Whole genome-scale assessment of gene fitness of Novosphingobium aromaticavorans during spaceflight.

In microgravity, bacteria undergo intriguing physiological adaptations. There have been few attempts to assess global bacterial physiological responses to microgravity, with most studies only focusing on a handful of individual systems. This study assessed the fitness of each gene in the genome of the aromatic compound-degrading Alphaproteobacterium Novosphingobium aromaticavorans during growth in spaceflight. This was accomplished using Comparative TnSeq, which involves culturing the same saturating transposon mutagenized library under two different conditions. To assess gene fitness, a novel comparative TnSeq analytical tool was developed, named TnDivA, that is particularly useful in leveraging biological replicates. In this approach, transposon diversity is represented numerically using a modified Shannon diversity index, which was then converted into effective transposon density. This transformation accounts for variability in read distribution between samples, such as cases where reads were dominated by only a few transposon inserts. Effective density values were analyzed using multiple statistical methods, including log2-fold change, least-squares regression analysis, and Welch's t-test. The results obtained across applied statistical methods show a difference in the number of significant genes identified. However, the functional categories of genes important to growth in microgravity showed similar patterns. Lipid metabolism and transport, energy production, transcription, translation, and secondary metabolite biosynthesis and transport were shown to have high fitness during spaceflight. This suggests that core metabolic processes, including lipid and secondary metabolism, play an important role adapting to stress and promoting growth in microgravity.

A rapid dynamic in vivo near-infrared fluorescence imaging assay to track lung vascular permeability after acute radiation injury.

To develop a dynamic in vivo near-infrared (NIR) fluorescence imaging assay to quantify sequential changes in lung vascular permeability-surface area product (PS) in rodents. Dynamic NIR imaging methods for determining lung vascular permeability-surface area product were developed and tested on non-irradiated and 13 Gy irradiated rats with/without treatment with lisinopril, a radiation mitigator. A physiologically-based pharmacokinetic (PBPK) model of indocyanine green (ICG) pulmonary disposition was applied to in vivo imaging data and PS was estimated. In vivo results were validated by five accepted assays: ex vivo perfused lung imaging, endothelial filtration coefficient (Kf) measurement, pulmonary vascular resistance measurement, Evan's blue dye uptake, and histopathology. A PBPK model-derived measure of lung vascular permeability-surface area product increased from 2.60 ± 0.40 [CL: 2.42-2.78] mL/min in the non-irradiated group to 6.94 ± 8.25 [CL: 3.56-10.31] mL/min in 13 Gy group after 42 days. Lisinopril treatment lowered PS in the 13 Gy group to 4.76 ± 6.17 [CL: 2.12-7.40] mL/min. A much higher up to 5× change in PS values was observed in rats exhibiting severe radiation injury. Ex vivo Kf (mL/min/cm H2O/g dry lung weight), a measure of pulmonary vascular permeability, showed similar trends in lungs of irradiated rats (0.164 ± 0.081 [CL: 0.11-0.22]) as compared to non-irradiated controls (0.022 ± 0.003 [CL: 0.019-0.025]), with reduction to 0.070 ± 0.035 [CL: 0.045-0.096] for irradiated rats treated with lisinopril. Similar trends were observed for ex vivo pulmonary vascular resistance, Evan's blue uptake, and histopathology. Our results suggest that whole body dynamic NIR fluorescence imaging can replace current assays, which are all terminal. The imaging accurately tracks changes in PS and changes in lung interstitial transport in vivo in response to radiation injury.

Host genetic modifiers of nonproductive angiogenesis inhibit breast cancer.

PURPOSE: Multiple aspects of the tumor microenvironment (TME) impact breast cancer, yet the genetic modifiers of the TME are largely unknown, including those that modify tumor vascular formation and function. METHODS: To discover host TME modifiers, we developed a system called the Consomic/Congenic Xenograft Model (CXM). In CXM, human breast cancer cells are orthotopically implanted into genetically engineered consomic xenograft host strains that are derived from two parental strains with different susceptibilities to breast cancer. Because the genetic backgrounds of the xenograft host strains differ, whereas the inoculated tumor cells are the same, any phenotypic variation is due to TME-specific modifier(s) on the substituted chromosome (consomic) or subchromosomal region (congenic). Here, we assessed TME modifiers of growth, angiogenesis, and vascular function of tumors implanted in the SSIL2Rγ and SS.BN3IL2Rγ CXM strains. RESULTS: Breast cancer xenografts implanted in SS.BN3IL2Rγ (consomic) had significant tumor growth inhibition compared with SSIL2Rγ (parental control), despite a paradoxical increase in the density of blood vessels in the SS.BN3IL2Rγ tumors. We hypothesized that decreased growth of SS.BN3IL2Rγ tumors might be due to nonproductive angiogenesis. To test this possibility, SSIL2Rγ and SS.BN3IL2Rγ tumor vascular function was examined by dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI), micro-computed tomography (micro-CT), and ex vivo analysis of primary blood endothelial cells, all of which revealed altered vascular function in SS.BN3IL2Rγ tumors compared with SSIL2Rγ. Gene expression analysis also showed a dysregulated vascular signaling network in SS.BN3IL2Rγ tumors, among which DLL4 was differentially expressed and co-localized to a host TME modifier locus (Chr3: 95-131 Mb) that was identified by congenic mapping. CONCLUSIONS: Collectively, these data suggest that host genetic modifier(s) on RNO3 induce nonproductive angiogenesis that inhibits tumor growth through the DLL4 pathway.

ADA3 regulates normal and tumor mammary epithelial cell proliferation through c-MYC.

BACKGROUND: We have established the critical role of ADA3 as a coactivator of estrogen receptor (ER), as well as its role in cell cycle progression. Furthermore, we showed that ADA3 is predominantly nuclear in mammary epithelium, and in ER+, but is cytoplasmic in ER- breast cancers, the latter correlating with poor survival. However, the role of nuclear ADA3 in human mammary epithelial cells (hMECs), and in ER+ breast cancer cells, as well as the importance of ADA3 expression in relation to patient prognosis and survival in ER+ breast cancer have remained uncharacterized. METHODS: We overexpressed ADA3 in hMECs or in ER+ breast cancer cells and assessed the effect on cell proliferation. The expression of ADA3 was analyzed then correlated with the expression of various prognostic markers, as well as survival of breast cancer patients. RESULTS: Overexpression of ADA3 in ER- hMECs as well as in ER+ breast cancer cell lines enhanced cell proliferation. These cells showed increased cyclin B and c-MYC, decreased p27 and increased SKP2 levels. This was accompanied by increased mRNA levels of early response genes c-FOS, EGR1, and c-MYC. Analysis of breast cancer tissue specimens showed a significant correlation of ADA3 nuclear expression with c-MYC expression. Furthermore, nuclear ADA3 and c-MYC expression together showed significant correlation with tumor grade, mitosis, pleomorphism, NPI, ER/PR status, Ki67 and p27 expression. Importantly, within ER+ cases, expression of nuclear ADA3 and c-MYC also significantly correlated with Ki67 and p27 expression. Univariate Kaplan Meier analysis of four groups in the whole, as well as the ER+ patients showed that c-MYC and ADA3 combinatorial phenotypes showed significantly different breast cancer specific survival with c-MYC-high and ADA3-Low subgroup had the worst outcome. Using multivariate analyses within the whole cohort and the ER+ subgroups, the significant association of ADA3 and c-MYC expression with patients' outcome was independent of tumor grade, stage and size, and ER status. CONCLUSION: ADA3 overexpression enhances cell proliferation that is associated with increased expression of c-MYC. Expression patterns with respect to ADA3/c-MYC can divide patients into four significantly different subgroups, with c-MYC High and ADA3 Low status independently predicting poor survival in patients.

Apoptosis-Mediated Chemoprevention by Different Ratios of Fish Oil in Experimental Colon Carcinogenesis.

Apoptosis plays an important role in prevention of colon cancer. In the present study, different ratios of fish oil and corn oil increased Fas expression in both phases and a decrease in FasL expression only in post initiation phase. Treatment with fish oil activated the intrinsic apoptotic pathway by increasing Bax expression and Cyt c release and decreasing Bcl-2 levels in both phases. This suggests that intrinsic pathway is upregulated by fish oil; however, Fas-FasL activity may be involved in inhibition of reversal of immune surveillance in tumor cells.

Documentation of Ultrastructural Changes in Nucleus and Microvilli by Fish Oil in Experimental Colon Carcinogenesis.

Fish oil (FO) exerts a chemopreventive effect by regulating apoptosis in colon carcinogenesis. The present study reports the ultrastructural changes in various organelles on supplementation of FO in experimental colon carcinogenesis. The carcinogen treatment led to abnormal nuclear shape and alteration in microvilli number indicating cancer establishment. On the other hand, different ratios of FO and corn oil increased chromatin condensation along with an extensive loss of microvilli in a dose- and time-dependent manner which depicts an increase in apoptosis. The associated ultrastuctural alterations support the facilitation of apoptosis by FO as a mechanism for its beneficial effect in colon carcinogenesis.

Alterations in mitochondrial membrane in chemopreventive action of fish oil.

Mitochondria are major regulators of pathways related to tumorigenesis; therefore, mitochondrial membrane characteristics and associated cell signaling events were evaluated with different ratios of fish oil (FO) and corn oil (CO) in experimental colon carcinogenesis. Treatment with carcinogen 1,2-dimethylhydrazine (DMH) altered reactive oxygen species (ROS), Ca(2+), and membrane characteristics, which resulted in an elevation in apoptosis in initiation phase and reduction in post-initiation phase. FO+CO(2.5:1)+DMH treatment, however, altered mitochondrial membrane parameters, ROS, and Ca(2+) to increase apoptosis in both phases, whereas FO+CO(1:1)+DMH treatment enhanced apoptosis only in post-initiation phase suggesting that FO supplementation in higher ratio has better chemopreventive efficacy.

X-ray and MR Contrast Bearing Nanoparticles Enhance the Therapeutic Response of Image-Guided Radiation Therapy for Oral Cancer.

INTRODUCTION: Radiation therapy for head and neck squamous cell carcinoma is constrained by radiotoxicity to normal tissue. We demonstrate 100 nm theranostic nanoparticles for image-guided radiation therapy planning and enhancement in rat head and neck squamous cell carcinoma models. METHODS: PEG conjugated theranostic nanoparticles comprising of Au nanorods coated with Gadolinium oxide layers were tested for radiation therapy enhancement in 2D cultures of OSC-19-GFP-luc cells, and orthotopic tongue xenografts in male immunocompromised Salt sensitive or SS rats via both intratumoral and intravenous delivery. The radiation therapy enhancement mechanism was investigated. RESULTS: Theranostic nanoparticles demonstrated both X-ray/magnetic resonance contrast in a dose-dependent manner. Magnetic resonance images depicted optimal tumor-to-background uptake at 4 h post injection. Theranostic nanoparticle + Radiation treated rats experienced reduced tumor growth compared to controls, and reduction in lung metastasis. CONCLUSIONS: Theranostic nanoparticles enable preprocedure radiotherapy planning, as well as enhance radiation treatment efficacy for head and neck tumors.

Clinical significance of promoter hypermethylation of ERß and RARß2 in tumor and serum DNA in Indian breast cancer patients.

PURPOSE: To determine concordance of promoter hypermethylation of ERß (estrogen receptor ß) and RARß2 (retinoic acid receptor ß2) in tumor and circulating DNA of Indian breast cancer patients and their association with clinicopathologic parameters and disease prognosis. METHODS: ERß and RARß2 methylation was analyzed by methylation-specific PCR in the tumors and circulating DNA of 100 patients with invasive ductal breast carcinoma. Promoter hypermethylation was associated with the expression of the encoded protein in tumors by immunohistochemistry, and their prognostic utility was explored in a follow-up study. RESULTS: Significant correlation was observed between promoter hypermethylation of ERß (r = + 0.77; p ≤ 0.001) and RARß2 (r = + 0.85; p ≤ 0.001) in tumors and paired sera. No association was found between ERß and RARß2 promoter hypermethylation and loss of protein expression. Kaplan-Meier survival curve showed loss of ERß expression, and RARß2 promoter hypermethylation was associated with poor overall survival (OS) (p = 0.03, p = 0.001). Breast cancer patients showing concurrent hypermethylation of ERß and RARß2 had a significantly shorter median OS (p = 0.02), underscoring that hypermethylation of these two genes may serve as an adverse prognosticator for breast carcinoma. CONCLUSIONS: Methylation status of ERß and RARß2 in serum could potentially be used to predict invasive ductal breast carcinoma. Furthermore, concurrent ERß and RARß2 methylation as well as loss of ERß expression may serve as a good prognostic marker.

DNA methylation of circulating DNA: a marker for monitoring efficacy of neoadjuvant chemotherapy in breast cancer patients.

Identification of biomarkers for monitoring efficacy of neoadjuvant chemotherapy in breast cancer patients is of utmost importance in individual tailoring of treatment and save from toxicity due to non-effective drugs. We hypothesized that methylation of circulating tumor-specific DNA may reflect changes in tumor burden in response to chemotherapy and help stratify responders from non-responders. The aim of this study was to evaluate the potential of methylation changes in circulating DNA to monitor treatment response of breast cancer patients. Six consecutive sera samples collected from 30 breast cancer patients undergoing neoadjuvant chemotherapy were analyzed for methylation status of a panel of five genes namely, BRCA1, MGMT, GSTP1, Stratifin, and MDR1. Among these five genes, BRCA1 methylation frequency was different among responders and non-responders groups. The correlation coefficients between total gene methylation with initial chemotherapy and tumor volume reduction were R(2) = 0.8 and R(2) = 0.05 in the responders and non-responders groups, respectively. Our findings warrant further development of this approach for monitoring response in patients undergoing neoadjuvant chemotherapy.

The Impacts of Microgravity on Bacterial Metabolism.

The inside of a space-faring vehicle provides a set of conditions unlike anything experienced by bacteria on Earth. The low-shear, diffusion-limited microenvironment with accompanying high levels of ionizing radiation create high stress in bacterial cells, and results in many physiological adaptations. This review gives an overview of the effect spaceflight in general, and real or simulated microgravity in particular, has on primary and secondary metabolism. Some broad trends in primary metabolic responses can be identified. These include increases in carbohydrate metabolism, changes in carbon substrate utilization range, and changes in amino acid metabolism that reflect increased oxidative stress. However, another important trend is that there is no universal bacterial response to microgravity, as different bacteria often have contradictory responses to the same stress. This is exemplified in many of the observed secondary metabolite responses where secondary metabolites may have increased, decreased, or unchanged production in microgravity. Different secondary metabolites in the same organism can even show drastically different production responses. Microgravity can also impact the production profile and localization of secondary metabolites. The inconsistency of bacterial responses to real or simulated microgravity underscores the importance of further research in this area to better understand how microbes can impact the people and systems aboard spacecraft.

The Use of TnSeq to Identify Essential Alphaproteobacterial Genes Reveals Operational Variability in Conserved Developmental and Cell Cycle Systems.

A powerful method for examining genetic fitness and function on a large scale is to couple saturating transposon mutagenesis with high-throughput sequencing (TnSeq). By mapping where transposon insertions can be tolerated in a genome, it is possible to analyze the fitness of every gene in a genome simultaneously under a given growth condition. While this technique can describe genes as essential or nonessential under those growth conditions, sufficient mutagenesis and sequencing depth can provide more subtle differences in fitness. In this paper, TnSeq was used to analyze gene fitness of two Alphaproteobacteria from different environments: the freshwater oligotroph Brevundimonas subvibrioides (Caulobacterales) and the soil plant pathogen Agrobacterium tumefaciens (Rhizobiales) for the purpose of comparing conservation of gene function.

Clinical significance of Maspin promoter methylation and loss of its protein expression in invasive ductal breast carcinoma: correlation with VEGF-A and MTA1 expression.

Maspin is a serine protease inhibitor with tumor-suppressor activity. Maspin can suppress tumor growth and metastasis in vivo and tumor cell motility and invasion in vitro. Previous studies indicate that the loss of Maspin expression is closely linked to aberrant methylation of the Maspin promoter. We examined the promoter methylation status of Maspin in tumor and corresponding serum of breast cancer patients. In addition, protein expression of this gene was also assessed to determine possible correlation between promoter hypermethylation and gene silencing. Further, we investigated the correlation of Maspin expression with vascular endothelial growth factor (VEGF-A) and MTA1 expression. Maspin methylation was analyzed by methylation-specific PCR in 100 invasive ductal breast carcinoma patients' tumors and circulating DNA in a prospective study. Promoter hypermethylation was correlated with expression of the encoded protein in tumors by immunohistochemistry. Significant correlation was observed between promoter hypermethylation of Maspin (r = +0.88; p ≤ 0.0001) in tumors and paired sera. Significant association was found between Maspin promoter hypermethylation and loss of its protein expression (p = 0.01, OR = 3.1, 95% CI = 1.3-7.4). The expression of VEGF-A and MTA1 was lower in tumors with high Maspin expression compared to tumors with loss of Maspin expression. Our results indicate that aberrant promoter methylation is associated with loss of Maspin immunoreactivity in breast cancer tissues. Further, loss of Maspin expression is significantly correlated with increased expression of VEGF-A and MTA1.

Evaluation of the role of oxidative stress in chemopreventive action of fish oil and celecoxib in the initiation phase of 7,12-dimethyl benz(α)anthracene-induced mammary carcinogenesis.

Cyclooxygenase-2 (COX-2) enzyme plays an important role in cancer development. COX-2 inhibition by non-steroidal anti-inflammatory drugs is a useful approach for cancer prevention, but its usage has been associated with side effects. n-3 polyunsaturated fatty acids also exhibit a chemopreventive effect mediated by COX-2 inhibition. Therefore, the present study was designed to evaluate the effect of combined dosage of celecoxib and fish oil in experimental mammary carcinogenesis. Female Wistar rats were distributed as control, 7,12-dimethyl benz(α)anthracene (DMBA) treated, celecoxib + fish oil (20 mg/kg b.w. + 0.5 ml), celecoxib + fish oil (30 mg/kg b.w. + 0.25 ml), and their corresponding controls treated with fish oil or celecoxib only. The treatment was given for 7 days, and on the 8th day animals of all the groups except the control group received DMBA orally and sacrificed after 90 days. The histopathology, DNA fragmentation, total sialic acid (TSA), lipid-associated sialic acid (LASA), and oxidative stress were measured in mammary tissue and liver mitochondrial fraction. The results showed ductal hyperplasia and an increase in TSA, LASA, lipid peroxidation, and nitrite levels with a decrease in the antioxidants on DMBA treatment. Pretreatment with celecoxib and fish oil in DMBA-treated animals led to normal histology, increase in DNA fragmentation, and decrease in TSA and LASA levels with reduced oxidative stress, and the effect was more pronounced than animals pretreated with either celecoxib/fish oil alone suggesting a synergistic effect of the two regimens. To conclude, a combination of celecoxib and fish oil is a better strategy for cancer chemoprevention than celecoxib/fish oil alone.

Demethylating agent 5-aza-2-deoxycytidine enhances susceptibility of breast cancer cells to anticancer agents.

DNA methylation plays an important role in regulation of gene expression and is increasingly being recognized as a determinant of chemosensitivity of human cancers. With the aim of improving the chemotherapeutic efficacy of breast carcinoma, the effect of DNA methyltransferase inhibitor, 5-Aza-2'-deoxycytidine (5-aza-CdR), on the chemosensitivity of anticancer drugs was investigated. The cytotoxicity of paclitaxel (PTX), adriamycin (ADR), and 5-fluorouracil (5-FU) was analyzed against human breast cancer cell lines, MDA MB 231 and MCF 7 cell lines using the MTT assay, and the synergy of 5-aza-CdR and these agents was determined by Drewinko's fraction method. The effects of each single agent or the combined treatment on cell cycle arrest were analyzed by flow cytometric analysis. We also investigated the effect of each single agent or the combined treatment of anticancer drugs with 5-aza-CdR on the methylation status of the selected genes by methylation specific PCR. In MDA MB 231 cells, a synergistic antiproliferative effect was observed with a combination of 10 microM 5-aza-CdR and these three anticancer drugs, while in MCF 7 cells, a semiadditive effect was observed. Treatment with 5-aza-CdR and anticancer drug resulted in partial demethylation of a panel of genes including RARbeta2, Slit2, GSTP1, and MGMT. Based on these findings, we propose that 5-aza-CdR enhances the chemosensitivity of anticancer drugs in breast cancer cells and may be a promising approach for increasing the chemotherapeutic potential of these anticancer agents for more effective management of breast carcinomas.

Experimental investigation of neural network estimator and transfer learning techniques for K-edge spectral CT imaging.

PURPOSE: Spectral computed tomography (CT) material decomposition algorithms require accurate physics-based models or empirically derived models. This study investigates a machine learning algorithm and transfer learning techniques for Spectral CT imaging of K-edge contrast agents using simulated and experimental measurements. METHODS: A feed forward multilayer perceptron was implemented and trained on data acquired using a step wedge phantom containing acrylic, aluminum, and gadolinium materials. The neural network estimator was evaluated by scanning a rod phantom with varying dilutions of gadolinium oxide nanoparticles and by scanning a rat leg specimen with injected nanoparticles on a bench-top photon-counting computed tomography system. The algorithm decomposed each spectral projection measurement into path lengths of acrylic and aluminum and mass lengths of gadolinium. Each basis material sinogram was reconstructed into basis material images using filtered backprojection. Machine learning techniques of data standardization, transfer learning from aggregated pixel data, and transfer learning from simulations were investigated to improve image quality. The algorithm was compared to a previously published empirical method based on a linear approximation and calibration error look-up tables. RESULTS: The combined transfer learning techniques did not improve quantification in the rod phantom and provided only a small qualitative improvement in ring artifacts. Transfer learning from aggregated pixel data and from simulations improved the qualitative image quality of the rat specimen, for which the calibration data were limited. Transfer learning from aggregated pixel data and simulations estimated 3.26, 6.26, and 12.45 mg/mL Gd concentrations compared to true 2.72, 5.44, and 10.88 mg/mL concentrations in the rod phantom. Additionally, the neural networks were able to separate the soft tissue, bone, and gadolinium nanoparticles of the ex vivo rat leg specimen into the different basis images. CONCLUSIONS: The results demonstrate that empirical K-edge imaging from calibration measurements using machine learning and transfer learning is possible without explicit models of material attenuations, incident spectra, or the detector response.

NIR-II window tracking of hyperglycemia induced intracerebral hemorrhage in cerebral cavernous malformation deficient mice.

Second near infrared (NIR-II) window fluorescence imaging between 1000 and 1700 nm with reduced scattering and autofluorescence and deep tissue light penetration allows early and non-invasive determination of vascular pathologies. Here, we demonstrate in vivo NIR-II imaging techniques for tracking hyperglycaemia-induced Intracerebral Hemorrhage (ICH) and Blood Brain Barrier (BBB) hyperpermeability in Cerebral Cavernous Malformation (CCM) deficient mice (CCM1+/-). We synthesised PEGylated Ag2S quantum dots (QDs) with a bright fluorescent emission peak centred at 1135 nm under an 808 nm NIR light for dynamic imaging of cerebral vasculature in mice and determined the development of ICH and BBB impairment in hyperglycaemic CCM1+/- mice. In vivo optical imaging was conducted with micro-CT (including k-mean cluster analysis) as well as in vivo permeability assays using FITC-dextran perfusion and IgG staining, respectively. The increased BBB permeability in CCM1+/- mice was further demonstrated to be associated with a high-glucose-caused decrease of CCM1 expressions. This study validates that deep-penetrating NIR-II QDs can be used for the tracking of ICH and BBB hyperpermeability in transgenic mice models of cerebral vascular anomalies.

Localized and triggered release of oxaliplatin for the treatment of colorectal liver metastasis.

Purpose: The aim of this study was to develop and evaluate a liposome formulation that deliver oxaliplatin under magnetic field stimulus in high concentration to alleviate the off-target effects in a rat model of colorectal liver metastases (CRLM). Materials and Methods: Hybrid liposome-magnetic nanoparticles loaded with Cy5.5 dye and oxaliplatin (L-NIR- Fe3O4/OX) were synthesized by using thermal decomposition method. CRLM (CC-531) cell viability was assessed and rats orthotopically implanted with CC-531 cells were treated with L-NIR-Fe3O4/OX or by drug alone via different routes, up to 3 cycles of alternating magnetic field (AMF). Optical and MR imaging was performed to assess the targeted delivery. Biodistribution and histology was performed to determine the distribution of oxaliplatin. Results: L-NIR-Fe3O4/OX presented a significant increase of oxaliplatin release (~18%) and lower cell viability after AMF exposure (p<0.001). Optical imaging showed a significant release of oxaliplatin among mesenteric vein injected (MV) group of animals. MR imaging on MV injected animals showed R2* changes in the tumor regions at the same regions immediately after infusion compared to the surrounding liver (p<0.001). Biodistribution analysis showed significantly higher levels of oxaliplatin in liver tissues compared to lungs (p<0.001) and intestines (p<0.001) in the MV animals that received AMF after L-NIR- Fe3O4/OX administration. Large tumor necrotic zones and significant improvement in the survival rates were noted in the MV animals treated with AMF. Conclusion: AMF triggers site selective delivery of oxaliplatin at high concentrations and improves survival outcomes in colorectal liver metastasis tumor bearing rats.

Heritable modifiers of the tumor microenvironment influence nanoparticle uptake, distribution and response to photothermal therapy.

We report the impact of notch-DLL4-based hereditary vascular heterogeneities on the enhanced permeation and retention (EPR) effect and plasmonic photothermal therapy response in tumors. Methods: We generated two consomic rat strains with differing DLL4 expression on 3rd chromosome. These strains were based on immunocompromised Salt-sensitive or SSIL2Rγ- (DLL4-high) and SS.BN3IL2Rγ- (DLL4-low) rats with 3rd chromosome substituted from Brown Norway rat. We further constructed three novel SS.BN3IL2Rγ- congenic strains by introgressing varying segments of BN chromosome 3 into the parental SSIL2Rγ- strain to localize the role of SSIL2Rγ- DLL4 on tumor EPR effect with precision. We synthesized multimodal theranostic nanoparticles (TNPs) based on Au-nanorods which provide magnetic resonance imaging (MRI), X-ray, and optical contrasts to assess image guided PTT response and quantify host specific therapy response differences in tumors orthotopically xenografted in DLL4-high and -low strains. We tested recovery of therapy sensitivity of PTT resistant strains by employing anti-DLL4 conjugated TNPs in two triple negative breast cancer tumor xenografts. Results: Host strains with high DLL4 allele demonstrated slightly increased tumor nanoparticle uptake but consistently developed photothermal therapy resistance compared to tumors in host strains with low DLL4 allele. Tumor micro-environment with low DLL4 expression altered the geographic distribution of nanoparticles towards closer proximity with vasculature which improved efficacy of PTT in spite of lower overall TNP uptake. Targeting TNPs to tumor endothelium via anti-DLL4 antibody conjugation improved therapy sensitivity in high DLL4 allele hosts for two triple negative human breast cancer xenografts. Conclusions: Inherited DLL4 expression modulates EPR effects in tumors, and molecular targeting of endothelial DLL4 via nanoparticles is an effective personalized nanomedicine strategy.

Epigenetic alterations of CDH1 and APC genes: relationship with activation of Wnt/beta-catenin pathway in invasive ductal carcinoma of breast.

Activation of canonical Wnt/beta-catenin pathway in Invasive Ductal Carcinoma of Breast (IDCs) was recently reported from our laboratory. Herein, we analyzed promoter methylation status of CDH1 and Adenomatous polyposis coli (APC) genes in 50 IDCs and correlated with expression of E-cadherin (E-CD) and APC proteins and with activation of oncogenic Wnt/beta-catenin signaling pathway components, Dvl, beta-catenin and CyclinD1. Further, Wnt/beta-catenin driven epithelial mesenchymal transition (EMT) was investigated by correlating the expression of Dvl, beta-catenin and CyclinD1 with vimentin expression in these IDCs. Promoter hypermethylation was observed in 25/50 (50%) IDCs for CDH1 and in 11/50 (22%) tumors for APC, associated with loss of expression of E-CD and APC proteins; concordant hypermethylation of these genes was observed in paired patients' sera. Further, 57% of tumors harboring CDH1 methylation and 50% tumors harboring the methylated APC gene showed nuclear localization of beta-catenin, suggesting activation of the canonical Wnt/beta-catenin pathway. Our study demonstrates significant association between vimentin expression and nuclear beta-catenin (p=0.001; Odds ratio (OR)=25.6) and Dvl (p=0.023; OR=8.0), suggesting that EMT may be driven by Wnt/beta-catenin activation in IDCs. In conclusion, we demonstrate correlation of CDH1 and APC promoter methylation with loss of E-CD and APC proteins and with activation of Wnt/beta-catenin signaling pathway. Association of nuclear Dvl and beta-catenin with vimentin expression suggests the importance of Wnt/beta-catenin pathway driven EMT in IDCs. The concordance between CDH1 and APC methylation in IDCs and paired circulating DNA underscores the utility of serum DNA as a non-invasive tool for methylation analysis in IDC patients.