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Dysregulation of cholesterol homeostasis is associated with many diseases such as cardiovascular disease and cancer. Liver X receptors (LXRs) are major upstream regulators of cholesterol homeostasis and are activated by endogenous cholesterol metabolites such as 27-hydroxycholesterol (27HC). LXRs and various LXR ligands such as 27HC have been described to influence several extra-hepatic biological systems. However, disparate reports of LXR function have emerged, especially with respect to immunology and cancer biology. This would suggest that, similar to steroid nuclear receptors, the LXRs can be selectively modulated by different ligands. Here, we use RNA-sequencing of macrophages and single-cell RNA-sequencing of immune cells from metastasis-bearing murine lungs to provide evidence that LXR satisfies the 2 principles of selective nuclear receptor modulation: (1) different LXR ligands result in overlapping but distinct gene expression profiles within the same cell type, and (2) the same LXR ligands differentially regulate gene expression in a highly context-specific manner, depending on the cell or tissue type. The concept that the LXRs can be selectively modulated provides the foundation for developing precision pharmacology LXR ligands that are tailored to promote those activities that are desirable (proimmune), but at the same time minimizing harmful side effects (such as elevated triglyceride levels).

Macrophages are white blood cells with diverse functions contributing to a healthy immune response as well as the pathogenesis of cancer, osteoarthritis, atherosclerosis, and obesity. Due to their pleiotropic and dynamic nature, tools for imaging and tracking these cells at scales spanning the whole body down to microns could help to understand their role in disease states. Here we report fluorescent and radioisotopic quantum dots (QDs) for multimodal imaging of macrophage cells in vivo, ex vivo, and in situ. Macrophage specificity is imparted by click-conjugation to dextran, a biocompatible polysaccharide that natively targets these cell types. The emission spectral band of the crystalline semiconductor core was tuned to the near-infrared for optical imaging deep in tissue, and probes were covalently conjugated to radioactive iodine for nuclear imaging. The performance of these probes was compared with all-organic dextran probe analogues in terms of their capacity to target macrophages in visceral adipose tissue using in vivo positron emission tomography/computed tomography (PET/CT) imaging, in vivo fluorescence imaging, ex vivo fluorescence, post-mortem isotopic analyses, and optical microscopy. All probe classes exhibited equivalent physicochemical characteristics in aqueous solution and similar in vivo targeting specificity. However, dextran-mimetic QDs provided enhanced signal-to-noise ratio for improved optical quantification, long-term photostability, and resistance to chemical fixation. In addition, the vascular circulation time for the QD-based probes was extended 9-fold compared with dextran, likely due to differences in conformational flexibility. The enhanced photophysical and photochemical properties of dextran-mimetic QDs may accelerate applications in macrophage targeting, tracking, and imaging across broad resolution scales, particularly advancing capabilities in single-cell and single-molecule imaging and quantification.

Breast cancer remains the second leading cause of cancer-related deaths among women. In recent years, immunotherapy has been tremendously successful in some metastatic cancers such as melanoma. However, a majority of breast cancer patients do not benefit from existing immunotherapy treatments, leaving many with an unmet need. Although undoubtedly multifactorial, one major obstacle to anti-cancer therapies, is the highly immunosuppressive breast tumor microenvironment. This phenomenon is strongly maintained by myeloid immune cells and immunosuppressive regulatory T cells (Tregs), which hinder anti-tumor immunosurveillance and promote tumor progression. Thus, strategies to ‘re-educate’ myeloid cells to inhibit Tregs is a potentially promising anti-cancer strategy. Mining clinical data, we have found that elevated mRNA expression of the nuclear receptor, NR0B2 within breast tumors is associated with an increased time to recurrence. Single cell RNA-sequencing indicates that NR0B2 is expressed within the macrophage populations of normal breast tissue, and various dendritic cell (DC) types in PBMCs. Overexpression of NR0B2 or activation with a small molecule agonist in murine bone marrow derived macrophages (BMDMs) or DCs resulted in a dichotomous T cell expansion - away from Tregs. Conversely, Treg expansion increased when NR0B2 was knocked-down. Tumor growth was markedly increased in mice lacking myeloid specific NR0B2 expression. We further investigated the downstream targets of NR0B2 mediating this anti-tumor phenotype and identified that NLRP3 inflammasome-IL1β activity is a likely modulator in re-educating myeloid cell-Treg function. Importantly, a putative small molecule agonist decreased established metastatic lesions and increased the efficacy of αPD-L1. Subsequent medicinal chemistry was used to develop a novel NR0B2 agonist with strong anti-metastatic properties when used as a single agent in a preclinical mouse model. Collectively, our data implicates NR0B2 within myeloid cells as a modulator of Tregs, a cell population that has thus far been therapeutically intractable. Therefore, NR0B2 may prove to be a promising therapeutic target to reshape the tumor microenvironment and improve breast cancer immunotherapy. This work was supported by the Era of Hope Scholar Award from the Department of Defense Breast Cancer Research Program grant (BC200206), National Cancer Institute (R01CA234025), and NIH Chemistry-Biology Interface Training Grant (T32-GM136629). Citation Format: Hashni Epa Vidana Gamage, Sayyed Hamed Shahoei, Tiffany Nguyen, Rachel Farmer, Samuel Albright, Erin Weisser, Rafael O. Bautista, Claire P. Schane, Yu Wang, Adam Nelczyk, Liqian Ma, Srishti Tiwari, Anasuya Das Gupta, Shruti Bendre, Lionel Apetoh, Paul J. Hergenrother, Erik R. Nelson. NR0B2 re-educates myeloid cells within the tumor microenvironment: Potential novel strategy for breast cancer immunotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2358.

Background: Breast cancer is the most common cancer among women in the United States, with elevated circulating cholesterol as an established risk factor for the onset and progression to metastatic disease. The cholesterol metabolite, 27-hydroxycholesterol (27HC) has been identified to establish an immunosuppressive tumor microenvironment via Liver X Receptor (LXR) in myeloid immune cells. Interestingly, 27HC treatment of different myeloid immune cell types results in increased secretion of extracellular vesicles (EVs) with altered cargo. Importantly, EVs from 27HC treated myeloid cells promote tumor growth and metastasis in murine models of breast cancer. Given that these altered EVs are involved in cancer progression, it is imperative that we understand how their biogenesis and secretion are regulated. Methods and Results: Immunofluorescence microscopy experiments revealed that 27HC increases the size and number of CD63+ multivesicular bodies (MVBs) but not EEA1+ endosomes in RAW 264.7 cells. Quantitative PCR analysis revealed no significant changes in mRNA expression of genes associated with EV biogenesis machinery. However, we did observe that 27HC increased the size and number of LAMP1+ lysosomes. This led to the hypothesis that 27HC increases EV secretion by skewing MVBs away from lysosomal degradation and towards secretion as EVs by i) impairing lysosomal integrity, and/or ii) altering the post-translational modifications on the MVBs. To study the effect of 27HC on lysosomal function, we stained the lysosomes with a pH sensing fluorescent dye and observed a decrease in fluorescent intensity, indicating an increase in lysosomal pH. In addition, treatment with 27HC increased levels of cathepsin B in cells. Treatment of cells with Bafilomycin A1, a lysosomal proton pump inhibitor phenocopies the 27HC mediated increase in EV secretion and co-treatment of cells with Bafilomycin A1 and 27HC has the same effect. Post-translational modifications such as ISGylation and NEDDylation have been previously implicated in modulating EV secretion and cargo, and we have observed that 27HC decreases overall ISGylation and NEDDylation of proteins in cells. Conclusion: 27HC increases the size of multivesicular bodies, impairs lysosomal integrity, and alters levels of ISG- and NEDDylated proteins in cells. These data suggest that a combination of these modulations leads to the increase in EV secretion. Collectively, our studies reveal novel mechanisms by which EV secretion is modulated by 27HC. By extension, this axis represents a novel avenue for intervening in EV secretion and ultimately in the development of breast cancer therapeutics. Funding: Department of Defense Breast Cancer Research Program Era of Hope Scholar Award (BC200206), and National Cancer Institute (R01CA234025). Citation Format: Anasuya Das Gupta, Natalia Krawczynska, Hashni Epa Vidana Gamage, Hannah Kim, Jaena Park, Janet E. Sorrells, Stephen A. Boppart, Erik R. Nelson. 27-Hydroxycholesterol impairs lysosomal integrity in myeloid immune cells, resulting in enhanced secretion of cancer promoting extracellular vesicles [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 1258.

Development of targeted therapies will be a critical step towards reducing the mortality associated with triple-negative breast cancer (TNBC). To achieve this, we searched for targets that met three criteria: (1) pharmacologically targetable, (2) expressed in TNBC, and (3) expression is prognostic in TNBC patients. Since nuclear receptors have a well-defined ligand-binding domain and are thus highly amenable to small-molecule intervention, we focused on this class of protein. Our analysis identified TLX (NR2E1) as a candidate. Specifically, elevated tumoral TLX expression was associated with prolonged recurrence-free survival and overall survival for breast cancer patients with either estrogen receptor alpha (ERα)-negative or basal-like tumors. Using two TNBC cell lines, we found that stable overexpression of TLX impairs in vitro proliferation. RNA-Seq analysis revealed that TLX reduced the expression of genes implicated in epithelial-mesenchymal transition (EMT), a cellular program known to drive metastatic progression. Indeed, TLX overexpression significantly decreased cell migration and invasion, and robustly decreased the metastatic capacity of TNBC cells in murine models. We identify SERPINB2 as a likely mediator of these effects. Taken together, our work indicates that TLX impedes the progression of TNBC. Several ligands have been shown to regulate the transcriptional activity of TLX, providing a framework for the future development of this receptor for therapeutic intervention.