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In the present work, the immobilized footprint of β-glucosidase (BGL) on silica-coated iron oxide was explored to produce reusable catalysts with flexible active sites for high activity and heightened storage stability. Synthesized iron oxide particles were coated with silica and functionalized with various densities of (3-aminopropyl)triethoxysilane (APTES) to obtain particles with amine densities ranging from 0 to 3 × 10

Supplemental data regarding cecum morphology, body weights, adhesion molecule expression in spleen and thymus, spleen size, tumor and stroma cell viability, and IFN-g and VEGF serum levels after induction of dysbiosis.

SF1: Tumor growth assays for lung and HN tumors. SF2: The complete set of MCR-derived pure component spectra and tables listing their peak assignments. SF3: Representative MCR score maps for lung and HN tumor datasets. SF4: Histologic images for HN tumor dataset. TS1-4: Results of binary leave-one-mouse-out SVM analyses.

Delay in the assessment of tumor response to radiotherapy continues to pose a major challenge to quality of life for patients with nonresponsive tumors. Here, we exploited label-free Raman spectroscopic mapping to elucidate radiation-induced biomolecular changes in tumors and uncovered latent microenvironmental differences between treatment-resistant and -sensitive tumors. We used isogenic radiation-resistant and -sensitive A549 human lung cancer cells and human head and neck squamous cell carcinoma (HNSCC) cell lines (UM-SCC-47 and UM-SCC-22B, respectively) to grow tumor xenografts in athymic nude mice and demonstrated the molecular specificity and quantitative nature of Raman spectroscopic tissue assessments. Raman spectra obtained from untreated and treated tumors were subjected to chemometric analysis using multivariate curve resolution-alternating least squares (MCR-ALS) and support vector machine (SVM) to quantify biomolecular differences in the tumor microenvironment. The Raman measurements revealed significant and reliable differences in lipid and collagen content postradiation in the tumor microenvironment, with consistently greater changes observed in the radiation-sensitive tumors. In addition to accurately evaluating tumor response to therapy, the combination of Raman spectral markers potentially offers a route to predicting response in untreated tumors prior to commencing treatment. Combined with its noninvasive nature, our findings provide a rationale for in vivo studies using Raman spectroscopy, with the ultimate goal of clinical translation for patient stratification and guiding adaptation of radiotherapy during the course of treatment.Significance:These findings highlight the sensitivity of label-free Raman spectroscopy to changes induced by radiotherapy and indicate the potential to predict radiation resistance prior to commencing therapy.

The overall use of antibiotics has increased significantly in recent years. Besides fighting infections, antibiotics also alter the gut microbiota. Commensal bacteria in the gastrointestinal tract are crucial to maintain immune homeostasis, and microbial imbalance or dysbiosis affects disease susceptibility and progression. We hypothesized that antibiotic-induced dysbiosis of the gut microbiota would suppress cytokine profiles in the host, thereby leading to changes in the tumor microenvironment. The induced dysbiosis was characterized by alterations in bacterial abundance, composition, and diversity in our animal models. On the host side, antibiotic-induced dysbiosis caused elongated small intestines and ceca, and B16-F10 melanoma and Lewis lung carcinoma progressed more quickly than in control mice. Mechanistic studies revealed that this progression was mediated by suppressed TNFα levels, both locally and systemically, resulting in reduced expression of tumor endothelial adhesion molecules, particularly intercellular adhesion molecule-1 (ICAM-1) and a subsequent decrease in the number of activated and effector CD8+ T cells in the tumor. However, suppression of ICAM-1 or its binding site, the alpha subunit of lymphocyte function-associated antigen-1, was not seen in the spleen or thymus during dysbiosis. TNFα supplementation in dysbiotic mice was able to increase ICAM-1 expression and leukocyte trafficking into the tumor. Overall, these results demonstrate the importance of commensal bacteria in supporting anticancer immune surveillance, define an important role of tumor endothelial cells within this process, and suggest adverse consequences of antibiotics on cancer control.Significance:Antibiotic-induced dysbiosis enhances distal tumor progression by altering host cytokine levels, resulting in suppression of tumor endothelial adhesion molecules and activated and effector CD8+ T cells in the tumor.

Antibiotic-induced microbial imbalance, or dysbiosis, has systemic and long-lasting effects on the host and response to cancer therapies. However, the effects on tumor endothelial cells are largely unknown. Therefore, the goal of the current study was to generate matched B16-F10 melanoma associated endothelial cell lines isolated from mice with and without antibiotic-induced dysbiosis. After validating endothelial cell markers on a genomic and proteomic level, functional angiogenesis assays (i.e., migration and tube formation) also confirmed their vasculature origin. Subsequently, we found that tumor endothelial cells derived from dysbiotic mice (TEC-Dys) were more sensitive to ionizing radiotherapy in the range of clinically-relevant hypofractionated doses, as compared to tumor endothelial cells derived from orthobiotic mice (TEC-Ortho). In order to identify tumor vasculature-associated drug targets during dysbiosis, we used tandem mass tag mass spectroscopy and focused on the statistically significant cellular membrane proteins overexpressed in TEC-Dys. By these criteria c-Met was the most differentially expressed protein, which was validated histologically by comparing tumors with or without dysbiosis. Moreover, in vitro, c-Met inhibitors Foretinib, Crizotinib and Cabozantinib were significantly more effective against TEC-Dys than TEC-Ortho. In vivo, Foretinib inhibited tumor growth to a greater extent during dysbiosis as compared to orthobiotic conditions. Thus, we surmise that tumor response in dysbiotic patients may be greatly improved by targeting dysbiosis-induced pathways, such as c-Met, distinct from the many targets suppressed due to dysbiosis.

Purpose Ongoing angiogenesis renders the tumor endothelium unresponsive to inflammatory cytokines and interferes with adhesion of leukocytes, resulting in escape from immunity. This process is referred to as tumor endothelial cell anergy. We aimed to investigate whether anti-angiogenic agents can overcome endothelial cell anergy and provide pro-inflammatory conditions. Experimental design Tissues of renal cell carcinoma (RCC) patients treated with VEGF pathway-targeted drugs and control tissues were subject to RNAseq and immunohistochemical profiling of the leukocyte infiltrate. Analysis of adhesion molecule regulation in cultured endothelial cells, in a preclinical model and in human tissues was performed and correlated to leukocyte infiltration. Results It is shown that treatment of RCC patients with the drugs sunitinib or bevacizumab overcomes tumor endothelial cell anergy. This treatment resulted in an augmented inflammatory state of the tumor, characterized by enhanced infiltration of all major leukocyte subsets, including T cells, regulatory T cells, macrophages of both M1- and M2-like phenotypes and activated dendritic cells. In vitro, exposure of angiogenic endothelial cells to anti-angiogenic drugs normalized ICAM-1 expression. In addition, a panel of tyrosine kinase inhibitors was shown to increase transendothelial migration of both non-adherent and monocytic leukocytes. In primary tumors of RCC patients, ICAM-1 expression was found to be significantly increased in both the sunitinib and bevacizumab-treated groups. Genomic analysis confirmed the correlation between increased immune cell infiltration and ICAM-1 expression upon VEGF-targeted treatment. Conclusion The results support the emerging concept that anti-angiogenic therapy can boost immunity and show how immunotherapy approaches can benefit from combination with anti-angiogenic compounds.

Nanoscale phenomena are under increasingly intense investigation both in academia and industry. The unique physical and chemical properties stemming from their high surface area and confined space ...

The crosstalk between the microbiota and the host is crucial to maintaining immune homeostasis. A growing body of evidence shows that microbial imbalance affects cancer susceptibility and progression. A common significant influence on the microbiota in the gastrointestinal (GI) tract is the use of antibiotics. Oral antibiotics severely alter the bacteria in the GI tract by destroying beneficial bacteria alongside pathogenic ones — producing a state of microbial imbalance called dysbiosis. The overall use of antibiotics has increased by more than 30% in recent years, and moreover, many cancer patients are prescribed antibiotics during treatment, as infection is frequently a complication. We hypothesized that antibiotic-induced dysbiosis of the gut microbiota would suppress cytokine profiles in the host, thereby causing stromal immune surveillance suppression. We found that antibiotics as well as tumorigenesis caused changes in the bacterial abundance, composition, or diversity in the GI tract. On the host side, we found that antibiotic-induced dysbiosis caused the small intestine and cecum to enlarge and elongate, yet the colon was not affected. Moreover, B16-F10 melanoma and Lewis Lung carcinoma progressed more quickly during dysbiosis. Mechanistically, tumor progression was mediated by suppressed TNF-α levels, both locally and systemically. In contrast, IFN-γ and VEGF did not change significantly during dysbiosis. The suppressed levels of TNF-α, a pleiotropic regulator of intercellular adhesion molecule-1 (ICAM-1), resulted in reduced expression of tumor endothelial cell associated ICAM-1, and a subsequent decrease in the number of activated and effector CD8+ T-cells in the tumor. However, suppression of ICAM-1 or its binding site, the alpha subunit of lymphocyte function-associated antigen-1 (LFA-1), was not seen in established vasculature during dysbiosis as assessed in the spleen or the thymus. While dysbiosis enhanced tumor growth in ICAM-1+/+ and immunocompetent mice, it did not affect tumor growth in ICAM-1-/- or T-cell deficient mice, highlighting the influence and impact of dysbiosis on T-cell trafficking and extravasation. TNF-α supplementation in dysbiotic mice was able to overcome the dysbiosis-induced suppression as evident by increased ICAM-1 expression, leukocyte trafficking and tumor growth inhibition. Overall, these results demonstrate the importance of commensal bacteria in supporting immune surveillance and expand our understanding of the crosstalk between the gut microbiome and the stroma in distal non-GI tract tumors. Moreover, this knowledge has the potential to influence clinical decision-making when treating patients for solid cancers with dysbiosis-inducing antibiotics and may further inform concurrent and (neo-)adjuvant treatments to overcome the adverse effects of the antibiotics. Citation Format: Samir V. Jenkins, Michael S. Robeson, Eric R. Siegel, Charles M. Quick, Robert J. Griffin, Martin J. Cannon, Kieng B. Vang, Ruud P. Dings. T-cell trafficking and extravasation is suppressed in distal tumors during gastrointestinal tract dysbiosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3529.

Background: The combination of biological variation and nanomaterial heterogeneity makes elucidating the mechanisms of interactions between cells and nanoparticles extremely complicated. Accurate nanoparticle quantification can be extremely challenging, and cellular response can change based on the location of the nanoparticle and the cell type under investigation. These complications are only augmented by the additional of external stimuli. These limitations have yielded a wide range of studies that show effects, but often provide little mechanistic insight. Results: Gold nanomaterials were stably immobilized onto glass coverslips treated with mercaptosilane to control both the average number of nanoparticles that interact with cells and their spatial orientation relative to the cell membrane. Surfaces were characterized optically and by electron microscopy to confirm their surface density and uniformity. The thermal response of Au nanocage-coated surfaces to near infrared laser irradiation was measured in cell culture medium and modelled computationally. The modelling showed a vastly higher thermal dose than would be predicted by bulk temperature measurements. Adherent or non-adherent cell lines were cultured directly on the nanocage-coated surface or in the medium, respectively, in culture wells and laser irradiation was applied. Survival of cells growing in suspension correlated with the bulk temperature increase in the culture medium, as measured by viability assay. Conversely, adherent cells exhibited a much greater susceptibility than expected from the bulk temperature measurement, which is ostensibly related to the close interaction with the nanoparticles on their growth substrate and induction of substantially greater thermal dose upon laser exposure. Conclusions: This platform is designed to be a new tool to determine how many particles need to be in contact with a cell to induce desired physical or biological effects. Here we demonstrate the delivery of precise thermal doses following laser irradiation. The anticipated biological effects based on bulk measurements vastly underestimated the effects that were observed, which is ascribed to the proximity of the nanoparticle to the cell and the extraordinary high surface temperature of the particle. This platform could be expanded to a variety of nanoparticles, external stimuli, and cell types to enable more deliberate and optimized application of nanomedicine.

The combination of radiotherapy and immunotherapy may generate synergistic anti-tumor host immune responses and promote abscopal effects. Spatial fractionation of a radiation dose has been found to promote unique physiological responses of tumors, which might promote synergy with immunotherapy. To determine whether spatial fractionation may augment immune activity, whole-tumor or spatial fractionation grid radiation treatment (GRID) alone or in combination with antibodies against immune checkpoints PD1 and CTLA-4 were tested in an immunocompetent mouse model using a triple negative breast tumor (4T1). Tumor growth delay, immuno-histochemistry and flow cytometry were used to characterize the effects of each treatment type. Whole-beam radiation with immune checkpoint inhibition significantly restrained tumor growth in the irradiated tumor, but not abscopal tumors, compared to either of these treatments alone. In mice that received spatially fractionated irradiation, evidence of abscopal immune responses were observed in contralateral tumors with markedly enhanced infiltration of both antigen-presenting cells and activated T cells, which were preceded by increased systemic IFNγ production and led to eventual tumor growth delay. These studies suggest that systemic immune activation may be triggered by employing GRID to a primary tumor lesion, promoting anti-tumor immune responses outside the treatment field. Interestingly, PD-L1 was found to be upregulated in abscopal tumors from GRID-treated mice. Combined radio-immunotherapy therapy is becoming a validated and novel approach in the treatment of cancer. With the potential increased benefit of GRID to augment both local and metastatic disease responses, further exploration of GRID treatment as a part of current standards of care is warranted.

Hypoxia, a hallmark characteristic of glioblastoma (GBM) induces changes in the transcriptome and the proteome of tumor cells. We discovered that hypoxic stress produces significant qualitative and quantitative changes in the protein content of secreted exosomes from GBM cells. Among the proteins found to be selectively elevated in hypoxic exosomes were protein-lysine 6-oxidase (LOX), thrombospondin-1 (TSP1), vascular derived endothelial factor (VEGF) and a disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1), well studied contributors to tumor progression, metastasis and angiogenesis. Our findings demonstrate that hypoxic exosomes induce differential gene expression in recipient glioma cells. Glioma cells stimulated with hypoxic exosomes showed a marked upregulation of small nucleolar RNA, C/D box 116–21 (SNORD116-21) transcript among others while significantly downregulated the potassium voltage-gated channel subfamily J member 3 (KCNJ3) message. This differential expression of certain genes is governed by the protein cargo being transferred via exosomes. Additionally, compared to normoxic exosomes, hypoxic exosomes increased various angiogenic related parameters vis-à-vis, overall tube length, branching intervals and length of isolated branches studied in tube formation assay with endothelial progenitor cells (EPCs). Thus, the intercellular communication facilitated via exosomes secreted from hypoxic GBM cells induce marked changes in the expression of genes in neighboring normoxic tumor cells and possibly in surrounding stromal cells, many of which are involved in cancer progression and treatment resistance mechanisms., Highlights • In GBM, hypoxic stress induces profound changes in the protein content of secreted exosomes. • Hypoxic exosomal contents induce angiogenesis and significant changes in recipient GBM cell transcriptome. • Hypoxic exosomes play a major role leading to tumor proliferation, tumor growth and cell survival.

Gold nanoparticles with a graft density of 0.09, 0.30 and 0.40 chains/nm2 of poly(N-isopropylacrylamide) were reproducibly synthesized by varying the ratio of disulfide terminated poly(N-isopropylacrylamide) to gold nanoparticle. The polymer coated nanoparticles were stable at room temperature in 50 mM NaCl, yet agglomerated at 37 °C. Previous studies have observed conflicting results as to the reversibility of this agglomeration. Particle agglomeration with three different graft densities was studied in 50 mM NaCl by measurements of their localized surface plasmon resonance and hydrodynamic diameter, and imaging with electron microscopy. Agglomerates with a polymer graft density of 0.30 and 0.40 chains/nm2 could be dispersed with sonication, while particles with a graft density of 0.09 chains/nm2 irreversibly aggregated. The graft density dependence on whether agglomeration or aggregation occurred is due to changes in collapsed polymer steric effects. Localized surface plasmon resonance measurements of agglomerates were discordant with hydrodynamic diameter measurements in determining agglomeration reversibility, which shed light on reasons previous reports yielded different interpretations on the reversibility of this agglomeration. This work demonstrates how polymer graft density affects thermoresponsive nanoparticle stability in salt solutions and the need for use of complementary techniques when determining agglomeration.

Strategies to increase the proportion of neural stem cells that differentiate into neurons are vital for therapy of neurodegenerative disorders. In vitro, the extracellular matrix composition and topography have been found to be important factors in stem cell differentiation. We have developed a novel artificial extracellular matrix (aECM) formed by attaching gold nanocages (AuNCs) to glass coverslips. After culturing rat neural stem cells (rNSCs) on these gold nanocage-coated surfaces (AuNC-aECMs), we observed that 44.6% of rNSCs differentiated into neurons compared to only 27.9% for cells grown on laminin-coated glass coverslips. We applied laser irradiation to the AuNC-aECMs to introduce precise amounts of photothermally induced heat shock in cells. Our results showed that laser-induced thermal stimulation of AuNC-aECMs further enhanced neuronal differentiation (56%) depending on the laser intensity used. Response to these photothermal effects increased the expression of heat shock protein 27, 70, and 90α in rNSCs. Analysis of dendritic complexity showed that this thermal stimulation promoted neuronal maturation by increasing dendrite length as thermal dose was increased. In addition, we found that cells growing on AuNC-aECMs post laser irradiation exhibited action potentials and increased the expression of voltage-gated Na+ channels compared to laminin-coated glass coverslips. These results indicate that the photothermal response induced in cells growing on AuNC-aECMs can be used to produce large quantities of functional neurons, with improved electrochemical properties, that can potentially be transplanted into a damaged central nervous system to provide replacement neurons and restore lost function.

Photodynamic therapy (PDT) of tumors relies on the delivery of a photosensitizer to the tumor site followed by external laser activation. The effectiveness of the therapy is determined by the fluence and irradiation of the laser, intratumoral photosensitizer retention, and availability of molecular oxygen. We hypothesized that retention and resulting therapeutic response may be improved with local hyperthermia. Tumors were grown in the rear limb then locally heated for 1 hour at 42.5 °C immediately following i.v. injection of the photosensitizer in an immunocompetent murine model. Laser exposure was applied at 2 h or 24 h after the heating session. Administration of heating caused a significant growth delay when a single laser treatment was applied only 2 hours following injection. However, in animals given photosensitizer only and irradiated 2 h later there was no measurable anti-tumor effect observed. The marked anti-tumor effects obtained with prior local hyperthermia were observed even as the dose was lowered from 10 mg/kg to 1.3 mg/kg. Additionally, histological analysis of our intial studies revealed that the majority of the tumor tissue (~75%) was necrotic after two days when heat was combined with PDT, while PDT alone resulted in only ~25% necrotic tissue. Additionally, a significant, though less notable, increase in the efficacy was observed if the laser treatment was applied 24 hours after hyperthermia and photosensitizer administration. This increase is in part ascribed to the increased retention of the photosensitizer in the tumor tissue and likely lasting effects on tumor blood flow and oxygenation in the heated vs. control groups.

Delay in the assessment of tumor response to radiotherapy continues to pose a major challenge to quality of life for patients with nonresponsive tumors. Here, we exploited label-free Raman spectroscopic mapping to elucidate radiation-induced biomolecular changes in tumors and uncovered latent microenvironmental differences between treatment-resistant and -sensitive tumors. We used isogenic radiation-resistant and -sensitive A549 human lung cancer cells and human head and neck squamous cell carcinoma (HNSCC) cell lines (UM-SCC-47 and UM-SCC-22B, respectively) to grow tumor xenografts in athymic nude mice and demonstrated the molecular specificity and quantitative nature of Raman spectroscopic tissue assessments. Raman spectra obtained from untreated and treated tumors were subjected to chemometric analysis using multivariate curve resolution-alternating least squares (MCR-ALS) and support vector machine (SVM) to quantify biomolecular differences in the tumor microenvironment. The Raman measurements revealed significant and reliable differences in lipid and collagen content postradiation in the tumor microenvironment, with consistently greater changes observed in the radiation-sensitive tumors. In addition to accurately evaluating tumor response to therapy, the combination of Raman spectral markers potentially offers a route to predicting response in untreated tumors prior to commencing treatment. Combined with its noninvasive nature, our findings provide a rationale for in vivo studies using Raman spectroscopy, with the ultimate goal of clinical translation for patient stratification and guiding adaptation of radiotherapy during the course of treatment. Significance: These findings highlight the sensitivity of label-free Raman spectroscopy to changes induced by radiotherapy and indicate the potential to predict radiation resistance prior to commencing therapy.

The overall use of antibiotics has increased significantly in recent years. Besides fighting infections, antibiotics also alter the gut microbiota. Commensal bacteria in the gastrointestinal tract are crucial to maintain immune homeostasis, and microbial imbalance or dysbiosis affects disease susceptibility and progression. We hypothesized that antibiotic-induced dysbiosis of the gut microbiota would suppress cytokine profiles in the host, thereby leading to changes in the tumor microenvironment. The induced dysbiosis was characterized by alterations in bacterial abundance, composition, and diversity in our animal models. On the host side, antibiotic-induced dysbiosis caused elongated small intestines and ceca, and B16-F10 melanoma and Lewis lung carcinoma progressed more quickly than in control mice. Mechanistic studies revealed that this progression was mediated by suppressed TNFα levels, both locally and systemically, resulting in reduced expression of tumor endothelial adhesion molecules, particularly intercellular adhesion molecule-1 (ICAM-1) and a subsequent decrease in the number of activated and effector CD8+ T cells in the tumor. However, suppression of ICAM-1 or its binding site, the alpha subunit of lymphocyte function-associated antigen-1, was not seen in the spleen or thymus during dysbiosis. TNFα supplementation in dysbiotic mice was able to increase ICAM-1 expression and leukocyte trafficking into the tumor. Overall, these results demonstrate the importance of commensal bacteria in supporting anticancer immune surveillance, define an important role of tumor endothelial cells within this process, and suggest adverse consequences of antibiotics on cancer control. Significance: Antibiotic-induced dysbiosis enhances distal tumor progression by altering host cytokine levels, resulting in suppression of tumor endothelial adhesion molecules and activated and effector CD8+ T cells in the tumor.

A major challenge in photothermal treatment is generating sufficient heat to eradicate diseased tissue while sparing normal tissue. Au nanomaterials have shown promise as a means to achieve highly localized photothermal treatment. Toward that end, the synthetic peptide anginex was conjugated to Au nanocages. Anginex binds to galectin-1, which is highly expressed in dividing endothelial cells found primarily in the tumor vasculature. The skin surface temperature during a 10 min laser exposure of subcutaneous murine breast tumors did not exceed 43°C and no normal tissue damage was observed, yet a significant anti-tumor effect was observed when laser was applied 24 h post-injection of targeted nanocages. Untargeted particles showed little effect in immunocompetent, tumor-bearing mice under these conditions. Photoacoustic, photothermal, and ICP-MS mapping of harvested tissue showed distribution of particles near the vasculature throughout the tumor. This uptake pattern within the tumor combined with a minimal overall temperature rise were nonetheless sufficient to induce marked photothermal efficacy and evidence of tumor control. Importantly, this evidence suggests that bulk tumor temperature during treatment does not correlate with treatment outcome, which implies that targeted nanomedicine can be highly effective when closely bound/distributed in and around the tumor endothelium and extensive amounts of direct tumor cell binding may not be a prerequisite of effective photothermal approaches.

Background Here we investigated the influence of different stabilization and storage strategies on the quality and composition of the fecal microbial community. Namely, same-day isolated murine DNA was compared to samples stored for 1 month in air at ambient temperature, with or without preservative buffers (i.e. EDTA and lysis buffer), different temperatures (i.e. 4 °C, − 20 °C, and − 80 °C), and hypoxic conditions. Results Only storage in lysis buffer significantly reduced DNA content, yet without integrity loss. Storage in EDTA affected alpha diversity the most, which was also reflected in cluster separation. Distinct changes were also seen in the phyla and bacterial species abundance per storage strategy. Metabolic function analysis showed 22 pathways not significantly affected by storage conditions, whereas the tyrosine metabolism pathway was significantly changed in all strategies except by EDTA. Conclusion Each long-term storage strategy introduced a unique post-collection bias, which is important to take into account when interpreting data. Electronic supplementary material The online version of this article (10.1186/s12866-018-1359-5) contains supplementary material, which is available to authorized users.

An improved technique for fractal characterization called the modified blanket method is introduced that can quantify surrounding fractal structures on a pixel by pixel basis without artifacts associated with scale-dependent image features such as object size. The method interprets images as topographical maps, obtaining information regarding the local surface area as a function of image resolution. Local fractal dimension (FD) can be quantified from the power law exponent derived from the surface area and image resolution relationship. We apply this technique on simulated cell images of known FD and compared the obtained values to power spectral density (PSD) analysis. Our method is sensitive to a wider FD range (2.0–4.5), having a mean error of 1.4% compared to 6% for PSD analysis. This increased sensitivity and an ability to compute regional FD properties enabled the discrimination of the differences in radiation resistant cancer cell responses that could not be detected using PSD analysis.

Resistance to conventional antibiotics is a growing public health concern that is quickly outpacing the development of new antibiotics. This has led the Infectious Diseases Society of America (IDSA) to designate Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species as "ESKAPE pathogens" on the basis of the rapidly decreasing availability of useful antibiotics. This emphasizes the urgent need for alternative therapeutic strategies to combat infections caused by these and other bacterial pathogens. In this study, we used Staphylococcus aureus (S. aureus) as a proof-of-principle ESKAPE pathogen to demonstrate that an appropriate antibiotic (daptomycin) can be incorporated into polydopamine-coated gold nanocages (AuNC@PDA) and that daptomycin-loaded AuNC@PDA can be conjugated to antibodies targeting a species-specific surface protein (staphylococcal protein A; Spa) as a means of achieving selective delivery of the nanoconstructs directly to the bacterial cell surface. Targeting specificity was confirmed by demonstrating a lack of binding to mammalian cells, reduced photothermal and antibiotic killing of the Spa-negative species Staphylococcus epidermidis, and reduced killing of S. aureus in the presence of unconjugated anti-Spa antibodies. We demonstrate that laser irradiation at levels within the current safety standard for use in humans can be used to achieve both a lethal photothermal effect and controlled release of the antibiotic, thus resulting in a degree of therapeutic synergy capable of eradicating viable S. aureus cells. The system was validated using planktonic bacterial cultures of both methicillin-sensitive and methicillin-resistant S. aureus strains and subsequently shown to be effective in the context of an established biofilm, thus indicating that this approach could be used to facilitate the effective treatment of intrinsically resistant biofilm infections.

OBJECTIVES/GOALS: Development of gold nanoparticles covalently linked to a photosensitizer for use to enhance radiation therapy. The particles will be thoroughly characterized structurally and mechanistically. The gold particles should enhance radiation activity by closer proximity to the photosensitizer and by increasing particle accumulation in the tumor.METHODS/STUDY POPULATION: Gold nanoparticles were synthesized and coated with amine-terminated poly(ethylene) glycol, then covalently conjugated to chlorin e6, a known FDA-approved photosensitizer. The system was characterized using UV-Vis spectroscopy, transmission electron microscopy, and nanoparticle tracking analysis. The generation of reactive oxygen species was measured after X-irradiation. Enhanced cell killing was evaluated clonogenically in addition to assessment of in vivo efficacy and tumor pathology.RESULTS/ANTICIPATED RESULTS: Conjugation of the particle to the photosensitizer was achieved, and the molecule was detected by UV-Vis spectroscopy. TEM and NTA showed no aggregation of the particles, and an increase in reactive oxygen species generation was observed. The conjugates increased cell killing during radiation treatment, whereas neither the particle alone nor the photosensitizer significantly affected clonogenic survival at the same concentrations. Breast tumors grown in immunocompetent mice showed increased necrotic tissue after a single 20 gy treatment in the presence of the conjugate.DISCUSSION/SIGNIFICANCE OF IMPACT: Radiation therapy is widely used clinically, but dosage is limited largely to prevent injury to adjacent normal tissue. By increasing the local effect of radiation therapy, our gold conjugate has the potential to augment the effective radiation dose in the tumor, thereby reducing damage to healthy tissue and providing a more effective therapy.

There is a critical need to identify patients with radiation-resistant tumors early after treatment commencement. In this study, we use diffuse reflectance spectroscopy (DRS) to investigate changes in vascular oxygenation and total hemoglobin concentration in A549 radiation-sensitive and resistant tumors treated with a clinically relevant dose fraction of 2 Gy. DRS spectra were acquired before, immediately after, 24, and 48 hours after radiation. Our data reveals a significantly higher reoxygenation (sO2) in the radiation-resistant tumors 24 and 48h after treatment, and provides promising evidence that DRS can discern between the reoxygenation trends of radiation-sensitive and resistant tumors.

We previously explored the use of antibody-conjugated, antibiotic-loaded gold nanocages for the treatment of bacterial infections. Using Staphylococcus aureus as a proof-of-principle pathogen, we confirmed that nanocages coated with polydopamine and loaded with daptomycin could be effectively targeted to bacterial cells using an antibody targeting S. aureus surface-associated protein A. We also confirmed that laser irradiation could then be used to achieve a lethal photothermal effect and localized release of the antibiotic, the synergistic effect of which was capable of eradicating viable bacteria even from a therapeutically recalcitrant biofilm. To assess the possibility that this comes at the cost of adverse side effects, we used multispectral optoacoustic tomography (MSOT) to track the biodistribution of our nanocages following intravenous administered and determined whether their administration was associated with toxic side effects. The results of our MSOT analysis confirmed that our nanocages accumulate primarily in the liver, spleen and kidney irrespective of infection status. However, in an infected animal, they also confirmed that nanocages ultimately do reach the site of infection. MSOT results were consistent with studies involving the direct analysis of these tissues, which confirmed the correlation between MSOT signals and the presence of gold nanocages. More importantly, they also demonstrated that the presence of nanocages was not associated with appreciable histopathology in the spleen, liver, kidney, lung or heart. This suggests that our use of antibody-conjugated, antibiotic-loaded gold nanocages for the treatment of infection offers significant promise that would not be compromised by systemic toxicity.

We previously demonstrated that a photoactivatable therapeutic approach employing antibiotic-loaded, antibody-conjugated, polydopamine (PDA)-coated gold nanocages (AuNCs) could be used for the synergistic killing of bacterial cells within a biofilm. The approach was validated with a focus on Staphylococcus aureus using an antibody specific for staphylococcal protein A (Spa) and an antibiotic (daptomycin) active against Gram-positive cocci including methicillin-resistant S. aureus (MRSA). However, an important aspect of this approach is its potential therapeutic versatility.In this report, we evaluated this versatility by examining the efficacy of AuNC formulations generated with alternative antibodies and antibiotics targeting S. aureus and alternative combinations targeting the Gram-negative pathogen Pseudomonas aeruginosa.The results confirmed that daptomycin-loaded AuNCs conjugated to antibodies targeting two different S. aureus lipoproteins (SACOL0486 and SACOL0688) also effectively kill MRSA in the context of a biofilm. However, our results also demonstrate that antibiotic choice is critical. Specifically, ceftaroline and vancomycin-loaded AuNCs conjugated to anti-Spa antibodies were found to exhibit reduced efficacy relative to daptomycin-loaded AuNCs conjugated to the same antibody. In contrast, gentamicin-loaded AuNCs conjugated to an antibody targeting a conserved outer membrane protein were highly effective against P. aeruginosa biofilms.These results confirm the therapeutic versatility of our approach. However, to the extent that its synergistic efficacy is dependent on the ability to achieve both a lethal photothermal effect and the thermally controlled release of a sufficient amount of antibiotic, they also demonstrate the importance of carefully designing appropriate antibody and antibiotic combinations to achieve the desired therapeutic synergy.

Recent advances in nanoparticle synthesis have created new potential avenues for aqueous catalysis of organic reactions. Morphological control of metal nanoparticles often involves the use of surface ligands, which in turn affect the catalytic activity of nanocatalysts. This Letter demonstrates that surface anchoring group, chain length, and configuration of the water-soluble, polymeric ligands influence the catalytic properties of alloyed AuCu 3 nanorods for metal-catalyzed organic reactions. Due to the binding affinity of the surface-anchoring groups, a thiol anchor was found to be detrimental the Au-catalyzed reduction of p -nitrophenol while the Cu-catalyzed azide–alkyne cycloaddition was severely inhibited by amine anchors. Furthermore, the catalytic activity of nanorods increased with increased dimension of the ligands with the same anchoring group due to the reduction of graft density. Elevated temperature facilitates the mobility of surface ligands in benzonitrile hydration to benzamide, resulting in the enhancement of catalytic activity. This work highlights the paramount importance of surface ligand selection in the design of nanocatalysts for catalytic organic reactions.

The rapid academic and industrial development of nanotechnology has led to its implementation in laboratory teaching for undergraduate-level chemistry and engineering students. This laboratory experiment introduces the galvanic replacement reaction for synthesis of hollow metal nanoparticles and investigates the optical properties of these nanoparticles. Changes of localized surface plasmon resonance from solid to hollow nanoparticles are monitored by UV–vis spectroscopy, while corresponding morphological changes are validated by transmission electron microscopy. The optical properties are further investigated by the use of a laser pointer, laser irradiation, and surface-enhanced Raman spectroscopy. By performing these experiments, students gain practical knowledge for the synthesis of hollow metal nanoparticles and the underlying structure-dependent optical properties.

An optical phenomena based on the incident light absorption and transduction to the detectable thermal signal by plasmonic bimetallic Ag and Au nanocages (Ag@AuNCs) has been researched on free standing layer-by-layer (LbL) films of poly(vinyl alcohol) (PVA) and poly(vinyl pyrrolidone) (PVPON), (PVA/PVPON_Ag@AuNCs). Unlike well-studied monometallic Au nanocages (NCs), which possess a photothermal response in the near-infrared region, the bimetallic nanocages show a pronounced photothermal response in the visible range (532 nm) and near-infrared range (780 nm) due to the presence of two characteristic peaks at both wavelength ranges. The photothermal response in the visible range (532 nm) is distinguishable. Specifically increasing the laser power to 100 mW led to visual burning of the free standing film (temperature increased greater than >150 °C). The photothermal response by (PVA/PVPON_Ag@AuNCs)n films increases in proportion to the number (n) of bilayers (bl). It also increases as the molar concentration of the Ag@AuNCs introduced to the PVPON layer is increased. Therefore the molar concentration of the plasmonic Ag@AuNCs in (PVA/PVPON_Ag@AuNCs)n films is a primary factor that affects the photothermal dynamic response along with Ag@AuNCs distribution. This is supposed to result from the Ag@AuNCs assembled in a layer that leads to electromagnetic field enhancement. The unusual observation in multilayered (PVA/PVPON_Ag@AuNCs)n films is that the UV-visible spectra (extinction efficiency) and photothermal response (Tmax) do not rely on the content of the adjacent layer of PVA and show a comparable (by value in magnitude) photothermal response at a different PVA composition of 2 mg ml−1 and 20 mg ml−1 at the same Ag@AuNCs concentration in the PVPON layer.

An ongoing need for new cancer therapeutics exists, especially ones that specifically home and target triple-negative breast cancer. Because triple-negative breast cancer express low or are devoid of estrogen, progesterone, or Her2/Neu receptors, another target must be used for advanced drug delivery strategies. Here, we engineered a nanodrug delivery system consisting of silver-coated gold nanorods (AuNR/Ag) targeting epithelial cell adhesion/activating molecule (EpCAM) and loaded with doxorubicin. This nanodrug system, AuNR/Ag/Dox-EpCAM, was found to specifically target EpCAM-expressing tumors compared to low EpCAM-expressing tumors. Namely, the nanodrug had an effective dose (ED50) of 3 μM in inhibiting 4T1 cell viability and an ED50 of 110 μM for MDA-MD-231 cells. Flow cytometry data indicated that 4T1 cells, on average, express two orders of magnitude more EpCAM than MDA-MD-231 cells, which correlates with our ED50 findings. Moreover, due to the silver coating, the AuNR/Ag can be detected simultaneously by surface-enhanced Raman spectroscopy and photoacoustic microscopy. Analysis by these imaging detection techniques as well as by inductively coupled plasma mass spectrometry showed that the targeted nanodrug system was taken up by EpCAM-expressing cells and tumors at significantly higher rates than untargeted nanoparticles (p, Targeted drug delivery for triple-negative breast cancer Silver-coated gold nanorods deliver drugs to a difficult-to-treat breast cancer by targeting an over-expressed antigen on its surface. Ruud Dings and colleagues at the University of Arkansas in the USA loaded the chemotherapeutic drug doxorubicin onto silver-coated gold nanorods that were conjugated with an antibody that specifically targets an over-expressed antigen on many types of ‘triple-negative breast cancers’ (TNBCs). Unlike other breast cancers, TNBCs lack certain receptors, making them difficult to target with cancer therapies. The team found that one of the two TNBC cell lines studied over-expressed the epithelial antigen EpCAM 100 times more than the other. Their drug-loaded silver-coated gold nanorods specifically targeted the EpCAM over-expressing cells over the low-expressing ones. The nanorods’ coatings also allowed them to be easily detected by two different imaging techniques: surfaced-enhanced Raman spectroscopy and photoacoustic microscopy.

Purpose: To demonstrate delivery of Au nanocages to cells using the galectin-1 binding peptide anginex (Ax) and to demonstrate the value of this targeting for selective in vitro photothermal cell killing. Materials and methods: Au nanocages were synthesised, coated with polydopamine (PDA), and conjugated with Ax. Tumour and endothelial cell viability was measured with and without laser irradiation. Photoacoustic (PA) mapping and PA flow cytometry were used to confirm cell targeting in vitro and in tissue slices ex vivo. Results: Cell viability was maintained at ≥50% at 100 pM suggesting low toxicity of the nanocage alone. Combining the targeted construct (25 pM) with low power 808 nm laser irradiation for 10–20 min (a duration previously shown to induce rapid and sustained heating of Au nanocages [AuNC] in solution), resulted in over 50% killing of endothelial and tumour cells. In contrast, the untargeted construct combined with laser irradiation resulted in negligible cell killing. We estimate approximately 6 × 104 peptides were conjugated to each nanocage, which also resulted in inhibition of cell migration. Binding of the targeted nanocage reached a plateau after three hours, and cell association was 20-fold higher than non-targeted nanocages both in vitro and ex vivo on tumour tissue slices. A threefold increase in tumour accumulation was observed in preliminary in vivo studies. Conclusions: These studies demonstrate Ax’s potential as an effective targeting agent for Au-based theranostics to tumour and endothelial cells, enabling photothermal killing. This platform further suggests potential for multimodal in vivo therapy via next-generation drug-loaded nanocages.

We have demonstrated that gold nanocage-photosensitizer conjugates can enable dual image-guided delivery of photosensitizer and significantly improve the efficacy of photodynamic therapy in a murine model. The photosensitizer, 3-devinyl-3-(1'-hexyloxyethyl)pyropheophorbide (HPPH), was noncovalently entrapped in the poly(ethylene glycol) monolayer coated on the surface of gold nanocages. The conjugate is stable in saline solutions, while incubation in protein rich solutions leads to gradual unloading of the HPPH, which can be monitored optically by fluorescence and photoacoustic imaging. The slow nature of the release in turn results in an increase in accumulation of the drug within implanted tumors due to the passive delivery of gold nanocages. Furthermore, the conjugate is found to generate more therapeutic singlet oxygen and have a lower IC50 value than the free drug alone. Thus the conjugate shows significant suppression of tumor growth as compared to the free drug in vivo. Short-term study showed neither toxicity nor phenotypical changes in mice at therapeutic dose of the conjugates or even at 100-fold higher than therapeutic dose of gold nanocages.

Background and Objective: Fecal microbiota transplantation helps restore beneficial bacteria in cancer patients after intense antibiotics administration. Here we investigated the influence of different stabilization and storage strategies on the quality and composition of the fecal microbial community. Methods: Same-day isolated murine DNA was compared to samples stored for one month in air at ambient temperature, with or without preservative buffers (i.e. EDTA and lysis buffer), different temperatures (i.e. 4°C, -20°C, and -80°C), and hypoxic conditions. Results: Only storage in lysis buffer significantly reduced DNA content, yet without integrity loss. Storage in EDTA affected alpha diversity the most, which was also reflected in cluster separation. Distinct changes were also seen in the phyla and bacterial species abundance per storage strategy. Metabolic function analysis showed 22 pathways not significantly affected by storage conditions, whereas the tyrosine metabolism pathway was significantly changed in all strategies except by EDTA. Conclusions: Overall, each long-term storage strategy introduced a unique post-collection bias, which is important to take into account when interpreting data. Funded by COBRE P20GM103625. Citation Format: Samir V. Jenkins, Kieng B. Vang, Allen Gies, Robert J. Griffin, Se-Ran Jun, Intawat Nookaew, Ruud Petrus Dings. Sample storage conditions alter microbiome profiles - its potential relevance for cancer patients undergoing fecal microbial transplants [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2823.

A general strategy to synthesize Cu–M (M = Au, Pt, or Pd) bimetallic nanorods has been demonstrated based on a seeded co-reduction method. In this approach, noble metal nanoparticles serve as seeds, and newly reduced Cu atoms are subsequently nucleated on one side of the seeds, resulting in Janus nanoparticles with an M-rich and a Cu-rich portion. The elongation of the particles originates from the site-specific deposition of Cu clusters on the Cu-rich side of these Janus nanoparticles by retarding reduction kinetics of Cu through galvanic replacement. Using this approach, Cu–M alloyed nanorods can be conveniently synthesized with tunable composition, crystal structure, and aspect ratio. These nanorods have also been demonstrated as a unique system for investigation of the structural and compositional effects on their optical and catalytic properties.

Resistance to conventional antibiotics is a growing public health concern that is quickly outpacing the development of new antibiotics. This has led the Infectious Diseases Society of America (IDSA) to designate Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species as “ESKAPE pathogens” on the basis of the rapidly decreasing availability of useful antibiotics. This emphasizes the urgent need for alternative therapeutic strategies to combat infections caused by these and other bacterial pathogens. In this study, we used Staphylococcus aureus (S. aureus) as a proof-of-principle ESKAPE pathogen to demonstrate that an appropriate antibiotic (daptomycin) can be incorporated into polydopamine-coated gold nanocages (AuNC@PDA) and that daptomycin-loaded AuNC@PDA can be conjugated to antibodies targeting a species-specific surface protein (staphylococcal protein A; Spa) as a means of achieving selective delivery of the nanoconstructs directly to the bacterial cell surface. Targeting specificity was confirmed by demonstrating a lack of binding to mammalian cells, reduced photothermal and antibiotic killing of the Spa-negative species Staphylococcus epidermidis, and reduced killing of S. aureus in the presence of unconjugated anti-Spa antibodies. We demonstrate that laser irradiation at levels within the current safety standard for use in humans can be used to achieve both a lethal photothermal effect and controlled release of the antibiotic, thus resulting in a degree of therapeutic synergy capable of eradicating viable S. aureus cells. The system was validated using planktonic bacterial cultures of both methicillin-sensitive and methicillin-resistant S. aureus strains and subsequently shown to be effective in the context of an established biofilm, thus indicating that this approach could be used to facilitate the effective treatment of intrinsically resistant biofilm infections.

The centrifugation method was proposed to extract Au nanocages (AuNCs) from a mixture of the bimetallic Au and Ag alloyed nanocages, Ag and Au core–shell structure, and Au-pitted silver nanocubes. The DDA simulation was found to be a powerful tool to characterize a colloidal dispersion of the plasmonic nanoparticles. The effect of the irregularity of Au hollow cages and presence of the polymer shells around the cages on the surface plasmon resonance spectra (SPR) had been suggested based on the DDA simulations.

The structure of both the free base and the hydrochloride of an unnatural analogue of prodigiosin, 4-methoxy-5-((4,5-dimethyl -2H-pyrrol-2-ylidene)methyl)- 2,2′-bi-1H-pyrrole, are reported. The free base crystallizes from methylene chloride as a hydrogen bonded dimer. The structure of the hemihydrate hydrochloride of the related 4-methoxy-5-((1-methyl -2H-pyrrol-2-ylidene)methyl)- 2,2′-bi-1H-pyrrole is also reported.

Non-covalent incorporation of hydrophobic drugs into polymeric systems is a commonly-used strategy for drug delivery because non-covalent interactions minimize modification of the drug molecules whose efficacy is retained upon release. The behaviors of the drug-polymer delivery system in the biological environments it encounters will affect the efficacy of treatment. In this report, we have investigated the interaction between a hydrophobic drug and its encapsulating polymer in model biological environment using a photosensitizer encapsulated in a polymer-coated nanoparticle system. The photosensitizer, 3-(1′-hexyloxyethyl)-3-devinylpyropheophorbide-a (HPPH), was non-covalently incorporated to the poly(ethylene glycol) (PEG) layer coated on Au nanocages (AuNCs) to yield AuNC-HPPH complexes. The non-covalent binding was characterized by Scatchard analysis, fluorescence lifetime, and Raman experiments. The dissociation constant (Kd) between PEG and HPPH was found to be ~35 μM with a maximum loading of ~2.5 × 105 HPPHs/AuNC. The release was studied in serum mimetic environment and in vesicles that model human cell membranes. The rate of protein-mediated drug release decreased when using a negatively-charged or cross-linked terminus of the surface-modified PEG. Furthermore, the photothermal effect of AuNCs can initiate burst release, and thus allow control of the release kinetics, demonstrating on-demand drug release. This study provides insights regarding the actions and release kinetics of non-covalent drug delivery systems in biological environments.

Due to their robust optical properties, biological inertness, and readily adjustable surface chemistry, gold nanostructures have been demonstrated as contrast agents in a variety of biomedical imaging applications. One application is dynamic imaging of live cells using bioconjugated gold nanoparticles to monitor molecule trafficking mechanisms within cells; for instance, the regulatory pathway of epidermal growth factor receptor (EGFR) undergoing endocytosis. In this paper, we have demonstrated a method to track endocytosis of EGFR in MDA-MB-468 breast adenocarcinoma cells using bioconjugated gold nanocages (AuNCs) and multiphoton microscopy. Dynamic imaging was performed using a time series capture of 4 images every minute for one hour. Specific binding and internalization of the bioconjugated AuNCs was observed while the two control groups showed non-specific binding at fewer surface sites, leading to fewer bound AuNCs and no internalization.

Plasmonic nanomaterials as drug delivery or bio-imaging agents are typically introduced to biological systems through intravenous administration. However, the potential for agglomeration of nanoparticles in biological systems could dramatically affect their pharmacokinetic profile and toxic potential. Development of rapid screening methods to evaluate agglomeration is urgently needed to monitor the physical nature of nanoparticles as they are introduced into blood. Here, we establish novel methods using darkfield microscopy with hyperspectral detection (hsDFM), single particle inductively-coupled plasma mass spectrometry (spICP-MS), and confocal Raman microscopy (cRM) to discriminate gold nanoparticles (AuNPs) and their agglomerates in blood. Rich information about nanoparticle agglomeration in situ is provided by hsDFM monitoring of the plasmon resonance of primary nanoparticles and their agglomerates in whole blood; cRM is an effective complement to hsDFM to detect AuNP agglomerates in minimally manipulated samples. The AuNPs and the particle agglomerates were further distinguished in blood for the first time by quantification of particle mass using spICP-MS with excellent sensitivity and specificity. Furthermore, the agglomeration status of synthesized and commercial NPs incubated in blood was successfully assessed using the developed methods. Together, these complementary methods enable rapid determination of the agglomeration status of plasmonic nanomaterials in biological systems, specifically blood.

Noble metal nanostructures are appealing for therapeutic, diagnostics, and sensing applications because of their unique optical properties and biocompatibility. The collective oscillation of electrons in a metal nanostructure resonates with particular wavelengths of light, generating the localized surface plasmon resonance (LSPR). The LSPR results in absorption and scattering of incoming photons. Absorption leads to photothermal generation of heat, photoluminescence, and quenching of fluorophores in close proximity. Scattering results in reflected photons and its amplification of the local electromagnetic field can enhance fluorescence, phosphorescence, and Raman scattering. These optical properties make plasmonic nanostructures ideal candidates for theranostic applications including light-induced thermal therapy, drug/gene delivery, and biomedical imaging. The use of plasmonic nanostructures for ex vivo detection of chemicals and biomolecules are also discussed in this chapter.

An ongoing need for new cancer therapeutics exists, especially ones that specifically home and target triple negative breast cancer (TNBC) tumors. Because TNBC does not express estrogen, progesterone, or Her2/Neu receptors, another target must be used for advanced drug delivery strategies. Here, we engineered a nanodrug delivery system consisting of silver-coated gold nanorods targeting EpCAM and loaded with doxorubicin. This novel nanodrug system, EpCAM-AuNR/Ag/Dox, was found to specifically target EpCAM-expressing tumor cells compared to low EpCAM-expressing tumors. Namely, the system had an inhibiting cell viability effective dose (ED50) of 3μM on 4T1 cells and an ED50 of 110μM for MDA-MD-231 cells. Flow cytometry indicated that 4T1 cells, on average, express two orders of magnitude more EpCAM than MDA-MD-231 cells, which correlates with our ED50 findings. Moreover, due to its thin silver coating, the AuNR/Ag allowed for detection by surface-enhanced Raman spectroscopy (SERS) and photoacoustics (PA). Analysis by both imaging detection techniques showed that the targeted nanodrug system was taken up by EpCAM-expressing cells and tumors at significantly higher rates than untargeted nanoparticles (p Citation Format: Samir V. Jenkins, Zeid A. Nima, Kieng B. Vang, Dmitry Nedosekin, Vladimir P. Zharov, Robert J. Griffin, Alexandru S. Biris, Ruud P. Dings. Breast cancer targeting by theranostic gold nanorods [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 LB-312. doi:10.1158/1538-7445.AM2017-LB-312

Radiation resistance remains a significant problem for cancer patients, especially due to the time required to definitively determine treatment outcome. For fractionated radiation therapy, nearly 7 to 8 weeks can elapse before a tumor is deemed to be radiation-resistant. We used the optical redox ratio of FAD / ( FAD + NADH ) to identify early metabolic changes in radiation-resistant lung cancer cells. These radiation-resistant human A549 lung cancer cells were developed by exposing the parental A549 cells to repeated doses of radiation (2 Gy). Although there were no significant differences in the optical redox ratio between the parental and resistant cell lines prior to radiation, there was a significant decrease in the optical redox ratio of the radiation-resistant cells 24 h after a single radiation exposure ( p = 0.01 ). This change in the redox ratio was indicative of increased catabolism of glucose in the resistant cells after radiation and was associated with significantly greater protein content of hypoxia-inducible factor 1 ( HIF - 1 ? ), a key promoter of glycolytic metabolism. Our results demonstrate that the optical redox ratio could provide a rapid method of determining radiation resistance status based on early metabolic changes in cancer cells.