Investigating the complex interplay between genotype and high-fat-diet feeding in the lactating mammary gland using the Tph1 and Ldlr knockout models.

Obesity is a prevailing problem across the globe. Women who are obese have difficulty initiating and sustaining lactation. However, the impact of genetics and diet on breastfeeding outcomes is understudied. Here we explore the effect of diet and genotype on lactation. We utilized the low-density lipoprotein receptor (Ldlr-KO) transgenic mouse model as an obesity and hypercholesterolemia model. Additionally, we used the tryptophan hydroxylase 1 (Tph1-KO) mouse, recently identified as a potential anti-obesogenic model, to investigate if addition of Tph1-KO could ameliorate negative effects of obesity in Ldlr-KO mice. We created a novel transgenic mouse line by combining the Ldlr and Tph1 [double knockout (DKO)] mice to study the interaction between the two genotypes. Female mice were fed a low-fat diet (LFD; 10% fat) or high-fat diet (HFD; 60% fat) from 3 wk of age through early [lactation day 3 (L3)] or peak lactation [lactation day 11 (L11)]. After 4 wk of consuming either LFD or HFD, female mice were bred. On L2 and L10, dams were milked to investigate the effect of diet and genotype on milk composition. Dams were euthanized on L3 or L11. There was no impact of diet or genotype on milk protein or triglycerides (TGs) on L2; however, by L10, Ldlr-KO and DKO dams had increased TG levels in milk. RNA-sequencing of L11 mammary glands demonstrated Ldlr-KO dams fed HFD displayed enrichment of genes involved in immune system pathways. Interestingly, the DKO may alter vesicle budding and biogenesis during lactation. We also quantified macrophages by immunostaining for F4/80+ cells at L3 and L11. Diet played a significant role on L3 (P = 0.013), but genotype played a role at L11 (P < 0.0001) on numbers of F4/80+ cells. Thus the impact of diet and genotype on lactation differs depending on stage of lactation, illustrating complexities of understanding the intersection of these parameters.NEW & NOTEWORTHY We have created a novel mouse model that is focused on understanding the intersection of diet and genotype on mammary gland function during lactation.

A covalent creatine kinase inhibitor ablates glioblastoma migration and sensitizes tumors to oxidative stress.

Glioblastoma is a Grade 4 primary brain tumor defined by therapy resistance, diffuse infiltration, and near-uniform lethality. The underlying mechanisms are unknown, and no treatment has been curative. Using a recently developed creatine kinase inhibitor (CKi), we explored the role of this inhibitor on GBM biology in vitro. While CKi minimally impacted GBM cell proliferation and viability, it significantly affected migration. In established GBM cell lines and patient-derived xenografts, CKi ablated both the migration and invasion of GBM cells. CKi also hindered radiation-induced migration. RNA-seq revealed a decrease in invasion-related genes, with an unexpected increase in glutathione metabolism and ferroptosis protection genes post-CKi treatment. The effects of CKi could be reversed by the addition of cell-permeable glutathione. Carbon-13 metabolite tracing indicated heightened glutathione biosynthesis post-CKi treatment. Combinatorial CKi blockade and glutathione inhibition or ferroptosis activation abrogated cell survival. Our data demonstrated that CKi perturbs promigratory and anti-ferroptotic roles in GBM, identifying the creatine kinase axis as a druggable target for GBM treatment.

Myeloid cell-derived creatine in the hypoxic niche promotes glioblastoma growth.

Glioblastoma (GBM) is a malignancy dominated by the infiltration of tumor-associated myeloid cells (TAMCs). Examination of TAMC metabolic phenotypes in mouse models and patients with GBM identified the de novo creatine metabolic pathway as a hallmark of TAMCs. Multi-omics analyses revealed that TAMCs surround the hypoxic peri-necrotic regions of GBM and express the creatine metabolic enzyme glycine amidinotransferase (GATM). Conversely, GBM cells located within these same regions are uniquely specific in expressing the creatine transporter (SLC6A8). We hypothesized that TAMCs provide creatine to tumors, promoting GBM progression. Isotopic tracing demonstrated that TAMC-secreted creatine is taken up by tumor cells. Creatine supplementation protected tumors from hypoxia-induced stress, which was abrogated with genetic ablation or pharmacologic inhibition of SLC6A8. Lastly, inhibition of creatine transport using the clinically relevant compound, RGX-202-01, blunted tumor growth and enhanced radiation therapy in vivo. This work highlights that myeloid-to-tumor transfer of creatine promotes tumor growth in the hypoxic niche.

STING agonist-loaded, CD47/PD-L1-targeting nanoparticles potentiate antitumor immunity and radiotherapy for glioblastoma.

As a key component of the standard of care for glioblastoma, radiotherapy induces several immune resistance mechanisms, such as upregulation of CD47 and PD-L1. Here, leveraging these radiotherapy-elicited processes, we generate a bridging-lipid nanoparticle (B-LNP) that engages tumor-associated myeloid cells (TAMCs) to glioblastoma cells via anti-CD47/PD-L1 dual ligation. We show that the engager B-LNPs block CD47 and PD-L1 and promote TAMC phagocytic activity. To enhance subsequent T cell recruitment and antitumor responses after tumor engulfment, the B-LNP was encapsulated with diABZI, a non-nucleotidyl agonist for stimulator of interferon genes. In vivo treatment with diABZI-loaded B-LNPs induced a transcriptomic and metabolic switch in TAMCs, turning these immunosuppressive cells into antitumor effectors, which induced T cell infiltration and activation in brain tumors. In preclinical murine models, B-LNP/diABZI administration synergized with radiotherapy to promote brain tumor regression and induce immunological memory against glioma. In summary, our study describes a nanotechnology-based approach that hijacks irradiation-triggered immune checkpoint molecules to boost potent and long-lasting antitumor immunity against glioblastoma.

The CXCL16-CXCR6 axis in glioblastoma modulates T-cell activity in a spatiotemporal context.

Introduction: Glioblastoma multiforme (GBM) pathobiology is characterized by its significant induction of immunosuppression within the tumor microenvironment, predominantly mediated by immunosuppressive tumor-associated myeloid cells (TAMCs). Myeloid cells play a pivotal role in shaping the GBM microenvironment and influencing immune responses, with direct interactions with effector immune cells critically impacting these processes. Methods: Our study investigates the role of the CXCR6/CXCL16 axis in T-cell myeloid interactions within GBM tissues. We examined the surface expression of CXCL16, revealing its limitation to TAMCs, while microglia release CXCL16 as a cytokine. The study explores how these distinct expression patterns affect T-cell engagement, focusing on the consequences for T-cell function within the tumor environment. Additionally, we assessed the significance of CXCR6 expression in T-cell activation and the initial migration to tumor tissues. Results: Our data demonstrates that CXCL16 surface expression on TAMCs results in predominant T-cell engagement with these cells, leading to impaired T-cell function within the tumor environment. Conversely, our findings highlight the essential role of CXCR6 expression in facilitating T-cell activation and initial migration to tumor tissues. The CXCL16-CXCR6 axis exhibits dualistic characteristics, facilitating the early stages of the T-cell immune response and promoting T-cell infiltration into tumors. However, once inside the tumor, this axis contributes to immunosuppression. Discussion: The dual nature of the CXCL16-CXCR6 axis underscores its potential as a therapeutic target in GBM. However, our results emphasize the importance of carefully considering the timing and context of intervention. While targeting this axis holds promise in combating GBM, the complex interplay between TAMCs, microglia, and T cells suggests that intervention strategies need to be tailored to optimize the balance between promoting antitumor immunity and preventing immunosuppression within the dynamic tumor microenvironment.

Targeting Obesity-Induced Macrophages during Preneoplastic Growth Promotes Mammary Epithelial Stem/Progenitor Activity, DNA Damage, and Tumor Formation.

Obesity enhances breast cancer risk in postmenopausal women and premenopausal women with genetic or familial risk factors. We have shown previously that within breast tissue, obesity increases macrophage-driven inflammation and promotes expansion of luminal epithelial cell populations that are hypothesized to be the cells of origin for the most common subtypes of breast cancer. However, it is not clear how these changes within the microenvironment of the breast alter cancer risk and tumor growth. Using a high-fat diet to induce obesity, we examined preneoplastic changes associated with epithelial cell-specific loss of Trp53. Obesity significantly enhanced the incidence of tumors of diverse histotypes and increased stromal cells within the tumor microenvironment. Obesity also promoted the growth of preneoplastic lesions containing elevated numbers of luminal epithelial progenitor cells, which were surrounded by macrophages. To understand how macrophage-driven inflammation due to obesity enhances tumor formation, mice were treated with IgG or anti-F4/80 antibodies to deplete macrophages during preneoplastic growth. Unexpectedly, depletion of macrophages in obese mice enhanced mammary epithelial cell stem/progenitor activity, elevated expression of estrogen receptor alpha, and increased DNA damage in cells. Together, these results suggest that in obesity, macrophages reduce epithelial cells with DNA damage, which may limit the progression of preneoplastic breast lesions, and uncovers complex macrophage function within the evolving tumor microenvironment. Understanding how obesity alters the function of macrophages during tumor formation may lead to chemoprevention options for at-risk obese women. SIGNIFICANCE: Understanding how obesity impacts early tumor growth and response to macrophage-targeted therapies may improve therapeutics for obese patients with breast cancer and identify patient populations that would benefit from macrophage-targeted therapies.