Stroma-regulated HMGA2 is an independent prognostic marker in PDAC and AAC.

BACKGROUND: The HMGA2 protein has experimentally been linked to EMT and cancer stemness. Recent studies imply that tumour-stroma interactions regulate these features and thereby contribute to tumour aggressiveness. METHODS: We analysed 253 cases of pancreatic ductal adenocarcinoma (PDAC) and 155 cases of ampullary adenocarcinoma (AAC) for HMGA2 expression by IHC. The data were correlated with stroma abundance and supplemented by experimental studies. RESULTS: HMGA2 acts as an independent prognostic marker associated with a significantly shorter overall survival in both tumour types. Overall, HMGA2-positivity was more frequent in patients with PDAC than with AAC. The HMGA2 status in tumour cells significantly correlated with the abundance of PDGFRß-defined stroma cells. In vivo co-injection of Panc-1 cancer cells with pancreatic stellate cells increased tumour growth in a manner associated with increased HMGA2 expression. Furthermore, in vitro treatment of Panc-1 with conditioned media from PDGF-BB-activated stellate cells increased their ability to form tumour spheroids. CONCLUSIONS: This study identifies HMGA2 expression in tumour cells as an independent prognostic marker in PDAC and AAC. Correlative data analysis gives novel tissue-based evidence for a heterotypic cross-talk with stroma cells as a possible mechanism for HMGA2 induction, which is further supported by experimental models.

Cancer immune therapy using engineered ?tail-flipping' nanoliposomes targeting alternatively activated macrophages.

Alternatively-activated, M2-like tumor-associated macrophages (TAM) strongly contribute to tumor growth, invasiveness and metastasis. Technologies to disable the pro-tumorigenic function of these TAMs are of high interest to immunotherapy research. Here we show that by designing engineered nanoliposomes bio-mimicking peroxidated phospholipids that are recognised and internalised by scavenger receptors, TAMs can be targeted. Incorporation of phospholipids possessing a terminal carboxylate group at the sn-2 position into nanoliposome bilayers drives their uptake by M2 macrophages with high specificity. Molecular dynamics simulation of the lipid bilayer predicts flipping of the sn-2 tail towards the aqueous phase, while molecular docking data indicates interaction of the tail with Scavenger Receptor Class B type 1 (SR-B1). In vivo, the engineered nanoliposomes are distributed specifically to M2-like macrophages and, upon delivery of the STAT6 inhibitor (AS1517499), zoledronic acid or muramyl tripeptide, these cells promote reduction of the premetastatic niche and/or tumor growth. Altogether, we demonstrate the efficiency and versatility of our engineered "tail-flipping" nanoliposomes in a pre-clinical model, which paves the way to their development as cancer immunotherapeutics in humans.

ITGA5 inhibition in pancreatic stellate cells attenuates desmoplasia and potentiates efficacy of chemotherapy in pancreatic cancer.

Abundant desmoplastic stroma is the hallmark for pancreatic ductal adenocarcinoma (PDAC), which not only aggravates the tumor growth but also prevents tumor penetration of chemotherapy, leading to treatment failure. There is an unmet clinical need to develop therapeutic solutions to the tumor penetration problem. In this study, we investigated the therapeutic potential of integrin α5 (ITGA5) receptor in the PDAC stroma. ITGA5 was overexpressed in the tumor stroma from PDAC patient samples, and overexpression was inversely correlated with overall survival. In vitro, knockdown of ITGA5 inhibited differentiation of human pancreatic stellate cells (hPSCs) and reduced desmoplasia in vivo. Our novel peptidomimetic AV3 against ITGA5 inhibited hPSC activation and enhanced the antitumor effect of gemcitabine in a 3D heterospheroid model. In vivo, AV3 showed a strong reduction of desmoplasia, leading to decompression of blood vasculature, enhanced tumor perfusion, and thereby the efficacy of gemcitabine in co-injection and patient-derived xenograft tumor models.

Reprogramming tumor stroma using an endogenous lipid lipoxin A4 to treat pancreatic cancer.

Pancreatic stellate cells (PSCs) are the precursors of cancer-associated fibroblasts (CAFs), which potentiate pancreatic tumor growth and progression. In this study, we investigated whether Lipoxin A4 (LXA4), an endogenous bioactive lipid, can inhibit the differentiation of human PSCs (hPSCs) into CAF-like myofibroblasts and thereby hPSC-induced pro-tumorigenic effects. LXA4 significantly inhibited TGF-ß-mediated differentiation of hPSCs by inhibiting pSmad2/3 signalling. Furthermore, treatment with LXA4 abolished the paracrine effects (proliferation and migration of Panc-1 tumor cells) of hPSCs in vitro. These data demonstrated that LXA4 can interrupt pro-tumoral paracrine signalling of hPSCs. Furthermore, LXA4 treatment significant decreased the size and growth rate of 3D-heterospheroids comprised of hPSC and Panc-1 and these effects were exhibited due to inhibition of hPSC-induced collagen1 expression. In vivo, we examined the therapeutic efficacy of LXA4 in a co-injection (Panc-1 and hPSCs) subcutaneous tumor model. Intriguingly, LXA4 significantly abolished the tumor growth (either injected intratumor or intraperitoneally), attributed to a significant reduction in fibrosis, shown with collagen1 expression. Altogether, this study proposes LXA4 as a potent inhibitor for hPSCs which can be applied to reprogram tumor stroma in order to treat pancreatic cancer.

Anti-microRNA targeting using peptide-based nanocomplexes to inhibit differentiation of human pancreatic stellate cells.

AIM: To develop novel peptide-based nanocomplexes (NCs) for delivery of anti-miRNA oligonucleotides to human-derived pancreatic stellate cells (hPSCs), precursors of cancer-associated fibroblasts. MATERIALS & METHODS: NCs of anti-miRNA oligonucleotides and cell-penetrating peptides (different variants) were formed and characterized. The effects of anti-miR-199a delivery on hPSC differentiation and 3D heterospheroid formation were investigated. RESULTS: Dimeric cell-penetrating peptide based NCs (NC-2) showed 130-fold higher uptake by hPSCs compared with monomer-based NCs (NC-1) and tenfold higher uptake compared with general fibroblasts and different pancreatic tumor cells. Interestingly, delivery of anti-miR-199a inhibited hPSC differentiation into cancer-associated fibroblasts and inhibited the size of 3D heterospheroids comprised of hPSCs and tumor cells. CONCLUSION: Our NCs present a highly efficient anti-miRNA delivery system to hPSCs to inhibit their protumorigenic activity.

MicroRNA Targeting to Modulate Tumor Microenvironment.

Communication between stromal cells and tumor cells initiates tumor growth, angiogenesis, invasion, and metastasis. Stromal cells include cancer-associated fibroblasts, tumor-associated macrophages, pericytes, endothelial cells, and infiltrating immune cells. MicroRNAs (miRNAs) in the tumor microenvironment have emerged as key players involved in the development of cancer and its progression. miRNAs are small endogenous non-protein-coding RNAs that negatively regulate the expression of multiple target genes at post-transcriptional level and thereby control many cellular processes. In this review, we provide a comprehensive overview of miRNAs dysregulated in different stromal cells and their impact on the regulation of intercellular crosstalk in the tumor microenvironment. We also discuss the therapeutic significance potential of miRNAs to modulate the tumor microenvironment. Since miRNA delivery is quite challenging and the biggest hurdle for clinical translation of miRNA therapeutics, we review various non-viral miRNA delivery systems that can potentially be used for targeting miRNA to stromal cells within the tumor microenvironment.

MicroRNA-199a and -214 as potential therapeutic targets in pancreatic stellate cells in pancreatic tumor.

Pancreatic stellate cells (PSCs) are the key precursor cells for cancer-associated fibroblasts (CAFs) in pancreatic tumor stroma. In this study, we explored miRNA as therapeutic targets in tumor stroma and found miR-199a-3p and miR-214-3p induced in patient-derived pancreatic CAFs and TGF-ß-activated human PSCs (hPSCs). Inhibition of miR-199a/-214 using hairpin inhibitors significantly inhibited TGFß-induced differentiation markers (e.g. α-SMA, collagen, PDGFßR), migration and proliferation. Furthermore, heterospheroids of Panc-1 and hPSCs attained smaller size with hPSCs transfected with anti-miR-199a/-214 compared to control anti-miR. The conditioned medium obtained from TGFß-activated hPSCs induced tumor cell growth and endothelial cell tube formation. Interestingly, these inductions were abrogated in hPSCs transfected with anti-miR-199a or miR-214. Moreover, IPA analyses revealed signaling pathways related to miR-199a (TP53, mTOR, Smad1) and miR-214 (PTEN, Bax, ING4). Taken together, this study reveals miR-199a-3p and miR-214-3p as major regulators of PSC activation and PSC-induced pro-tumoral effects, representing them as key therapeutic targets in pancreatic cancer.

SAINT-liposome-polycation particles, a new carrier for improved delivery of siRNAs to inflamed endothelial cells.

Interference with acute and chronic inflammatory processes by means of delivery of siRNAs into microvascular endothelial cells at a site of inflammation demands specific, non-toxic and effective siRNA delivery system. In the current work we describe the design and characterization of siRNA carriers based on cationic pyridinium-derived lipid 1-methyl-4-(cis-9-dioleyl)methyl-pyridinium-chloride) (SAINT-C18) and the transfection enhancer protamine, complexed with siRNA/carrier DNA or siRNA only. These carriers, called SAINT-liposome-polycation-DNA (S-LPD) and SAINT-liposome-polycation (S-LP), have a high efficiency of siRNA encapsulation, low cellular toxicity, and superior efficacy of gene downregulation in endothelial cells in vitro as compared to DOTAP-LPD. Incorporation of 10 mol% PEG and anti-E-selectin antibody in these formulations resulted in selective siRNA delivery into activated endothelial cells. Furthermore, we showed that the physicochemical characteristics of S-LPD and S-LP, including size-stability and maintenance of the siRNA integrity in the presence of serum at 37 °C, comply with requirements for in vivo application.