Abstract Extracellular vesicles (EVs) derived from immune cells (e.g., NK cells, dendritic cells) may be able to treat malignancies that are resistant to other treatments (e.g., chemotherapy) relying on the hypothesis that EVs carry almost the same cargo of the parent cells. In this study, we evaluated IL-15 stimulated natural killer cell-derived EVs (NK-EVs) as therapeutic agents in vitro and in vivo in Osimertinib resistant lung cancer (H1975R) with EGFR mutations(L858R). NK-EVs were isolated by ultracentrifugation and nanoparticle tracking analysis revealed a size distribution of 89.5 ± 3.4 nm, zeta potential of -31.38 ± 0.25 mV. Atomic force microscopy imaging revealed vesicles with a spherical form and comparable sizes, meeting the criteria of exosomal EVs. Further, western blot studies demonstrated the presence of regular EV markers along with specific NK markers (perforin and granzyme). EVs were also characterized by using proteomic analysis which demonstrated that EVs had proteins for natural killer cell mediated cytotoxicity (Granzyme B) and T cell activation (Perforin and Plastin-2). Gene oncology (GO) analysis also showed that these are differentially expressed proteins (DEPs) that are involved in programmed cell death and positive regulation of cell death. Further, isolated NK-EVs (1*1011 particles/mL) were cytotoxic to H1975R cells in vitro with 55% and 68% cell viability in 2D and 3D cell cultures respectively against control. Carboplatin’s IC50 was reduced by approximately 1.9 and 1.7-fold (p less than 0.001) in 2D and 3D cell culture respectively when combined with NK-EVs. The EVs were then combined with carboplatin (25 mg/kg) and administered by i.p. route to H1975R tumor xenografts and significant reduction in tumor volume in vivo (≈1000 mm3 compared to ≈2000 mm3 of control group, p less than 0.001) was observed after 10 days. Our findings show for the first time that NK-EVs target the PD-L1/PD-1 immunological checkpoint and induce apoptosis and anti-inflammatory response by downregulation of SOD2, PARP, BCL2, SET, NF-κB and TGF-ß. MicroRNAs regulating cytotoxic proteins like perforin, granzyme and plastin were also identified using sequencing and the miRNAs: hsa-miR-5193/149-5P/3133/193-5p are picked for further studies. These miRNAs have been encapsulated in NK-EVs by electroporation and will be evaluated against Lung PDX xenografts along with carboplatin loaded NK-EVs. These miRNAs are directly involved in T cell or NK cell mediated cytotoxicity and are being currently investigated. The ability to isolate functional NK-EVs on a large scale and using them with platinum-based drugs may lead to new clinical applications. The results of the present study suggest the possibility of the combination of NK-cell-derived EVs and carboplatin as a viable immunochemotherapeutic strategy for resistant cancers. Citation Format: Aakash Nathani, Li Sun, Islauddin Khan, Mounika Aare, Arvind Bagde, Yan Li, Mandip Sachdeva. Combined role of interleukin-15 stimulated natural killer cell-derived extracellular vesicles and carboplatin in Osimertinib resistant H1975 lung cancer with EGFR mutations [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 1346.

Pharmaceutical development of cancer therapeutics is a dynamic area of research. Even after decades of intensive work, cancer continues to be a dreadful disease with an ever-increasing global incidence. The progress of nanotechnology in cancer research has overcome inherent limitations in conventional cancer chemotherapy and fulfilled the need for target-specific drug carriers. Nanotechnology uses the altered patho-physiological microenvironment of malignant cells and offers various advantages like improved solubility, reduced toxicity, prolonged drug circulation with controlled release, circumventing multidrug resistance, and enhanced biodistribution. Early cancer detection has a crucial role in selecting the best drug regime, thus, diagnosis and therapeutics go hand in hand. Furthermore, nanobots are an amazing possibility and promising innovation with numerous significant applications, particularly in fighting cancer and cleaning out blood vessels. Nanobots are tiny robots, ranging in size from 1 to 100 nm. Moreover, the nanobots would work similarly to white blood cells, watching the bloodstream and searching for indications of distress. This review articulates the evolution of various organic and inorganic nanoparticles and nanobots used as therapeutics, along with their pros and cons. It also highlights the shift in diagnostics from conventional methods to more advanced techniques. This rapidly growing domain is providing more space for engineering desired nanoparticles that can show miraculous results in therapeutic and diagnostic trials.

The epidermal growth factor receptor (EGFR) is highly expressed in many non-small cell lung cancers (NSCLC) that necessitates EGFR-tyrosine kinase inhibitors (TKIs) role as first-line treatments. Osimertinib (OSM), a third generation TKI, is being routinely used in clinic and resistance (T790M mutation in exon 20) to it develops, which requires the development of improved and effective therapeutics. In this study, we evaluated the anti-cancer effects of osimertinib (OSM), Cell cycle and apoptosis regulatory protein 1 (CARP-1) functional mimetic (CFM4.17) formulation, and Telmisartan (TLM) combination in experimental NSCLC. Briefly, H1975 and HCC827 NSCLC cell lines were used to investigate the effectiveness of a combination of OSM and CFM4.17 lipid formulation (CFM-F) in vitro utilizing 2D and 3D models. Tumor growth was investigated in an H1975 xenograft model. Next-generation RNA sequencing, proteomics, RT-PCR, and western blotting was used to analyze Gene and protein expression. In vitro investigations demonstrated that the combination was more effective than a single treatment to reduce tumor cell growth and trigger cell death. In H1975 tumor xenografts, TLM, CFM-F, and OSM combination (TLM_CFM-F_OSM) significantly inhibited tumor growth compared to CFM 4.17, OSM, or their combination. RNA seq and proteomics data revealed that TLM_CFM-F_OSM induced downregulation of Lamin B2, STAT3, SOD, NFKB, MMP-1, TGF beta, Sox-2, and PD-L1 and upregulated AMPK proteins which was validated by RT-PCR and western blotting. Our results suggest that TLM_CFM-F_OSM shows a superior anti-cancer effect in the treatment of NSCLC by affecting multiple pathways which regulate mitochondrial homeostasis, inflammation, oxidative stress, and apoptosis.

The objective of the present study was to develop and evaluate 3D printed microneedles (MNs) containing Lithium phenyl-2,4,6- trimethylbenzoylphosphinate (LAP) as a photoinitiator to deliver lipophilic active pharmaceutical ingredients (APIs) transdermally for 72 hours (hrs) using quality by design (QbD) and artificial intelligence (AI) algorithms. In the present study, dissolvable MN patch of ibuprofen (IBU) as a model drug was successfully fabricated using digital light projector (DLP) printing technology with ~750 μm height, ~250 μm base diameter and tip with radius of curvature (Roc) of ~15 μm. MN patches comprised of IBU, LAP, poly (ethylene glycol) dimethacrylate 550 (PEGDAMA 550) and distilled water were developed using QbD optimization approach and validating using AI and machine learning algorithms. Mechanical strength studies demonstrated that IBU MNs formed pores both on parafilm and human cadaver skin. IBU MNs consisting of 0.23% and 0.49% LAP with 10% water in each showed ~ 2mg/cm2 sustained drug permeation at the end of 72 hrs in vitro skin permeation testing studies (IVPT) with flux of ~ 20 μg/ cm2 /hr in first 24 hrs. Further, pharmacokinetic (PK) studies conducted in rats for 48 hrs showed ~ 62812.02 ± 11128.39 (ng/ml*hr) of Area under curve (AUC) (0-48hr), 2.66 ± 1.12 h of Tmax and 3717.43 ± 782.25(ng/ml) of Cmax from the IBU MN patch containing 0.23% LAP with no significant difference compared to the patch containing 0.49% LAP. In conclusion, in the current study, biocompatible dissolvable IBU MN patch was successfully fabricated using DLP printing technology to deliver lipophilic API in a sustained manner for 72 hrs.

Cernumidine (CER) is a guanidinic alkaloid isolated from Solanum cernuum leaves. In this work, we investigated the cytotoxicity, chemosensitizing effect of cernumidine to cisplatin (cDDP) and the possible mechanism of action of the combination on bladder cancer cells. Cernumidine showed cytotoxicity and could sensitize bladder cancer cells to cisplatin. The combination of CER+cDDP inhibited cell migration on T24 cells. CER+cDDP down-regulated MMP-2/9 and p-ERK1/2, while it increased EGFR activity corroborating the observed cell migration inhibition. Down-regulation of Bcl-2 and up-regulation pro-apoptotic Bax and further depletion of the mitochondrial membrane potential (Delta psi m) indicates that mitochondria play a central role in the combination treatment inducing the mitochondrial signaling pathway of apoptosis in T24 cells. Our data showed that the alkaloid cernumidine is worthy of further studies as a chemosensitizing agent to be used in complementary chemotherapy.

Abstract Background: Bleb transformation is an initial induction of apoptosis leading to cellular compartmentalization into vesicles, increased glycolysis/mitochondrial activity combined with dynamin mediated endocytosis allowing the cell to tether vesicles to form stem like spheroids protecting exposed genetic material. Our laboratory has found that not only do both Erlotinib sensitive and resistant cell lines undergo blebbishield transformation in response to treatment, but the addition of Glycyrrhetinic acid derivative (CDODA-Me) overwhelms this pathway leading to cellular apoptosis. Methods: NSCLC cell lines HCC827 (both erlotinib resistant and sensitive), were treated with CDODA-Me and Erlotinib (combination and single treatment). MicroRNA expression profiling was used to identify and validate reference genes involved with the synergistic effects comparing combination treatment to erlotinib single treatment groups. Spheroids were collected after 24hr treatment using blebbishield extraction media (BEM) consisting of 12µM erlotinib alone and in combination with 2µM CDODA-Me. Western Blot analysis was used to confirm identified pathways as well as to quantify the effects of CDODA-Me on VEGF and TGF-β cellular release levels during treatment as well as MET and EGFR kinase activation. Results: Genomic studies showed an increase in microRNA has-miR-711 (approximately 4-fold) which is responsible for inhibiting dynamin mediated trafficking, various growth factor expression levels (including VEGF and TGF-β), as well as the expression of receptors associated with these growth factors (EGFR, MET, VEGFR). This inhibition was confirmed through western blot analysis showing a decrease in receptor activation but no effects on receptor expression (6 fold and 8 fold decrease when compared to erlotinib single treatment for EGFR and MET respectively). Sphere reattachment was found to be dependent on the presence of growth factors and other unknown cell signaling molecules through the fact that spheres did not attach in fresh media, but attached in as little as 3hrs in media obtained after 24hr incubation in parental cells. Media obtained after incubation in parental cells provided higher levels of VEGF and TGF-β when compared to media obtained after 24hr treatment with (resistant to non-resistant expression ratios of 3.94 and 4.06 for HCC827 (4µM erlotinib resistant) and HCC827 Cl4 respectively). Actin fluorescent staining 48hrs after reseeding spheroids showed that cells grown in untreated parental media regained normal branch like morphology while those grown in CDODA-Me treated parental media remained rounded and died. Conclusions: Based on the results of these studies, CDODA-Me has been shown to inhibit NSCLC cell types from overcoming apoptosis thereby inhibiting increased drug resistance as well as tumorgenicity. Citation Format: Ebony Nottingham, Elizabeth Mazzio, Arindam Mondal, Mandip Sachdeva. The use of glycyrrhetinic acid derivatives to inhibit blebbishield emergency program pathogenesis overcoming erlotinib resistance in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4893.

Abstract Background:Erlotinib, an EGFR selective reversible inhibitor, improved response of patients diagnosed with non-small cell lung cancer (NSCLC) with 70% of patients showing significant tumor regression upon treatment. Unfortunately,most patients eventually relapse due to development of resistance. Our research goal is to quantify the effects of combination treatment of erlotinib with a glycyrrhetinic acid analogs and to improve the oral bioavailability of this treatment using self nano-emulsifying drug delivery systems. Methods: NSCLC cell lines HCC827, HCC827 (4µM erlotinib resistant), HCC827 Cl4 (second site EGFR mutated), HCC827BEAS2B, and H1975 (4µMerlotinib resistant) were treated in combination with CDODA-Me, CF3CDODA-Me and Erlotinib. The cell viability assays were performed and combination index (CI) values were calculated by isobolographic analysis. Self nano-emulsifying drug delivery systems (SNEDDs) were formulated and characterized through in vitro and in vivo studies. Results: CF3CDODA and improved efficacy for erlotinib in all cell lines with IC50 values of 6.0µM, 7.8µM, 4.6µM and 4.2±1.56µM for HCC827(RESISTANT), H1975(RESISTANT), HCC827CL4 and HCC827 respectively compared to IC50values of erlotinib alone (25µM, 21µM, 23µM, and 8µM). This was superior when compared to IC50 values of CDODA-Me combination treatment (6.66µM, 7.32µM, 12.06µM, and 5.45µM respectively). Combination treatment showed strong synergism with a constant concentration of 0.5µM CF3CDODA and CI values of 0.461, 0.548, 0.389, 0.550 for HCC827 4µM, H1975µM, HCC827CL4, and HCC827 respectively and antagonism in HCC827BEAS2B (1.508) cells. This is comparable to the synergism observed at a minimum concentration of 2µM CDODA-Me with CI values of 0.337, 0.67, 0.64, and 0.657 respectively and antagonism CI of 1.175 for HCC827BEAS2B. CF3CDODA Combination therapy showed a 2.5-fold decrease in colony formation in resistant cells making them comparable to wild type. This was comparable to CDODA-Me combination treatment with a 1.46-fold decrease in colony formation. Both combination treatments showed little effect on HCC827BEAS2B. Western Blot analysis showed decrease MET expression and decreased apoptosis inhibiting markers BCL2 and Survivin. SNEDD formulations increased maximum drug plasma concentrations for Erlotinib (281ng/ml to 442ng/ml) and circulating time as well (12hrs - 21hrs). Conclusions: Based on the results of these studies, CDODA-Me and CF3CDODA have shown to overcome erlotinib resistance in non-small cell lung cancer and when combining these treatments with the use of SNEDDS, they have shown to be a promising treatment regimen. Citation Format: Ebony Nottingham, Vasanth Sekar, Arindam Mondal, Mandip Sachdeva. The use of self nano-emulsifying drug delivery systems to improve bioavailability of combination therapy and overcome erlotinib resistance in non-small cell lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2039. doi:10.1158/1538-7445.AM2017-2039

Purpose: The use of Noscapine (Nos) as a chemosensitizer followed by docetaxel (DTX) treatment therapy could be a novel approach for the treatment for breast cancer and possibly reduce the adverse side effects associated with DTX based chemotherapy. The goal of this study was to examine the chemo-sensitizing effect of Nos to DTX and also tumor stromal disruption effect of Nos in mice bearing xenograft TNBC tumors. Methods: Effect of Nos chemosensitization on DTX cytotoxicity was evaluated in MDA-MB-231 cells by trypan blue dye method. Apoptosis was measured by AnnexinV/FITC method using flow cytometer. Expression of different proteins like phospho-p38, pJNK, bcl-2, α-tubulin, Akt, pAkt, survivin was evaluated by immunoblot. Alpha-tubulin binding assay was done by fluorescent microscopy. In vivo antifibrotic efficacy of Nos and uptake of coumarin-6 loaded fluorescent liposomes was evaluated by picro-sirius red staining and fluorescent microscopy respectively in xenograft breast tumors. Results: MDA-MB-231 TNBC cells were exposed at sub-therapeutic dose of Nos (4 μM) which increased the cytotoxicity of DTX by 3.0-fold. Flow-cytometric analysis showed significant increase (30 percent) of late apoptotic cells in Nos chemosensitized, DTX-treated MDA-MB-231 cells compared with DTX alone treatment. Further, chemosensitization of TNBC cells with Nos at different time intervals (6 h, 12 h and 24 h), the effect on stress transducer p38 stress activator protein kinase was significantly activated (p Conclusion: In conclusion, chemosensitization with sub-therapeutic dose of oral noscapine could be a promising approach to increase anticancer activity of DTX and can further enhance uptake of liposomes suggesting that this approach may have potential in TNBC treatment. Citation Format: Ravi Doddapaneni, Ketan Patel, Nusrat Chowdhury, Mandip Sachdeva. Noscapine chemosensitization enhances docetaxel anticancer activity and tumor stroma disruption against triple negative breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2086.

Abstract Purpose: The purpose of this research is to determine the effects of the combination of (CDODA-Me), a derivative of glycyrrhetinic acid with chemotherapeutic agents like docetaxel (DTX) and Erlotinib (a TKI inhibitor) to treat triple negative breast cancer (TNBC) and TKI resistant lung cancer. Methods: TNBC lines MDA-MB-231 MDA-MB-468 and DTX resistant (MDA231) cells were treated in combination with CDODA-Me (nontoxic dose of 2 μM) and DTX. Wildtype (HCC827) and resistant (Erlotinib resistant) lung cancer cells were treated with Erlotinib and CDODA-Me. The cell viability of MDA-MB-231, MDA-MB-468, Docetaxel resistant (MDA231) cells and HCC 827 Erlotinib resistant (4 μM) cells in each treatment group was determined by crystal violet assay. Combination index values were calculated by isobolographic analysis. Western blot annalysis was used to investigate the influence of CDODA-Me combinations on drug resistance and key apoptotic proteins such as bcl2, survivin, SP1, SP3, and SP4. 2’,7’ -dichlorofluorescin diacetate (DCFDA) was used to measure reactive oxygen species (ROS) levels in all cell lines and examined by flowcytometry. Results: Breast Cancer cells (MDA-MB-468 and MDA-MB-231) showed increased cytotoxicity with a ten fold decrease in IC50 concentration (0.3 μM DTX and 0.03 μM DTX in combination with 2 μM CDODA-Me). DTX resistant (MDA231) cells showed IC50 values comparable to wildtype cells at 0.29 μM DTX in combination with CDODA-Me. HCC827 (Erlotinib resistant) showed a three fold decrease in cytotoxicity (IC 50 values of 4.9 μM and 16.9 μM Erlotinib for the combination and Erlotinib alone respectively). The combination treatment also showed higher response in resistant cells with IC50 values comparable to wildtypes. The expression of bcl2, survivin, specific transcription factors (like SP1, SP3 and SP4) was downregulated in cells treated with CDODA-ME and DTX combination compared to CDODA-ME alone, DTX alone and control cells. Similar results were observed with CDODA-Me and Erlotinib combination in HCC827 erlotonib resistant (4 μM) cells. CDODA-Me alone treatment showed ROS levels in HCC 827 (4 μM) resistant cell lines were increased (8-fold) significantly as compared to other breast cancer cells. Conclusion: In conclusion, CDODA-Me inhibits growth of TNBC cells and Erlotinib resistant lung cancer cells and downregulates the SP proteins and anti-apoptotic proteins. Therefore, these results indicate that CDODA-Me is a promising anticancer agent and can overcome resistance of chemotherapeutic agents against breast and lung cancer. Citation Format: Ebony Nottingham, Ravi Doddapaneni, Mandip Sachdeva. CDODA-Me augments the efficacy of chemotherapeutic agents and overcomes chemo-resistance in breast and lung cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4833.

The objective of the study was to investigate the pharmacokinetics and efficacy of 5-FU entrapped pH-sensitive liposomal nanoparticles with surface-modified anti-epidermal growth factor receptor (EGFR) antibody (pHLNps-5-FU) delivery system. Cytotoxicity of 5-FU and pHLNps-5-FU was determined in vitro against HCT-116 cells. The biodistribution and pharmacokinetic parameters of the administered 5-FU and pHLNps-5-FU as well as efficacy of 5-FU and pHLNps-5-FU were determined in HCT-116 subcutaneous mouse model. Mean size of pHLNp-5-FU was 164.3 ± 8.4 nm with entrapment efficiency (E.E) of 54.17%. While cytotoxicity of 5-FU and pHLNps-5-FU showed a strong dose-dependent, pHLNps-5-FU proved to be more effective (2-3 fold high) than that of 5-FU against HCT-116 cells. Pharmacokinetic study showed a prolonged plasma circulation of pHLNps-5-FU and a more significant body exposure while accumulation of pHLNps-5-FU in tumor was significantly higher than that of free 5-FU. Further, the efficacy of pHLNps-5-FU, was greater than free 5-FU at equivalent 5-FU dose. The study suggests that pHLNps may be an effective drug delivery system to enhance the anticancer activity of 5-FU against colorectal tumor growth.

Abstract The objective of this study was to demonstrate the chemopreventive potential of 1,1-bis(3′-indolyl)-1-(p-chlorophenyl methane) (DIM-D) in skin cancer using an in vitro and in vivo models. In vitro cell cytotoxicity and viability assays were carried out in A431 human epidermoid carcinoma cell line (A431) and normal human epidermal keratinocytes (NHEK) respectively. Apoptosis induction and accumulation of ROS following UVB exposure in DIM-D pretreated NHEK cells (2 hr prior) was also evaluated. Immunocytochemistry and western blot analysis were performed to determine proapoptotic and proinflammatory markers expressions in DIM-D treated A431 cells and in UVB irradiated NHEK cells. For in vivo experiments, DIM-D was encapsulated within nanocarriers by hot melt homogenization using Nano DeBEE. Nanocarriers were surface modified with oleic acid and incorporated into a gel using established procedures (G-NOD) and characterized for particle size, zeta potential, entrapment efficiency, rheology, skin permeation and drug release. The chemo-preventive efficiency of G-NOD was evaluated using UVB-induced skin cancer model in SKH hairless mice and skin samples were collected and investigated for lipid peroxidation, protein carbonyls, and molecular markers by western blot. The IC50 values of DIM-D were 24.5±2.6, 17.2±3.6and 4.1±1.1 whilst for EGCG were 192.1±3.8, 85.3±2.4 and 26.0 ±1.4 for 24, 48 and 72 hr treatments respectively. DIM-D induced higher expression in A431 cells compared to EGCG of cleaved caspase 3 (3.0-fold vs. 2.4-fold changes), Nurr1 (2.7-fold vs. 1.7-fold changes) and NFκB (1.3-fold vs. 1.1-fold changes). Nanocarriers containing DIM-D were 188.00±8.00 nm in size with polydispersity of 0.59±0.01. The zeta potential and entrapment efficiency were 37.17±0.90mV and 89±0.50%, respectively. There was significant (p<0.05) reduction in tumor size/number for G-NOD pretreated group (2 tumors/mouse, average diameter, 2±0.50 mm) compared to DIM-PG (4 tumors/mouse, average diameter, 4±0.45mm) and EGCG PEG-gel (EGCG-PG; 6 tumors/mouse, average diameter, 5±0.25mm). IHC and western blot results indicated significant increase (p<0.05) in expression of Nurr1 in G-NOD pretreated group compared to EGCG-PG. There was however pronounced reduction in expression of STAT3, NFkB, cleaved Caspase-3, BCl2 and 8-OHdG for G-NOD pretreated group compared to DIM-PG and EGCG-PG. Lipid peroxidation assay revealed decrease in amount of Malondialehyde (MDA) produced in G-NOD pretreated group prior to UV exposure by 2 fold compared to DIM-PG and 1.3 fold compared to EGCG-PG treatments. Therefore, findings in both in vitro and in vivo studies suggest that the enhanced percutaneous delivery of DIM-D reduces UVB-induced damage to skin lipids and protein and inhibits initiation and progression of skin photocarcinogenesis in SKH mice via the transactivation of Nurr1. Citation Format: Ravi Doddapaneni, Cedar Boakye, Punit Shah, Apurva R. Patel, Chandraiah Godugu, Stephen Safe, Santosh Katiyar, Mandip Sachdeva. Nurr1, a novel target of 1,1-bis(3’-indolyl)-1-(p-chlorophenyl) methane for inhibition of the initiation and progression of skin cancer tumorigenesis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3139. doi:10.1158/1538-7445.AM2014-3139

Abstract Recent studies support the existence of small populations of pluripotent stem-like cancer cells capable of self-renewal. These cancer stem cells (CSCs) are attributed to the resistance phenotype occurring in many tumors such as invasion and metastasis. Annexin A2 (Anxa2) overexpression is associated with invasion, metastasis and drug resistance in many cancers. We investigated the effect of Anxa2 knockdown by a liposomal (nanosome) formulation of Anxa2 shRNA (shAnxa2) on growth and metastasis in H1650 CSCs. H1650 CSCs were maintained on basement membrane-coated supports in DMEM:F12 media enriched with growth factors. Nanosomes were prepared by solvent evaporation and bath sonication. H1650 CSCs were transfected with lipoplexes of shAnxa2 and varying amounts of nanosome in serum-free media. After 4 hrs of incubation, the medium was replaced with regular media and knockdown of Anxa2 analyzed after 48 hrs by Western blot. Inhibition of H1650 CSC sphere formation over 7 days was investigated in lipidure® plates following treatment with lipoplexes for 4 hrs. CSCs were assessed for induction of apoptosis after treatment with lipoplexes (4 hrs) by acridine orange-ethidium bromide staining. The effect of Anxa2 lipoplexes on proliferation, migration and capillary-like tube formation was investigated in human primary pulmonary artery endothelial cells (HPAEC). The effect of Anxa2 lipoplexes on the expression of Anxa2, EGFR, VEGF, and NFkβ was evaluated by Western blot. Efficient loading of shAnxa2 in nanosomes was achieved with high transfection efficiency compared to lipofectamine. Anxa2 shRNA lipoplex was cytotoxic to H1650 CSC spheroids, inhibiting spheroid growth by 30-60% between day 3 and 7. Early and late apoptosis induction was observed after treatment with shAnxa2 with a 2-fold increase of cells undergoing apoptosis compared to shAnxa2-lipofectamine 2000 complex. Anxa2 knockdown (90%) was observed in H1650 CSCs parallel to decreases in EGFR, VEGF, and NFkβ expression as shown by Western blot. High loading and enhanced transfection of shAnxa2 was achieved with the nanosome formulation. Anxa2 knockdown was achieved in parallel with inhibition of H1650 CSC spheroid growth, angiogenesis and induction of apoptosis. These results altogether support evidence that Anxa2 could be a therapeutic target for treatment of non-small cell lung cancer and that nanosome formulation enhances delivery of shAnxa2 as an anticancer agent. Citation Format: Terrick A. Andey, Srujan Marepally, Pomila Singh, Mandip S. Sachdeva. Knockdown of Annexin A2 by liposomal formulation of Annexin A2 small hairpin RNA induces apoptosis and inhibits angiogenesis in lung cancer stem cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3738. doi:10.1158/1538-7445.AM2013-3738

Abstract Introduction: The intratumoral distribution of tumor targeted nanoparticles (NP) is hindered by dense collagen network and highly fibrous interstitium. Use of antifibrotic agents can decrease tumor interstitial fibrosis and promote NP intratumoral distribution. The objective of this study was to evaluate the effect of Telmisartan (TEL, AT1 blocker/PPAR-γ agonist) and Losartan (LOS, AT1 blocker) on NPs intratumoral distribution and anticancer effects in TNBC and lung cancer. Methods: Anticancer effect of TEL (5 mg/kg) and LOS (20 mg/kg) by inhalation and oral route were evaluated using Non-small cell lung cancer (A549 orthotopic and metastatic model) and triple negative breast cancer (TNBC, MDA-MB-231 orthotopic model) in Nu/nu mice. Also, to study intratumoral distribution, fluorescent polystyrene NP (FPNP) were administered intravenously followed by fluorescent microscopy. Tumor fibrous nature was characterized by estimating collagen-1, transforming growth factor beta1 (TGF-β;1) expression by IHC and ELISA. Collagen deposition in the tumor interstitium was studied by Masson's Trichome staining. To correlate the AT1 receptor blockage to anticancer effects, expression of VEGF, cleaved caspase-3, MMP-9 and microvessel densities (CD31) were quantified by IHC and western blotting. Epithelial Mesenchymal transition (EMT) mediated tumor metastasis was characterized by expressions of E-Cadherin and Vimentin. Results: TEL showed significant antitumor effects in both lung and TNBC tumor models. TEL at 1/4th dose produced superior anticancer effects than LOS. Survival rate was extended with TEL compare to LOS & untreated control. Further, Paclitaxel-NP produced better tumor regression in inhalation with TEL compared to single agent treatment and control. The fibrous nature of lung tumors and TNBC was decreased significantly (p<0.05) with TEL and LOS treatment based on expression of collagen-1 and TGF-β;1. This allowed better FPNP distribution into the tumors compared to control. Further, compared to control tumors, LOS and TEL treated group resulted in 5.33 and 14.33 fold increase in NP distribution. Similarly, Paclitaxel-NP distribution was superior in combination with TEL & LOS treatment, which in turn exhibited synergistic anticancer effects. TEL treatment resulted in the decreased metastatic tumor nodules and tumor volumes. The metastatic markers MMP-9 & vimentin levels were significantly decreased (p<0.001) in TEL group compared to LOS & control, suggesting the promising effects of TEL in tumor anti-metastasis. Further, our study also demonstrated the safety of TEL upon inhalation route. Conclusion: Telmisartan showed significant anticancer effects in Lung cancer and TNBC. Telmisartan could be an ideal candidate for combination therapy to improve the NPs intratumoral distribution and anticancer effects in various cancer types. Citation Format: Chandraiah Godugu, Apurva R. Patel, Srujan Marepally, Ravi Doddapaneni, Mandip Singh Sachdeva, Mahavir B. Chougule. Effect of telmisartan on triple negative breast cancer (TNBC) and lung cancer tumor progression and intratumoral distribution of nanoparticles. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2139. doi:10.1158/1538-7445.AM2013-2139

Abstract Purpose: Lung cancer is one of the leading cause of death (1.3 million deaths annually) worldwide and non-small cell lung cancer (NSCLC) accounts for 85 % of all lung cancers. Vascular endothelial growth factor (VEGF) over-expression (61% to 92% of NSCLC) is associated with poor survival. Recently, new approaches in the treatment of lung cancer with novel drugs that selectively inhibit tumor blood supply thus controlling cancer cell survival, proliferation and/or metastasis in combination with conventional anticancer or antiangiogenic drugs have generated clinical interest, e.g. DIM-C-pPhC6H5 (DIM-P); a c-substituted diindolylmethanes which is novel anti-cancer agent. Objective of this study are: 1) to formulate tumor homing pegylated CREKA peptide coated nanoparticle of DIM-P (PCNCs-D)/ D-luciferin (PCNCs-DL)/ XenolightDiR (PCNCs-Di), 2) In-vivo imaging of tumor progression / tumor vasculature and tracking of PCNCs-Di. Methods: Nanoparticles were prepared with DIM-P (NCs-D)/ D-luciferin (NCs-DL)/XenolightDiR(NCs-Di), Compritol, Miglyol, DOGS-NTA-Ni and sodium taurocholate using high pressure homogenizer (Nano-DeBEE). PCNCs-D and PCNCs-DL/PCNCs-Di were prepared by conjugating NCs-D and NCs-DL/ NCs-Di with 6His-PEG2K-CREKA peptide and characterized for physical properties, clot binding assay and tube formation assay. Pharmacokinetic parameters of formulations were evaluated in BALB/c mice. In-vivo imaging of tumor and tracking of nanoparticles was carried out with IVIS® Spectrum CT (Caliper life Sciences) by using fluorescent dye (Xenolight DIR) and bioluminescence (luciferin) following intravenous and inhalation delivery of nanoparticles. Results: Particle size of PCNCs-D was 190-210 nm. The PCNCs-D formulation showed an initial burst release followed by a slow release up to 72 hr (90%) of DIM-P. PCNCs-D showed (p<0.001) 3 fold higher binding to the clotted plasma proteins compared to NCs-D. PCNCs-D decreased average branching point by 68 ± 5 percent compared to 39 ± 4 percent by DIM-P alone in a tube formation assay suggesting anti-angiogenic activity. Pharmacokinetic parameters showed that PCNCs-D increased plasma half life of DIM-P from 0.83 ± 0.26 hr to 2.34 ± 0.39 hr. In-vivo imaging following exposure of PCNCs-DL/PCNCs-Di demonstrated their targeting to the tumor vasculature, where the PCNCs-Di were found to migrate more in newly formed blood vessels and total radiant efficiency [p/s] / [µW/cm*2] was 2.1*1012 ± 0.5*1012 over the period of 0.5 hr to 3hr. NCs-Di didn't show any specific migration to tumor vasculature confirming the specific targeting of PCNCs-Di and total radiant efficiency [p/s] / [µW/cm*2] was 0.6*1012 ± 0.18*1012. Conclusion: The results emanating from these studies demonstrate an anti-angiogenic potential of DIM-P and the role of PCNCs-D as an effective tumor homing drug delivery systems for lung cancer treatment. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 382. doi:1538-7445.AM2012-382