Proinflammatory activity of VEGF-targeted treatment through reversal of tumor endothelial cell anergy.

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.

Exosomal MicroRNA and Protein Profiles of Hepatitis B Virus-Related Hepatocellular Carcinoma Cells.

Infection with hepatitis B virus (HBV) is a main risk factor for hepatocellular carcinoma (HCC). Extracellular vesicles, such as exosomes, play an important role in tumor development and metastasis, including regulation of HBV-related HCC. In this study, we have characterized exosome microRNA and proteins released in vitro from hepatitis B virus (HBV)-related HCC cell lines SNU-423 and SNU-182 and immortalized normal hepatocyte cell lines (THLE2 and THLE3) using microRNA sequencing and mass spectrometry. Bioinformatics, including functional enrichment and network analysis, combined with survival analysis using data related to HCC in The Cancer Genome Atlas (TCGA) database, were applied to examine the prognostic significance of the results. More than 40 microRNAs and 200 proteins were significantly dysregulated (p < 0.05) in the exosomes released from HCC cells in comparison with the normal liver cells. The functional analysis of the differentially expressed exosomal miRNAs (i.e., mir-483, mir-133a, mir-34a, mir-155, mir-183, mir-182), their predicted targets, and exosomal differentially expressed proteins (i.e., POSTN, STAM, EXOC8, SNX9, COL1A2, IDH1, FN1) showed correlation with pathways associated with HBV, virus activity and invasion, exosome formation and adhesion, and exogenous protein binding. The results from this study may help in our understanding of the role of HBV infection in the development of HCC and in the development of new targets for treatment or non-invasive predictive biomarkers of HCC.

Liposome Formulation for Tumor-Targeted Drug Delivery Using Radiation Therapy.

Targeted delivery of drugs or other therapeutic agents through internal or external triggers has been used to control and accelerate the release from liposomal carriers in a number of studies, but relatively few utilize energy of therapeutic X-rays as a trigger. We have synthesized liposomes that are triggered by ionizing radiation (RTLs) to release their therapeutic payload. These liposomes are composed of natural egg phosphatidylethanolamine (PE), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), cholesterol, and 1,2-disteroyl-sn-glycero-3-phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (DSPE-PEG-2000), and the mean size of the RTL was in the range of 114 to 133 nm, as measured by nanoparticle tracking analysis (NTA). The trigger mechanism is the organic halogen, chloral hydrate, which is known to generate free protons upon exposure to ionizing radiation. Once protons are liberated, a drop in internal pH of the liposome promotes destabilization of the lipid bilayer and escape of the liposomal contents. In proof of principle studies, we assessed RTL radiation-release of fluorescent tracers upon exposure to a low pH extracellular environment or exposure to X-ray irradiation. Biodistribution imaging before and after irradiation demonstrated a preferential uptake and release of the liposomes and their cargo at the site of local tumor irradiation. Finally, a potent metabolite of the commonly used chemotherapy irinotecan, SN-38, was loaded into RTL along with near infrared (NIR) fluorescent dyes for imaging studies and measuring tumor cell cytotoxicity alone or combined with radiation exposure, in vitro and in vivo. Fully loaded RTLs were found to increase tumor cell killing with radiation in vitro and enhance tumor growth delay in vivo after three IV injections combined with three, 5 Gy local tumor radiation exposures compared to either treatment modality alone.

Dysbiotic stress increases the sensitivity of the tumor vasculature to radiotherapy and c-Met inhibitors.

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.

Sample storage conditions induce post-collection biases in microbiome profiles.

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.

Galectin-1-based tumour-targeting for gold nanostructure-mediated photothermal therapy.

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.

Versatility of targeted antibiotic-loaded gold nanoconstructs for the treatment of biofilm-associated bacterial infections.

BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Bystander Effects in Spatially Fractionated Radiation Therapy: From Molecule To Organism To Clinical Implications.

The standard of care for radiation therapy is numerous, low-dose fractions that are distributed homogeneously throughout the tumor. An alternative strategy under scrutiny is to apply spatially fractionated radiotherapy (high and low doses throughout the tumor) in one or several fractions, either alone or followed by conventional radiation fractionation . Spatial fractionation allows for significant sparing of normal tissue, and the regions of tumor or normal tissue that received sublethal doses can give rise to beneficial bystander effects in both cases. Bystander effects are broadly defined as biological responses that are significantly greater than would be anticipated based on the radiation dose received. Typically these effects are initiated by diffusion of reactive oxygen species and secretion of various cytokines. As demonstrated in the literature, spatial fractionation related bystander effects can occur locally from cell to cell and in what are known as "cohort effects," which tend to take the form of restructuring of the vasculature, enhanced immune infiltration, and development of immunological memory. Other bystander effects can take place at distant sites in what are known as "abscopal effects." While these events are rare, they are mediated by the immune system and can result in the eradication of secondary and metastatic disease. Currently, due to the complexity and variability of these bystander effects, they are not thoroughly understood, but as knowledge improves they may present significant opportunities for improved clinical outcomes.

Acquired Radiation Resistance Induces Thiol-dependent Cisplatin Cross-resistance.

Resistance to radiation remains a significant clinical challenge in non-small cell lung carcinoma (NSCLC). It is therefore important to identify the underlying molecular and cellular features that drive acquired resistance. We generated genetically matched NSCLC cell lines to investigate characteristics of acquired resistance. Murine Lewis lung carcinoma (LLC) and human A549 cells acquired an approximate 1.5-2.5-fold increase in radiation resistance as compared to their parental match, which each had unique intrinsic radio-sensitivities. The radiation resistance (RR) was reflected in higher levels of DNA damage and repair marker γH2AX and reduced apoptosis induction after radiation. Morphologically, we found that radiation resistance A549 (A549-RR) cells exhibited a greater nucleus-to-cytosol (N/C) ratio as compared to its parental counterpart. Since the N/C ratio is linked to the differentiation state, we next investigated the epithelial-to-mesenchymal transition (EMT) phenotype and cellular plasticity. We found that A549 cells had a greater radiation-induced plasticity, as measured by E-cadherin, vimentin and double-positive (DP) modulation, as compared to LLC. Additionally, migration was suppressed in A549-RR cells, as compared to A549 cells. Subsequently, we confirmed in vivo that the LLC-RR and A549-RR cells are also more resistance to radiation than their isogenic-matched counterpart. Moreover, we found that the acquired radiation resistance also induced resistance to cisplatin, but not carboplatin or oxaliplatin. This cross-resistance was attributed to induced elevation of thiol levels. Gamma-glutamylcysteine synthetase inhibitor buthionine sulfoximine (BSO) sensitized the resistant cells to cisplatin by decreasing the amount of thiols to levels prior to obtaining acquired radiation resistance. By generating radiation-resistance genetically matched NSCLC we were able to identify and overcome cisplatin cross-resistance. This is an important finding arguing for combinatorial treatment regimens including glutathione pathway disruptors in patients with the potential of improving clinical outcomes in the future.

Proteomic analysis of transcription factors involved in the alteration of ischemic mouse heart as modulated by MSC exosomes.

Mesenchymal stem cell (MSC) exosomes have been found to attenuate cardiac systolic and diastolic dysfunction in animal models of ischemia. Exosomes carry a plethora of active and inactive proteins as their cargo, which are readily available to the recipient cell for use in intracellular signaling pathways-depending on the stresses, such as ischemia or hypoxia. Among the exosomal proteins are the often-overlooked cargo of transcriptional regulators. These transcriptional regulators influence the transcriptome and subsequently the proteome of recipient cell. Here, we report the transcriptional factors and regulators differentially modulated and their potential role in modulating cardiac function in MSC exosome treated ischemic mice hearts. Our analysis shows ischemic stress modulating transcriptional regulators and factors such as HSF1 and HIF1A in the infarct and peri-infarct areas of ischemic hearts which is mitigated by MSC exosomes. Similarly, STAT3 and SMAD3 are also modulated by MSC exosomes. Interestingly, NOTCH1 and ß-catenin were detected in the ischemic hearts. The differential expression of these regulators and factors drives changes in various biological process governed in the ischemic cardiac cells. We believe these studies will advance our understanding of cardiac dysfunction occurring in the ischemic hearts and lay the groundwork for further studies on the modulation of cardiac function during ischemia by MSC exosomes.

Morphological Effects and In Vitro Biological Mechanisms of Radiation-Induced Cell Killing by Gold Nanomaterials.

Gold nanomaterials have been shown to augment radiation therapy both in vitro and in vivo. However, studies on these materials are mostly phenomenological due to nanoparticle heterogeneity and the complexity of biological systems. Even accurate quantification of the particle dose still results in bulk average biases; the effect on individual cells is not measured but rather the effect on the overall population. To perform quantitative nanobiology, we coated glass coverslips uniformly at varying densities with Au nanoparticle preparations with different morphologies (45 nm cages, 25 nm spheres, and 30 nm rods). Consequently, the effect of a specific number of particles per unit area in contact with breast cancer cells growing on the coated surfaces was ascertained. Gold nanocages showed the highest degree of radiosensitization on a per particle basis, followed by gold nanospheres and gold nanorods, respectively. All three materials showed little cytotoxic effect at 0 Gy, but clonogenic survival decreased proportionally with the radiation dose and particle coverage density. A similar trend was seen in vivo in the combined treatment antitumor response in 4T1 tumor-bearing animals. The presence of gold affected the type and quantity of reactive oxygen species generated, specifically superoxide and hydroxyl radicals, and the concentration of nanocages correlated with the development of more numerous double-stranded DNA breaks and increased protein oxidation as measured by carbonylation. This work demonstrates the dependence on morphology and concentration of radiation enhancement by gold nanomaterials and may lead to a novel method to differentiate intra- and extracellular functionalities of gold nanomedicine treatment strategies. It further provides insights that can guide the rational development of gold nanomaterial-based radiosensitizers for clinical use.

ß-Glucosidase Immobilized on Magnetic Nanoparticles: Controlling Biomolecule Footprint and Particle Functional Group Density to Navigate the Activity-Stability Tradeoff.

In the present work, the immobilized footprint of ß-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 × 10-5 mol/g particle. The amine-modified particles were activated with glutaraldehyde, and subsequently, BGL was immobilized using either a 0.1 or 1 mg/mL enzyme solution to produce biomolecules with a large or small footprint on the particle surface. The initial activity, activity for subsequent hydrolysis cycles, activity after extended storage, and biomolecule footprint were studied as a function of APTES density and concentration of enzyme used for immobilization. At high immobilization amounts, the specific activity and footprint were reduced, but the immobilized biomolecules were stable during storage. However, at low enzyme immobilizations, the activity of the enzymes was retained, the immobilized enzymes adopted large footprints, and the storage stability increased with APTES density relative to the free enzyme. These results highlight how controlling both the protein load and functional group density can yield immobilized enzymes possessing high activity, which are stable during storage.

Photothermal Response Induced by Nanocage-Coated Artificial Extracellular Matrix Promotes Neural Stem Cell Differentiation.

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.

Evidence for Early Stage Anti-Tumor Immunity Elicited by Spatially Fractionated Radiotherapy-Immunotherapy Combinations.

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, immunohistochemistry 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.

Enhanced Photothermal Treatment Efficacy and Normal Tissue Protection via Vascular Targeted Gold Nanocages.

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.

Gastrointestinal Tract Dysbiosis Enhances Distal Tumor Progression through Suppression of Leukocyte Trafficking.

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.

Label-Free Raman Spectroscopy Reveals Signatures of Radiation Resistance in the Tumor Microenvironment.

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.

Quantitative diffuse reflectance spectroscopy of short-term changes in tumor oxygenation after radiation in a matched model of radiation resistance.

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.

Rapid quantification of mitochondrial fractal dimension in individual cells.

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.

A Radiosensitizing Inhibitor of HIF-1 alters the Optical Redox State of Human Lung Cancer Cells In Vitro.

Treatment failure caused by a radiation-resistant cell phenotype remains an impediment to the success of radiation therapy in cancer. We recently showed that a radiation-resistant isogenic line of human A549 lung cancer cells had significantly elevated expression of hypoxia-inducible factor (HIF-1α), and increased glucose catabolism compared with the parental, radiation-sensitive cell line. The objective of this study was to investigate the longitudinal metabolic changes in radiation-resistant and sensitive A549 lung cancer cells after treatment with a combination of radiation therapy and YC-1, a potent HIF-1 inhibitor. Using label-free two-photon excited fluorescence microscopy, we determined changes in the optical redox ratio of FAD/(NADH and FAD) over a period of 24 hours following treatment with YC-1, radiation, and both radiation and YC-1. To complement the optical redox ratio, we also evaluated changes in mitochondrial organization, glucose uptake, reactive oxygen species (ROS), and reduced glutathione. We observed significant differences in the optical redox ratio of radiation-resistant and sensitive A549 cells in response to radiation or YC-1 treatment alone; however, combined treatment eliminated these differences. Our results demonstrate that the optical redox ratio can elucidate radiosensitization of previously radiation-resistant A549 cancer cells, and provide a method for evaluating treatment response in patient-derived tumor biopsies.