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Fig S1. Phospho-aspirin inhibits the growth of human PC cells. Figure S2. Phospho-aspirin induces apoptosis in human PC cells. Figure S3. Phospho-aspirin induces p53 acetylation in human PC cells and xenografts. Figure S4: PA does not inhibit PGE2 levels in PC cells. Figure S5. Expression levels of phospho-EGFR (p-EGFR) in multiple human PC cells. Figure S6. Phospho-aspirin inhibits EGFR phosphorylation in PC cells. Figure S7. Serum amylase and lipase levels. Figure S8. Representative images of acinar cells explants at day 0 and the resulting metaplasia (day 5).

Pancreatic cancer is a deadly disease with a dismal 5-year survival rate of 50 values. In a Panc-1 xenograft model, phospho-aspirin, at a dose of 100 mg/kg/d 5 times per week for 30 days, reduced tumor growth by 78% (P < 0.01 vs. vehicle control). Furthermore, phospho-aspirin prevented pancreatitis-accelerated acinar-to-ductal metaplasia in mice with activated Kras. In p48-Cre;KrasG12D mice, cerulein treatment (6 hourly injections two times per week for 3 weeks) led to a significant increase in ductal metaplasia, replacing the majority of the exocrine compartment. Administration of phospho-aspirin 100 mg/kg/day five times per week for 21 days (starting on the first day of cerulein injection) inhibited the acinar-to-ductal metaplasia, reducing it by 87% (P < 0.01, vs. cerulein-treated control). Phospho-aspirin appeared to be safe, with the animals showing no signs of toxicity during treatment. Mechanistically, phospho-aspirin inhibited EGFR activation in pancreatic cancer, an effect consistently observed in pancreatic cancer cells, primary acinar explants and in vivo. In conclusion, our findings indicate that phospho-aspirin has strong anticancer efficacy in preclinical models of pancreatic cancer, warranting its further evaluation. Cancer Prev Res; 9(7); 624–34. ©2016 AACR.

This study aims to identify pathway involvement in the development of cisplatin (cis-diamminedichloroplatinum (II); CDDP) resistance in A549 lung cancer (LC) cells by utilizing advanced bioinformatics software. We developed CDDP-resistant A549 (A549/DDP) cells through prolonged incubation with the drug and performed RNA-seq on RNA extracts to determine differential mRNA and miRNA expression between A549/DDP and A549 cells. We analyzed the gene dysregulation with Ingenuity Pathway Analysis (IPA; QIAGEN) software. In contrast to prior research, which relied on the clustering of dysregulated genes to pathways as an indication of pathway activity, we utilized the IPA software for the dynamic evaluation of pathway activity depending on the gene dysregulation levels. We predicted 15 pathways significantly contributing to the chemoresistance, with several of them to have not been previously reported or analyzed in detail. Among them, the PKR signaling, cholesterol biosynthesis, and TEC signaling pathways are included, as well as genes, such as PIK3R3, miR-34c-5p, and MDM2, among others. We also provide a preliminary analysis of SNPs and indels, present exclusively in A549/DDP cells. This study's results provide novel potential mechanisms and molecular targets that can be explored in future studies and assist in improving the understanding of the chemoresistance phenotype.

Lung cancer (LC) is among the leading causes of death from malignancies worldwide. miRNAs (miRs), small noncoding RNAs capable of regulating gene expression through the cell’s RNAi mechanism, have emerged as promising molecules for cancer treatment. In our previous work, we identified two miRs, miR-143 and miR-506, whose transient transfection induced apoptosis, cell cycle inhibition, and cyclin-dependent kinase (CDK) 1, 4, and 6 downregulations. Here, we developed stable deregulations of the miRs’ expressions in A549 cells, individually or in combination, to evaluate their effect on LC cell behavior. To establish homogeneous populations of deregulated cells, A549 cells with stable up and downregulated miR-143 and miR-506, alone or in combination, were sorted according to marker expression using fluorescence-activated cell sorting (FACS). miR deregulations were confirmed with quantitative real-time polymerase chain reaction (qRT -PCR) analysis, and the cells were analyzed for changes in cell proliferation through cell cycle analysis using flow cytometry and cell doubling time using Quantitative Phase Imaging (QPI). With the same method, morphometric characteristics were quantified. Finally, we evaluated the motility of the cells using a scratch assay and determined the gene expression of CDKs. Taqman qPCR analysis indicated a >5-fold increase of the respective miR’s basal expression due to the stable transfections. The cell cycle analysis indicated a complex behavior for the individual miRs, while, in contrast, the combined upregulation of the two miRs indicated a G2 inhibition, representing a well-defined behavior in comparison to both the control and downregulation groups. Furthermore, the combined miR upregulation increased the cell doubling time compared to the control and respective downregulations. Similarly, using QPI microscopy, we observed that the miR combination upregulation had higher cellular sphericity, perimeter, and area compared to the control group. The wound healing assay did not indicate statistically significant differences among the different groups, with miR-506 upregulation demonstrating the slowest closure rate, followed by miR -143. In contrast, the differences between the miR-combination and control group were small, although the anti-miR-combination indicated an accelerated closure. In conclusion, the combined upregulation of both miRs inhibited cell proliferation through a G2 arrest, while the deregulation of the individual miRs demonstrated a complex behavior. Thus, the miR-combination’s consistent, promising behavior would indicate potential benefits using the two miRs against LC that merits further evaluation. Citation Format: Archana Shrestha, Behnaz Lahooti, Mahboubeh Madadi, Constantinos M. Mikelis, George Mattheolabakis. Impact of individual and combined deregulation of miR-143 and miR-506 on cytokinetic and morphometric parameters in A549 lung cancer cells. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3759.

Purpose Nucleic acid-based therapies are a promising therapeutic tool. The major obstacle in their clinical translation is their efficient delivery to the desired tissue. We developed a novel nanosized delivery system composed of conjugates of α-tocopherol, polyethyleneimine, and polyethylene glycol (TPP) to deliver nucleic acids. Methods We synthesized a panel of TPP molecules using different molecular weights of PEG and PEI and analyzed with various analytical approaches. The optimized version of TPP (TPP111 - the 1:1:1 molecular ratio) was self-assembled in water to produce nanostructures and then evaluated in diversified in vitro and in vivo studies. Results Through a panel of synthesized molecules, TPP111 conjugate components self-assembled in water, forming globular shaped nanostructures of ~90 nm, with high nucleic acid entrapment efficiency. The polymer had low cytotoxicity in vitro and protected nucleic acids from nucleases. Using a luciferase-expressing plasmid, TPP111-plasmid nano-complexes were rapidly up-taken by cancer cells in vitro and induced strong transfection, comparable to PEI. Colocalization of the nano-complexes and endosomes/lysosomes suggested an endosome-mediated uptake. Using a subcutaneous tumor model, intravenously injected nano-complexes preferentially accumulated to the tumor area over 24 h. Conclusion These results indicate that we successfully synthesized the TPP111 nanocarrier system, which can deliver nucleic acids in vitro and in vivo and merits further evaluation.

Background and study aims Patients with pancreatic cancer often have locally advanced or metastatic disease and are not candidates for curative surgery. Polymer-based microparticles (MPs) represent a drug delivery system that offers sustained release of a chemotherapeutic drug after intralesional injection for local tumor management. The aim of this study was to determine the feasibility of endoscopic ultrasound-guided fine-needle injection (EUS-FNI) of drug-loaded MPs tagged with a fluorophore and fiducial markers for locating the injection site. Secondary aims were to determine the tissue-specific effects of MPs. Methods Five pigs underwent EUS with selection of an injection site within the pancreas that was marked by placing fiducial markers prior to the MPs injection. EUS-FNI of either blank microparticles (BMPs), containing no drug, or gemcitabine-loaded microparticles (GMPs) was performed. A saline flush containing Spot Endoscopic Marker was used to expel any residual MPs in the needle shaft and tattoo the injection site. Results A green fluorescent protein flashlight was used to successfully identify the site of MP injection sites in the dissected pancreas. Frozen sections of pig pancreas demonstrated a defined deposit, confirming the delivery of the MPs. Finally, fluorescence microscopy showed activation of caspase-mediated cell death in pancreases of animals that received injections of GMPs. Conclusions This pilot study demonstrated that fiducial marker placement and pancreatic EUS-FNI of MPs was successful in all pigs with no animals demonstrating pancreatitis. Further studies are needed to determine the role for intralesional injection of drug-loaded MPs in borderline resectable or unresectable pancreatic cancer.

Lung cancer (LC) and pancreatic cancer (PC) are the first and fourth leading causes of cancer‑related deaths in the US. Deregulated cell cycle progression is the cornerstone for rapid cell proliferation, tumor development, and progression. Here, we provide evidence that a novel combinatorial miR treatment inhibits cell cycle progression at two phase transitions, through their activity on the CDK4 and CDK1 genes. Following transfection with miR‑143 and miR‑506, we analyzed the differential gene expression of CDK4 and CDK1, using qPCR or western blot analysis, and evaluated cell cycle inhibition, apoptosis and cytotoxicity. The combinatorial miR‑143/506 treatment downregulated CDK4 and CDK1 levels, and induced apoptosis in LC cells, while sparing normal lung fibroblasts. Moreover, the combinatorial miR treatment demonstrated a comparable activity to clinically tested cell cycle inhibitors in inhibiting cell cycle progression, by presenting substantial inhibition at the G1/S and G2/M cell cycle transitions. More importantly, the miR‑143/506 treatment presented a broader application, effectively downregulating CDK1 and CDK4 levels, and reducing cell growth in PC cells. These findings suggest that the miR‑143/506 combination acts as a promising approach to inhibit cell cycle progression for cancer treatment with minimal toxicity to normal cells.

Among the lung cancers, approximately 85% are histologically classified as non-small-cell lung cancer (NSCLC), a leading cause of cancer deaths worldwide. Epidermal growth factor receptors (EGFRs) are known to play a crucial role in lung cancer. HER2 overexpression is detected by immunohistochemistry in 2.4%–38% of NSCLC samples. EGFRs have been targeted with three generations of tyrosine kinase inhibitors (TKIs), and drug resistance has become a major issue; HER2 dimerization with EGFR also plays a major role in the development of resistance to TKI therapy. We have designed grafted peptides to bind to the HER2 extracellular domain (ECD) and inhibit protein-protein interactions of EGFR:HER2 and HER2:HER3. A sunflower trypsin inhibitor (SFTI-1) template was used to graft a peptidomimetic compound. Among several grafted peptides, SFTI-G5 exhibited antiproliferative activity in HER2-positive NSCLC cell lines such as Calu-3 cells with an IC50 value of 0.073 μM. SFTI-G5 was shown to bind to ECD of HER2 and inhibit EGFR:HER2 and HER2:HER3 dimerization and inhibit the phosphorylation of HER2 and downstream signaling proteins. As a proof-of-concept, the in vivo activity of SFTI-G5 was evaluated in two NSCLC mouse models. SFTI-G5 was able to inhibit tumor growth in both models. Furthermore, SFTI-G5 was shown to inhibit EGFR dimerization in tissue samples obtained from in vivo models. These grafted peptides can be used as novel dual inhibitors of EGFR dimerization in NSCLC.

Wound regeneration is a complex process, which necessitates proper coordination among the inflammatory response, vascularization, matrix formation, and reformation of epithelial tissue. It is a unique process, where healing and regeneration take place simultaneously. Matrix formation is the first critical stage that starts the communication between the keratinocytes, fibroblasts, and integrins. This, in turn, stimulates the differentiation of monocytes into macrophages, to produce cytokines for fibroblasts. This phenomenon is the crucial part for the keratinocyte migration and epithelialization to fill the wound. To understand the complex procedure of wound regeneration, there is a need for easy, convenient, and low-cost approaches that will simulate the wound-repairing process. Scratch assay or cellular migration assay is one of the most convenient and affordable approaches, commonly used by the scientific community. In this chapter, we present the fundamental principles of the experimental procedures required for the Scratch assay.

Wound regeneration is a complex process, which necessitates proper coordination among the inflammatory response, vascularization, matrix formation, and reformation of epithelial tissue. It is a unique process, where healing and regeneration take place simultaneously. Matrix formation is the first critical stage that starts the communication between the keratinocytes, fibroblasts, and integrins. This, in turn, stimulates the differentiation of monocytes into macrophages, to produce cytokines for fibroblasts. This phenomenon is the crucial part for the keratinocyte migration and epithelialization to fill the wound. To understand the complex procedure of wound regeneration, there is a need for easy, convenient, and low-cost approaches that will simulate the wound-repairing process. Scratch assay or cellular migration assay is one of the most convenient and affordable approaches, commonly used by the scientific community. In this chapter, we present the fundamental principles of the experimental procedures required for the Scratch assay.

miRNAs are promising therapeutic targets or tools for the treatment of numerous diseases, with most prominently, cancer. The inherent capacity of these short nucleic acids to regulate multiple cancer-related pathways simultaneously has prompted strong research on understanding miR functions and their potential use for therapeutic purposes. A key determinant of miR therapeutics’ potential for treatment is their delivery. Viral and non-viral vectors attempt to address the major limitations associated with miR delivery, but several hurdles have been identified. Here, we present an overview on the general limitations of miR delivery, and the delivery strategies exploited to overcome them. We provide an introduction on the advantages and disadvantages of viral and non-viral vectors, and we go into detail to analyze the most prominently used non-viral systems. We provide with an update on the most recent research on this topic and we describe the mechanism and limitations of the lipid-, polymer- and inorganic material- based miR delivery systems.

Despite the apparent advantages for long-term treatment and local therapies against intestinal diseases, the oral delivery of nucleic acids has been challenging due to unfavorable physiological conditions for their stability. In this study, a novel nanodelivery system of PEG-PCL nanoparticles with encapsulated nucleic acids–mannosylated PEI (Man-PEI) complexes was developed for intestinal delivery. We complexed model nucleic acids with Man-PEI at the optimal N/P ratio of 20:1 for in vitro and in vivo analyses. Cells were transfected in vitro and analyzed for gene expression, receptor-mediated uptake, and PEG-PCL nanoparticles’ toxicity. We also evaluated the nucleic acid’s stability in the nanocarrier during formulation, and under simulated gastrointestinal environments or the presence of nucleases. Finally, we assessed the biodistribution for the PEG-PCL nanoparticles with encapsulated complexes and their ability to transfect intestinal cells in vivo. Nucleic acids complexed with Man-PEI were protected from degradation against nucleases. In comparison to the parent compound PEI, Man-PEI transfected the cells with an overall higher potency. Competition assay indicated receptor-mediated endocytosis promoted by mannose receptors. The PEG-PCL nanoparticles with Man-PEI/plasmid complexes indicated minimal cytotoxicity. The nanocarrier successfully protected the complexes in a simulated gastric fluid environment and released them in a simulated intestinal fluid environment, promoted by the presence of lipases. The oral administration of the PEG-PCL nanoparticles with encapsulated Man-PEI/plasmid complexes transfected intestinal cells with the plasmid in vivo, while presenting a time-dependent progression through the intestines. Conclusively, our carrier system can deliver genetic material to the GI tract and actively target mannose receptor overexpressing cells.

Oral delivery of nucleic acids has been challenging, due to unfavorable physiological factors that do not comply with the nucleic acids' properties. Firstly, nucleic acids break down in the harsh acidic gastric environment. Secondly, they are hydrophilic, negatively charged, large-molecular-weight molecules, unable to efficiently penetrate through the mucus membrane and enter the epithelial intestinal wall. We developed a novel nano delivery system of nucleic acids complexed with mannosylated PEI encapsulated in PEG-PCL matrix. We will use this carrier to overcome the above-mentioned limitations of oral nucleic acid delivery aiming for colon cancer treatment. We synthesized the mannosylated PEI with the objective to target colon cancer cells. We will use active targeting as these cells overexpress mannose receptors. We complexed a model nucleic acid, the PGL-3 luciferase expressing plasmid, with mannosylated PEI at the optimal N/P ratio of 20:1. We transfected cancer cells in vitro and analyzed the luciferase expression. Furthermore, we analyzed the cytotoxicity of PCL-PEG nanoparticles containing mannosylated PEI/PGL-3 complexes, as well as the nanoparticles capacity to protect nucleic acids and release their load. Mannosylated PEI successfully complexed with the nucleic acids, protecting from degradation against nucleases. Mannosylated PEI/PGL-3 complexes transfected colon cancer cells and the luciferase expression was significantly higher when compared to PEI alone, at 24 and 48 h. The complexes were successfully up taken by cell lines in a time dependent manner. Competitive transfection assay with free mannose demonstrated the active targeting effect caused due to the mannose receptors. Similarly, the PCL-PEG nanoparticles with mannose PEI/PGL-3 complexes had a limited cytotoxicity. Most importantly, the carrier successfully protected the mannose PEI/PGL-3 complexes in a simulated gastric fluid environment and released them in a simulated intestinal fluid environment. This indicates that the nanocarriers can potentially protect the nucleic acids in an acidic environment, such as the stomach, and release their mannosylated PEI/PGL-3 in a neutral environment, such as in the intestines. Such a behavior would indicate a passive targeting to the small and large intestines. We can conclude that the formulated polymeric nanoparticles were successful in protecting the nucleic acids. The mannosylated PEI was able to completely complex with nucleic acids and actively target colon cancer. This promising nanocarrier and our approach merits further evaluation for the oral administration of nucleic acids in therapeutic applications of passive and active targeting against colon cancer. Citation Format: Sagun Poudel, George Mattheolabakis. Passive and active nucleic acid delivery against colon cancer cells using a novel nanocarrier aimed for oral administration [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 302.

Tumor-induced angiogenesis has been a significant focus of anti-cancer therapies for several decades. The immature and "leaky" tumor vasculature leads to significant cancer cell intravasation, increasing the metastatic potential, while the disoriented and hypo-perfused tumor vessels hamper the anti-tumor efficacy of immune cells and prevent the efficient diffusion of chemotherapeutic drugs. Therefore, tumor vascular normalization has emerged as a new treatment goal, aiming to provide a mature tumor vasculature, with higher perfusion, decreased cancer cell extravasation, and higher efficacy for anti-cancer therapies. Here we propose an overview of the nanodelivery approaches that target tumor vasculature, aiming to achieve vascular normalization. At the same time, abnormal vascular architecture and leaky tumor vessels have been the cornerstone for nanodelivery approaches through the enhanced permeability and retention (EPR) effect. Vascular normalization presents new opportunities and requirements for efficient nanoparticle delivery against the tumor cells and overall improved anti-cancer therapies.

Nucleic acid-based therapeutics are evaluated for their potential of treating a plethora of diseases, including cancer and inflammation. Short nucleic acids, such as miRNAs, have emerged as versatile regulators for gene expression and are studied for therapeutic purposes. However, their inherent instability in vivo following enteral and parenteral administration has prompted the development of novel methodologies for their delivery. Although research on the oral delivery of siRNAs is progressing, with the development and utilization of promising carrier-based methodologies for the treatment of a plethora of gastrointestinal diseases, research on miRNA-based oral therapeutics is lagging behind. In this review, we present the potential role of miRNAs in diseases of the GI tract, and analyze current research and the cardinal features of the novel carrier systems used for nucleic acid oral delivery that can be expanded for oral miRNA administration.

Cisplatin, is recognized as a first line therapeutic for the treatment of non-small cell lung cancer (NSCLC). Cisplatin resistance is identified as the most detrimental complication during treatment and has been associated with upregulation of several genes, such as the anti-apoptotic gene survivin. In this study, we have evaluated the cytotoxic activity of lipid (C6 and C8)-modified platinum compounds in combination with a survivin-silencing siRNA against cisplatin resistant tumors.We synthesized and characterized several lipid-modified platinum compounds and evaluated their cytotoxic activity alone or in combination with survivin-silencing siRNA in vitro and in vivo against A549The lipid-modified compounds exhibited significantly stronger cytotoxic activity in vitro compared to cisplatin, with CDDP-C6 and CDDP-C8 producing the most pronounced effect, in both A549 and A549Co-treatment of lipid-modified compounds and survivin-silencing siRNA can constitute a reliable alternative to cisplatin treatment for cisplatin-resistant lung tumors that merit further evaluation.

Dual stimuli-sensitive mixed polymeric micelles (MM) are developed for co-delivery of the endogenous tumor suppressor miRNA-34a and the chemotherapeutic agent doxorubicin (Dox) into cancer cells. The novelty of the system resides in two stimuli-sensitive prodrugs, a matrix metalloproteinase 2 (MMP2)-sensitive Dox conjugate and a reducing agent (glutathione, GSH)-sensitive miRNA-34a conjugate, self-assembled in a single particle decorated with a polyethylene glycol corona for longevity, and a cell-penetrating peptide (TATp) for enhanced intracellular delivery. The MMP2-sensitivity of the system results in threefold higher cytotoxicity in MMP2-overexpressing HT1080 cells compared to low MMP2-expressing MCF7 cells. Cellular internalization of Dox increases by more than 70% after inclusion of TATp to the formulation. MMP2-sensitive MM also inhibits proliferation and migration of HT1080 cells. Moreover, GSH-sensitive MM allows for an efficient downregulation of Bcl2, survivin, and notch1 (65%, 55%, and 46%, respectively) in HT1080 cells. Combination of both conjugates in dual sensitive MM reduces HT1080 cell viability to 40% and expression of Bcl2 and survivin. Finally, 50% cell death is observed in 3D models of tumor mass. The results confirm the potential of the MM to codeliver miRNA-34a and doxorubicin triggered by dual stimuli inherent of tumor tissues.

Lung cancer (LC) is the leading cause of cancer-related deaths worldwide. Among the 18.1 million of total cancer patients last year, 2.1 million of the new cases were of LC worldwide. The poor prognosis, higher incidence, and mortality rate make this disease a devastating type of cancer. Cell cycle inhibition is one of the prominent choices among researchers in cancer treatment. Different cell cycle inhibitors are developed by scientists where the cyclin-dependent kinase (CDK) inhibitors are among the most frequently researched. Just recently, 3 CDK inhibitors received FDA-approval for treatment of specific breast cancer subtypes, but no CDK inhibitors currently exist for LC patient treatment. Thus, there is a need for molecules that regulate cell cycle progression and CDK expression. Micro RNAs (miRNAs) are gene-expression regulatory molecules. Among several miRs, we selected miR-143 and miR-506 to regulate CDK1 and CDK4/6, respectively. qPCR and RNA-sequencing indicated that this combinatorial miR therapy caused downregulation of the respective CDK1, 4, and 6 genes in H358 and H1975 lung cancer cell lines. This RNA inhibition corresponded to protein levels downregulation, as detected by western blot analysis. Further investigation found that this treatment caused significant halt of the cell cycle progression, which is markedly stronger than the commercially available CDK inhibitors, Ribociclib and Flavopiridol. We also identified the combined miR-143 and miR-506 caused significant increase of the apoptotic cell population at both 24 and 48 h post-treatment, as detected by an Annexin V/PI analysis. Interestingly, low or no apoptotic impact was observed in HFL1 normal lung fibroblast cells by the combinatorial miR treatment, indicating a cancer-cell specific activity. Upstream pathway analysis through RNA-sequencing using Ingenuity Pathway Analysis software (IPA; Qiagen) indicated a potential ERK-mediation on the activity of the combinatorial miR treatment. Our analysis of the ERK pathway using MAPK (ERK) inhibitors indicated an ERK-independent cell cycle inhibitory activity of the two miRs. Through further analysis of the RNA-Seq data, we observed a potential inhibition of the cellular migration and invasion activity of the cells, which was confirmed by a cell migration assay. In summary, combinatorial treatment of miR-143 and miR-506 has a significant impact on the cell cycle, overall survival, and migration of LC cells, which still merits further study. Citation Format: A K M Nawshad Hossian. Combinatorial micro RNA therapy can be used as potential cell cycle inhibitors to treat lung cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3710.

IntroductionAs one of the most aggressive cancers worldwide, pancreatic cancer is associated with an extremely poor prognosis. The pancreatic tumor microenvironment consists of cancer cells and other tumor associated cells. Cross-talk between these different cell types through various signaling molecules results in the development of a more aggressive and malignant phenotype. Additionally, due to the highly dysregulated vasculature of tumors, the inner tumor core becomes hypoxic and eventually necrotic. Therefore, there is a need for the development of a physiologically relevant in vitro model that recapitulates these dynamic cell-cell interactions and the 3-dimensional (3D) structure of pancreatic tumors.MethodsFour different 3D co-culture spheroid models using different combinations of Panc-1 tumor cells, J774.A1 macrophages, and NIH-3T3 fibroblast cell lines were reproducibly developed using the hanging drop technique in order to mimic the tumor microenvironment and to evaluate the differences in expression of various inflammatory, hypoxia, and cancer stem cell markers, including IL-8, TNF-α, TGF-β, HIF-1α HIF-2α, SCF, and LDH-A. Additionally, immunofluorescence studies were employed to investigate whether these spheroids tested positive for a cancer stem cell population.ResultsPronounced differences in morphology as well as expression of signalling markers were observed using qPCR, indicative of strong influences of co-culturing different cell lines. These models also tested positive for cancer stem cell (CSCs) markers based on immunofluorescence and qPCR analysis.ConclusionOur results demonstrate the potential of 3D co-culture spheroid models to capture the inflammatory and hypoxic markers of pancreatic tumor microenvironment. We further demonstrate the presence of cancer cells with stem cell markers, similar to actual pancreatic cancer tumor. These spheroids present excellent in vitro system to study tumor-immune-stromal cell interactions as well as test deliverability of potential therapeutics in the tumor microenvironment with accurate physical and physiological barriers.

Lung cancer (LC) is the leading cause of cancer-related deaths. Downregulation of CDK1, 4 and 6, key regulators of cell cycle progression, correlates with decreased LC cell proliferation. Enforced expression of miRNAs (miRs) is a promising approach to regulate genes. Here, we study the combinatorial treatment of miR-143 and miR-506 to target the CDK1, 4/6 genes, respectively. We analyzed the differential expression of CDK genes by qPCR, and western blot, and evaluated changes in the cell cycle distribution upon combinatorial treatment. We used an antibody microarray analysis to evaluate protein expression, focusing on the cell cycle pathway, and performed RNA-sequencing for pathway analysis. The combinatorial miR treatment significantly downregulated CDK1, 4 and 6 expression, and induced a shift of the cell cycle populations, indicating a G1 and G2 cell cycle block. The two miRs induces strong cytotoxic activity, with potential synergism, and a significant Caspase 3/7 activation. We identified a strong inhibition of tube formation in the presence or absence VEGF in an in vitro angiogenesis model. Together with the pathways analysis of the RNA-sequencing data, our findings establish the combinatorial miR transfection as a viable strategy for lung cancer treatment that merits further investigation.

Curcumin reduces Hodgkin's lymphoma (HL) cell growth in vitro, but its unfavorable pharmacokinetics highlight the need for novel in vivo delivery systems. Thus, we explored whether formulation of curcumin in solid lipid nanoparticles (SLN-curc) or d-α-Tocopheryl polyethylene glycol 1000 succinate (TPGS) nanoparticles (TPGS-curc) could enhance its efficacy in mice. Curcumin formulated in SLN and in TPGS resulted in higher curcumin plasma levels in mice. Compared to vehicle-treated controls, SLN-curc and TPGS-curc reduced HL xenograft growth by 50.5% (p

Phospho-non-steroidal anti-inflammatory drugs (phospho-NSAIDs) are a novel class of NSAID derivatives with potent antitumor activity. However, phospho-NSAIDs have limited stability in vivo due to their rapid hydrolysis by carboxylesterases at their carboxylic ester link. Here, we synthesized phospho-ibuprofen amide (PIA), a metabolically stable analog of phospho-ibuprofen, formulated it in nanocarriers, and evaluated its pharmacokinetics and anticancer efficacy in pre-clinical models of human lung cancer. PIA was 10-fold more potent than ibuprofen in suppressing the growth of human non-small-cell lung cancer (NSCLC) cell lines, an effect mediated by favorably altering cytokinetics and inducing oxidative stress. Pharmacokinetic studies in rats revealed that liposome-encapsulated PIA exhibited remarkable resistance to hydrolysis by carboxylesterases, remaining largely intact in the systemic circulation, and demonstrated selective distribution to the lungs. The antitumor activity of liposomal PIA was evaluated in a metastatic model of human NSCLC in mice. Liposomal PIA strongly inhibited lung tumorigenesis (>95%) and was significantly (p

Lung cancer is the leading cause of cancer related deaths worldwide. Although targeting the cell cycle of cancer cells has been a promising approach, no such therapy has translated for the treatment of lung cancer. In our previous work, we identified that the combinatorial treatment of two miRNAs, miR-143 and miR-506, inhibited the cell cycle of lung cancer cells in vitro. These miRs downregulated the CDK1 and CDK4/6 genes, which are involved in the G1/S and G2/M transitions, respectively. Nucleic acid delivery has met formidable challenges, such as short in vivo half-life and rapid degradation in the circulation, as well as limited cell penetration or endosomal escape. We developed a novel nanocarrier, composed of tocopherol (vitamin E), polyethylenenimine, and polyethylene glycol (TPP) in 1:1:1 molar ratio for the delivery of miRNAs. Proper characterization with NMR and FTIR confirmed the synthesis of this molecule. We found it can form nanomicelles, and the positively charged of polyethyleneimine allowed for the complexation of miRNAs, as was detected by a gel retardation assay. Fluorescently tagged TPP micelles were uptaken by cancer cells, and the carrier successfully transfected cancer cells with the combinatorial miRNA treatment inducing gene downregulation. In vivo pharmacokinetics study using a subcutaneous mouse model of lung cancer indicated prolonged accumulation of the TPP-miRNA micelles in the tumor area. In vivo administration of the miR-143/506 in TPP micelles, along with cisplatin co-treatment, inhibited tumor growth in a subcutaneous mouse model of lung cancer. Overall, the promising role of miR-143 and 506 for the regulation of LC growth may be achieved by this novel TPP-based nanocarrier, which merits further evaluation. Citation Format: A K M Nawshad Hossian, Chandra Mohan Reddy Muthumula, George Mattheolabakis. Delivery of miR-143 and miR-506 with novel nano carrier to arrest cell cycle in lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4429.

Pancreatic cancer is a deadly disease with a dismal 5-year survival rate of

Tumor cells utilize many different mechanisms to desensitize themselves to the cytotoxic effects of drugs, but it has recently been recognized that alterations in epigenetic control of gene expression underly many of them. As master regulators of gene expression, microRNAs (miRNAs) present a promising therapeutic strategy for the reversal of epigenetic changes that lead to drug resistance phenotypes in tumor cells.Effects of epigenetic changes on drug resistance in a variety of solid tumors are discussed. Specific miRNAs that are involved with the regulation of epigenetic machinery are highlighted. Further, we consider how delivery of miRNA or antagomirs may be utilized to resensitize drug-resistant tumor cells.Reversal of epigenetically controlled tumor drug resistance mechanisms via miRNA delivery presents a novel strategy for enhancing the efficacy of chemotherapeutics. Further, the ability to target delivery of miRNAs may provide the opportunity to go beyond reversal of resistance to hyper-sensitization of tumor cells to the cytotoxic effects of drugs. However, understanding of the role of miRNA in epigenetic regulation is still in its early stages and further research is critical for potential utility in improving therapeutic efficacy in cancer patients.

Pancreatic cancer (PC) is one of the most difficult cancers to treat. Since the current chemotherapy is inadequate and various biological approaches have failed, the need for agents that have a potential to treat PC is pressing. Phospho-valproic acid (P-V), a novel anticancer agent, is efficacious in xenograft models of human PC and is apparently safe. In the present study, we evaluated whether formulating P-V in nanoparticles could enhance its anticancer efficacy. In a mouse model of Kras/pancreatitis-associated PC, P-V, orally administered, inhibited the incidence of acinar-to-ductal metaplasia by 60%. To improve its efficacy, we formulated P-V in five different polymeric nanoparticles. Poly-(L)-lactic acid- poly(ethylene glycol) (PLLA-PEG) nanoparticles proved the optimal formulation. PLLA-PEG improved P-V's pharmacokinetics in mice enhancing the levels of P-V in blood. Compared to control, P-V formulated in PLLA-PEG suppressed the growth of MIA PaCa-2 xenografts by 81%, whereas P-V alone reduced it by 51% (p

Cell therapy offers an innovative approach for treating enteric neuropathies. Postnatal gut-derived enteric neural stem/progenitor cells (ENSCs) represent a potential autologous source, but have a limited capacity for proliferation and neuronal differentiation. Since serotonin (5-HT) promotes enteric neuronal growth during embryonic development, we hypothesized that serotonin receptor agonism would augment growth of neurons from transplanted ENSCs. Postnatal ENSCs were isolated from 2 to 4 week-old mouse colon and cultured with 5-HT4 receptor agonist (RS67506)-loaded liposomal nanoparticles. ENSCs were co-cultured with mouse colon explants in the presence of RS67506-loaded (n = 3) or empty nanoparticles (n = 3). ENSCs were also transplanted into mouse rectum in vivo with RS67506-loaded (n = 8) or blank nanoparticles (n = 4) confined in a thermosensitive hydrogel, Pluronic F-127. Neuronal density and proliferation were analyzed immunohistochemically. Cultured ENSCs gave rise to significantly more neurons in the presence of RS67506-loaded nanoparticles. Similarly, colon explants had significantly increased neuronal density when RS67506-loaded nanoparticles were present. Finally, following in vivo cell delivery, co-transplantation of ENSCs with 5-HT4 receptor agonist-loaded nanoparticles led to significantly increased neuronal density and proliferation. We conclude that optimization of postnatal ENSCs can support their use in cell-based therapies for neurointestinal diseases.

Cluster of differentiation-44 (CD44) is a ubiquitously present glycoprotein on the surface of mammalian cells that plays a significant role in a number of biological functions. Since the discovery that the receptor is over-expressed in a variety of solid tumors, such as pancreatic, breast and lung cancer, many studies have focused on methods for targeting CD44 in an attempt to improve drug delivery and discrimination between healthy and malignant tissue, while reducing residual toxicity and off-target accumulation. In this review, we describe CD44 receptor biology and its involvement in the different stages of tumor growth and metastasis, as well as methods currently used for targeting the receptor. Hyaluronic acid, the primary CD44 binding molecule, has proved a significant ally in developing nanocarriers that demonstrate preferential tumor accumulation and increased cell uptake. We outline a number of research approaches from the current literature that take advantage of hyaluronic acid's targeting ability and describe the possible advantages for each approach. The value of CD44 targeting can be easily appreciated from the number of different approaches that have reached clinical trials.

In this study, we have formulated redox-responsive epidermal growth factor receptor (EGFR)-targeted type B gelatin nanoparticles as a targeted vector for systemic delivery of gemcitabine therapy in pancreatic cancer. The gelatin nanoparticles were formed by ethanol-induced desolvation process to encapsulate the bound drug. The surface of the nanoparticles was decorated either with poly(ethylene glycol) (PEG) chains to impart enhanced circulation time or with EGFR targeting peptide to confer target specificity. Our in vitro studies in Panc-1 human pancreatic ductal adenocarcinoma cells confirm that gemcitabine encapsulated in EGFR-targeted gelatin nanoparticles, released through disulfide bond cleavage, had a significantly improved cytotoxic profile. Further, the in vivo anticancer activity was evaluated in an orthotopic pancreatic adenocarcinoma tumor bearing SCID beige mice, which confirmed that EGFR-targeted gelatin nanoparticles could efficiently deliver gemcitabine to the tumor leading to higher therapeutic benefit as compared to the drug in solution. From the Clinical Editor The treatment of pancreatic cancer remains unsatisfactory, with an average 5-year survival of less than 5%. New treatment modalities are thus urgently needed. In this study, the authors presented their formulation of redox-responsive epidermal growth factor receptor (EGFR)-targeted type B gelatin nanoparticles as a carrier for gemcitabine. In-vitro and in-vivo experiments showed encouraging results. It is hoped that the findings would provide a novel and alternative drug delivery platform for the future.

It is clear that exosomes (endosome derived vesicles) serve important roles in cellular communication both locally and distally and that the exosomal process is abnormal in cancer. Cancer cells are not malicious cells; they are cells that represent 'survival of the fittest' at its finest. All of the mutations, abnormalities, and phenomenal adaptations to a hostile microenvironment, such as hypoxia and nutrient depletion, represent the astute ability of cancer cells to adapt to their environment and to intracellular changes to achieve a single goal - survival. The aberrant exosomal process in cancer represents yet another adaptation that promotes survival of cancer. Cancer cells can secrete more exosomes than healthy cells, but more importantly, the content of cancer cells is distinct. An illustrative distinction is that exosomes derived from cancer cells contain more microRNA than healthy cells and unlike exosomes released from healthy cells, this microRNA can be associated with the RNA-induced silencing complex (RISC) which is required for processing mature and biologically active microRNA. Cancer derived exosomes have the ability to transfer metastatic potential to a recipient cell and cancer exosomes function in the physical process of invasion. In this review we conceptualize the aberrant exosomal process (formation, content selection, loading, trafficking, and release) in cancer as being partially attributed to cancer specific differences in the endocytotic process of receptor recycling/degradation and plasma membrane remodeling and the function of the endosome as a signaling entity. We discuss this concept and, to advance comprehension of exosomal function in cancer as mediators of communication, we detail and discuss exosome biology, formation, and communication in health and cancer; exosomal content in cancer; exosomal biomarkers in cancer; exosome mediated communication in cancer metastasis, drug resistance, and interfacing with the immune system; and discuss the therapeutic manipulation of exosomal content for cancer treatment including current clinical trials of exosomal therapeutics. Often referred to as cellular nanoparticles, understanding exosomes, and how cancer cells use these cellular nanoparticles in communication is at the cutting edge frontier of advancing cancer biology.

Cell secreted exosomes (30-100nm vesicles) play a major role in intercellular communication due to their ability to transfer proteins and nucleic acids from one cell to another. Depending on the originating cell type and the cargo, exosomes can have immunosuppressive or immunostimulatory effects, which have potential application as immunotherapies for cancer and autoimmune diseases. Cellular components shed from tumor cells or antigen presenting cells (APCs), such as dendritic cells, macrophages and B cells, have been shown to be efficiently packaged in exosomes. In this review, we focus on the application of exosomes as nanocarriers and immunological agents for cancer and autoimmune immunotherapy. APC-derived exosomes demonstrate effective therapeutic efficacy for the treatment of cancer and experimental autoimmune diseases such as rheumatoid arthritis, inflammatory bowel disease, and multiple sclerosis. In addition to their intrinsic immunomodulating activity, exosomes have many advantages over conventional nanocarriers for drug and gene delivery.