Significance: New approaches to prevention and treatment of drug-resistant bacterial infections are needed. Here we show how synthetic molecular evolution, screening of iterative libraries, can identify membrane permeabilizing antimicrobial peptides (AMPs) that do not have the usual impediments to activity in vivo. Screening in the presence of concentrated human erythrocytes to mimic a generic cell-rich environment enabled identification of peptides with potent, broad-spectrum bactericidal activity under conditions where most known AMPs have little or no activity. One optimized peptide was tested in the cell- and protein-rich environment of an infected, purulent wound in mice. Infection was significantly reduced, and biofilm formation in the wound was prevented, demonstrating the power of synthetic molecular evolution of AMPs.

Inflammatory bowel disease (IBD), characterized by infiltration of polymorphonuclear neutrophils (PMNs), increases the risk of colon cancer. PMN activation corresponds to the accumulation of intracellular Lipid Droplets (LDs). As increased LDs are negatively regulated by transcription factor Forkhead Box O3 (FOXO3), we aim to determine the significance of this regulatory network in PMN-mediated IBD and tumorigenesis. Affected tissue of IBD and colon cancer patients, colonic and infiltrated immune cells, have increased LDs’ coat protein, PLIN2. Mouse peritoneal PMNs with stimulated LDs and FOXO3 deficiency have elevated transmigratory activity. Transcriptomic analysis of these FOXO3-deficient PMNs showed differentially expressed genes (DEGs; FDR < 0.05) involved in metabolism, inflammation, and tumorigenesis. Upstream regulators of these DEGs, similar to colonic inflammation and dysplasia in mice, were linked to IBD and human colon cancer. Additionally, a transcriptional signature representing FOXO3-deficient PMNs (PMN-FOXO3₃₈₉) separated transcriptomes of affected tissue in IBD (p = 0.00018) and colon cancer (p = 0.0037) from control. Increased PMN-FOXO3₃₈₉ presence predicted colon cancer invasion (lymphovascular p = 0.015; vascular p = 0.046; perineural p = 0.03) and poor survival. Validated DEGs from PMN-FOXO3₃₈₉ (P2RX1, MGLL, MCAM, CDKN1A, RALBP1, CCPG1, PLA2G7) are involved in metabolism, inflammation, and tumorigenesis (p < 0.05). These findings highlight the significance of LDs and FOXO3-mediated PMN functions that promote colonic pathobiology.

The obesity epidemic is associated with increased colorectal cancer (CRC) risk and progression, the mechanisms of which remain unclear. In obese individuals, hypertrophic epiploic adipose tissue (EPAT), attached to the colon, has unique characteristics compared to other fats. We hypothesized that this understudied fat could serve as a tumor-promoting tissue and developed a novel microphysiological system (MPS) for human EPAT-dependent colorectal cancer (CRC-MPS). In CRC-MPS, obese EPAT, unlike lean EPAT, considerably attracted colon cancer HT29-GFP cells and enhanced their growth. Conditioned media (CM) from the obese CRC-MPS significantly increased the growth and migration of HT29 and HCT116 cells (p < 0.001). In HT29 cells, CM stimulated differential gene expression (hOEC₈₆₇) linked to cancer, tumor morphology, and metabolism similar to those in the colon of high-fat-diet obese mice. The hOEC₈₆₇ signature represented pathways found in human colon cancer. In unsupervised clustering, hOEC₈₆₇ separated transcriptomes of colon cancer samples from normal with high significance (PCA, p = 9.6 × 10⁻¹¹). These genes, validated in CM-treated HT29 cells (p < 0.05), regulate the cell cycle, cancer stem cells, methylation, and metastasis, and are similarly altered in human colon cancer (TCGA). These findings highlight a tumor-promoting role of EPAT in CRC facilitated with obesity and establishes a platform to explore critical mechanisms and develop effective treatments.