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Developing a delivery vehicle to protect siRNA from degradation is a significant challenge. To solve this challenge, researchers attempted to use protein-based nanoparticles to deliver siRNA with limited to moderate success. However, a systematic investigation of comparing the ability of different protein-based nanoparticles as vehicles to deliver siRNA stably within cells is still unavailable. Therefore, in this study we synthesized a library of both non-targeted (proteinsiRNA) nanoparticles (NPs) and targeted (antibody conjugated protein-siRNA) NPs and evaluated ability to stably deliver siRNA in to cells to silence the gene of interest. We investigated nanoparticles of casein, bovine serum albumin, and gelatin for the delivery of siRNA. We synthesized and characterized a total of 12 nanoconjugates; in these conjugates, we either encapsulated, electrostatically attached, or covalently conjugated siRNA. We evaluated the efficiency of attaching siRNA to nanoconjugates, stability, and cellular delivery. The ability of siRNA to silence the protein of interest in cancer cells was also investigated. Among non-targeted conjugates, BSA matrix imparted relatively high stability to siRNA when encapsulated. Among targeted nanoconjugates, gelatin nanoparticles rendered high stability to siRNA upon covalent conjugation to the surface. On comparing with both targeted and non-targeted NPs for release of siRNA within cells, antibody-gelatin-siRNA conjugate exhibited high release and functional activity (down-regulation of target protein levels) within the cells as confirmed by both fluorescence imaging and Western blotting. In summary, our investigations show that targeted gelatin nanoparticles and non-targeted BSA nanoparticles possess high stability and excellent gene suppression capabilities and warrants further studies. We can extend the results from this study to develop stable siRNA delivery vehicles to specifically silence the protein of interest.

Modern agricultural activities and concentrated animal feeding operations (CAFOs) are two of the major sources of groundwater contamination that affect the quality of drinking water. Conventional water quality improvement methods include direct filtration, coagulation/settling treatment techniques, membrane-based systems, and absorption-based systems. However, to date, there are no efficient and cost-effective processes available for water treatment. This study developed an innovative nanotechnology-based technique to improve groundwater quality. Magnetic nanosponges (MNSs) were synthesized and characterized using two different magnetic nanoparticles along with polymeric coagulants. The efficiency of MNSs in removing pollutants in wastewater collected from local diary and swine CAFO lagoons was investigated. Standard water quality evaluation parameters, such as the total organic content (TOC), turbidity, total suspended solids (TSS), and biological oxygen demand (BOD), were measured prior to and after treatment with MNSs. The results demonstrate the potential of MNSs to improve the quality of groundwater and support the development of a cost-effective best management practice (BMP) that also employs traditional coagulants at CAFOs and other wastewater treatment plants.

India is very rich in fauna and diversified flora. It is established that synthetic drugs could pose serious problems; besides this, they are toxic and costly. In contrast to this, herbal medicines are relatively nontoxic, economical, and eco-friendly. Moreover, people have been using them for generations. Herbal feed additives, enzymes, probiotics and prebiotics vitamins, and trace minerals with herbal extracts are commonly used as an herbal animal health product. The range of herbal supplements offered is well known for its nutritious value, effectiveness, and ability to resist different diseases, etc. They have also been used in managing problems of health-related issues of animals. Animal health products protect the sustainable food supply chain and are dynamic enough to prevent disease, etc., in animals. They are a wide variety of herbs, and products are derived from them. Although several reports have confirmed antimicrobial and antioxidative and stimulation of immune efficacy in vitro, a particular experiment under vivo conditions is still not sufficient. Less number of experimental comparisons of herbal plants has suggested similar effects on the animal gut microflora. It is mandatory to use herbal products to keep fit our farm animals and to get healthy and quality products. However, the future of using herb products in animal feed will in a great degree depend on the information of chemical structure, value, and features of practical herbs.

Current methods for capturing circulating tumor cells (CTCs) are based on the overexpression of cytokeratin (CK) or epithelial cell-adhesion molecule (EpCAM) on cancer cells. However, during the process of metastasis, tumor cells undergo epithelial to mesenchymal transition (EMT) that can lead to the loss of CK/EpCAM expression. Therefore, it is vital to develop a capturing technique independent of CK/EpCAM expression on the cancer cell. To develop this technique, it is important to identify common secondary oncogenic markers overexpressed on tumor cells before and after EMT. We analyzed the biomarker expression levels in tumor cells, before and after EMT, and found two common proteins human epidermal growth factor receptor 2 (Her2) and epidermal growth factor receptor (EGFR) whose levels remained unaffected. So, we synthesized immunomagnetic iron nanocubes covalently conjugated with antibodies of Her2 or EGFR to capture cancer cells irrespective of the EMT status. The nanocubes showed high specificity (6 to 9 fold) in isolating the cancer cells of interest from a mixture of cells spiked in serum. We characterized the captured cells for identifying their EMT status. Thus, we believe the results presented here would help in the development of novel strategies for capturing both primary and metastatic cancer cells from patients blood to develop an effective treatment plan., Comment: 12 pages, 9 figures

Non-small cell lung cancer (NSCLC) is the leading cause of cancer mortality with the 5-year survival rate at a dismal 16% for the past 40 years. Drug resistance is a major obstacle to achieving long-term patient survival. Identifying and validating molecular biomarkers responsible for resistance and thereby adopting multi-directional therapy is necessary to improve the survival rate. Previous studies indicated ~20% of tyrosine kinase inhibitor (TKI) resistant NSCLC patients overexpress AXL with increase in EMT and decrease in p53 expression. To overcome the resistance, we designed gelatin nanoparticles covalently conjugated with EGFR targeting antibody and siRNA (GAbsiAXL). GAbsiAXL efficiently silences AXL, decreases mTOR and EMT signaling with concomitant increase in p53 expression. Because of the molecular changes, the AXL silencing sensitizes the cells to TKI. Our results show AXL overexpression has an important role in driving TKI resistance through close association with energy-dependent mitochondrial pathways.

Due to their antimicrobial properties, silver nanoparticles (AgNPs) are being used in non-edible and edible consumer products. It is not clear though if exposure to these chemicals can exert toxic effects on the host and gut microbiome. Conflicting studies have been reported on whether AgNPs result in gut dysbiosis and other changes within the host. We sought to examine whether exposure of Sprague-Dawley male rats for two weeks to different shapes of AgNPs, cube (AgNC) and sphere (AgNS) affects gut microbiota, select behaviors, and induces histopathological changes in the gastrointestinal system and brain. In the elevated plus maze (EPM), AgNS-exposed rats showed greater number of entries into closed arms and center compared to controls and those exposed to AgNC. AgNS and AgNC treated groups had select reductions in gut microbiota relative to controls. Clostridium spp., Bacteroides uniformis, Christensenellaceae, and Coprococcus eutactus were decreased in AgNC exposed group, whereas, Oscillospira spp., Dehalobacterium spp., Peptococcaeceae, Corynebacterium spp., Aggregatibacter pneumotropica were reduced in AgNS exposed group. Bacterial reductions correlated with select behavioral changes measured in the EPM. No significant histopathological changes were evident in the gastrointestinal system or brain. Findings suggest short-term exposure to AgNS or AgNC can lead to behavioral and gut microbiome changes., 14 figures, 15 pages

A tri-block nanoparticle (TBN) comprising of an enzymatically cleavable porous gelatin nanocore encapsulated with gefitinib (tyrosine kinase inhibitor (TKI)) and surface functionalized with cetuximab-siRNA conjugate has been synthesized. Targeted delivery of siRNA to undruggable KRAS mutated non-small cell lung cancer cells would sensitize the cells to TKI drugs and offers an efficient therapy for treating cancer; however, efficient delivery of siRNA and releasing it in cytoplasm remains a major challenge. We have shown TBN can efficiently deliver siRNA to cytoplasm of KRAS mutant H23 Non-Small Cell Lung Cancer (NSCLC) cells for oncogene knockdown; subsequently, sensitizing it to TKI. In the absence of TKI, the nanoparticle showed minimal toxicity suggesting that the cells adapt a parallel GAB1 mediated survival pathway. In H23 cells, activated ERK results in phosphorylation of GAB1 on serine and threonine residues to form GAB1-p85 PI3K complex. In the absence of TKI, knocking down the oncogene dephosphorylated ERK, and negated the complex formation. This event led to tyrosine phosphorylation at Tyr627 domain of GAB1 that regulated EGFR signaling by recruiting SHP2. In the presence of TKI, GAB1-SHP2 dissociation occurs, leading to cell death. The outcome of this study provides a promising platform for treating NSCLC patients harboring KRAS mutation., 14 pages, 9 figures, research article

Summary The CN represents runoff potential is estimated using three different methods for three watersheds namely Barureva, Sher and Umar watershed located in Narmada basin. Among three watersheds, Sher watershed has gauging site for the runoff measurements. The CN computed from the observed rainfall-runoff events is termed as CN (PQ) , land use and land cover (LULC) is termed as CN (LU) and the CN based on land slope is termed as SACN 2 . The estimated annual CN (PQ) varies from 69 to 87 over the 26 years data period with median 74 and average 75. The range of CN (PQ) from 70 to 79 are most significant values and these truly represent the AMC II condition for the Sher watershed. The annual CN (LU) was computed for all three watersheds using GIS and the years are 1973, 1989 and 2000. Satellite imagery of MSS, TM and ETM+ sensors are available for these years and obtained from the Global Land Cover Facility Data Center of Maryland University USA. The computed CN (LU) values show rising trend with the time and this trend is attributed to expansion of agriculture area in all watersheds. The predicted values of CN (LU) with time (year) can be used to predict runoff potential under the effect of change in LULC. Comparison of CN (LU) and CN (PQ) values shows close agreement and it also validates the classification of LULC. The estimation of slope adjusted SA-CN 2 shows the significant difference over conventional CN for the hilly forest lands. For the micro watershed planning, SCS-CN method should be modified to incorporate the effect of change in land use and land cover along with effect of land slope.

Silver nanoparticles (AgNPs) have antimicrobial and insecticidal properties and they have been considered for their potential use as insecticides. While they do, indeed, kill some insects, two broader issues have not been considered in a critical way. First, reports of insect-lethal AgNPs are often based on simplistic methods that yield nanoparticles of nonuniform shapes and sizes, leaving questions about the precise treatments test insects experienced. Second, we do not know how AgNPs influence beneficial insects. This work addresses these issues. We assessed the influence of AgNPs on life history parameters of two agricultural pest insect species, Heliothis virescens (tobacco budworm) and Trichoplusia ni (cabbage looper) and a beneficial predatory insect species, Podisus maculiventris (spined soldier bug), all of which act in agroecosystems. Rearing the two pest species on standard media amended with AgNPs led to negligible influence on developmental times, pupal weights, and adult emergence, however, they led to retarded development, reductions in adult weight and fecundity, and increased mortality in the predator. These negative effects on the beneficial species, if also true for other beneficial insect species, would have substantial negative implications for continued development of AgNPs for insect pest management programs.

Three-dimensional nanocomposites prepared using two different families of nanomaterials holds significant relevance pertaining to biological applications. However, integration of the two distinct nanomaterials with precision to control the overall compositional homogeneity of the resulting 3D nanocomposite is a synthetic challenge. Conventional reactions result in nanocomposites with heterogeneous composition and render useless. To address this challenge, we have developed a fluidics-mediated process for controlling the interaction of nanoparticles to yield a compositional uniform multidimensional nanoparticle; as an example, we demonstrated the integration of gold nanoparticles on gelatin nanoparticles. The composition of the nanocomposite is controlled by reacting predetermined number of gold nanoparticles to a known number of thiolated gelatin nanoparticles at any given time within a defined cross-sectional area. Using the fluidics process, we developed nanocomposites of different composition: [gelatin nanoparticles-(gold nanoparticles)x] where xaverage = 2, 12, or 25. The nanocomposites were further surface conjugated with organic molecules such as fluorescent dye or polyethylene glycol (PEG) molecules. To study the biological behavior of nanocomposite, we investigated the cellular internalization and trafficking characteristics of nanocomposites in two human cancer cell lines. The nanocomposites exhibited a three-stage cellular release mechanism that enables the translocation of gold nanoparticles within various cellular compartments. In summary, the three-dimensional nanocomposite serves as a novel platform for developing well-defined protein-metal nanocomposites for potential drug delivery, sensory, and molecular imaging applications.

Silver plasmonic gratings with a thin corrosion protection film enable enhanced fluorescence-based detection, including single molecule, over a much wider fluorescent dye concentration range, 100 μM-1fM, and deeper field penetration than traditional sensor substrates.

The study area consists of three adjacent watersheds namely Barureva, Sher and Umar which conjoin together to form an important southern sub-basin of Narmada basin, MP, India. Morphological parameters of the three watersheds and their corresponding fourth order subwatersheds have been calculated with the help of data attributes generated from relevant GIS analysis. A Geomorphological Permeability Index (GPI) has been proposed which is comprised of watershed morphological parameters such as length ratio (Rt), drainage density (Dd), drainage frequency (Df) and relief ratio (Rh) to assess the nature of permeability of geological formation and ground water recharge potential in 89 subwatersheds of study area. Result and analysis conclude that the high GPI values (>10) indicate the presence of permeable or soft geological formation and consequently assures the presence of good ground water storage. The comparison GPI values with geohydrological data conclude GPI index can also be successfully used to evaluate geohydrological condition of small watersheds in absence of observed field data. On the basis of GPI values ranges, subwatersheds of the study area can be sorted out for identification of ground water recharge areas, rain water harvesting areas and areas require erosion control measures.

Non-small Cell Lung Cancer (NSCLC) patients who express EGFR activating mutations are treated with tyrosine kinase inhibitors (TKI) and have a median overall survival time of 22 months. TKI therapy showed only moderate success with poor (37%) survival rate. On the other hand, TKI therapy is ineffective in patients with active KRAS mutation. Acquired drug resistance is one of the major reasons for the failure of the TKI therapy. Studies have shown that overexpression of AXL is one of the possible reasons for the drug resistance. In our previous studies, we have shown that AXL inhibition in NSCLC drug-resistant cells resulted in FN14 survival activation. Subsequently, we have demonstrated that co-inhibition of both AXL and FN14 resulted in increased therapeutic efficacy. Based on these studies, we recognize that both AXL and FN14 play important role in proliferation, invasion and survival pathways. Therefore, synergistic co-inhibition of AXL-FN14 is a viable therapeutic strategy for treating NSCLC. In the present study, we compared the efficacy of inhibition of AXL and FN14 by siRNA (using nanoparticle delivery vehicles) and small-molecule inhibitors in A549 mouse xenografts. Our data further confirms that co-inhibition is effective and results in tumor volume reduction. The details of this study provide a rationale for enhanced treatment of drug resistant NSCLC. Citation Format: Dhananjay Suresh, Soumavo Mukherjee, Ajit Zambre, Shreya Ghoshdastidar, Sairam Yadavilli, Anandhi Upendran, Raghuraman Kannan. Co-inhibition of AXL and FN14 by pharmacologic inhibitors or genetic inhibition in NSCLC mice models [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 4891.

Background and Objectives: NSCLC patients undergoing Cisplatin therapy often develop resistance within 9-12 months that decreases treatment efficacy and is the major reason for the low median survival of 10-14 months among stage IV patients. Therefore, resistance is the main therapeutic limitation in the later part of the progressive disease. It has been suggested that suppressed Caspase-3 is one of the major reasons for failure to induce apoptosis by the cell during cisplatin resistance. We hypothesized this resistance to be related to EMT and other survival pathways. Recently, AXL and TWEAK/FN14 pathways have been shown to be upregulated in cisplatin resistant cells causing activation of EMT and survival pathways. Therefore, we examined whether dual inhibition of AXL and TWEAK/FN14 successfully reverses the cisplatin resistance in NSCLC. Materials and Methods: Drug resistant EGFR mutant H1975 and KRAS mutant A549 cells were tested for cisplatin desensitization following knock down of AXL and FN14 inhibition. We studied resensitization by determining the IC50 values in the drug resistant cell lines by treatment with both AXL and FN14 siRNA. We investigated detailed cellular mechanisms imparting resistance by detecting relative levels of Caspase 3, cleaved Caspase 3, PARP, cleaved PARP, p-SRC, p-EGFR, AXL and FN14 by western blotting. Cytochrome-C induced apoptosis was detected by monitoring cytochrome-c released from mitochondria using fluorescence microscopy. Results: Suppression of AXL and FN14 by siRNA treatment resensitized NSCLC to cisplatin and induced apoptosis. IC50 values were decreased significantly following dual inhibition (by more than 200%). A marked increase in cleaved caspase 3 levels was also observed in both mutant cell lines. A subsequent increase in PARP and cleaved PARP levels verified the fact that caspase 3 is indeed responsible for the apoptosis in these cells. Upregulation of p-SRC and eventually p-EGFR failed to suppress the apoptosis through PI3K/AKT pathway. Cytochrome c release from mitochondria further confirmed cellular apoptosis. Conclusion: Our results show that dual inhibition of AXL and TWEAK/FN14 can reverse cisplatin resistance by inducing higher caspase 3 cleavage that can enhance survival of NSCLC patients undergoing chemotherapy. Citation Format: Soumavo Mukherjee, Dhananjay Suresh, Ajit P. Zambre, Anandhi Upendran, Raghuraman Kannan. Dual inhibition of AXL and FN14 reverses cisplatin resistance in non-small cell lung carcinoma by inducing higher caspase 3 cleavage [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 5512.

Lung cancer accounts for about 27 percent of all cancer deaths in the United States. Approximately 85 percent of lung cancers are non-small cell type (NSCLC) and, when treated with both chemotherapy and targeted therapies, have a five-year overall survival rate of approximately 16 percent. These statistics clearly emphasize the need for developing treatment methodologies more effective than the current standard of care. Here, we evaluated the efficacy of combined inhibition of SRC and AXL in NSCLC.Dasatinib, a BCR/abl and src family tyrosine kinase inhibitor (TKI) is a drug that has a potential to be used against both EGFR and KRAS mutated lung cancer patients, alone (phase II clinical trial) and in combination with cetuximab (in phase III clinical trial), osimertinib and erlotinib (both in phase II clinical trials) by exploiting its SRC inhibition capabilities. However, NSCLC patients treated with dasatinib often develop resistance and is the major reason for the low median survival in stage IV patients. Recently, AXL pathway has been shown to be upregulated in Dasatinib resistant cells causing activation of EMT, AKT and survival pathways. It has been suggested that auto-phosphorylation of DDR2 and subsequent activation of SHP2/RAS/AKT pathway as a result of AXL upregulation is one of the major reasons for failure to induce apoptosis in cells during dasatinib resistance. Therefore, we examined whether inhibition of AXL resensitized TKI resistant NSCLC to dasatinib. Our results show that AXL knockdown can resensitize cells to dasatinib. IC50 values decreased significantly following AXL inhibition. We have also demonstrated that nanoparticle-mediated AXL knockdown also produces similar resensitization. The details of the study will be presented. Citation Format: Soumavo Mukherjee, Dhananjay Suresh, Ajit P. Zambre, Anandhi Upendran, Raghuraman Kannan. Overcoming drug resistance in NSCLC with SRC inhibitor [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 1820.

The morphological analysis has been used to identify the erosion-prone sites in the Sher river basin which is one of the sub-basins of the greater Narmada basin, Madhya Pradesh, India. The Sher river basin has been divided into the forty five sub-basins of fourth order and fourteen sub-basin areas representing fifth, sixth and seventh order streams. The various morphological parameters have been derived for Sher river basin and its derived sub-basins to describe their topographical conditions, drainage patterns and their probable response to the runoff. The erosional risk prone map for the study area has been prepared using Texture-Slope Index and the erosional scale of the basin has been prepared using the same index. The derived erosional scale has been arranged into ‘0’ to ‘100’ numerical scale in which the base values ‘0’ and ‘100’ denote the least and the highest erosion prone areas respectively. Comparative study of the sub-basins has been carried out by use of developed erosional scale. Classified erosional hazard map has been categorized into low, medium, high and very high sediment erosion. The classified map of erosional hazard may be useful to suggest various soil water conservation measures for water resources development and management.

Motivation: Lung cancer is the leading cause of cancer death, and lung adenocarcinoma (LUAD) is its predominant histological subtype. Fifteen single nucleotide polymorphisms (SNPs) have been significantly associated with LUAD risk in genome-wide association studies (GWAS). However, because most GWAS SNPs reside in non-coding regions and are co-inherited with hundreds of SNPs in linkage disequilibrium (LD), which SNPs play a causal role in disease development remains largely unknown. We hypothesized that some of these SNPs affect oncogenic transcriptional programs by modulating the activity of gene enhancers in alveolar epithelial cells (AECs), the purported cells of origin for LUAD. Methods: To test this hypothesis, we overlaid epigenomic features of primary human AECs over the locations of index LUAD risk SNPs and associated high LD SNPs. Luciferase assays for enhancer activity were performed for candidate SNPs that were predicted to disrupt transcription factor (TF) binding sites in loci marked by features of active enhancers. Expression quantitative trait loci (eQTL) analysis was also performed using The Cancer Genome Atlas (TCGA) dataset and the online Genotype-Tissue Expression (GTEx) Portal to identify potential target genes of each SNP-enhancer pair. Results: Thirty-three LUAD risk-associated SNPs mapped to putative AEC enhancer regions. TF binding site prediction suggests that numerous SNPs might alter the binding affinity of TFs implicated in lung cancer, including RXRA and NKX2-1. Luciferase assays indicate that two of the SNPs significantly affect enhancer activity in lung cancer cell lines. eQTL analyses link each of the putative enhancers to candidate target genes, including both known oncogenes and genes not previously associated with lung cancer. Conclusions: Taken together, our analyses provide new mechanistic insight into long-known associations between non-coding SNPs and LUAD outcomes, and may ultimately yield more effective and personalized strategies for lung cancer risk assessment, prevention, and treatment. Magnetic nanocubes for selective capture of circulating tumor cells in NSCLC

are highly magnetic, and stable in serum solutions for extended periods of time. The MNPs are incubated with cells in 1X PBS for 3h for receptor binding at 37 C after which particles bound to cells are magnetically separated with a pull force of 57 lbs. Cells are then washed and counted using an automated algorithm. Thewhole process has been optimized to a minimum cell population of 100 and can be theoretically reduced to a 2h process which makes this extremely effective for clinical evaluations and straightforward. Similar protocol was used when the cells were spiked in blood plasma and captured. Our data suggests strong correlation between number of A549 cell captured when Herceptin conjugated MNPs are used (96%difference vs HCC827), while Cetuximab conjugated MNPs pull both HCC827 aswell as A549 (31%difference). We expect to reduce the cell capture limit to less than 10 cells in further experiments as required for patient testing. In conclusion, our results show that MNP based sensing allows both cell marker characterization as well as capture simultaneously. The nanocubes allow better characterization of HER2 and EGFR positive metastatic cell subpopulations and provide easier prediction of tumor heterogeneity without resorting to invasive procedures.

Lung cancer is the number one cause of cancer-related deaths in both men and women with a median survival time of 8-10 months’ post treatment. Non Small Cell Lung Cancer (NSCLC) accounts for 80% of the lung cancers and the treatment plan is determined based on the active mutations (EGFR, ALK/ROS, and KRAS) present in the tumor. Patients bearing EGFR mutation initially respond to targeted tyrosine kinase inhibitor (TKI) therapy and after 10-14 months of treatment acquire TKI resistance. Consequently, the median PFS for NSCLC patients with EGFR mutation is only 9.5 months. The reason for this acquired drug resistance is not yet fully understood. Recent studies have reported oncogenes such as AXL could be responsible for TKI resistance. Therefore, understanding the mechanism of drug resistance is key in developing a solution to overcome the problem. For this study, we first examined the resistance mechanism and developed a biodegradable targeted nanoparticle based solution to systematically investigate the role of AXL in resistant NSCLC cell lines. In this study we (1) downregulated AXL using siRNA and separately (2) knocked out the AXL gene using crRNA (CRISPR) and treated with TKI. Our results show that AXL is responsible for activation of several EMT related proteins and upregulation of mTOR pathway. We believe the upregulation of these proteins is essential for cancer cells to switch pathways for proliferation and regulating miRNAs linked to mutations. Our results further confirm that AXL is responsible for regulating MMP-2 that is associated with cell invasion. Based on data from multiple cell lines such as H820 and A549, we demonstrate that AXL upregulation is responsible for resistance independent of EGFR activating mutations. The elucidated pathway for drug resistance was further confirmed in cell lines generated by knocking-out the AXL gene. Interpretation for AXL signaling and drug resensitization was confirmed by Western blotting, Zymography, Invasion & Migration assay, Apoptosis assay and MTT toxicity assay. We performed mRNA and miRNA analysis using qRT-PCR to understand gene expression post treatment. During the process of our investigation, we found that NSCLC cells undergo further survival cross talk with other biomarkers. Indeed, we report the first experimental evidence of a survival cross talk between AXL and FN14, a wound healing gene, that enhance cell survival post treatment. Down regulation of both AXL and FN14 dramatically reduced the IC50 of TKI. Based on the mechanism, we designed a gelatin nanoparticle that can carry both AXL and FN14 to deliver it in the tumor cell. The nanoparticle is targeted to tumor using EGFR-antibody and releases the silencing RNA within cytoplasm. We further demonstrated that dual-inhibition of AXL and FN14 in A549 mice xenografts showed tumor reduction compared to controls. In conclusion, inhibition of AXL and FN14 can maximize therapeutic response of TKI, wherein AXL is upregulated before or during drug treatment. Citation Format: Dhananjay Suresh, Ajit Zambre, Soumavo Mukherjee, Shreya Ghoshdastidar, Jennifer L. Schnabel, Sarah Chapman, W. Matthew Leevy, Anandhi Upendran, Raghuraman Kannan. Combined AXL/FN14 inhibition sensitize drug-resistant NSCLC in vitro and in vivo [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 4171. doi:10.1158/1538-7445.AM2017-4171

The 5-year survival rate for early to advanced NSCLC patients is 10-5%. Once the tumor metastasizes, the survival rate often drops below 1%. One of the major challenges is to select the patient for appropriate therapy, and the selection process involves invasive biopsy that is difficult to recurrently perform during treatment. Recent studies have shown that liquid biopsy is an attractive alternative. Liquid biopsy involves analysis of either CTCs or ctDNA to understand the tumor therapy. Isolating entire tumor cells provide an opportunity to perform whole genome sequencing for in depth understanding of the tumor. However, frequent changes in cancer signaling and acquired mutations during treatment lead to drug resistance that cannot be diagnosed using current CTC techniques. These oncogenic biochemical modifications that are often associated with metastasis involve upregulation of epithelial to mesenchymal transition (EMT) pathway that change the elasticity of tumor cells for easy shedding into the blood stream. Consequently, EMT transition leads to depletion in epithelial markers such as EpCAM and cytokeratin that are to be targeted. This effect largely limits the use of present technologies utilizing EpCAM as a marker to isolate the CTCs. We hypothesized that these cells can be captured by targeting surface markers that are overexpressed in EM transitioned CTCs. For example, markers such as EGFR and HER2 have been shown to be overexpressed pre and post EMT tumors and therefore can present as a new strategy for non-invasive diagnosis before and during treatment. In this study we have shown that EGFR and HER2 receptors are overexpressed in EGFR and KRAS mutated NSCLC cells. We artificially activated EMT in NSCLC cells and compared the surface biomarker concentrations. Furthermore, we used protein analysis data using western blotting to identify our targets after EMT. Based on the biomarker concentrations, we designed and developed magnetic nanocubes (MNC) surface attached with antibodies to target the selected biomarkers to capture EMT CTCs. The capture efficiency of these magnetic nanocubes was compared in multiple cell lines (HCC827 and A549). Overall we could achieve capture as low as 10 spiked cells in our study. In conclusion, cancer markers such as EGFR and HER-2 that are highly expressed in tumors, once shed as CTCs can be targeted for diagnosis. Therefore, we have developed a novel method to capture EMT CTCs with high selectivity and this method presents a minimally-invasive method for real-time monitoring of patients during drug treatment. Citation Format: Dhananjay Suresh, Shreya Ghoshdastidar, Soumavo Mukherjee, Abilash Gangula, Anandhi Upendran, Raghuraman Kannan. Selective isolation of epithelial to mesenchymal transitioned circulating tumor cells in NSCLC using novel magnetic nanocubes [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 3794. doi:10.1158/1538-7445.AM2017-3794

The nature of interaction and mechanism of internalization of receptor-avid peptide nanoparticles with cells is not yet completely understood. This article describes the cellular internalization mechanism and intracellular trafficking of peptide conjugated receptor targeted porous Gold nanocages (AuNCs) in cancer cells. We synthesized and characterized a library of AuNCs conjugated with bombesin (BBN) peptide. Evidence of selective affinity of AuNC-BBN toward gastrin releasing peptide receptors (GRPR) was obtained using radiolabeled competitive cell binding assay. Endocytic mechanism was investigated using cell inhibitor studies and monitored using optical and transmission electron microscopy (TEM). Results show AuNC-BBN uptake in PC3 cells is mediated by clathrin mediated endocytosis (CME). Indeed, in the presence of CME inhibitors, AuNC-BBN uptake in cells is reduced up to 84%. TEM images further confirm CME characteristic clathrin coated pits and lysosomal release of AuNCs. These results demonstrate that peptide ligands conjugated to the surface of nanoparticles maintain their target specificity. This bolsters the case for peptide robustness and its persisting functionality in intracellular vehicular delivery systems.

Objective: The purpose of this study was to apply the enhanced surface chemistry and imaging properties of gold nanorods to the area of tumor protein diagnostics in order to give an accurate, quantifiable interpretation of a patient's biomarker status. The epidermal growth factor receptor (EGFR; HER1) is a cell surface receptor that regulates cell migration, adhesion, and proliferation. EGFR is commonly overexpressed in a variety of cancers. EGFR expression testing has been mandated by insurance companies in many areas before therapy can be given. Current methods of EGFR evaluation have been shown to be unreliable, thus EGFR was chosen as a target of interest for our studies. Methods: Gold nanorods (GNR) of aspect ratio 3:4 were prepared using an established seed-mediated growth method. The GNR were then PEGylated and conjugated with a unique modified peptide that has shown specific affinity for the EGF receptor. The product was characterized to show stability and presence of surface-bound peptide. The compound known as GNR-1070 was used to examine correlations between EGFR expression and gold present on the membranes of human cell lines and paraffin-embedded tissue samples. Results: In cell lines that are known to express EGFR, gold particles were identified bound to the membranes. The amount of bound gold correlated with the degree of EGFR expression of each cell line. Histochemical analysis of paraffin-embedded human tissue lines also correlated with the amount of gold bound to the membranes of the tissues. Conclusion: Gold nanorods conjugated with receptor-specific peptides can be used to give an accurate evaluation of protein expression in human cell lines and tissue samples. Citation Format: Chuck Caldwell, Ajit Zambre, Dhananjay Suresh, Gerald Arthur, Raghuraman Kannan. Personalized diagnostics: Receptor specific gold nanorods used for accurate diagnosis of EGFR expression in human tumor tissues. [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 1903. doi:10.1158/1538-7445.AM2014-1903

We utilized different super-resolution techniques to image silver nanostructures with the assist of grating-based surface plasmon resonance. We can identify the shape and size of nanoparticles in epi-fluorescence mode and image single-molecule using a smartphone.