Purpose Local tumor growth is a major cause of morbidity and mortality in nearly 30% of patients with pancreatic ductal adenocarcinoma (PDAC). Radiotherapy (RT) is commonly used for local disease control in PDAC, but efficacy is limited. We studied the impact of selectively intervening on RT-induced inflammation as an approach to overcome resistance to RT in PDAC. Experimental Design PDAC cell lines derived from primary pancreatic tumors arising spontaneously in KrasLSL-G12D/+;Trp53LSL-R172H/+;Pdx-1 Cre (KPC) mice were implanted into syngeneic mice and tumors were focally irradiated using the Small Animal Radiation Research Platform (SARRP). We determined the impact of depleting T cells and Ly6C+ monocytes as well as inhibiting the chemokine CCL2 on RT efficacy. Tumors were analyzed by flow cytometry and immunohistochemistry to detect changes in leukocyte infiltration, tumor viability and vascularity. Assays were performed on tumor tissues to detect cytokines and gene expression. Results Ablative RT alone had minimal impact on PDAC growth but led to a significant increase in CCL2 production by tumor cells and recruitment of Ly6C+CCR2+ monocytes. A neutralizing anti-CCL2 antibody selectively inhibited RT-dependent recruitment of monocytes/macrophages and delayed tumor growth but only in combination with RT (p<0.001). This anti-tumor effect was associated with decreased tumor proliferation and vascularity. Genetic deletion of CCL2 in PDAC cells also improved RT efficacy. Conclusions PDAC responds to RT by producing CCL2, which recruits Ly6C+CCR2+ monocytes to support tumor proliferation and neovascularization after RT. Disrupting the CCL2-CCR2 axis in combination with RT holds promise for improving RT efficacy in PDAC.

Macrophages enforce anti-tumor immunity by engulfing and killing tumor cells. Although these functions are determined by a balance of stimulatory and inhibitory signals, the role of macrophage metabolism is unknown. Here, we study the capacity of macrophages to circumvent inhibitory activity mediated by CD47 on cancer cells. We show that stimulation with CpG, a TLR9 agonist, evokes changes in the central carbon metabolism of macrophages that enable anti-tumor activity, including engulfment of CD47 + cancer cells. CpG activation engenders a metabolic state, that requires fatty acid oxidation and shunting of tricarboxylic acid cycle intermediates for de novo lipid biosynthesis. This integration of metabolic inputs is underpinned by carnitine palmitoyltransferase 1A and ATP citrate lyase, which together, impart macrophages with anti-tumor potential capable of overcoming inhibitory CD47 on cancer cells. Our findings identify central carbon metabolism to be a novel determinant and potential therapeutic target for stimulating anti-tumor activity by macrophages.

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Keratinocytes undergo significant structural remodeling during epidermal differentiation, including a broad transformation of the proteome coupled with a reduction in total cellular biomass. This suggests that intracellular digestion of proteins and organelles is necessary for keratinocyte differentiation. Here, we use both genetic and pharmacologic approaches to demonstrate that autophagy and lysosomal functions are required for keratinocyte differentiation in organotypic human skin. Lysosomal activity was required for mechanistic target of rapamycin signaling and mitochondrial oxidative metabolism. In turn, mitochondrial reactive oxygen species, produced as a natural byproduct of oxidative phosphorylation, were necessary for keratinocyte differentiation. Finally, treatment with exogenous reactive oxygen species rescued the differentiation defect in lysosome-inhibited keratinocytes. These findings highlight a reciprocal relationship between lysosomes and mitochondria, in which lysosomes support mitochondrial metabolism and the associated production of mitochondrial reactive oxygen species. The mitochondrial reactive oxygen species released to the cytoplasm in suprabasal keratinocytes triggers autophagy and lysosome-mediated degradation necessary for epidermal differentiation. As defective lysosome-dependent autophagy is associated with common skin diseases including psoriasis and atopic dermatitis, a better understanding of the role of lysosomes in epidermal homeostasis may guide future therapeutic strategies.

Barrett's esophagus (BE) is defined as an incomplete intestinal metaplasia characterized generally by the presence of columnar and goblet cells in the formerly stratified squamous epithelium of the esophagus. BE is known as a precursor for esophageal adenocarcinoma. Currently, the cell of origin for human BE has yet to be clearly identified. Therefore, we investigated the role of Notch signaling in the initiation of BE metaplasia. Affymetrix gene expression microarray revealed that BE samples express decreased levels of Notch receptors (NOTCH2 and NOTCH3) and one of the the ligands (JAG1). Furthermore, BE tissue microarray showed decreased expression of NOTCH1 and its downstream target HES1. Therefore, Notch signaling was inhibited in human esophageal epithelial cells by expression of dominant-negative-Mastermind-like (dnMAML), in concert with MYC and CDX1 overexpression. Cell transdifferentiation was then assessed by 3D organotypic culture and evaluation of BE-lineage specific gene expression. Notch inhibition promoted transdifferentiation of esophageal epithelial cells toward columnar-like cells as demonstrated by increased expression of columnar keratins (K8, K18, K19, K20) and glandular mucins (MUC2, MUC3B, MUC5B, MUC17) and decreased expression of squamous keratins (K5, K13, K14). In 3D culture, elongated cells were observed in the basal layer of the epithelium with Notch inhibition. Furthermore, we observed increased expression of KLF4, a potential driver of the changes observed by Notch inhibition. Interestingly, knockdown of KLF4 reversed the effects of Notch inhibition on BE-like metaplasia. Overall, Notch signaling inhibition promotes transdifferentiation of esophageal cells toward BE-like metaplasia in part via upregulation of KLF4. These results support a novel mechanism through which esophageal epithelial transdifferentiation promotes the evolution of BE.