Red cabbage juice-mediated gut microbiota modulation improves intestinal epithelial homeostasis and ameliorates colitis.

Gut microbiota plays a crucial role in inflammatory bowel disease (IBD) and has therapeutic benefits. Thus, targeting the gut microbiota is a promising therapeutic approach for IBD treatment. We recently found that red cabbage juice (RCJ) ameliorates dextran sulfate sodium (DSS)-induced colitis in mice. However, the underlying mechanisms remain unknown. The current study investigated the modulation of gut microbiota in response to treatment with RCJ to ameliorate the DSS colitis. The initial results demonstrated that mice treated with DSS + RCJ showed increased body weight and decreased diarrhea and blood in feces compared to the DSS alone group. RCJ ameliorated colitis by regulating the intestinal barrier function by reducing the number of apoptotic cells, improving colonic protective mucin, and increasing tight junction protein in RCJ + DSS groups compared to the DSS group. Short-gun metagenomic analysis revealed significant enrichment of short-chain fatty acid (SCFAs)-producing bacteria (Butyrivibrio, Ruminococcaceae, Acetatifactor muris, Rosburia Sp. CAG:303 , Dorea Sp. 5-2) increased PPAR-© activation, leading to repression of the nuclear factor κB (NFκB) signaling pathway, thus decreasing the production of crucial inflammatory cytokines and chemokines in the RCJ + DSS groups compared to the DSS group. Pathway abundance analysis showed an increased abundance of the SCFA pathway, reduced histidine degradation ( Bacteroides sartorii, and Bacteroides caecimuris ), and LCFA production in the RCJ+DSS treated group, suggesting the promotion of good colonic health. Furthermore, increased T-reg (FOXP3+) cells in the colon were due to SCFAs produced by the gut microbiota, which was corroborated by an increase in IL-10, a vital anti-inflammatory cytokine. Thus, our study provides the first evidence that RCJ ameliorates colonic inflammation by modulating the gut microbiota.

Small molecule antagonist of CXCR2 and CXCR1 inhibits tumor growth, angiogenesis, and metastasis in pancreatic cancer.

Pancreatic cancer (PC) has a poor prognosis, and current therapeutic strategies are ineffective in advanced diseases. We and others have shown the aberrant expression of CXCR2 and its ligands in PC development and progression. Our objective for this study was to evaluate the therapeutic utility of CXCR2/1 targeting using an small molecule antagonist, SCH-479833, in different PC preclinical murine models (syngeneic or xenogeneic). Our results demonstrate that CXCR2/1 antagonist had both antitumor and anti-metastatic effects in PC. CXCR2/1 antagonist treatment inhibited tumor cell proliferation, migration, angiogenesis, and recruitment of neutrophils, while it increased apoptosis. Treatment with the antagonist enhanced fibrosis, tumor necrosis, and extramedullary hematopoiesis. Together, these findings suggest that selectively targeting CXCR2/1 with small molecule inhibitors is a promising therapeutic approach for inhibiting PC growth, angiogenesis, and metastasis.

Red Cabbage Juice-Mediated Gut Microbiota Modulation Improves Intestinal Epithelial Homeostasis and Ameliorates Colitis.

Gut microbiota plays a crucial role in inflammatory bowel diseases (IBD) and can potentially prevent IBD through microbial-derived metabolites, making it a promising therapeutic avenue. Recent evidence suggests that despite an unclear underlying mechanism, red cabbage juice (RCJ) alleviates Dextran Sodium Sulfate (DSS)-induced colitis in mice. Thus, the study aims to unravel the molecular mechanism by which RCJ modulates the gut microbiota to alleviate DSS-induced colitis in mice. Using C57BL/6J mice, we evaluated RCJ's protective role in DSS-induced colitis through two cycles of 3% DSS. Mice were daily gavaged with PBS or RCJ until the endpoint, and gut microbiota composition was analyzed via shotgun metagenomics. RCJ treatment significantly improved body weight (p ≤ 0.001), survival in mice (p < 0.001) and reduced disease activity index (DAI) scores. Further, RCJ improved colonic barrier integrity by enhancing the expression of protective colonic mucins (p < 0.001) and tight junction proteins (p ≤ 0.01) in RCJ + DSS-treated mice compared to the DSS group. Shotgun metagenomic analysis revealed an enrichment of short-chain fatty acids (SCFAs)-producing bacteria (p < 0.05), leading to increased Peroxisome Proliferator-Activated Receptor Gamma (PPAR-γ) activation (p ≤ 0.001). This, in turn, resulted in repression of the nuclear factor κB (NFκB) signaling pathway, causing decreased production of inflammatory cytokines and chemokines. Our study demonstrates colitis remission in a DSS-induced mouse model, showcasing RCJ as a potential modulator for gut microbiota and metabolites, with promising implications for IBD prevention and treatment.

Differential expression profile of CXC-receptor-2 ligands as potential biomarkers in pancreatic ductal adenocarcinoma.

The discovery of early detection markers of pancreatic cancer (PC) disease is highly warranted. We analyzed the expression profile of different CXC-receptor-2 (CXCR2) ligands in PC cases for the potential of biomarker candidates. Analysis of different PDAC microarray datasets with matched normal and pancreatic tumor samples and next-generation sequenced transcriptomics data using an online portal showed significantly high expression of CXCL-1, 3, 5, 6, 8 in the tumors of PC patients. High CXCL5 expression was correlated to poor PC patient survival. Interestingly, mRNA and protein expression analysis of human PC cell lines showed higher CXCL2, 3, and 5 expressions in cell lines derived from metastatic sites than primary tumors. Furthermore, we utilized immunohistochemistry (IHC) to evaluate the expression of CXCR2 ligands in the human PC tumors and observed positive staining for CXCL1, 3, and 8 with a higher average IHC composite score of CXCL3 in the PC tissue specimens than the normal pancreas. We also observed an increase in the expression of mouse CXCL1, 3, and 5 in the pre-cancerous lesions of tumors and metastasis tissues derived from the PDX-cre-LSL-KrasG12D mouse model. Together, our data suggest that different CXCR2 ligands show the potential of being utilized as a diagnostic biomarker in PC patients.

Plexin-B3 Regulates Cellular Motility, Invasiveness, and Metastasis in Pancreatic Cancer.

The Plexins family of proteins are well-characterized transmembrane receptors of semaphorins, axon guidance cue molecules, that mediate the cell attraction or repelling effects for such cues. Plexins and their ligands are involved in numerous cellular activities, such as motility, invasion, and adhesion to the basement membrane. The detachment of cells and the gain in motility and invasion are hallmarks of the cancer metastasis cascade, thus generating interest in exploring the role of plexins in cancer metastasis. Semaphorin-plexin complexes can act as tumor promoters or suppressors, depending upon the cancer type, and are under investigation for therapeutic purposes. Our group has identified Semaphorin-5A (SEMA5A)/Plexin-B3 as an attractive targetable complex for pancreatic cancer (PC) metastasis. However, our understanding of the Plexin-B3 function and pathological expression in PC is limited, and our present study delineates the role of Plexin-B3 in PC malignancy. We examined the pathological expression of Plexin-B3 in PC tumors and metastasis using a human tissue microarray, disease progression model of PDX-Cre-Kras(G12D) (KC) mice, and different metastatic sites obtained from the KrasG12D; Trp53R172H; Pdx1-Cre (KPC) mice model. We observed a higher Plexin-B3 expression in PC tumor cores than the normal pancreas, and different metastatic sites were positive for Plexin-B3 expression. However, in the KC mice model, the Plexin-B3 expression increased initially and then decreased with the disease progression. Next, to evaluate the functional role of Plexin-B3, we utilized T3M-4- and CD18/HPAF-Control and -Plexin B3 knockdown cells for different in vivo and in vitro studies. The knockdown of Plexin-B3 enhanced the in vitro cellular migration, invasiveness, and impaired colony formation in three-dimensional culture, along with an increase in cellular spread and remodeling of the actin filaments. We also observed a higher metastasis in nude mice injected with T3M-4- and CD18/HPAF-shPlexin-B3 cells compared to their respective control cells. Furthermore, we observed a lower number of proliferating Ki-67-positive cells and higher ALDH1-A1-positive cells in the tumors formed by Plexin-B3 knockdown cells compared to tumors formed by the control cells. Together, our data suggest that the loss of Plexin-B3 is associated with the interference of cell division machinery and the induction of stem cell-like characteristics in PC cells.

Host Cxcr2-Dependent Regulation of Pancreatic Cancer Growth, Angiogenesis, and Metastasis.

Pancreatic ductal adenocarcinoma (PDAC) manifests aggressive tumor growth and early metastasis. Crucial steps in tumor growth and metastasis are survival, angiogenesis, invasion, and immunosuppression. Our prior research showed that chemokine CXC- receptor-2 (CXCR2) is expressed on endothelial cells, innate immune cells, and fibroblasts, and regulates angiogenesis and immune responses. Here, we examined whether tumor angiogenesis, growth, and metastasis of CXCR2 ligands expressing PDAC cells are regulated in vivo by a host CXCR2-dependent mechanism. C57BL6 Cxcr2-/- mice were generated following crosses between Cxcr2-/+ female and Cxcr2-/- male. Cxcr2 ligands expressing Kirsten rat sarcoma (KRAS-PDAC) cells were orthotopically implanted in the pancreas of wild-type or Cxcr2-/- C57BL6 mice. No significant difference in PDAC tumor growth was observed. Host Cxcr2 loss led to an inhibition in microvessel density in PDAC tumors. Interestingly, an enhanced spontaneous and experimental liver metastasis was observed in Cxcr2-/- mice compared with wild-type mice. Increased metastasis in Cxcr2-/- mice was associated with an increase in extramedullary hematopoiesis and expansion of neutrophils and immature myeloid precursor cells in the spleen of tumor-bearing mice. These data suggest a dynamic role of host CXCR2 axis in regulating tumor immune suppression, tumor growth, and metastasis.

Metabolic programming of distinct cancer stem cells promotes metastasis of pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) metastasizes to distant organs, which is the primary cause of mortality; however, specific features mediating organ-specific metastasis remain unexplored. Emerging evidence demonstrates that cancer stem cells (CSCs) and cellular metabolism play a pivotal role in metastasis. Here we investigated the role of distinct subtypes of pancreatic CSCs and their metabolomic signatures in organ-specific metastatic colonization. We found that PDAC consists of ALDH+/CD133+ and drug-resistant (MDR1+) subtypes of CSCs with specific metabolic and stemness signatures. Human PDAC tissues with gemcitabine treatment, autochthonous mouse tumors from KrasG12D; Pdx1-Cre (KC) and KrasG12D; Trp53R172H; Pdx-1 Cre (KPC) mice, and KPC- Liver/Lung metastatic cells were used to evaluate the CSC, EMT (epithelial-to-mesenchymal transition), and metabolic profiles. A strong association was observed between distinct CSC subtypes and organ-specific colonization. The liver metastasis showed drug-resistant CSC- and EMT-like phenotype with aerobic glycolysis and fatty acid ß-oxidation-mediated oxidative (glyco-oxidative) metabolism. On the contrary, lung metastasis displayed ALDH+/CD133+ and MET-like phenotype with oxidative metabolism. These results were obtained by evaluating FACS-based side population (SP), autofluorescence (AF+) and Alde-red assays for CSCs, and Seahorse-based oxygen consumption rate (OCR), extracellular acidification rate (ECAR), and fatty acid ß-oxidation (FAO)-mediated OCR assays for metabolic features along with specific gene signatures. Further, we developed in vitro human liver and lung PDAC metastasis models by using a combination of liver or lung decellularized scaffolds, a co-culture, and a sphere culture methods. PDAC cells grown in the liver-mimicking model showed the enrichment of MDR1+ and CPT1A+ populations, whereas the PDAC cells grown in the lung-mimicking environment showed the enrichment of ALDH+/CD133+ populations. In addition, we observed significantly elevated expression of ALDH1 in lung metastasis and MDR1/LDH-A expression in liver metastasis compared to human primary PDAC tumors. Our studies elucidate that distinct CSCs adapt unique metabolic signatures for organotropic metastasis, which will pave the way for the development of targeted therapy for PDAC metastasis.

Breast Cancer Cell-Neutrophil Interactions Enhance Neutrophil Survival and Pro-Tumorigenic Activities.

Breast cancer remains the most prevalent cancer in women with limited treatment options for patients suffering from therapy-resistance and metastatic disease. Neutrophils play an important role in breast cancer progression and metastasis. We examined the pro-tumorigenic nature of the breast cancer cell-neutrophil interactions and delineated the differences in neutrophil properties between the chemotherapy-resistant and the parent tumor microenvironment. Our data demonstrated that high neutrophil infiltration is associated with disease aggressiveness and therapy resistance. In the human breast cancer dataset, expression of neutrophil-related signature gene expression was higher in tumors from therapy-resistant patients than therapy-sensitive patients. We observed that breast cancer-derived factors significantly enhanced neutrophil survival, polarization, and pro-inflammatory cytokine expression. Breast cancer cell-derived supernatant treated neutrophils significantly expressed high levels of interleukin-1ß (IL-1ß), CC-chemokine ligand-2-4 (CCL2, CCL3, CCL4), inducible nitric oxide synthase (iNOS), and matrix metallopeptidase-9 (MMP9), and formed extracellular traps (NETs). Moreover, neutrophils showed increased secretion of MMP9 when cultured with the supernatant of chemotherapy-resistant Cl66-Doxorubicin (Cl66-Dox) and Cl66-Paclitaxel (Cl66-Pac) cells in comparison with the supernatant of Cl66-parent cells. Together, these data suggest an important role of breast cancer cell-neutrophil interactions in regulating pro-tumor characteristics in neutrophils and its modulation by therapy resistance.

CXCR2 signaling promotes secretory cancer-associated fibroblasts in pancreatic ductal adenocarcinoma.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most challenging malignancies. Desmoplasia and tumor-supporting inflammation are hallmarks of PDAC. The tumor microenvironment contributes significantly to tumor progression and spread. Cancer-associated fibroblasts (CAFs) facilitate therapy resistance and metastasis. Recent reports emphasized the concurrence of multiple subtypes of CAFs with diverse roles, fibrogenic, and secretory. C-X-C motif chemokine receptor 2 (CXCR2) is a chemokine receptor known for its role during inflammation and its adverse role in PDAC. Oncogenic Kras upregulates CXCR2 and its ligands and, thus, contribute to tumor proliferation and immunosuppression. CXCR2 deletion in a PDAC syngeneic mouse model produced increased fibrosis revealing a potential undescribed role of CXCR2 in CAFs. In this study, we demonstrate that the oncogenic Kras-CXCR2 axis regulates the CAFs function in PDAC and contributes to CAFs heterogeneity. We observed that oncogenic Kras and CXCR2 signaling alter CAFs, producing a secretory CAF phenotype with low fibrogenic features; and increased secretion of pro-tumor cytokines and CXCR2 ligands, utilizing the NF-κB activity. Finally, using syngeneic mouse models, we demonstrate that oncogenic Kras is associated with secretory CAFs and that CXCR2 inhibition promotes activation of fibrotic cells (myofibroblasts) and impact tumors in a mutation-dependent manner.

Correction: Global analysis of human glycosyltransferases reveals novel targets for pancreatic cancer pathogenesis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Global analysis of human glycosyltransferases reveals novel targets for pancreatic cancer pathogenesis.

BACKGROUND: Several reports have shown the role of glycosylation in pancreatic cancer (PC), but a global systematic screening of specific glycosyltransferases (glycoTs) in its progression remains unknown. METHODS: We demonstrate a rigorous top-down approach using TCGA-based RNA-Seq analysis, multi-step validation using RT-qPCR, immunoblots and immunohistochemistry. We identified six unique glycoTs (B3GNT3, B4GALNT3, FUT3, FUT6, GCNT3 and MGAT3) in PC pathogenesis and studied their function using CRISPR/Cas9-based KD systems. RESULTS: Serial metastatic in vitro models using T3M4 and HPAF/CD18, generated in house, exhibited decreases in B3GNT3, FUT3 and GCNT3 expression on increasing metastatic potential. Immunohistochemistry identified clinical significance for GCNT3, B4GALNT3 and MGAT3 in PC. Furthermore, the effects of B3GNT3, FUT3, GCNT3 and MGAT3 were shown on proliferation, migration, EMT and stem cell markers in CD18 cell line. Talniflumate, GCNT3 inhibitor, reduced colony formation and migration in T3M4 and CD18 cells. Moreover, we found that loss of GCNT3 suppresses PC progression and metastasis by downregulating cell cycle genes and ß-catenin/MUC4 axis. For GCNT3, proteomics revealed downregulation of MUC5AC, MUC1, MUC5B including many other proteins. CONCLUSIONS: Collectively, we demonstrate a critical role of O- and N-linked glycoTs in PC progression and delineate the mechanism encompassing the role of GCNT3 in PC.

Stromal Modulation and Treatment of Metastatic Pancreatic Cancer with Local Intraperitoneal Triple miRNA/siRNA Nanotherapy.

Nanomedicines achieve tumor-targeted delivery mainly through enhanced permeability and retention (EPR) effect following intravenous (IV) administration. Unfortunately, the EPR effect is severely compromised in pancreatic cancer due to hypovascularity and dense desmoplastic stroma. Intraperitoneal (IP) administration may be an effective EPR-independent local delivery approach to target peritoneal tumors. Besides improved delivery, effective combination delivery strategies are needed to improve pancreatic cancer therapy by targeting both cancer cells and cellular interactions within the tumor stroma. Here, we described simple cholesterol-modified polymeric CXCR4 antagonist (PCX) nanoparticles (to block cancer-stroma interactions) for codelivery of anti-miR-210 (to inactivate stroma-producing pancreatic stellate cells (PSCs)) and siKRASG12D (to kill pancreatic cancer cells). IP administration delivered the nanoparticles to an orthotopic syngeneic pancreatic tumors as a result of preferential localization to the tumors and metastases with disrupted mesothelium and effective tumor penetration. The local IP delivery resulted in nearly 15-fold higher tumor accumulation than delivery by IV injection. Through antagonism of CXCR4 and downregulation of miR-210/KRASG12D, the triple-action nanoparticles favorably modulated desmoplastic tumor microenvironment via inactivating PSCs and promoting the infiltration of cytotoxic T cells. The combined therapy displayed improved therapeutic effect when compared with individual therapies as documented by the delayed tumor growth, depletion of stroma, reduction of immunosuppression, inhibition of metastasis, and prolonged survival. Overall, we present data that a local IP delivery of a miRNA/siRNA combination holds the potential to improve pancreatic cancer therapy.