROVIDER: S-EPMC8071875 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:TGFβ signaling plays crucial role during development and cancer, however the role for TGFβ signaling in regulating the noncoding part of the human genome in triple negative breast cancer (TNBC) is still being unraveled. Herein, we provide the transcriptional landscape of TNBC in response to TGFβ activation and subsequent inhibition employing SB431542, selective TGFβ1 Receptor ALK5 Inhibitor. Our data revealed 72 commonly upregulated [fold change (FC) ≥ 2.0], including PLAU, TPM1, TAGLN, COL1A1, TGFBI, and SNAI1, and 53 downregulated (FC ≤ 2.0) protein coding genes in BT-549 and MDA-MB-231 models in response to TGFβ1 activation. Alignment to the geocode (V33) identified 41 upregulated (FC ≥ 2.0) and 22 downregulated (FC ≤ 2.0) long non-coding RNA (lncRNA) in response to TGFβ1 activation, which were inhibited by concurrent treatment with SB431542. To place our data from the in vitro models into their clinical context, we identified AC015909.1, AC013451.1, CYP1B1-AS1, AC004862.1, LINC01824, AL138828.1, B4GALT1-AS1, AL353751.1, AC090826.3, AC104695.4, ADORA2A-AS1, PTPRG-AS1, LINC01943, AC026954.3, TPM1-AS, ZFPM2-AS1, AC007362.1, AC112721.2, MALAT1, AL513314.2, AC112721.1, AC010343.3, LINC01711, and MAP3K2-DT lncRNA expression to positively correlate with TGFβ1 expression in a cohort of 360 TNBC patients. To provide mechanistic insight into lncRNA regulation by TGFβ signaling, SMAD2/3 ChIp-Seq data from BT-549 TNBC model retrieved from Gene Expression Omnibus (GEO) revealed direct binding of SMAD2/SMAD3 to the promoter of AC112721.1, AC112721.2, MALAT1, HHIP-AS1, LINC00472, and SLC7A11, suggesting their direct regulation by TGFβ1/SMAD2/SMAD3 pathway. Interestingly, AC112721.1, AC112721.2 exhibited higher expression in TNBC compared to normal breast tissue suggesting a possible role for those lncRNA in TNBC biology. Our miRNA analysis in the BT-549 model in response to exogenous TGFB1 revealed several affected miRNAs (2.0 ≤ FC ≤ 2.0), whose expression pattern was reversed in the presence of SB431542, suggesting those miRNA as plausible targets for TGFβ regulation. In particular, we observed hsa-miR-1275 to be downregulated in response to TGFB1 which was highly predicted to regulate PCDH1, FIBCD1, FXYD7, GDNF, STC1, EDN1, ZSWIM4, FGF1, PPP1R9B, NUAK1, PALM2AKAP2, IGFL3, and SPOCK1 whose expression were upregulated in response to TGFβ1 stimulus. On the other hand, hsa-miR-181b-5p was among the top upregulated miRNAs in response to TGFB1, which is also predicted to regulate CDKN1B, TNFRSF11B, SIM1, and ARSJ in the BT-549 model. Taken together, our data is the first to provide such in depth analysis of lncRNA and miRNA epigenetic changes in response to TGFβ signaling in TNBC.

Project description:Tumor normalization strategies aim to improve tumor blood vessel functionality (i.e., perfusion) by reducing the hyper-permeability of tumor vessels or restoring compressed vessels. Despite progress in strategies to normalize the tumor microenvironment (TME), their combinatorial antitumor effects with nanomedicine and immunotherapy remain unexplored. Methods: Here, we re-purposed the TGF-β inhibitor tranilast, an approved anti-fibrotic and antihistamine drug, and combined it with Doxil nanomedicine to normalize the TME, increase perfusion and oxygenation, and enhance anti-tumor immunity. Specifically, we employed two triple-negative breast cancer (TNBC) mouse models to primarily evaluate the therapeutic and normalization effects of tranilast combined with doxorubicin and Doxil. We demonstrated the optimized normalization effects of tranilast combined with Doxil and extended our analysis to investigate the effect of TME normalization to the efficacy of immune checkpoint inhibitors. Results: Combination of tranilast with Doxil caused a pronounced reduction in extracellular matrix components and an increase in the intratumoral vessel diameter and pericyte coverage, indicators of TME normalization. These modifications resulted in a significant increase in tumor perfusion and oxygenation and enhanced treatment efficacy as indicated by the notable reduction in tumor size. Tranilast further normalized the immune TME by restoring the infiltration of T cells and increasing the fraction of T cells that migrate away from immunosuppressive cancer-associated fibroblasts. Furthermore, we found that combining tranilast with Doxil nanomedicine, significantly improved immunostimulatory M1 macrophage content in the tumorigenic tissue and improved the efficacy of the immune checkpoint blocking antibodies anti-PD-1/anti-CTLA-4. Conclusion: Combinatorial treatment of tranilast with Doxil optimizes TME normalization, improves immunostimulation and enhances the efficacy of immunotherapy.

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Triple-Negative Breast Cancer Cells Recruit Neutrophils by Secreting TGF-β and CXCR2 Ligands.

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