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The fundamental role that NAD(P)H/quinone oxidoreductase 1 (NQO1) plays, in normal cells, as a cytoprotective enzyme guarding against stress induced by reactive oxygen species (ROS) is well documented. However, what is not known is whether the observed overexpression of NQO1 in neoplastic cells contributes to their survival. The current study discovered that depleting NQO1 expression in A549 and H292 lung adenocarcinoma cells caused an increase in ROS formation, inhibited anchorage-independent growth, increased anoikis sensitization, and decreased three-dimensional tumor spheroid invasion. These in vivo data further implicate tumor-NQO1 expression in a protumor survival role, because its depletion suppressed cell proliferation and decreased lung tumor xenograft growth. Finally, these data reveal an exploitable link between tumor-NQO1 expression and the survival of lung tumors because NQO1 depletion significantly decreased the percentage of ALDH(high) cancer cells within the tumor population.Implications: Loss of tumor-NQO1 expression inhibits tumor growth and suggests that novel therapeutics directed at tumor-NQO1 may have clinical benefit. Mol Cancer Res; 14(1); 14–25. ©2015 AACR.

Supplemental Figure Legends 1-14

Supplemental Figure 1: Tumor-NQO1 overexpression leads to poor prognosis in lung cancer patients. Supplemental Figure 2: shNQO1-B knockdown of NQO1 leads to decreased growth in soft agar. Supplemental Figure 3: shNQO1-B knockdown of NQO1 in H292 cells leads to decreased growth in soft agar. Supplemental Figure 4: Dicoumarol inhibits growth of A549 cells in soft agar. Supplemental Figure 5: Mac220 inhibits growth of A549 cells in soft agar. Supplemental Figure 6: Stable overexpression of NQO1 in H596 (NQO1 null) lung cancer cells causes increased growth in soft agar. Supplemental Figure 7: Transient knockdown of NQO1 in H596 LPC-NQO1 cells causes decreased growth in soft agar. Supplemental Figure 8: shNQO1-B knockdown of NQO1 in A549 cells decreases cell invasion. Supplemental Figure 9: Stable overexpression of NQO1 in H596 NQO1 null cells causes increased invasion. Supplementary Figure 10: Transient knockdown of NQO1 using siRNA inhibits invasion of HCC1171 cells. Supplemental Figure 11: Stable knockdown of NQO1 in A549 cells causes loss of PARP- 1 protein expression. Supplemental Figure 12: Knockdown of NQO1 reduces ALDH (high) activity in Pancreas cancer cells. Supplemental Figure 13: Knockdown of NQO1 reduces ALDH (high) activity in Prostate cancer cells. Supplemental Figure 14: NQO1 knockdown does not inhibit short-term viability our long-term survival of non-transformed human bronchial epithelial cells.

Distribution of targeted (cRGD) and non-targeted (cRAD) C' dots at early (3 hr) timepoints in a genetically-engineered mouse model of glioma.

All supplemental data

Scheme depicting the experimental approach for in vivo studies

Supplemental Video 1a from Ultrasmall Core-Shell Silica Nanoparticles for Precision Drug Delivery in a High-Grade Malignant Brain Tumor Model

Supplementary Data from Ultrasmall Nanoparticle Delivery of Doxorubicin Improves Therapeutic Index for High-Grade Glioma

Word document including all supplemental figures and text

Purpose:Despite dramatic growth in the number of small-molecule drugs developed to treat solid tumors, durable therapeutic options to control primary central nervous system malignancies are relatively scarce. Chemotherapeutic agents that appear biologically potent in model systems have often been found to be marginally effective at best when given systemically in clinical trials. This work presents for the first time an ultrasmall (Experimental Design:This work presents first-in-kind renally clearable ultrasmall (Results:Optimal drug-per-particle ratios of as-developed nanoparticle–drug conjugates were established and used to obtain favorable pharmacokinetic profiles. The in vivo efficacy results showed significantly improved biological, therapeutic, and toxicological properties over the native drug after intravenous administration in platelet-derived growth factor–driven genetically engineered mouse model, and an EGF-expressing patient-derived xenograft (EGFR PDX) model.Conclusions:Ultrasmall C′ dot–drug conjugates showed great translational potential over DOX for improving the therapeutic outcome of patients with high-grade gliomas, even without a cancer-targeting moiety.

Table reviewing nanoparticle drug delivery systems in brain tumor models

Purpose:Small-molecule inhibitors have revolutionized treatment of certain genomically defined solid cancers. Despite breakthroughs in treating systemic disease, central nervous system (CNS) metastatic progression is common, and advancements in treating CNS malignancies remain sparse. By improving drug penetration across a variably permeable blood–brain barrier and diffusion across intratumoral compartments, more uniform delivery and distribution can be achieved to enhance efficacy.Experimental Design:Ultrasmall fluorescent core-shell silica nanoparticles, Cornell prime dots (C' dots), were functionalized with αv integrin-binding (cRGD), or nontargeting (cRAD) peptides, and PET labels (124I, 89Zr) to investigate the utility of dual-modality cRGD-C' dots for enhancing accumulation, distribution, and retention (ADR) in a genetically engineered mouse model of glioblastoma (mGBM). mGBMs were systemically treated with 124I-cRGD- or 124I-cRAD-C' dots and sacrificed at 3 and 96 hours, with concurrent intravital injections of FITC-dextran for mapping blood–brain barrier breakdown and the nuclear stain Hoechst. We further assessed target inhibition and ADR following attachment of dasatinib, creating nanoparticle–drug conjugates (Das-NDCs). Imaging findings were confirmed with ex vivo autoradiography, fluorescence microscopy, and p-S6RP IHC.Results:Improvements in brain tumor delivery and penetration, as well as enhancement in the ADR, were observed following administration of integrin-targeted C' dots, as compared with a nontargeted control. Furthermore, attachment of the small-molecule inhibitor, dasatinib, led to its successful drug delivery throughout mGBM, demonstrated by downstream pathway inhibition.Conclusions:These results demonstrate that highly engineered C' dots are promising drug delivery vehicles capable of navigating the complex physiologic barriers observed in a clinically relevant brain tumor model.

Supplementary Figure from Ultrasmall Nanoparticle Delivery of Doxorubicin Improves Therapeutic Index for High-Grade Glioma

Purpose:Small-molecule inhibitors have had a major impact on cancer care. While treatments have demonstrated clinically promising results, they suffer from dose-limiting toxicities and the emergence of refractory disease. Considerable efforts made to address these issues have more recently focused on strategies implementing particle-based probes that improve drug delivery and accumulation at target sites, while reducing off-target effects.Experimental Design:Ultrasmall (in vivo biological and therapeutic properties of a prototype epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, gefitinib. Novel surface chemical components were used to conjugate gefitinib–dipeptide drug-linkers and deferoxamine (DFO) chelators for therapeutic delivery and PET imaging labels, respectively.Results:Gefitinib-bound C′ dots (DFO-Gef-C′ dots), synthesized using the gefitinib analogue, APdMG, at a range of drug-to-particle ratios (DPR; DPR = 11–56), demonstrated high stability for DPR values≤ 40, bulk renal clearance, and enhanced in vitro cytotoxicity relative to gefitinib (LD50 = 6.21 nmol/L vs. 3 μmol/L, respectively). In human non–small cell lung cancer mice, efficacious Gef-C′ dot doses were at least 200-fold lower than that needed for gefitinib (360 nmoles vs. 78 μmoles, respectively), noting fairly equivalent tumor growth inhibition and prolonged survival. Gef-C′ dot–treated tumors also exhibited low phosphorylated EFGR levels, with no appreciable wild-type EGFR target inhibition, unlike free drug.Conclusions:Results underscore the clinical potential of DFO-Gef-C′ dots to effectively manage disease and minimize off-target effects at a fraction of the native drug dose.

Synthesis scheme of cRGDY-Das-NDCs

Images of p-S6RP IHC performed on additional treatment and control mouse brain tissues

Representative images of I-124-cRGD-C' dot uptake and distribution in primary human glioma; part of an ongoing Phase 1 trial

Cellular binding and internalization of cRGD-C' dots in glioma cells

Identifying cellular drivers responsible for enhancing cancer cell resistance to therapeutics provides critical information for designing more effective drugs. Populations of slowly growing, self-renewing, chemo-resistant cells purportedly contribute to the development of therapeutic resistance in many solid tumors. In the current study, we implemented a tumor spheroid model to determine whether NAD(P)H quinone oxidoreductase-1 (NQO1) was requisite for self-renewal and promotion of the drug-resistant phenotype in non-small cell lung cancer (NSCLC). We found that stable depletion of NQO1 in A549 and H358 human NSCLC tumor models inhibits self-renewal capabilities, as demonstrated by a reduced ability to form primary, secondary, and tertiary spheroids. In contrast, the rescue of NQO1 expression restored the tumor cells’ ability to form spheroids. Additionally, we discovered that NQO1 depletion renders cisplatin-refractory tumor spheroids highly susceptible to drug treatment. Together, these results suggest that NQO1 loss reduces the self-renewing capabilities of NSCLC cells and enhances their susceptibility to clinically relevant therapeutics. These findings describe a novel role for NQO1 and suggest that combining NQO1-inhibitors with conventional chemotherapeutics may enhance anti-tumor effects.

Purpose: Despite dramatic growth in the number of small-molecule drugs developed to treat solid tumors, durable therapeutic options to control primary central nervous system malignancies are relatively scarce. Chemotherapeutic agents that appear biologically potent in model systems have often been found to be marginally effective at best when given systemically in clinical trials. This work presents for the first time an ultrasmall ( Experimental Design: This work presents first-in-kind renally clearable ultrasmall ( Results: Optimal drug-per-particle ratios of as-developed nanoparticle–drug conjugates were established and used to obtain favorable pharmacokinetic profiles. The in vivo efficacy results showed significantly improved biological, therapeutic, and toxicological properties over the native drug after intravenous administration in platelet-derived growth factor–driven genetically engineered mouse model, and an EGF-expressing patient-derived xenograft (EGFR PDX) model. Conclusions: Ultrasmall C′ dot–drug conjugates showed great translational potential over DOX for improving the therapeutic outcome of patients with high-grade gliomas, even without a cancer-targeting moiety.

Although important advances have been achieved in the development of radiolabeled prostate-specific membrane antigen (PSMA)-targeting ligand constructs for both diagnosis and therapy of prostate cancer (PCa) over the past decade, challenges related to off-target effects and limited treatment responses persist. In this study, which builds upon the successful clinical translation of a series of ultrasmall, dye-encapsulating core-shell silica nanoparticles, or Cornell Prime Dots (C′ dots), for cancer management, we sought to address these limitations by designing a dual-modality, PSMA-targeting platform that evades undesirable accumulations in the salivary glands, kidneys, and reticuloendothelial system, while exhibiting bulk renal clearance. This versatile PCa-targeted particle imaging probe offers significant clinical potential to improve future theranostic applications in a variety of patient care settings.

Lung cancer is the leading cause of cancer related deaths worldwide. The five-year survival rates for those patients suffering from non-small cell lung cancer (NSCLC), continues to be an abysmal 15%. One of the major reasons for the poor survival rate amongst NSCLC patients is the lack of early detection and subsequent late stage initial diagnosis. Tumors discovered at later stages are often refractory toward chemotherapy and radiation regimens. One theory as to why tumors become resistant to therapy relies heavily on the cells that make up the cancer stem cell (CSC) niche. This small percentage of cells within the heterogeneous tumor has been reported to be responsible for drug resistance, tumor recurrence, and metastasis. In general, CSCs have been isolated using a number of different markers, including cluster differentiation markers, somatic stem cell markers, as well as a number of functional markers such as the side population and aldehyde dehydrogenase (ALDH) activity. While some cancer types, such as breast and hematologic cancers, have been significantly investigated to identify and define their CSC population, lung cancer researchers have only recently begun to identify CSC markers in lung tumors. In addition to the CSC population, malignant cells can also alter their expression of a number of cytoprotective genes that promote tumorigenesis. NAD(P)H:quinone oxidoreductase 1 (NQO1) is a detoxifying enzyme that has been demonstrated to be highly overexpressed in a number of different malignancies. This overexpression has been utilized as a drug target, as the enzyme is expressed at low levels in normal tissue. To this point, there has been success in using NQO1 as a drug target, however little research has been conducted on understanding why NQO1 is overexpressed in these malignancies. The work presented here investigated the role of NQO1 in tumorigenesis as well as its role in maintaining the CSC population in NSCLC. We demonstrate that NQO1 promotes anchorage-independent growth, invasion, reactive oxygen species regulation, anoikis resistance, proliferation, in vivo tumor growth, survival, and ALDH activity. Secondly, we demonstrate that NQO1 also promotes spheroid formation, both in initial and serial contexts, enhances the CSC frequency, and protects spheroid-cultured cells from chemotherapy. Finally, we provide preliminary data that indicates that NQO1 mRNA may be playing an important signaling role in the promotion of the CSC phenotype. This was demonstrated by CRISPR-Cas9 genetic knockout of NQO1 that resulted in a reemergence of the CSC phenotype that can be reversed with transient knockdown of NQO1 mRNA. In summary, our data demonstrate that NQO1 is playing a vital role in the promotion of NSCLC tumorigenesis, as well as supporting the cancer stem cell population. Interestingly, these results may be due to a novel signaling mechanism by NQO1 mRNA, and not the enzyme itself. Further research will be needed to completely understand the role of NQO1 mRNA in NSCLC tumorigenesis and the CSC phenotype.

Key Points Question Can the favorable properties of an ultrasmall fluorescent core-shell silica nanoparticle aid real-time image-guided detection, localization, and surgical management of sentinel lymph nodes (SLNs) in patients with head and neck melanoma? Findings In this nonrandomized clinical trial of 24 patients, real-time, particle-based fluorescence imaging of SLNs was feasible and safe at the microdosing level and enabled deep-tissue nodal detection. There was high concordance in identifying nodes between preoperative lymphoscintigraphy and particle-based fluorescence-guided biopsy. Meaning The findings of this study suggest that ultrabright, optical contrast–conferring particles for SLN identification hold promise for overcoming current probe limitations and improving surgical outcomes., This nonrandomized clinical trial evaluates whether an ultrasmall, molecularly targeted core-shell silica nanoparticle can safely and reliably identify optically avid SLNs in head and neck melanoma during fluorescence-guided biopsy., Importance Sentinel lymph node (SLN) mapping agents approved for current surgical practice lack sufficient brightness and target specificity for high-contrast, sensitive nodal visualization. Objective To evaluate whether an ultrasmall, molecularly targeted core-shell silica nanoparticle (Cornell prime dots) can safely and reliably identify optically avid SLNs in head and neck melanoma during fluorescence-guided biopsy. Design, Setting, and Participants This nonrandomized clinical trial enrolled patients aged 18 years or older with histologically confirmed melanoma in whom SLN mapping was indicated. Exclusion criteria included known pregnancy, breast-feeding, or medical illness unrelated to the tumor. The trial was conducted between February 2015 and March 2018 at Memorial Sloan Kettering Cancer Center, with postoperative follow-up of 2 years. Data analysis was conducted from February 2015 to March 2018. Interventions Patients received standard-of-care technetium Tc 99m sulfur colloid followed by a microdose administration of integrin-targeting, dye-encapsulated nanoparticles, surface modified with polyethylene glycol chains and cyclic arginine-glycine–aspartic acid–tyrosine peptides (cRGDY-PEG-Cy5.5-nanoparticles) intradermally. Main Outcomes and Measures The primary end points were safety, procedural feasibility, lowest particle dose and volume for maximizing nodal fluorescence signal, and proportion of nodes identified by technetium Tc 99m sulfur colloid that were optically visualized by cRGDY-PEG-Cy5.5-nanoparticles. Secondary end points included proportion of patients in whom the surgical approach or extent of dissection was altered because of nodal visualization. Results Of 24 consecutive patients enrolled (median [interquartile range] age, 64 [51-71] years), 18 (75%) were men. In 24 surgical procedures, 40 SLNs were excised. Preoperative localization of SLNs with technetium Tc 99m sulfur colloid was followed by particle dose-escalation studies, yielding optimized doses and volumes of 2 nmol and 0.4 mL, respectively, and maximum SLN signal-to-background ratios of 40. No adverse events were observed. The concordance rate of evaluable SLNs by technetium Tc 99m sulfur colloid and cRGDY-PEG-Cy5.5-nanoparticles was 90% (95% CI, 74%-98%), 5 of which were metastatic. Ultrabright nanoparticle fluorescence enabled high-sensitivity SLN visualization (including difficult-to-access anatomic sites), deep tissue imaging, and, in some instances, detection through intact skin, thereby facilitating intraoperative identification without extensive dissection of adjacent normal tissue or nerves. Conclusions and Relevance This study found that nanoparticle-based fluorescence-guided SLN biopsy in head and neck melanoma was feasible and safe. This technology holds promise for improving lymphatic mapping and SLN biopsy procedures, while potentially mitigating procedural risks. This study serves as a first step toward developing new multimodal approaches for perioperative care. Trial Registration ClinicalTrials.gov Identifier: NCT02106598

Purpose: Small-molecule inhibitors have had a major impact on cancer care. While treatments have demonstrated clinically promising results, they suffer from dose-limiting toxicities and the emergence of refractory disease. Considerable efforts made to address these issues have more recently focused on strategies implementing particle-based probes that improve drug delivery and accumulation at target sites, while reducing off-target effects. Experimental Design: Ultrasmall ( Results: Gefitinib-bound C′ dots (DFO-Gef-C′ dots), synthesized using the gefitinib analogue, APdMG, at a range of drug-to-particle ratios (DPR; DPR = 11–56), demonstrated high stability for DPR values≤ 40, bulk renal clearance, and enhanced in vitro cytotoxicity relative to gefitinib (LD50 = 6.21 nmol/L vs. 3 μmol/L, respectively). In human non–small cell lung cancer mice, efficacious Gef-C′ dot doses were at least 200-fold lower than that needed for gefitinib (360 nmoles vs. 78 μmoles, respectively), noting fairly equivalent tumor growth inhibition and prolonged survival. Gef-C′ dot–treated tumors also exhibited low phosphorylated EFGR levels, with no appreciable wild-type EGFR target inhibition, unlike free drug. Conclusions: Results underscore the clinical potential of DFO-Gef-C′ dots to effectively manage disease and minimize off-target effects at a fraction of the native drug dose.

The poor prognosis associated with malignant melanoma has not changed substantially over the past 30 years. Targeted molecular therapies, such as immunotherapy, have shown promise but suffer from resistance and off-target toxicities, underscoring the need for alternative therapeutic strategies that can be used in combination with existing protocols. Moreover, peptides targeting melanoma-specific markers, like the melanocortin-1 receptor (MC1-R), for imaging and therapy exhibit high renal uptake that limits clinical translation. In the current study, the application of ultrasmall fluorescent (Cy5) silica nanoparticles (C' dots), conjugated with MC1-R targeting alpha melanocyte stimulating hormone (αMSH) peptides on the polyethylene glycol (PEG) coated surface, is examined for melanoma-selective imaging. αMSH peptide sequences, evaluated for conjugation to the PEG-Cy5-C' dot nanoparticles, bound to MC1-R with high affinity and targeted melanoma in syngenetic and xenografted melanoma mouse models. Results demonstrated a 10-fold improvement in MC1-R affinity over the native peptide alone following surface attachment of the optimal αMSH peptide. Systematic in vivo studies further demonstrated favorable in vivo renal clearance kinetics as well as receptor-mediated tumor cell internalization of as-developed radiolabeled particle tracers in B16F10 melanoma bearing mice. These findings highlight the ability of αMSH-PEG-Cy5-C' dots to overcome previous hurdles that prevented clinical translation of peptide and antibody-based melanoma probes and reveal the potential of αMSH-PEG-Cy5-C' dots for melanoma-selective imaging, image-guided surgery, and therapeutic applications.

Spectrally distinct ultrasmall fluorescence silica particles as multiplexing tools address cancer heterogeneity and aid surgery., Accurate detection and quantification of metastases in regional lymph nodes remain a vital prognostic predictor for cancer staging and clinical outcomes. As intratumoral heterogeneity poses a major hurdle to effective treatment planning, more reliable image-guided, cancer-targeted optical multiplexing tools are critically needed in the operative suite. For sentinel lymph node mapping indications, accurately interrogating distinct molecular signatures on cancer cells in vivo with differential levels of sensitivity and specificity remains largely unexplored. To address these challenges and demonstrate sensitivity to detecting micrometastases, we developed batches of spectrally distinct 6-nm near-infrared fluorescent core-shell silica nanoparticles, each batch surface-functionalized with different melanoma targeting ligands. Along with PET imaging, particles accurately detected and molecularly phenotyped cancerous nodes in a spontaneous melanoma miniswine model using image-guided multiplexing tools. Information afforded from these tools offers the potential to not only improve the accuracy of targeted disease removal and patient safety, but to transform surgical decision-making for oncological patients.

Purpose: Small-molecule inhibitors have revolutionized treatment of certain genomically defined solid cancers. Despite breakthroughs in treating systemic disease, central nervous system (CNS) metastatic progression is common, and advancements in treating CNS malignancies remain sparse. By improving drug penetration across a variably permeable blood–brain barrier and diffusion across intratumoral compartments, more uniform delivery and distribution can be achieved to enhance efficacy. Experimental Design: Ultrasmall fluorescent core-shell silica nanoparticles, Cornell prime dots (C' dots), were functionalized with αv integrin-binding (cRGD), or nontargeting (cRAD) peptides, and PET labels (124I, 89Zr) to investigate the utility of dual-modality cRGD-C' dots for enhancing accumulation, distribution, and retention (ADR) in a genetically engineered mouse model of glioblastoma (mGBM). mGBMs were systemically treated with 124I-cRGD- or 124I-cRAD-C' dots and sacrificed at 3 and 96 hours, with concurrent intravital injections of FITC-dextran for mapping blood–brain barrier breakdown and the nuclear stain Hoechst. We further assessed target inhibition and ADR following attachment of dasatinib, creating nanoparticle–drug conjugates (Das-NDCs). Imaging findings were confirmed with ex vivo autoradiography, fluorescence microscopy, and p-S6RP IHC. Results: Improvements in brain tumor delivery and penetration, as well as enhancement in the ADR, were observed following administration of integrin-targeted C' dots, as compared with a nontargeted control. Furthermore, attachment of the small-molecule inhibitor, dasatinib, led to its successful drug delivery throughout mGBM, demonstrated by downstream pathway inhibition. Conclusions: These results demonstrate that highly engineered C' dots are promising drug delivery vehicles capable of navigating the complex physiologic barriers observed in a clinically relevant brain tumor model.

Controlling the biodistribution of nanoparticles upon intravenous injection is the key to achieving target specificity. One of the impediments in nanoparticle-based tumor targeting is the inability to limit the trafficking of nanoparticles to liver and other organs leading to smaller accumulated amounts in tumor tissues, particularly via passive targeting. Here we overcome both these challenges by designing nanoparticles that combine the specificity of antibodies with favorable particle biodistribution profiles, while not exceeding the threshold for renal filtration as a combined vehicle. To that end, ultrasmall silica nanoparticles are functionalized with anti-human epidermal growth factor receptor 2 (HER2) single-chain variable fragments to exhibit high tumor-targeting efficiency and efficient renal clearance. This ultrasmall targeted nanotheranostics/nanotherapeutic platform has broad utility, both for imaging a variety of tumor tissues by suitably adopting the targeting fragment and as a potentially useful drug delivery vehicle., One of the major obstacles in nanoparticle-based therapy is to achieve tumour targeting, limiting non-specific accumulation of the nanoparticles. Here the authors propose the conjugation of anti-HER2 scFv fragments to the silica nanoparticles, increasing specificity and limiting the final size of the immunoconjugates below the renal clearance threshold.

The fundamental role that NAD(P)H/quinone oxidoreductase 1 (NQO1) plays, in normal cells, as a cytoprotective enzyme guarding against stress induced by reactive oxygen species (ROS) is well documented. However, what is not known is whether the observed overexpression of NQO1 in neoplastic cells contributes to their survival. The current study discovered that depleting NQO1 expression in A549 and H292 lung adenocarcinoma cells caused an increase in ROS formation, inhibited anchorage-independent growth, increased anoikis sensitization, and decreased three-dimensional tumor spheroid invasion. These in vivo data further implicate tumor-NQO1 expression in a protumor survival role, because its depletion suppressed cell proliferation and decreased lung tumor xenograft growth. Finally, these data reveal an exploitable link between tumor-NQO1 expression and the survival of lung tumors because NQO1 depletion significantly decreased the percentage of ALDH(high) cancer cells within the tumor population. Implications: Loss of tumor-NQO1 expression inhibits tumor growth and suggests that novel therapeutics directed at tumor-NQO1 may have clinical benefit. Mol Cancer Res; 14(1); 14–25. ©2015 AACR.

Designing a nanomaterials platform with high target-to-background ratios has long been one of the major challenges in the field of nanomedicine. Here, we introduce a “target-or-clear” multifunctional nanoparticle platform that demonstrates high tumor-targeting efficiency and retention while minimizing off-target effects. Encouraged by the favorable preclinical and clinical pharmacokinetic profiles derived after fine-tuning surface chemical properties of radioiodinated (124I, t1/2 = 100.2 h) ultrasmall cRGDY-conjugated fluorescent silica nanoparticles (C dots), we sought to investigate how the biological properties of these radioconjugates could be influenced by the conjugation of radiometals such as zirconium-89 (89Zr, t1/2 = 78.4 h) using two different strategies: chelator-free and chelator-based radiolabeling. The attachment of 89Zr to newer, surface-aminated, integrin-targeting C′ dots using a two-pot synthesis approach led to favorable pharmacokinetics and clearance profiles as well as high tumor uptake and target-to-background ratios in human melanoma models relative to biological controls while maintaining particle sizes below the effective renal glomerular filtration size cutoff

Purpose: Surgical resection remains the most effective therapy for solid tumors worldwide. The most important prognostic indicator for cure following cancer surgery is a complete resection with no residual disease. However, intraoperative detection of retained cancer cells after surgery is challenging, and residual disease continues to be the most common cause of local failure. We hypothesized that visual enhancement of tumors using near-infrared imaging could potentially identify tumor deposits in the wound after resection. Experimental Design: A small animal model of surgery and retained disease was developed. Residual tumor deposits in the wound were targeted using an U.S. Food and Drug Administration–approved imaging agent, indocyanine green, by the enhanced permeability and retention effect. A novel handheld spectrometer was used to optically visualize retained disease after surgery. Results: We found residual disease using near-infrared imaging during surgery that was not visible to the naked eye or micro-CT. Furthermore, examination of tumor nodules was remarkably precise in delineating margins from normal surrounding tissues. This approach was most successful for tumors with increased neovasculature. Conclusions: The results suggest that near-infrared examination of the surgical wound after curative resection can potentially enable the surgeon to locate residual disease. The data in this study is the basis of an ongoing Phase I/II clinical trial in patients who undergo resection for lung and breast cancer. Clin Cancer Res; 18(20); 5741–51. ©2012 AACR.

The poor prognosis associated with malignant melanoma has not changed substantially over the past 30 years. Targeted molecular therapies, such as immunotherapy have shown promise, but suffer from resistance and off-target toxicities, underscoring the need for alternative therapeutic strategies that can be used in combination with existing protocols. Moreover, peptides targeting melanoma-specific markers, like the melanocortin-1 receptor (MC1-R), for imaging and therapy exhibit high renal uptake that limits clinical translation. In the current study, the application of ultrasmall fluorescent (Cy5) silica nanoparticles (C′ dots), conjugated with MC1-R targeting alpha melanocyte stimulating hormone (αMSH) peptides on the polyethylene glycol (PEG) coated surface, is examined for melanoma-selective imaging. αMSH peptide sequences, evaluated for conjugation to the PEG-Cy5-C′ dot nanoparticles, bound to MC1-R with high affinity, and targeted melanoma in syngenetic and xenografted melanoma mouse models. Results demonstrated a 10-fold improvement in MC1-R affinity over the native peptide alone following surface attachment of the optimal αMSH peptide. Systematic in vivo studies further demonstrated favorable in vivo renal clearance kinetics as well as receptor-mediated tumor cell internalization of as-developed radiolabeled particle tracers in B16F10 melanoma bearing mice. These findings highlight the ability of αMSH-PEG-Cy5-C′ dots to overcome previous hurdles that prevented clinical translation of peptide and antibody-based melanoma probes, and reveal the potential of αMSH-PEG-Cy5-C′ dots for melanoma selective imaging, image-guided surgery, and therapeutic applications.

Surgery is the most effective method to cure patients with solid tumors, and 50% of all cancer patients undergo resection. Local recurrences are due to tumor cells remaining in the wound, thus we explore near-infrared (NIR) fluorescence spectroscopy and imaging to identify residual cancer cells after surgery. Fifteen canines and two human patients with spontaneously occurring sarcomas underwent intraoperative imaging. During the operation, the wounds were interrogated with NIR fluorescence imaging and spectroscopy. NIR monitoring identified the presence or absence of residual tumor cells after surgery in 14/15 canines with a mean fluorescence signal-to-background ratio (SBR) of ∼16. Ten animals showed no residual tumor cells in the wound bed (mean SBR1-year follow-up. In five animals, the mean SBR of the wound was >15, and histopathology confirmed tumor cells in the postsurgical wound in four/five canines. In the human pilot study, neither patient had residual tumor cells in the wound bed, and both remain disease free at >1.5-year follow up. Intraoperative NIR fluorescence imaging and spectroscopy identifies residual tumor cells in surgical wounds. These observations suggest that NIR imaging techniques may improve tumor resection during cancer operations.

Fluorescence guided surgery (FGS) is a developing field of surgical and oncologic research. Practically, FGS has shown useful applications in urologic surgery, benign biliary surgery, colorectal cancer liver metastasis resection, and ovarian cancer debulking. Most notably in in cancer surgery, FGS allows for the clear delineation of cancerous tissue from benign tissue. FGS requires the utilization of a fluorescent contrast agent and an intraoperative fluorescence imaging device (IFID). Currently available IFIDs are expensive, unable to work with multiple fluorophores, and can be cumbersome. This study aims to describe the development and utility of a small, cost-efficient, and interchangeable IFID made from commercially available components. Extensive research was done to design and construct a light-weight, portable, and cost-effective IFID. We researched the capabilities, size, and cost of several camera types and eventually decided on a near-infrared (NIR) charged couple device (CCD) camera for its overall profile. The small portable interchangeable imager of fluorescence (SPIIF) is a “scout” IFID system for FGS. The main components of the SPIIF are a NIR CCD camera with an articulating light filter. These components and a LED light source with an attached heat sink are mounted on a small metal platform. The system is connected to a laptop by a USB 2.0 cable. Pixielink © software on the laptop runs the system by controlling exposure time, gain, and image capture. After developing the system, we evaluated its utility as an IFID. The system weighs less than two pounds and can cover a large area. Due to its small size, it is easily made sterile by covering it with any sterile plastic sheet. To determine the system’s ability to detect fluorescent signal, we used the SPIIF to detect indocyanine green under ex and in-vivo conditions and fluorescein under ex-vivo conditions. We found the SPIIF was able to detect both ICG and fluorescein under different depths of a semi-opaque colloid. Second, we found that a concentration as low as 0.5 g/ml of indocyanine green dissolved in plasma was detectable. Lastly, in a murine and human cancer model, the SPIIF was able to detect indocyanine green signal within tumors and generate a signal-to-background ratio (SBR) of 3.75. This study shows that a low-cost IFID can be made from commercially available parts. Second, this IFID is capable of in and ex-vivo detection of multiple fluorophores without sacrificing its small size or favorable ergonomics.

Background Over 80,000 people undergo pulmonary resection for a lung nodule in the United States each year. Small nodules are frequently missed or difficult to find despite preoperative imaging. We hypothesized that near-infrared (NIR) imaging technology could be used to identify and locate lung nodules during surgery. Methods We enrolled 18 patients who were diagnosed with a pulmonary nodule that required resection. All patients had a fine-cut 1-mm computed tomography scan preoperatively. The patients were given systemic 5 mg/kg indocyanine green and then underwent an open thoracotomy 24 hours later. The NIR imaging was used to identify the primary nodule and search for additional nodules that were not found by visual inspection or manual palpation of the ipsilateral lung. Results Manual palpation and visual inspection identified all 18 primary pulmonary nodules and no additional lesions. Intraoperative NIR imaging detected 16 out of the 18 primary nodules. The NIR imaging also identified 5 additional subcentimeter nodules; 3 metastatic adenocarcinomas and 2 metastatic sarcomas. This technology could identify nodules as small as 0.2 cm and as deep as 1.3 cm from the pleural surface. This approach discovered 3 nodules that were in different lobes than the primary tumor. Nodule fluorescence was independent of size, metabolic activity, histology, tumor grade and vascularity. Conclusions This is the first-in-human demonstration of identifying pulmonary nodules during thoracic surgery with NIR imaging without a priori knowledge of their location or existence. The NIR imaging can detect pulmonary nodules during lung resections that are poorly visualized on computed tomography and difficult to discriminate on finger palpation.

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a phase II detoxifying enzyme responsible for quinone reduction, where it scavenges quinone induced reactive oxygen species. In recent studies, NQO1 has been investigated as a possible drug target due to its overexpression in a number of solid tumors. To this end, treatment with the quinone-analog drug, β-lapachone (ARQ-761), has progressed to Phase II clinical trials. In an effort to better understand the necessity of NQO1 in the overall lifecycle of cancer, our earlier work demonstrated that NQO1 played a vital role in a number of tumorigenic processes including anoikis resistance, and most interestingly alteration of aldehyde dehydrogenase (ALDH) activity. Given that ALDH is a widely reported cancer stem cell marker, and loss of NQO1 leads to a decrease in ALDH activity, we have begun to investigate the effect of NQO1 expression on the non-small cell lung cancer (NSCLC) stem cell population. Here, we present data that demonstrates NQO1 is vital to tumorsphere formation as demonstrated by decreased spheroid formation following NQO1 knocked down with shRNA. We also show that NQO1 appears to be necessary for cancer stem cell renewal as illustrated by decreased serial tumorsphere formation. In addition to these aforementioned data, in extreme limited dilution assays we demonstrate a reduced cancer stem cell frequency in NQO1 knockdown cells as compared to controls. Interestingly, in NQO1 knockdown populations, those cells that do form spheres show a remarkable re-expression of NQO1 as well as a rescue of ALDH activity, further supporting NQO1's role in tumorsphere formation and stem cell maintenance. Future work on this project will involve use of the CRISPR-Cas9 system to generate NQO1 knockout cell lines, drug resistance studies, cancer stem cell marker validation, and in vivo limited dilution assays to definitively demonstrate NQO1's necessity in maintaining the NSCLC stem cell population. Citation Format: Brian Madajewski, Erik A. Bey. NQO1's role in maintaining the cancer stem cell phenotype in NSCLC. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2503.

Lung cancer is the leading cause of cancer related deaths in the world. In 2015, roughly 160,000 people in the U.S. alone will succumb to the disease. The high mortality is mainly due to late diagnosis and therapeutic resistance, followed by disease progression. One mechanism by which lung cancer is able to circumvent treatment is through the existence of cancer stem cells (CSCs). CSCs are a subpopulation of cells within the heterogenous tumor that are capable of self-renewal, are less sensitive to therapeutics and are believed to be responsible for disease progression, metastasis, and relapse. Aldehyde dehydrogenase (ALDH) high activity has been linked to the cancer stem cell phenotype in various cancers; and the elimination of cells within the heterogenous tumor that have ALDH (high) activity has been associated with decreased tumorigenicity. NADPH quinone oxidoreductase-1 (NQO1) is a two-electron oxidoreductase that is highly overexpressed in many cancers including non-small cell lung cancer. In normal cells NQO1 is expressed only at low levels, but plays a significant role in regulating oxidative stress. Thus, we hypothesized that the noted overexpression of NQO1 in most cancers may be due to its critical role in tumor survival. Specifically, in balancing the noted elevated levels of oxidative stress observed in cancer cells. Thus, reducing NQO1 expression in tumor cells may tip the balance towards pro-death instead of pro-survival. In our studies we used two different shRNA constructs to deplete NQO1 expression and assayed the ability of the knockdown cells to perform in assays that define a tumors ability to participate in disease progression. In brief, our data showed that NQO1 depleted non-small cell lung cancer cell lines had decreased colony formation in soft agar assays, increased detachment induced cell death (anoikis), increased oxidative stress and increased apoptosis. In addition, shNQO1 knockdown caused a depletion in ALDH (high) activity, suggesting that the cancer stem cell population was reduced due to the loss of NQO1 expression. Our in vivo data supported our in vitro data since athymic mice bearing shQNO1 tumors had significantly longer long-term survival and less tumor growth as compared to mice bearing vector-control tumors. In conclusion, our data strongly suggest that NQO1 depletion is a viable target for lung cancer stem cell reduction. Furthermore, our data suggest that implementation of NQO1-directed therapies to reduce cancer stem cells will lead to improved survival in patients whose tumors overexpress NQO1. Citation Format: Brian Madajewski, Michael A. Boatman, Erik A. Bey. NQO1 depletion in non-small lung cancer cells decreases their tumorigenicity by reducing the ALDH (high) cancer stem cell population. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr A64.

Up to 30% of cancer patients undergoing curative surgery develop local recurrences due to positive margins. Patients typically receive adjuvant chemotherapy, immunotherapy and/or radiation to prevent such relapses. Interestingly, evidence supporting these therapies is traditionally derived in animal models of primary tumors, thus failing to consider surgically induced tumor microenvironment changes that may influence adjuvant therapy efficacy. To address this consideration, we characterized a murine model of local cancer recurrence. This model was reproducible and generated a postoperative inflammatory tumor microenvironment that resembles those observed following human cancer surgery. To further validate this model, antagonists of two pro-inflammatory mediators, TGFβ and COX-2, were tested and found to be effective in decreasing the growth of recurrent tumors. We appreciated that preoperative TGFβ inhibition led to wound dehiscence, while postoperative initiation of COX-2 inhibition resulted in a loss of efficacy. In summary, although not an exact replica of all human cancer surgeries, our proposed local recurrence approach provides a biologically relevant and reliable model useful for preclinical evaluation of novel adjuvant therapies. The use of this model yields results that may be overlooked using traditional preclinical cancer models that fail to incorporate a surgical component.