Regulation of pancreatic cancer therapy resistance by chemokines.

Pancreatic ductal adenocarcinoma (PDAC) is a highly lethal malignancy characterized by high resistance and poor response to chemotherapy. In addition, the poorly immunogenic pancreatic tumors constitute an immunosuppressive tumor microenvironment (TME) that render immunotherapy-based approaches ineffective. Understanding the mechanisms of therapy resistance, identifying new targets, and developing effective strategies to overcome resistance can significantly impact the management of PDAC patients. Chemokines are small soluble factors that are significantly deregulated during PDAC pathogenesis, contributing to tumor growth, metastasis, immune cell trafficking, and therapy resistance. Thus far, different chemokine pathways have been explored as therapeutic targets in PDAC, with some promising results in recent clinical trials. Particularly, immunotherapies such as immune check point blockade therapies and CAR-T cell therapies have shown promising results when combined with chemokine targeted therapies. Considering the emerging pathological and clinical significance of chemokines in PDAC, we reviewed major chemokine-regulated pathways leading to therapy resistance and the ongoing endeavors to target chemokine signaling in PDAC. This review discusses the role of chemokines in regulating therapy resistance in PDAC and highlights the continuing efforts to target chemokine-regulated pathways to improve the efficacy of various treatment modalities.

Immune checkpoint blockade in pancreatic cancer: Trudging through the immune desert.

Pancreatic cancer (PC) has exceptionally high mortality due to ineffective treatment strategies. Immunotherapy, which mobilizes the immune system to fight against cancer, has been proven successful in multiple cancers; however, its application in PC has met with limited success. In this review, we articulated that the pancreatic tumor microenvironment is immuno-suppressive with extensive infiltration by M2-macrophages and myeloid-derived suppressive cells but low numbers of cytotoxic T-cells. In addition, low mutational load and poor antigen processing, presentation, and recognition contribute to the limited response to immunotherapy in PC. Immune checkpoints, the critical targets for immunotherapy, have high expression in PC and stromal cells, regulated by tumor microenvironmental milieu (cytokine and metabolites) and cell-intrinsic mechanisms (epigenetic regulation, oncogenic signaling, and post-translational modifications). Combining immunotherapy with modulators of the tumor microenvironment may facilitate the development of novel therapeutic regimens to manage PC.

Endothelin-axis antagonism enhances tumor perfusion in pancreatic cancer.

Delivery of therapeutic agents in pancreatic cancer (PC) is impaired due to its hypovascular and desmoplastic tumor microenvironment. The Endothelin (ET)-axis is the major regulator of vasomotor tone under physiological conditions and is highly upregulated in multiple cancers. We investigated the effect of dual endothelin receptor antagonist bosentan on perfusion and macromolecular transport in a PC cell-fibroblast co-implantation tumor model using Dynamic Contrast-Enhanced Magnetic Resonance Imaging (DCE-MRI). Following bosentan treatment, the contrast enhancement ratio and wash-in rates in tumors were two- and nine times higher, respectively, compared to the controls, whereas the time to peak was significantly shorter (7.29 ± 1.29 min v/s 22.08 ± 5.88 min; p = 0.04). Importantly, these effects were tumor selective as the magnitudes of change for these parameters were much lower in muscles. Bosentan treatment also reduced desmoplasia and improved intratumoral distribution of high molecular weight FITC-dextran. Overall, these findings support that targeting the ET-axis can serve as a potential strategy to selectively enhance tumor perfusion and improve the delivery of therapeutic agents in pancreatic tumors.

Preferential binding of DAP-PGs by major peptidoglycan recognition proteins found in cell-free hemolymph of Manduca sexta.

Peptidoglycan recognition proteins (PGRPs) detect invading bacteria to trigger or modulate immune responses in insects. While these roles are established in Drosophila, functional studies are not yet achieved at the PGRP family level in other insects. To attain this goal, we selected Manduca sexta PGRP12 and five of the nine secreted PGRPs for recombinant expression and biochemical characterization. We cloned PGRP2-5, 12 and 13 cDNAs, produced the proteins in full (PGRP2-5, 13) or in part (PGRP3s, 12e, 13N, 13C) in Sf9 cells, and tested their bindings of two muramyl pentapeptides by surface plasmon resonance, two soluble peptidoglycans by competitive ELISA, and four insoluble peptidoglycans and eight whole bacteria by a pull-down assay. Preferential binding of meso-diaminopimelic acid-peptidoglycans (DAP-PGs) was observed in all the proteins containing a peptidoglycan binding domain and, since PGRP6, 7 and 9 proteins were hardly detected in cell-free hemolymph, the reportoire of PGRPs (including PGRP1 published previously) in M. sexta hemolymph is likely adapted to mainly detect Gram-negative bacteria and certain Gram-positive bacteria with DAP-PGs located on their surface. After incubation with plasma from naïve larvae, PGRP2, 3f, 4, 5, 13f and 13N considerably stimulated prophenoloxidase activation in the absence of a bacterial elicitor. PGRP3s and 12e had much smaller effects. Inclusion of the full-length PGRPs and their regions in the plasma also led to proHP8 activation, supporting their connections to the Toll pathway, since HP8 is a Spӓtzle-1 processing enzyme in M. sexta. Together, these findings raised concerns on the common belief that the Toll-pathway is specific for Gram-positive bacteria in insects.

Nanocarriers for pancreatic cancer imaging, treatments, and immunotherapies.

Pancreatic tumors are highly desmoplastic and immunosuppressive. Delivery and distribution of drugs within pancreatic tumors are compromised due to intrinsic physical and biochemical stresses that lead to increased interstitial fluid pressure, vascular compression, and hypoxia. Immunotherapy-based approaches, including therapeutic vaccines, immune checkpoint inhibition, CAR-T cell therapy, and adoptive T cell therapies, are challenged by an immunosuppressive tumor microenvironment. Together, extensive fibrosis and immunosuppression present major challenges to developing treatments for pancreatic cancer. In this context, nanoparticles have been extensively studied as delivery platforms and adjuvants for cancer and other disease therapies. Recent advances in nanotechnology have led to the development of multiple nanocarrier-based formulations that not only improve drug delivery but also enhance immunotherapy-based approaches for pancreatic cancer. This review discusses and critically analyzes the novel nanoscale strategies that have been used for drug delivery and immunomodulation to improve treatment efficacy, including newly emerging immunotherapy-based approaches. This review also presents important perspectives on future research directions that will guide the rational design of novel and robust nanoscale platforms to treat pancreatic tumors, particularly with respect to targeted therapies and immunotherapies. These insights will inform the next generation of clinical treatments to help patients manage this debilitating disease and enhance survival rates.

Characterization of recombinant ß subunit of human MUC4 mucin (rMUC4ß).

MUC4 is a transmembrane mucin expressed on various epithelial surfaces, including respiratory and gastrointestinal tracts, and helps in their lubrication and protection. MUC4 is also aberrantly overexpressed in various epithelial malignancies and functionally contributes to cancer development and progression. MUC4 is putatively cleaved at the GDPH site into a mucin-like α-subunit and a membrane-tethered growth factor-like ß-subunit. Due to the presence of several functional domains, the characterization of MUC4ß is critical for understanding MUC4 biology. We developed a method to produce and purify multi-milligram amounts of recombinant MUC4ß (rMUC4ß). Purified rMUC4ß was characterized by Far-UV CD and I-TASSER-based protein structure prediction analyses, and its ability to interact with cellular proteins was determined by the affinity pull-down assay. Two of the three EGF-like domains exhibited typical ß-fold, while the third EGF-like domain and vWD domain were predominantly random coils. We observed that rMUC4ß physically interacts with Ezrin and EGFR family members. Overall, this study describes an efficient and simple strategy for the purification of biologically-active rMUC4ß that can serve as a valuable reagent for a variety of biochemical and functional studies to elucidate MUC4 function and generating domain-specific antibodies and vaccines for cancer immunotherapy.

Polyanhydride nanoparticles stabilize pancreatic cancer antigen MUC4ß.

Pancreatic cancer (PC) is one of the most lethal malignancies and represents an increasing and challenging threat, especially with an aging population. The identification of immunogenic PC-specific upregulated antigens and an enhanced understanding of the immunosuppressive tumor microenvironment have provided opportunities to enable the immune system to recognize cancer cells. Due to its differential upregulation and functional role in PC, the transmembrane mucin MUC4 is an attractive target for immunotherapy. In the current study we characterized the antigen stability, antigenicity and release kinetics of a MUC4ß-nanovaccine to guide further optimization and, in vivo evaluation. Amphiphilic polyanhydride copolymers based on 20 mol % 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane and 80 mol % 1,6-bis(p-carboxyphenoxy)hexane were used to synthesize nanoparticles. Structurally stable MUC4ß protein was released from the particles in a sustained manner and characterized by gel electrophoresis and fluorescence spectroscopy. Modest levels of protein degradation were observed upon release. The released protein was also analyzed by MUC4ß-specific monoclonal antibodies using ELISA and showed no significant loss of epitope availability. Further, mice immunized with multiple formulations of combination vaccines containing MUC4ß-loaded nanoparticles generated MUC4ß-specific antibody responses. These results indicate that polyanhydride nanoparticles are viable MUC4ß vaccine carriers, laying the foundation for evaluation of this platform for PC immunotherapy.

Irreversible and sustained upregulation of endothelin axis during oncogene-associated pancreatic inflammation and cancer.

Endothelin-1 (ET-1) and its two receptors, endothelin receptor A (ETAR) and endothelin receptor B (ETBR) exhibit deregulated overexprerssion in pancreatic ductal adenocarcinoma (PDAC) and pancreatitis. We examined the expression pattern of endothelin (ET) axis components in the murine models of chronic and acute inflammation in the presence or absence of oncogenic K-ras. While the expression of endothelin converting enzyme-1 (ECE-1), ET-1, ETAR and ETBR in the normal pancreas is restricted predominantly to the islet cells, progressive increase of ET receptors in ductal cells and stromal compartment is observed in the KC model (Pdx-1 Cre; K-rasG12D) of PDAC. In the murine pancreas harboring K-rasG12D mutation (KC mice), following acute inflammation induced by cerulein, increased ETAR and ETBR expression is observed in the amylase and CK19 double positive cells that represent cells undergoing pancreatic acinar to ductal metaplasia (ADM). As compared to the wild type (WT) mice, cerulein treatment in KC mice resulted in significantly higher levels of ECE-1, ET-1, ETAR and ETBR, transcripts in the pancreas. Similarly, in response to cigarette smoke-induced chronic inflammation, the expression of ET axis components is significantly upregulated in the pancreas of KC mice as compared to the WT mice. In addition to the expression in the precursor pancreatic intraepithelial neoplasm (PanIN lesions) in cigarette smoke-exposure model and metaplastic ducts in cerulein-treatment model, ETAR and ETBR expression is also observed in infiltrating F4/80 positive macrophages and α-SMA positive fibroblasts and high co-localization was seen in the presence of oncogenic K-ras. In conclusion, both chronic and acute pancreatic inflammation in the presence of oncogenic K-ras contribute to sustained upregulation of ET axis components in the ductal and stromal cells suggesting a potential role of ET axis in the initiation and progression of PDAC.

Serine protease-related proteins in the malaria mosquito, Anopheles gambiae.

Insect serine proteases (SPs) and serine protease homologs (SPHs) participate in digestion, defense, development, and other physiological processes. In mosquitoes, some clip-domain SPs and SPHs (i.e. CLIPs) have been investigated for possible roles in antiparasitic responses. In a recent test aimed at improving quality of gene models in the Anopheles gambiae genome using RNA-seq data, we observed various discrepancies between gene models in AgamP4.5 and corresponding sequences selected from those modeled by Cufflinks, Trinity and Bridger. Here we report a comparative analysis of the 337 SP-related proteins in A. gambiae by examining their domain structures, sequence diversity, chromosomal locations, and expression patterns. One hundred and ten CLIPs contain 1 to 5 clip domains in addition to their protease domains (PDs) or non-catalytic, protease-like domains (PLDs). They are divided into five subgroups: CLIPAs (22) are clip1-5-PLD; CLIPBs (29), CLIPCs (12) and CLIPDs (14) are mainly clip-PD; most CLIPEs (33) have a domain structure of PD/PLD-PLD-clip-PLD0-1. While expression of the CLIP genes in group-1 is generally low and detected in various tissue- and stage-specific RNA-seq libraries, some putative GPs/GPHs (i.e. single domain gut SPs/SPHs) in group-2 are highly expressed in midgut, whole larva or whole adult libraries. In comparison, 46 SPs, 26 SPHs, and 37 multi-domain SPs/SPHs (i.e. PD/PLD-PLD≥1) in group-3 do not seem to be specifically expressed in digestive tract. There are 16 SPs and 2 SPH containing other types of putative regulatory domains (e.g. LDLa, CUB, Gd). Of the 337 SP and SPH genes, 159 were sorted into 46 groups (2-8 members/group) based on similar phylogenetic tree position, chromosomal location, and expression profile. This information and analysis, including improved gene models and protein sequences, constitute a solid foundation for functional analysis of the SP-related proteins in A. gambiae.