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Background: Lymphovascular invasion (LVI) is a major route of metastatic dissemination and recent studies indicate its value as an independent prognostic indicator for advanced breast, colorectal, squamous cell, prostate, brain cancers. LVI is a clinicopathological hallmark of inflammatory breast cancer (IBC), an understudied and most lethal breast cancer. IBC is often misdiagnosed due to an absence of a solid mass and its unique presentation of diffuse tumor cell clusters/emboli in the dermal lymphatics. Widely used mammary tumor implantation models coupled with bioluminescence or fluorescence imaging to monitor tumor growth kinetics are ineffective for evaluating spatial and temporal changes in growth and migration patterns of individual tumor cells and clusters within their microenvironmental context. The goal of this study was to develop a murine model to simulate the unique clinicopathological features of IBC patients and to assess both qualitatively and quantitatively local tumor growth, motility, and LVI. Methods: To specifically facilitate visualization of lymphatic and endothelial vessels along with tumor-vessel interactions, we generated a transgenic nude mice model (ProxTom RFP Nu/Nu) wherein, the mice exhibit red, fluorescent lymphatics [tdTomato fluorophore under control of a Prox1 promoter, which encodes a transcription factor (prospero-related homeobox 1) necessary for the formation and maintenance of lymphatic vessels]. Next, we employed a surgical technique, wherein a window chamber is placed on the dorsal skinfold of mice, which allows for microscopic examination of implanted tumor cells and ability to track dynamic changes of the tumor in its local microenvironment from the time of implantation up to 10 days. Patient-derived IBC or PDX stably transfected to express green, red fluorescent and/or dual tagged with luciferase reporters were transplanted in mice bearing window chambers. Intravital fluorescence microscopy and IVIS imaging were used to serially quantify local tumor growth, motility, length density of lymph and blood vessels, and degree of tumor cell lymphatic invasion over 0-140h. Results: Multichannel optical imaging of the window chamber in the ProxTom RFP Nu/Nu mice demonstrated co-localization of IBC tumor cells and lymphatics. Diffuse tumor cells were observed along regions of lymphatic vessels both proximal and distal to the primary tumor site. However, measurement of blood and lymph vessel density showed no significant change over time. Next, these datasets were used for quantitative analysis by setting the tumor cell channel (GFP) at a threshold to count any clusters greater than 50 pixels2 (~0.0013mm2) and with greater than the mean + 2 standard deviations of the background signal while avoiding noise/artifacts from very small regions ( Citation Format: Gayathri R Devi, Dorababu Sannareddy, Alexandra Bennion, Ashlyn Rickard, Douglas C Rouse, Mark W Dewhirst, Gregory M Palmer. Monitoring Lymphovascular Invasion and Tumor Growth of Inflammatory Breast Cancer in a Murine Lymphatic Reporter Window Chamber Model [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P3-08-06.

Inflammatory breast cancer (IBC), an understudied and lethal breast cancer, is often misdiagnosed due to its unique presentation of diffuse tumor cell clusters in the skin and dermal lymphatics. Here, we describe a window chamber technique in combination with a novel transgenic mouse model that has red fluorescent lymphatics (ProxTom RFP Nu/Nu) to simulate IBC clinicopathological hallmarks. Various breast cancer cells stably transfected to express green or red fluorescent reporters were transplanted into mice bearing dorsal skinfold window chambers. Intravital fluorescence microscopy and the in vivo imaging system (IVIS) were used to serially quantify local tumor growth, motility, length density of lymph and blood vessels, and degree of tumor cell lymphatic invasion over 0–140 h. This short-term, longitudinal imaging time frame in studying transient or dynamic events of diffuse and collectively migrating tumor cells in the local environment and quantitative analysis of the tumor area, motility, and vessel characteristics can be expanded to investigate other cancer cell types exhibiting lymphovascular invasion, a key step in metastatic dissemination. It was found that these models were able to effectively track tumor cluster migration and dissemination, which is a hallmark of IBC clinically, and was recapitulated in these mouse models.

Gene expression profiles identified 933 differentially expressed genes between control and wtXIAP tumors

Primers used in this study, alignment of probesets used for detection of XIAP expression on XIAP mRNA, GSEA network graphic of XIAP-dependent mRNA expression, heat maps of genes positively and negatively correlated with XIAP expression, and analysis of in vivo tumor tissue from xenograft studies.

Cancer is the second leading cause of death and there is an urgent need to improve cancer management. We have developed an innovative cancer therapy named Synergistic Immuno Photothermal Nanotherapy (SYMPHONY) by combining gold nanostars (GNS)-mediated photothermal ablation with checkpoint inhibitor immunotherapy. Our previous studies have demonstrated that SYMPHONY photoimmunotherapy not only treats the primary tumor but also dramatically amplifies anticancer immune responses in synergy with checkpoint blockade immunotherapy to treat remote and unresectable cancer metastasis. The SYMPHONY treatment also induces a ‘cancer vaccine’ effect leading to immunologic memory and prevents cancer recurrence in murine animal models. This manuscript provides an overview of our research activities on the SYMPHONY therapy with plasmonic GNS for cancer treatment.

Less than 20% of Triple Negative Breast Cancer (TNBC) patients experience long-term responses to mainstay chemotherapy, as tumors develop chemo-resistance. While combinations of chemotherapies and targeted therapies show potential improvements in TNBC clinical outcomes, patient stratification and prediction of treatment response is critical. Spatio-temporal metabolic reprogramming holds promise as a biomarker of therapy response as resistant tumor subpopulations utilize alternate metabolic pathways to escape therapy, enter minimum residual disease (MRD) and recur. Currently, there are limited tools to temporally evaluate heterogeneous changes along distinct metabolic axes in vivo at a spatial resolution capable of resolving vulnerabilities of residual tumor subpopulations. Here, we utilized an optical imaging-based platform to identify in vivo, longitudinal differences in metabolic reprogramming between a resistant and sensitive tumor model at high resolutions along three metabolic axes of TNBC chemoresistance (oxidative phosphorylation, glycolysis, and fatty acid oxidation). Xenografts were established by orthotopic cell injection and mice were treated with Paclitaxel (PTX), a commonly used chemotherapeutic drug in TNBC treatment, under a conventional maximum dose density regimen once the tumor reached a volume ~150mm3. MDA-MB-231 xenografts were resistant to PTX, defined as an initial response to PTX, a period of minimal residual disease, and a resurgence in tumor volume at ~60 days post drug withdrawal (n=3). HCC-1806 xenografts were sensitive to PTX, defined as an initial response to PTX in all mice and a complete cure in 7/10 mice. A separate cohort of mice for each tumor line was implanted with window chambers and imaged longitudinally at distinct stages of the tumor’s lifecycle with previously validated fluorophores 2-NBDG, TMRE, and Bodipy to directly report on glucose uptake, mitochondrial membrane potential, or fatty acid uptake, respectively. Wide field fluorescence imaging of MDA-MB-231 mice showed a significant increase in TMRE as early as two days after the 3rd PTX dose (n=5, p< 0.05), a significant decrease in 2-NBDG as early as two days after the 5th PTX dose (n=5, p< 0.05) and no significant changes in bodipy uptake. This increase in non-glucose-driven mitochondrial respiration was sustained during MRD. An increase in heterogeneity of TMRE uptake was seen during disease regression, MRD, and recurrence (n=5, p< 0.05). HCC-1806 tumors showed increased glucose uptake, decreased fatty acid uptake, and no significant changes in mitochondrial membrane potential during acute treatment. Metabolic changes were transient, with no significant changes in probe uptakes after drug withdrawal and during MRD. Unlike the MDA-MB-231 tumors, no significant changes in the heterogeneity of TMRE uptake were seen following paclitaxel withdrawal in HCC-1806 tumors (n=5, p>0.05). Our results point towards a metabolic switch from glycolysis to non-glucose, non-fat-driven mitochondrial respiration in MDA-MB-231 mice, possibly suggesting amino acid catabolism as a fuel during MRD. In the sensitive HCC-1806 line, we observed changes in glucose and fatty acid uptake during acute treatment, but no significant changes in metabolism during MRD. Consistent with the literature, our results point toward an increase in metabolic plasticity and heterogeneity in a chemo-resistant model compared to a chemo-sensitive one. Together, our results show the potential of using metabolic changes following therapy as a biomarker of therapy response, while highlighting the importance of tracking the change (or lack of change) in metabolism longitudinally. We aim to use this system to visualize and exploit early, in vivo metabolic vulnerabilities of disease regression that accompany local and distal recurrences. Citation Format: Enakshi D. Sunassee, Elizabeth Maydew, Brian Crouch, Megan Madonna, Gregory M Palmer, Nimmi Ramanujam. Validation of an Optical Imaging Platform to Identify Metabolic Vulnerabilities in Chemo-Resistant and Sensitive Tumors [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-01-01.

Bladder cancer has been ranked as one of the most commonly occurring cancers in men and women with approximately half of the diagnoses being the late stage and/or metastatic diseases. We have developed a novel cancer treatment by combining gold nanostar-mediated photothermal therapy with checkpoint inhibitor immunotherapy to treat bladder cancer. Experiment results with a murine animal model demonstrated that our developed photoimmunotherapy therapy is more efficacious than any individual studied treatment. In addition, we used intravital optical imaging with a dorsal skinfold window chamber animal model to study immune responses and immune cell accumulation in a distant tumor following our photoimmunotherapy. The mice used have the CX3CR1-GFP receptor on monocytes, natural killer cells, and dendritic cells allowing us to dynamically track their presence by fluorescence imaging. Our proof-of-principle study results showed that the photoimmunotherapy triggered anti-cancer immune responses to generate anti-cancer immune cells which accumulate in metastatic tumors. Our study results illustrate that intravital optical imaging is an efficient and versatile tool to investigate immune responses and mechanisms of photoimmunotherapy in future studies.

Background: Lymphovascular invasion (LVI), a critical feature of advanced cancers, is a major route of metastatic dissemination. Of the clinically-distinct types of breast cancer, the most lethal variant is inflammatory breast cancer (IBC) where LVI is a histopathological hallmark. Majority of IBC patients lack a distinct, solid tumor and instead present with diffuse tumor cell clusters cells, also termed as tumor emboli, in the breast and dermal lymphatics, which contributes to the clinical breast-skin symptoms and postulated to provide an efficient path to metastatic spread. The steps in lymphatic dissemination are challenging to study in vitro, and in vivo models are limited. Our goal was to develop new in vivo models that would enable direct visualization and quantitation of local tumor cell growth and tumor-lymphatic vessel interactions for improved understanding of the unique IBC tumor biology and for drug discovery. Methods: mCherry/GFP-expressing non-IBC (4T1, E0771) and IBC cells derived from patient untreated primary tumors (SUM149/basal and SUM190/Her2+) were implanted into the fascia in the center of a window chamber in the skin-fold or mammary fat pad. In order to allow direct study of tumor cell–lymphatic vessel interaction, we generated transgenic nude mice with fluorescent lymphatics [tdTomato fluorophore under control of a Prox1 promoter, encodes a transcription factor (prospero-related homeobox 1) necessary for the formation and maintenance of lymphatic vessels]. Multichannel microscopy was employed to serially quantify tumor growth, tumor spread and lymph-tumor interactions. Using this model, we also optimized implantation and live imaging of pre-formed IBC-cell derived tumor emboli generated by simulating the lymphatic sheer stress in culture. Results: Compared to non-IBC cells, which formed solid tumors that increased in size over a 14 day period, IBC models exhibited a diffuse and disseminating phenotype, within 24h, rather than a centralized tumor mass, similar to the disease presentation in patients. Using a threshold analysis in ImageJ, we quantified tumor area (as a metric of tumor growth), the tumor motility, and the linear density of lymph and blood vessels. Briefly, we binarized each fluorescent channel image such that the tumor or lymph signal was positive; then, an algorithm drew regions of interest (ROIs) around each defined tumor-cell cluster. The ROIs were used to calculate the parameters describing tumor growth and motility (distance of the tumor from the center of mass (CM)), and the area moment (the movement of the clusters relative to the CM) at each timepoint. The linear vascular density was calculated from the distance of segments drawn through the center of large vessels for each mouse, which provided quantitative data describing tumor cell or IBC emboli and lymphovascular interactions, suggesting that there is a specific microenvironment necessary for the unique phenotype and dissemination pattern exhibited by IBC tumor cells. Conclusions: Despite the poor prognosis of IBC, the clinical implications of how and why tumor emboli form and how they survive to migrate into the lymphatics is not defined. These in vivo models provide in-depth imaging and quantitative measurements of locoregional invasion, tumor cell-lymphatic or endothelial vessel interactions of implanted tumor cells or tumor emboli. This model has the potential to be extended to study other cancer cell types exhibiting LVI as well as a screening metric for IBC therapies. Supported by DOD-Breakthrough-W81XWH-17-1-029, IBC Research Foundation, DCI pilot funds, IBC Network Foundation, Duke Surgery Gardner Award. Citation Format: Ashlyn Rickard, Pranalee Patel, Scott J Sauer, Mark W Dewhirst, Gregory M Palmer, Gayathri R Devi. Multichannel serial imaging of transgenic, preclinical murine models provides the first quantitative analysis of the unusual growth kinetics and lymph-vascular invasion of patient-derived inflammatory breast cancer cells and tumor emboli [abstract]. In: Proceedings of the 2019 San Antonio Breast Cancer Symposium; 2019 Dec 10-14; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2020;80(4 Suppl):Abstract nr P1-03-03.

Plasmonics–active gold nanostars provide an excellent multifunctional platform for cancer photo-immunotherapy. Their multiple sharp branches acting like “lightning rods” can convert safely and efficiently light into heat. As with other nanoparticles, GNS sizes can be controlled so that they passively accumulate in tumors due to the enhanced permeability and retention (EPR) effect of tumor vasculature. GNS therefore can act as nano-sources of heat that can ablate tumor cells in their microenvironment (i.e., from the inside) with high spatial accuracy. This feature improves tumor-targeting precision and permits the use of reduced laser energy required to destroy the targeted cancer cells. GNS have a tunable plasmonic band in the near infrared region, where there is low tissue absorption and autofluorescence, and therefore they are quite suitable for in vivo applications. The ability to selectively heat tumor areas where GNS are located while keeping surrounding healthy tissues at significantly lower temperatures offers significant advantages over other thermal therapies. GNS-mediated photothermal therapy combined with checkpoint immunotherapy was shown to reverse tumor-mediated immunosuppression, leading to the treatment of not only primary tumors but also cancer metastasis as well as inducing effective long-lasting immunity against cancer, i.e. an anticancer ‘vaccine’ effect.

Intravital microscopy is a powerful technique that is uniquely suitable for gaining insight into complex and dynamic interactions between tumor cells, the tumor microenvironment, immune system, other stromal cells, and therapy. There has recently been an expanding interest in understanding these key interactions as they relate to novel and successful immune and targeted therapies for treating cancer. In particular, how these can aid in our understanding of how these complex systems interact and how to understand the determinants of treatment response.

Significance: Decreasing the oxygen consumption rate (OCR) of tumor cells is a powerful method for ameliorating tumor hypoxia. However, quantifying the change in OCR is challenging in complex experimental systems. Aim: We present a method for quantifying the OCR of two tumor cell lines using oxygen-sensitive dual-emissive boron nanoparticles (BNPs). We hypothesize that our BNP results are equivalent to the standard Seahorse assay. Approach: We quantified the spectral emissions of the BNP and accounted for external oxygen diffusion to quantify OCR over 24 h. The BNP-computed OCR of two breast cancer cell lines, E0771 and 4T07, were compared with their respective Seahorse assays. Both cell lines were also irradiated to quantify radiation-induced changes in the OCR. Results: Using a Bland–Altman analysis, our BNPs OCR was equivalent to the standard Seahorse assay. Moreover, in an additional experiment in which we irradiated the cells at their 50% survival fraction, the BNPs were sensitive enough to quantify 24% reduction in OCR after irradiation. Conclusions: Our results conclude that the BNPs are a viable alternative to the Seahorse assay for quantifying the OCR in cells. The Bland–Altman analysis showed that these two methods result in equivalent OCR measurements. Future studies will extend the OCR measurements to complex systems including 3D cultures and in vivo models, in which OCR measurements cannot currently be made.

The discovery of new treatments for cancer is imperative. Recently, we showed in a proof-of-concept study that SYnergistic IMmuno PHOtothermal NanotherapY (SYMPHONY) is a powerful treatment for metastatic bladder cancer and brain tumor in mouse models. Our work has recently demonstrated that combining immunotherapy checkpoint inhibitors and gold nanostar (AuNS) photothermal therapy (PTT) is more effective in killing primary tumors and activating the immune system to eradicate tumors at distant sites, than each individual treatment alone. When the tumor is being ablated via PTT in mice models, using low intensity heat from a near infrared laser, the dying tumor releases proteins that trigger the immune system to destroy remaining tumor cells. Immune checkpoint inhibitors prevent the tumor cells from hiding from the immune system’s mechanisms; thus, the immune system becomes capable of attacking distant secondary tumors, after the primary tumor has been eradicated using AuNS mediated PTT. The data shows that after the cured mice were rechallenged with bladder cancer cells, no tumor formation was observed after 60 days; hence these results indicate that the SYMPHONY treatment can function as a cancer vaccine and lead to long-lasting immunity.

PurposeReal-time monitoring of physiological changes of tumor tissue during radiation therapy (RT) could improve therapeutic efficacy and predict therapeutic outcomes. Cherenkov radiation is a normal byproduct of radiation deposited in tissue. Previous studies in rat tumors have confirmed a correlation between Cherenkov emission spectra and optical measurements of blood-oxygen saturation based on the tissue absorption coefficients. The purpose of this study is to determine if it is feasible to image Cherenkov emissions during radiation therapy in larger human-sized tumors of pet dogs with cancer. We also wished to validate the prior work in rats, to determine if Cherenkov emissions have the potential to act an indicator of blood-oxygen saturation or water-content changes in the tumor tissue-both of which have been correlated with patient prognosis.MethodsA DoseOptics camera, built to image the low-intensity emission of Cherenkov radiation, was used to measure Cherenkov intensities in a cohort of cancer-bearing pet dogs during clinical irradiation. Tumor type and location varied, as did the radiation fractionation scheme and beam arrangement, each planned according to institutional standard-of-care. Unmodulated radiation was delivered using multiple 6 MV X-ray beams from a clinical linear accelerator. Each dog was treated with a minimum of 16 Gy total, in ≥3 fractions. Each fraction was split into at least three subfractions per gantry angle. During each subfraction, Cherenkov emissions were imaged.ResultsWe documented significant intra-subfraction differences between the Cherenkov intensities for normal tissue, whole-tumor tissue, tissue at the edge of the tumor and tissue at the center of the tumor (pConclusionHere we demonstrate that it is possible to obtain Cherenkov emissions from canine cancers within a fraction of radiotherapy. The entire optical spectrum was obtained which includes the window for imaging changes in water and hemoglobin saturation. This lends credence to the goal of using this method during radiotherapy in human patients and client-owned pets.

Purpose Radiotherapy (RT) is commonly used to treat most pelvic malignancies. While treatment is often effective, curative radiation doses to the rectum can result in chronic radiation-induced proctitis, which is characterized by diarrhea, tenesmus, and/or rectal bleeding, recently termed pelvic radiation disease. An animal model of chronic radiation-induced proctitis would be useful to test both preventative and therapeutic strategies to limit this morbidity but has been elusive because of the high rodent mortality associated with acute bowel RT injury. The objective of this research was to develop a novel mouse model of chronic radiation-induced proctitis using advanced technology. Methods and Materials Using an X-RAD 225-Cx (Precision X-Ray) small animal irradiator, multiple plan configurations were evaluated for planning treatment volume and organ-at-risk avoidance to deliver a 15 Gy 3D conformal treatment plan. The final plan was verified by high resolution 3D dosimetry (PRESAGE/optical-CT), and delivered using a single arc. Mice were monitored for mortality for 250 days, followed by histopathological correlates including mucicarmine, Masson's trichrome, and fecal pellet length. Results Six beam arrangements were considered: single and parallel-opposed fields with whole-pelvis coverage, and collimated fields in parallel-opposed, 3-field, 4-field, and arc geometries. A collimated arc plan offered superior planning treatment volume coverage and organ-at-risk avoidance compared to whole-pelvis irradiation. Treatment verification with PRESAGE 3D dosimetry (Heuris Inc) showed >99% of voxels passing gamma analysis with 2%/2 mm criteria. Our treatment resulted in no acute mortality and 40% mortality at 250 days. Histopathological analysis showed increased mucous production and fibrosis of the irradiated colon, but no change in fecal pellet length. Conclusions Our model was able to target successfully lower colon and rectum with lower mortality than other published models. This permitted measurement of late effects that recapitulate some features of rectal damage in humans.

Although nanotechnology has led to important advances in in vitro diagnostics, the development of nanosensors for in vivo detection remains very challenging. Here, we demonstrated the proof-of-principle of in vivo detection of nucleic acid targets using a promising type of surface-enhanced Raman scattering (SERS) nanosensor implanted in the skin of a large animal model (pig). The in vivo nanosensor used in this study involves the “inverse molecular sentinel” detection scheme using plasmonics-active nanostars, which have tunable absorption bands in the near infrared region of the “tissue optical window”, rendering them efficient as an optical sensing platform for in vivo optical detection. Ex vivo measurements were also performed using human skin grafts to demonstrate the detection of SERS nanosensors through tissue. In this study, a new core–shell nanorattle probe with Raman reporters trapped between the core and shell was utilized as an internal standard system for self-calibration. These results illustrate the usefulness and translational potential of the SERS nanosensor for in vivo biosensing.

Objective Optical spectroscopy offers a noninvasive alternative to biopsy as a first-line screening tool for suspicious skin lesions. This study sought to define several optical parameters across malignant and benign tissue types. Study design Prospective pilot trial utilizing the Zenalux IM1 optical spectroscopy device from April 2016 to February 2017. For each skin lesion, provider pre-biopsy probability of malignancy was compared to histolopathologic diagnosis. Optical data were characterized across basal cell carcinoma (BCC; n = 9), squamous cell carcinoma (SCC; n = 5), actinic keratosis (AK; n = 4), scar tissue (n = 6), nevus (n = 2), and neurofibroma (NF; n = 1). Across all patients, agreement was determined between control measurements collected adjacent to the lesion and from the upper extremity. Methods Prospective single center pilot study. The optical properties of 27 cutaneous lesions were collected from 18 adult patients presenting to Otolaryngology and Dermatology clinics with suspicious skin lesions warranting biopsy. Spectroscopy measurements were recorded for each lesion: two at the lesion site, two at an adjacent site (internal control), and one at the central medial upper extremity (arm control). Variables of interest included absolute oxygenated hemoglobin (Hb), Hb saturation, total Hb concentration, and Eumelanin concentration. For each lesion, internal control averages were subtracted from lesion averages to provide delta parameter values, and lesion averages were divided by internal control averages to provide ratio parameter values. Results Mean percent difference between pre-biopsy probability of malignancy and histology was 29%, with a difference of 75% or greater seen in 5 of 25 lesions. Mean values for BCC, SCC, AK, and scar tissue varied most between extracted mean reduced scatter estimate (μa'; cm- ) delta values (BCC: -2.2 ± 3.8; SCC: -3.9 ± 2.0; AK: -3.3 ± 4.2, Scar: -1.7 ± 1.2) and total Hb (µM) ratio (BCC: 2.0 ± 3.3; SCC: 3.0 ± 1.3; AK: 1.1 ± 0.6; Scar: 1.4 ± 1.1). Agreement between local and arm controls was poor. Conclusion This pilot trial utilizes optical spectroscopy as a noninvasive method for determining cutaneous lesion histology. Effect sizes observed across optical parameters for benign and malignant tissue types will guide larger prospective studies that may ultimately lead to prediction of lesional histology without need for invasive biopsy. Lasers Surg. Med. 50:246-252, 2018. © 2018 Wiley Periodicals, Inc.

Metastatic spread is the mechanism in more than 90 percent of cancer deaths and current therapeutic options, such as systemic chemotherapy, are often ineffective. Here we provide a proof of principle for a novel two-pronged modality referred to as Synergistic Immuno Photothermal Nanotherapy (SYMPHONY) having the potential to safely eradicate both primary tumors and distant metastatic foci. Using a combination of immune-checkpoint inhibition and plasmonic gold nanostar (GNS)–mediated photothermal therapy, we were able to achieve complete eradication of primary treated tumors and distant untreated tumors in some mice implanted with the MB49 bladder cancer cells. Delayed rechallenge with MB49 cancer cells injection in mice that appeared cured by SYMPHONY did not lead to new tumor formation after 60 days observation, indicating that SYMPHONY treatment induced effective long-lasting immunity against MB49 cancer cells.

Hypoxia, a prevalent characteristic of most solid malignant tumors, contributes to diminished therapeutic responses and more aggressive phenotypes. The term hypoxia has two definitions. One definition would be a physiologic state where the oxygen partial pressure is below the normal physiologic range. For most normal tissues, the normal physiologic range is between 10 and 20 mmHg. Hypoxic regions develop when there is an imbalance between oxygen supply and demand. The impact of hypoxia on cancer therapeutics is significant: hypoxic tissue is 3× less radiosensitive than normoxic tissue, the impaired blood flow found in hypoxic tumor regions influences chemotherapy delivery, and the immune system is dependent on oxygen for functionality. Despite the clinical implications of hypoxia, there is not a universal, ideal method for quantifying hypoxia, particularly cycling hypoxia because of its complexity and heterogeneity across tumor types and individuals. Most standard imaging techniques can be modified and applied to measuring hypoxia and quantifying its effects; however, the benefits and challenges of each imaging modality makes imaging hypoxia case-dependent. In this chapter, a comprehensive overview of the preclinical and clinical methods for quantifying hypoxia is presented along with the advantages and disadvantages of each.

INTRODUCTION AND OBJECTIVES:We developed a novel treatment for bladder cancer (BC): a combination of gold nanoparticle-based immune- and photothermal therapy called SYMPHONY, which ablates primary ...

Although nanotechnology has led to important advances in in vitro diagnostics, the development of nanosensors for in vivo molecular detection remains very challenging. Here, we demonstrated the proof‐of‐principle of in vivo detection of nucleic acid targets using a promising type of surface‐enhanced Raman scattering (SERS) nanosensor implanted in the skin of a large animal model (pig). The in vivo “smart tattoo” nanosensor used in this study employs the “inverse molecular sentinel” (iMS) detection scheme, which is a label-free homogeneous biosensing system based on a non-enzymatic DNA strand-displacement process and conformational change of stem-loop (hairpin) oligonucleotide probes upon target binding. In this study, plasmonics‐active nanostar was utilized as an efficient in vivo SERS sensing platform due to their tunable absorption bands in the near infrared region of the “tissue optical window. The results of this study illustrate the usefulness of SERS iMS nanosensors as an implantable skin‐based in vivo biosensing platform, providing a foundation for developments in continuous health status sensing, disease biomarker monitoring, and other clinical translation applications.

The increased expression of vascular endothelial growth factor (VEGF) and its receptors is associated with angiogenesis in a growing tumor, presenting potential targets for tumor-selective imaging by way of targeted tracers. Though fluorescent tracers are used for targeted in vivo imaging, the lack of photostability and biocompatibility of many current fluorophores hinder their use in several applications involving long-term, continuous imaging. To address these problems, fluorescent nanodiamonds (FNDs), which exhibit infinite photostability and excellent biocompatibility, were explored as fluorophores in tracers for targeting VEGF receptors in growing tumors. To explore FND utility for imaging tumor VEGF receptors, we used click-chemistry to conjugate multiple copies of an engineered single-chain version of VEGF site-specifically derivatized with trans-cyclooctene (scVEGF-TCO) to 140 nm FND. The resulting targeting conjugates, FND-scVEGF, were then tested for functional activity of the scVEGF moieties through biochemical and tissue culture experiments and for selective tumor uptake in Balb/c mice with induced 4T1 carcinoma. We found that FND-scVEGF conjugates retain high affinity to VEGF receptors in cell culture experiments and observed preferential accumulation of FND-scVEGF in tumors relative to untargeted FND. Microspectroscopy provided unambiguous determination of FND within tissue by way of the unique spectral shape of nitrogen-vacancy induced fluorescence. These results validate and invite the use of targeted FND for diagnostic imaging and encourage further optimization of FND for fluorescence brightness.

Hypoxia, a prevalent characteristic of most solid malignant tumors, contributes to diminished therapeutic responses and more aggressive phenotypes. The term hypoxia has two definitions. One definition would be a physiologic state where the oxygen partial pressure is below the normal physiologic range. For most normal tissues, the normal physiologic range is between 10 and 20 mmHg. Hypoxic regions develop when there is an imbalance between oxygen supply and demand. The impact of hypoxia on cancer therapeutics is significant: hypoxic tissue is 3× less radiosensitive than normoxic tissue, the impaired blood flow found in hypoxic tumor regions influences chemotherapy delivery, and the immune system is dependent on oxygen for functionality. Despite the clinical implications of hypoxia, there is not a universal, ideal method for quantifying hypoxia, particularly cycling hypoxia because of its complexity and heterogeneity across tumor types and individuals. Most standard imaging techniques can be modified and applied to measuring hypoxia and quantifying its effects; however, the benefits and challenges of each imaging modality makes imaging hypoxia case-dependent. In this chapter, a comprehensive overview of the preclinical and clinical methods for quantifying hypoxia is presented along with the advantages and disadvantages of each.

We describe the development of a highly tunable, physiologically stable, and ultra-bright Raman probe, named as TARGET (Tunable and Amplified Raman Gold Nanoprobes for Effective Tracking), for in vitro and in vivo surface-enhanced Raman scattering (SERS) applications. The TARGET structure consists of a gold core inside a larger gold shell with a tunable interstitial gap similar to a "nanorattle" structure. The combination of galvanic replacement and the seed mediated growth method was employed to load Raman reporter molecules and subsequently close the pores to prevent leaking and degradation of reporters under physiologically extreme conditions. Precise tuning of the core-shell gap width, core size, and shell thickness allows us to modulate the plasmonic effect and achieve a maximum electric-field (E-field) intensity. The interstitial gap of TARGET nanoprobes can be designed to exhibit a plasmon absorption band at 785 nm, which is in resonance with the dye absorption maximum and lies in the "tissue optical window", resulting in ultra-bright SERS signals for in vivo studies. The results of in vivo measurements of TARGETs in laboratory mice illustrated the usefulness of these nanoprobes for medical sensing and imaging.

Checkpoint inhibitor immunotherapy has been a revolutionary milestone in cancer treatment because it can counter the tumor immunosuppressive environment to improve patients' overall survival and even result in a durable complete response. However, only a fraction of patients benefit from checkpoint inhibitor immunotherapy. Improving the response rate and maximizing efficacy for immunotherapy are still unmet needs of great clinical significance. We have developed a novel combination cancer therapy referred to as Synergistic Immuno Photothermal Nanotherapy (SYMPHONY) by integrating plasmonic gold nanostars (GNS)-mediated photothermal therapy with anti-PD-L1 checkpoint inhibitor immunotherapy to improve the therapeutic efficacy of cancer treatment. GNS nanoparticles are superior photon-to-heat conversion transducers for photothermal therapy because they have tip-enhanced plasmonics from multiple sharp spikes and tunable plasmonic absorption in the near-infrared tissue optical window. Our in vivo experimental results with bladder cancer and brain cancer murine animal models have demonstrated that photothermal therapy with GNS in combination with anti-PD-L1 checkpoint inhibitor immunotherapy not only effectively ablates primary tumor with laser irradiation but also stimulates the immune response to treat distant tumors without laser irradiation. Furthermore, a rechallenge study performed three months later using cancer cells injected into long-term survivors demonstrates that SYMPHONY therapy can generate a long-term memorized immune response and prevent cancer recurrences in cured mice. The SYMPHONY combination therapy results in synergistic treatment surpassing what either single treatment can achieve, and shows the great promise for future translational medicine studies to treat aggressive metastatic cancer diseases. Citation Format: Yang Liu, Paolo Maccarini, Gregory M. Palmer, Brant A. Inman, Peter E. Fecci, Tuan Vo-Dinh. Synergistic immuno photothermal nanotherapy (SYMPHONY) for combination cancer therapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6630.

Therapeutically exploiting vascular and metabolic endpoints becomes critical to translational cancer studies because altered vascularity and deregulated metabolism are two important cancer hallmarks. The metabolic and vascular phenotypes of three sibling breast tumor lines with different metastatic potential are investigated in vivo with a newly developed quantitative spectroscopy system. All tumor lines have different metabolic and vascular characteristics compared to normal tissues, and there are strong positive correlations between metabolic (glucose uptake and mitochondrial membrane potential) and vascular (oxygen saturations and hemoglobin concentrations) parameters for metastatic (4T1) tumors but not for micrometastatic (4T07) and nonmetastatic (67NR) tumors. A longitudinal study shows that both vascular and metabolic endpoints of 4T1 tumors increased up to a specific tumor size threshold beyond which these parameters decreased. The synchronous changes between metabolic and vascular parameters, along with the strong positive correlations between these endpoints suggest that 4T1 tumors rely on strong oxidative phosphorylation in addition to glycolysis. This study illustrates the great potential of our optical technique to provide valuable dynamic information about the interplay between the metabolic and vascular status of tumors, with important implications for translational cancer investigations.

Biocompatible gold nanostars (GNS) with tip-enhanced electromagnetic and optical properties have been developed and applied for multifunctional cancer diagnostics and therapy (theranostics). Their multiple sharp branches acting like “lightning rods” can convert safely and efficiently light into heat. As with other nanoparticles, GNS sizes can be controlled so that they passively accumulate in tumors due to the enhanced permeability and retention (EPR) effect of tumor vasculature. This feature improves tumor-targeting precision and permits the use of reduced laser energy required to destroy the targeted cancer cells. The ability to selectively heat tumor areas where GNS are located while keeping surrounding healthy tissues at significantly lower temperatures offers significant advantages over other thermal therapies. GNS-mediated photothermal therapy combined with checkpoint immunotherapy was shown to reverse tumor-mediated immunosuppression, leading to the treatment of not only primary tumors but also cancer metastasis as well as inducing effective long-lasting immunity, i.e. an anticancer ‘vaccine’ effect.

Hyperactivation of the NFκB pathway is a distinct feature of inflammatory breast cancer (IBC), a highly proliferative and lethal disease. Gene expression studies in IBC patient tissue have linked EGFR (EGFR/HER2)-mediated MAPK signaling to NFκB hyperactivity, but the mechanism(s) by which this occurs remain unclear. Here, we report that the X-linked inhibitor of apoptosis protein (XIAP) plays a central role in linking these two pathways. XIAP overexpression correlated with poor prognoses in breast cancer patients and was frequently observed in untreated IBC patient primary tumors. XIAP drove constitutive NFκB transcriptional activity, which mediated ALDH positivity (a marker of stem-like cells), in vivo tumor growth, and an IBC expression signature in patient-derived IBC cells. Using pathway inhibitors and mathematical models, we defined a new role for the MAPK interacting (Ser/Thr)-kinase (MNK) in enhancing XIAP expression and downstream NFκB signaling. Furthermore, targeted XIAP knockdown and treatment with a MNK inhibitor decreased tumor cell migration in a dorsal skin fold window chamber murine model that allowed for intravital imaging of local tumor growth and migration. Together, our results indicate a novel role for XIAP in the molecular cross-talk between MAPK and NFκB pathways in aggressive tumor growth, which has the potential to be therapeutically exploited. Significance: Signaling by the MNK kinase is essential in inflammatory breast cancer, and it can be targeted to inhibit XIAP–NFκB signaling and the aggressive phenotype of this malignancy. Cancer Res; 78(7); 1726–38. ©2018 AACR.

BACKGROUND Transfusion of red blood cells (RBCs) is a frequent health care practice. However, unfavorable consequences may occur from transfusions of stored RBCs and are associated with RBC changes during storage. Loss of S-nitrosohemoglobin (SNO-Hb) and other S-nitrosothiols (SNOs) during storage is implicated as a detriment to transfusion efficacy. It was hypothesized that restoring SNOs within banked RBCs would improve RBC functions relevant to successful transfusion outcomes, namely, increased deformability and decreased adhesivity. STUDY DESIGN AND METHODS Stored human RBCs were incubated with nitric oxide (NO) donors PROLI/NO and DEA/NO (disodium 1-[2-(carboxylato)-pyrrolidin-1-yl]diazen-1-ium-1,2-diolate and diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate) under varying experimental conditions (e.g., aerobic/anaerobic incubation, NO donor to RBC ratio). SNO restoration was evaluated in vitro and in vivo as a means to improve RBC function after storage. RESULTS Incubation of RBCs with the NO donors resulted in 10-fold greater levels of SNO-Hb versus untreated control or sham RBCs, with significantly higher Hb-bound NO yields from an NO dose delivered by DEA/NO. RBC incubation with DEA/NO at a stoichiometry of 1:62.5 NO:Hb significantly increased RBC deformabilty and reduced adhesion to cultured endothelial cells. RBC incubation with DEA/NO also increased S-nitrosylation of RBC cytoskeletal and membrane proteins, including the β-spectrin chain. Renitrosylation attenuated both RBC sequestration in the lung and the mild blood oxygen saturation impairments seen with banked RBCs in a mouse model of transfusion. CONCLUSIONS RBC renitrosylation using NO donors has promise for correcting deficient properties (e.g., adhesivity, rigidity, and SNO loss) of banked RBCs and in turn improving transfusion outcomes.

Surface modification of nanoparticles and biosensors is a dynamic, expanding area of research for targeted delivery in vivo. For more efficient delivery, surfaces are PEGylated to impart stealth properties, long circulation and enable enhanced permeability and retention (EPR) in tumor tissues. Previously, BF2dbm(I)PLA was proven to be a good oxygen nanosensor material for tumor hypoxia imaging in vivo, though particles were applied directly to the tumor and surrounding region. Further surface modification is needed for this dual-emissive oxygen sensitive material for effective intravenous (IV) administration and passive and active delivery to tumors. In this paper an efficient synthesis of a new dual-emissive material BF2dbm(I)PLA-mPEG is presented and in vitro stability studies were conducted. It is found that fabricated nanoparticles are stable for 24 weeks as a suspension, while after 25 weeks the nanoparticles swell and both dye and polymer degradation escalates. Preliminary studies show BF2dbm(I)PLA-mPEG nanoparticle accumulation in a window chamber mammary tumor 24 hours after IV injection into mice (C57Bl/6 strain) enabling tumor oxygen imaging.

Motivated by the goal of developing a fully biodegradable optical contrast agent with translational clinical potential, a nanoparticle delivery vehicle was generated from the self-assembly of poly(ethylene-glycol)-block-poly(trimethylene carbonate-co-caprolactone) (PEG-b-TCL) copolymers. Cryogenic transmission electron microscopy verified that PEG-b-TCL-based micelles were formed at low solution temperatures (~ 38 °C). Detailed spectroscopic experiments validated facile loading of large quantities of the high emission dipole strength, tris(porphyrin)-based fluorophore PZn3 within their cores, and the micelles displayed negligible in vitro and in vivo toxicities in model systems. The pharmacokinetics and biodistribution of PZn3-loaded PEG-b-TCL-based micelles injected intravenously were determined via ex vivo near-infrared (NIR) imaging of PZn3 emission in microcapillary tubes containing minute quantities of blood, to establish a novel method for minimally invasive pharmacokinetic monitoring. The in vivo circulatory half-life of the PEG-b-TCL-based micelles was found to be ~19.6 h. Additionally, longitudinal in vivo imaging of orthotopically transplanted breast tumors enabled determination of micelle biodistribution that correlated to ex vivo imaging results, demonstrating accumulation predominantly within the tumors and livers of mice. The PEG-b-TCL-based micelles quickly extravasated within 4T1 orthotopic mammary carcinomas, exhibiting peak accumulation at ~48 h following intravenous tail-vein injection. In summary, PEG-b-TCL-based micelles demonstrated favorable characteristics for further development as in vivo optical contrast agents for minimally invasive imaging of breast tumors.

Luminescent difluoroboron β-diketonate poly(lactic acid) (BF2bdkPLA) materials serve as biological imaging agents. In this study, dye structures were modified to achieve emission colors that span the visible region with potential for multiplexing applications. Four dyes with varying π−conjugation (phenyl, naphthyl) and donor groups (-OMe, -NMe2) were coupled to PLLA-PEG block copolymers (~11 kDa) by a post-polymerization Mitsunobu reaction. The resulting dye-polymer conjugates were fabricated as nanoparticles (~55 nm diameter) to produce nanomaterials with a range of emission colors (420–640 nm). For increased stability, dye-PLLA-PEG conjugates were also blended with dye-free PDLA-PEG to form stereocomplex nanoparticles of smaller size (~45 nm diameter). The decreased dye loading in the stereoblocks blue-shifted the emission, generating a broader range of fluorescence colors (410–620 nm). Tumor accumulation was confirmed in a murine model through biodistribution studies with a red emitting dimethyl amino-substituted dye-polymer analogue. The synthesis, optical properties, oxygen-sensing capabilities, and stability of these block copolymer nanoparticles are presented.

Hypoxia correlates with reduced radiosensitivity and therapy response. Imaging anesthisized mice using fluorescent nanoparticles causes problems in modeling hypoxia. Therefore, a mobile imaging apparatus was developed to image un-anesthetized mice.

Tissue-engineered skeletal muscle can serve as a physiological model of natural muscle and a potential therapeutic vehicle for rapid repair of severe muscle loss and injury. Here, we describe a platform for engineering and testing highly functional biomimetic muscle tissues with a resident satellite cell niche and capacity for robust myogenesis and self-regeneration in vitro. Using a mouse dorsal window implantation model and transduction with fluorescent intracellular calcium indicator, GCaMP3, we nondestructively monitored, in real time, vascular integration and the functional state of engineered muscle in vivo. During a 2-wk period, implanted engineered muscle exhibited a steady ingrowth of blood-perfused microvasculature along with an increase in amplitude of calcium transients and force of contraction. We also demonstrated superior structural organization, vascularization, and contractile function of fully differentiated vs. undifferentiated engineered muscle implants. The described in vitro and in vivo models of biomimetic engineered muscle represent enabling technology for novel studies of skeletal muscle function and regeneration.

Intact red blood cells (RBCs) are required for phenotypic analyses. In order to allow separation (time and location) between subject encounter and sample analysis, we developed a research-specific RBC cryopreservation protocol and assessed its impact on data fidelity for key biochemical and physiological assays. RBCs drawn from healthy volunteers were aliquotted for immediate analysis or following glycerol-based cryopreservation, thawing, and deglycerolization. RBC phenotype was assessed by (1) scanning electron microscopy (SEM) imaging and standard morphometric RBC indices, (2) osmotic fragility, (3) deformability, (4) endothelial adhesion, (5) oxygen (O2) affinity, (6) ability to regulate hypoxic vasodilation, (7) nitric oxide (NO) content, (8) metabolomic phenotyping (at steady state, tracing with [1,2,3-13C3]glucose ± oxidative challenge with superoxide thermal source; SOTS-1), as well as in vivo quantification (following human to mouse RBC xenotransfusion) of (9) blood oxygenation content mapping and flow dynamics (velocity and adhesion). Our revised glycerolization protocol (40% v/v final) resulted in >98.5% RBC recovery following freezing (-80°C) and thawing (37°C), with no difference compared to the standard reported method (40% w/v final). Full deglycerolization (>99.9% glycerol removal) of 40% v/v final samples resulted in total cumulative lysis of ~8%, compared to ~12–15% with the standard method. The post cryopreservation/deglycerolization RBC phenotype was indistinguishable from that for fresh RBCs with regard to physical RBC parameters (morphology, volume, and density), osmotic fragility, deformability, endothelial adhesivity, O2 affinity, vasoregulation, metabolomics, and flow dynamics. These results indicate that RBC cryopreservation/deglycerolization in 40% v/v glycerol final does not significantly impact RBC phenotype (compared to fresh cells).

Hypoxia is a significant therapeutic problem for solid tumors because hypoxic cells are treatment-resistant and more aggressive. Hypoxia-activated prodrugs such as SN30000 use a mechanism of activation in hypoxic cells similar to that of 2-nitroimidazole hypoxia PET tracers. Therefore, we have evaluated the usefulness of 2-(2-nitro-1H-imidazol-1-yl)-N-(2,2,3,3,3-(18)F-pentafluoropropyl)-acetamide ((18)F-EF5) PET to monitor and predict tumor response to SN30000 plus radiation treatment (RT).Human non-small cell lung cancer xenografts (H460) in athymic rats were imaged with (18)F-EF5 PET before and after treatment with SN30000 (90 mg/kg), with or without 15-Gy RT. The feasibility of imaging early changes in hypoxia in response to SN30000 was examined 24 h after treatment, followed by ex vivo γ-counting and immunohistochemical examination to study drug-induced apoptosis. Subsequently, the therapeutic effects of SN30000 with or without RT were evaluated in tumor growth delay studies and compared with early treatment-induced changes observed by (18)F-EF5 PET. Changes in tumor hemoglobin oxygen saturation as a function of time after treatment measured by optical spectroscopy were compared with PET data.The uptake of (18)F-EF5 was significantly lower in SN30000-treated tumors than in saline controls 24 h after treatment (mean standardized uptake value, 0.44 ± 0.08 vs. 0.56 ± 0.08 for control group; P0.05). Apoptosis was significantly higher in SN30000-treated tumors than in controls. Early treatment-induced changes in (18)F-EF5 uptake were indicative of tumor response in growth delay studies at the group level. SN30000 plus RT significantly decreased (18)F-EF5 uptake relative to baseline and resulted in complete tumor remission in 5 of 7 animals. SN30000 alone decreased (18)F-EF5 uptake, generally in tumors with high initial standardized uptake values, and showed a minor tumor growth delay effect. The changes induced by SN30000 with or without RT in (18)F-EF5 uptake correlated with baseline hypoxia levels. RT caused significant increases in tumor oxygen concentration and hemoglobin oxygen saturation.A hypoxia PET imaging agent can measure changes in tumor hypoxic fraction in response to SN30000. These results suggest the utility of (18)F-EF5 PET for monitoring early response to tumor treatment with SN30000 plus RT in the clinical development of this novel hypoxia-activated prodrug.

Intravital microscopy of the pulmonary microcirculation in research animals is of great scientific interest for its utility in identifying regional changes in pulmonary microcirculatory blood flow. Although feasibility studies have been reported, the pulmonary window can be further refined into a practical tool for pharmaceutical research and drug development. We have established a method to visualize and quantify dynamic changes in three key features of lung function: microvascular red blood cell velocity, flow direction, and hemoglobin saturation. These physiological parameters were measured in an acute closed-chest pulmonary window, which allows real-time images to be captured by fluorescence and multispectral absorption microscopy; images were subsequently quantified using computerized analysis. We validated the model by quantifying changes in microcirculatory blood flow and hemoglobin saturation in two ways: 1) after changes in inspired oxygen content and 2) after pharmacological reduction of pulmonary blood flow via treatment with the β1 adrenergic receptor blocker metoprolol. This robust and relatively simple system facilitates pulmonary intravital microscopy in laboratory rats for pharmacological and physiological research.

Hypoxia and angiogenesis can significantly influence the efficacy of cancer therapy and the behavior of surviving tumor cells. There is a growing demand for technologies to measure tumor hypoxia and angiogenesis temporally in vivo to enable advances in drug development and optimization. This paper reports the use of frequency-domain photon migration with a side-firing probe to quantify tumor oxygenation and hemoglobin concentrations in nude rats bearing human head/neck tumors administered with carbogen gas, cycling hypoxic gas or just room air. Significant increase (with carbogen gas breathing) or decrease (with hypoxic gas breathing) in tumor oxygenation was observed. The trend in tumor oxygenation during forced cycling hypoxia (CH) followed that of the blood oxygenation measured with a pulse oximeter. Natural CH was also observed in rats under room air. The studies demonstrated the potential of the technology for longitudinal monitoring of tumor CH during tumor growth or in response to therapy.

High-resolution reflectance-derived optical parameter maps of the ratio of the β-carotene concentration to the reduced scattering coefficient can be used to rapidly survey tissue morphology for large tissue areas on intra-operative time scales.

Decreased physical performance is a well-known consequence of rapid ascent to high altitude. Hypoxic pulmonary vasoconstriction (HPV) potentially limits cardiac output and systemic blood flow, thus preventing successful adaptation to rapid ascent. We hypothesized that pharmacological enhancement of the heart rate with theophylline, combined with reversal of HPV via endothelin blockade, could increase exercise performance at high altitude. Female Sprague-Dawley rats were treated with combinations of 1) theophylline, 2) the endothelin receptor antagonists sitaxsentan/ambrisentan, and/or 3) phosphodiesterase-5 inhibitor sildenafil and exposed to either a simulated high altitude (4,267 m) or 12% oxygen. Exercise capacity, peripheral blood flow, hemodynamics, and pulmonary leak were examined. Combination treatment with theophylline and endothelin blockade, but not with the respective single compounds, significantly prolonged run-to-fatigue time under simulated high altitude. No such efficacy was found when theophylline was combined with sildenafil. Neither theophylline nor sitaxsentan or their combination influenced breathing rates and hemoglobin oxygen saturation. Whereas under hypoxia, theophylline significantly increased muscular blood flow, and sitaxsentan increased tissue oxygenation, the combination improved both parameters but in a reduced manner. Under hypoxia, the combination treatment but not the single compounds significantly enhanced pulmonary arterial pressure compared with controls (13.1 ± 6.3 vs. 11.9 ± 5.2 mmHg), whereas mean arterial pressure remained unaffected. Pulmonary wet-to-dry weight ratios were unaffected by combination treatment. We conclude that concomitant dosing with a cardiac stimulant and endothelin antagonist can partially reverse loss of physical performance capacity under hypobaric hypoxia, independent from improving blood oxygen saturation.

BACKGROUND AND PURPOSE Pathological angiogenesis is associated with various human diseases, such as cancer, autoimmune diseases and retinopathy. The angiopoietin (Ang)–Tie2 system plays critical roles in several steps of angiogenic remodelling. Here, we have investigated the anti-angiogenic effect of a novel angiopoietin-derived peptide. EXPERIMENTAL APPROACH Using computational methods, we identified peptides from helical segments within angiopoietins, which were predicted to inhibit their activity. These peptides were tested using biochemical methods and models of angiogenesis. The peptide with best efficacy, A11, was selected for further characterization as an anti-angiogenic compound. KEY RESULTS The potent anti-angiogenic activity of A11 was demonstrated in a multicellular assay of angiogenesis and in the chorioallantoic membrane model. A11 bound to angiopoietins and reduced the binding of Ang-2 to Tie2. A11 was also significantly reduced vascular density in a model of tumour-induced angiogenesis. Its ability to inhibit Ang-2 but not Ang-1-induced endothelial cell migration, and to down-regulate Tie2 levels in tumour microvessels, suggests that A11 targets the Ang–Tie2 pathway. In a rat model of oxygen-induced retinopathy, A11 strongly inhibited retinal angiogenesis. Moreover, combination of A11 with an anti-VEGF antibody showed a trend for further inhibition of angiogenesis, suggesting an additive effect. CONCLUSIONS AND IMPLICATIONS Our results indicate that A11 is a potent anti-angiogenic compound, through modulation of the Ang–Tie2 system, underlining its potential as a therapeutic agent for the treatment of ocular and tumour neovascularization, as well as other pathological conditions that are dependent on angiogenesis.

A wide variety of imaging approaches have been developed in the past few decades for monitoring tumor oxygenation and hypoxia in vivo. In particular, nuclear medicine has seen the development of several radiolabeled hypoxia markers and is the preferred method for imaging of tumor hypoxia. Hypoxia imaging is increasingly being used in the clinical setting and is progressing from a mere detection method to application in individualization of chemoradiotherapy.

Cancer has become a severe thread to human health resulting in more than eight million deaths each year. Cancer metastasis is the main mechanism in more than 90 percent of cancer deaths. However, current therapeutic options including chemotherapy and radiation therapy have limited therapeutic effects to treat cancer metastasis. Therefore, there is an urgent need to develop novel methods for cancer treatment. Our group has developed a unique method to synthesize star-shaped gold nanoparticles, gold nanostars (GNS), without using toxic surfactant, which is very suitable for in vivo applications. The synthesized GNS has tip-enhanced plasmonics and tunable strong in the near-infrared “tissue optical window”, enabling it a superior photon-to-heat nanotransducer. The GNS nanoparticles accumulate preferably into tumor with enhanced permeability and retention (EPR) effect after intravenous administration. We have applied GNS for photothermal therapy with 808 nm laser to specifically ablate tumor. Here we report a new cancer treatment method by combing nanophotothermal therapy and anti-PD-L1 immunotherapy. Immunogenic cell death is often insufficient to trigger effective anti-cancer immunity due to the presence of immunosuppressive factors occurring in the tumor microenvironment such as hypoxia, impaired leukocyte trafficking and inhibitory immune checkpoint signaling. The GNS-mediated photothermal ablation can reduce hypoxia and improve leukocyte trafficking to the tumor. In addition, the administration of anti-PD-L1 antibody reverses another critical immunosuppression factor. Experiment results using a mouse bladder cancer model demonstrated that the combined photothermal and immunotherapy have synergistic effect and can not only eradicate primary tumors with laser treatment but also distant metastasis without laser treatment. Furthermore, delayed rechallenge in the cured mice didn't result in new tumor generation, indicating that our combined therapy induced memorized immune response against cancer. The combined GNS-mediated photothermal therapy and anti-PD-L1 immunotherapy has promise to improve therapeutic effect on not only primary tumor but also cancer metastasis. Citation Format: Yang Liu, Paolo Maccarini, Gregory M. Palmer, Brant A. Inman, Tuan Vo-Dinh. Synergistic gold nanostar-mediated photothermal and immunotherapy for cancer metastasis treatment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4152.

The shifting metabolic landscape of aggressive tumors, with fluctuating oxygenation conditions and temporal changes in glycolysis and mitochondrial metabolism, is a critical phenomenon to study in order to understand negative treatment outcomes. Recently, we have demonstrated near-simultaneous optical imaging of mitochondrial membrane potential (MMP) and glucose uptake in non-tumor window chambers, using the fluorescent probes tetramethylrhodamine ethyl ester (TMRE) and 2-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG). Here, we demonstrate a complementary technique to perform near-simultaneous in vivo optical spectroscopy of tissue vascular parameters, glucose uptake, and MMP in a solid tumor model that is most often used for therapeutic studies. Our study demonstrates the potential of optical spectroscopy as an effective tool to quantify the vascular and metabolic characteristics of a tumor, which is an important step towards understanding the mechanisms underlying cancer progression, metastasis, and resistance to therapies.