Optical imaging reveals chemotherapy-induced metabolic reprogramming of residual disease and recurrence.

Fewer than 20% of triple-negative breast cancer patients experience long-term responses to mainstay chemotherapy. Resistant tumor subpopulations use alternative metabolic pathways to escape therapy, survive, and eventually recur. Here, we show in vivo, longitudinal metabolic reprogramming in residual disease and recurrence of triple-negative breast cancer xenografts with varying sensitivities to the chemotherapeutic drug paclitaxel. Optical imaging coupled with metabolomics reported an increase in non-glucose-driven mitochondrial metabolism and an increase in intratumoral metabolic heterogeneity during regression and residual disease in resistant MDA-MB-231 tumors. Conversely, sensitive HCC-1806 tumors were primarily reliant on glucose uptake and minimal changes in metabolism or heterogeneity were observed over the tumors' therapeutic life cycles. Further, day-matched resistant HCC-1806 tumors revealed a higher reliance on mitochondrial metabolism and elevated metabolic heterogeneity compared to sensitive HCC-1806 tumors. Together, metabolic flexibility, increased reliance on mitochondrial metabolism, and increased metabolic heterogeneity are defining characteristics of persistent residual disease, features that will inform the appropriate type and timing of therapies.

Evaluating Tumor Hypoxia Radiosensitization Via Electron Paramagnetic Resonance Oxygen Imaging (EPROI).

PURPOSE: Tumor hypoxia contributes to aggressive phenotypes and diminished therapeutic responses to radiation therapy (RT) with hypoxic tissue being 3-fold less radiosensitive than normoxic tissue. A major challenge in implementing hypoxic radiosensitizers is the lack of a high-resolution imaging modality that directly quantifies tissue-oxygen. The electron paramagnetic resonance oxygen-imager (EPROI) was used to quantify tumor oxygenation in two murine tumor models: E0771 syngeneic transplant breast cancers and primary p53/MCA soft tissue sarcomas, with the latter autochthonous model better recapitulating the tumor microenvironment in human malignancies. We hypothesized that tumor hypoxia differs between these models. We also aimed to quantify the absolute change in tumor hypoxia induced by the mitochondrial inhibitor papaverine (PPV) and its effect on RT response. PROCEDURES: Tumor oxygenation was characterized in E0771 and primary p53/MCA sarcomas via EPROI, with the former model also being quantified indirectly via diffuse reflectance spectroscopy (DRS). After confirming PPV's effect on hypoxic fraction (via EPROI), we compared the effect of 0 versus 2 mg/kg PPV prior to 20 Gy on tumor growth delay and survival. RESULTS: Hypoxic sarcomas were more radioresistant than normoxic sarcomas (p=0.0057, 2-way ANOVA), and high baseline hypoxic fraction was a significant (p=0.0063, Cox Regression Model) hazard in survivability regardless of treatment. Pre-treatment with PPV before RT did not radiosensitize tumors in the sarcoma or E0771 model. In the sarcoma model, EPROI successfully identified baseline hypoxic tumors. DRS quantification of total hemoglobin, saturated hemoglobin, changes in mitochondrial potential and glucose uptake showed no significant difference in E0771 tumors pre- and post-PPV. CONCLUSION: EPROI provides 3D high-resolution pO2 quantification; EPR is better suited than DRS to characterize tumor hypoxia. PPV did not radiosensitize E0771 tumors nor p53/MCA sarcomas, which may be related to the complex pattern of vasculature in each tumor. Additionally, understanding model-dependent tumor hypoxia will provide a much-needed foundation for future therapeutic studies with hypoxic radiosensitizers.

A Novel Preclinical Murine Model to Monitor Inflammatory Breast Cancer Tumor Growth and Lymphovascular Invasion.

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.

A Clinical Study to Assess Diffuse Reflectance Spectroscopy with an Auto-Calibrated, Pressure-Sensing Optical Probe in Head and Neck Cancer.

Diffuse reflectance spectroscopy (DRS) is a powerful tool for quantifying optical and physiological tissue properties such as hemoglobin oxygen saturation and vascularity. DRS is increasingly used clinically for distinguishing cancerous lesions from normal tissue. However, its widespread clinical acceptance is still limited due to uncontrolled probe-tissue interface pressure that influences reproducibility and introduces operator-dependent results. In this clinical study, we assessed and validated a pressure-sensing and automatic self-calibration DRS in patients with suspected head and neck squamous cell carcinoma (HNSCC). The clinical study enrolled nineteen patients undergoing HNSCC surgical biopsy procedures. Patients consented to evaluation of this improved DRS system during surgery. For each patient, we obtained 10 repeated measurements on one tumor site and one distant normal location. Using a Monte Carlo-based model, we extracted the hemoglobin saturation data along with total hemoglobin content and scattering properties. A total of twelve cancer tissue samples from HNSCC patients and fourteen normal tissues were analyzed. A linear mixed effects model tested for significance between repeated measurements and compared tumor versus normal tissue. These results demonstrate that cancerous tissues have a significantly lower hemoglobin saturation compared to normal controls (p < 0.001), which may be reflective of tumor hypoxia. In addition, there were minimal changes over time upon probe placement and repeated measurement, indicating that the pressure-induced changes were minimal and repeated measurements did not differ significantly from the initial value. This study demonstrates the feasibility of conducting optical spectroscopy measurements on intact lesions prior to removal during HNSCC procedures, and established that this probe provides diagnostically-relevant physiologic information that may impact further treatment.

Quantifying the effects of anesthesia on intracellular oxygen via low-cost portable microscopy using dual-emissive nanoparticles.

Intracellular oxygenation is an important parameter for numerous biological studies. While there are a variety of methods available for acquiring in vivo measurements of oxygenation in animal models, most are dependent on indirect oxygen measurements, restraints, or anesthetization. A portable microscope system using a Raspberry Pi computer and Pi Camera was developed for attaching to murine dorsal window chambers. Dual-emissive boron nanoparticles were used as an oxygen-sensing probe while mice were imaged in awake and anesthetized states. The portable microscope system avoids altered in vivo measurements due to anesthesia or restraints while enabling increased continual acquisition durations.

Intravital optical imaging for immune cell tracking after photoimmunotherapy with plasmonic gold nanostars.

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.

Plasmonic gold nanostars for synergistic photoimmunotherapy to treat cancer.

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.

Dual-emissive, oxygen-sensing boron nanoparticles quantify oxygen consumption rate in breast cancer cells.

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.

Cherenkov emissions for studying tumor changes during radiation therapy: An exploratory study in domesticated dogs with naturally-occurring cancer.

PURPOSE: Real-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. METHODS: A 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. RESULTS: We 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 (p<0.05). Additionally, intra-subfraction changes suggest that Cherenkov emissions may have captured fluctuating absorption properties within the tumor. CONCLUSION: Here 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.

Clinical and Pre-clinical Methods for Quantifying Tumor Hypoxia.

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.

Targeting Fluorescent Nanodiamonds to Vascular Endothelial Growth Factor Receptors in Tumor.

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.

Simultaneous in vivo optical quantification of key metabolic and vascular endpoints reveals tumor metabolic diversity in murine breast tumor models.

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.

Red blood cell phenotype fidelity following glycerol cryopreservation optimized for research purposes.

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).

Development and Preliminary Evaluation of a Murine Model of Chronic Radiation-Induced Proctitis.

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.

Near-simultaneous quantification of glucose uptake, mitochondrial membrane potential, and vascular parameters in murine flank tumors using quantitative diffuse reflectance and fluorescence spectroscopy.

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.

Assessing effects of pressure on tumor and normal tissue physiology using an automated self-calibrated, pressure-sensing probe for diffuse reflectance spectroscopy.

Diffuse reflectance spectroscopy (DRS) represents a quantitative, noninvasive, nondestructive means of assessing vascular oxygenation, vascularity, and structural properties. However, it is known that such measurements can be influenced by the effects of pressure, which is a major concern for reproducible and operator-independent assessment of tissues. Second, regular calibration is a necessary component of quantitative DRS to account for factors such as lamp decay and fiber bending. Without a means of reliably controlling for these factors, the accuracy of any such assessments will be reduced, and potentially biased. To address these issues, a self-calibrating, pressure-controlled DRS system is described and applied to both a patient-derived xenograft glioma model, as well as a set of healthy volunteers for assessments of oral mucosal tissues. It was shown that pressure had a significant effect on the derived optical parameters, and that the effects on the optical parameters were magnified with increasing time and pressure levels. These findings indicate that not only is it critical to integrate a pressure sensor into a DRS device, but that it is also important to do so in an automated way to trigger a measurement as soon as possible after probe contact is made to minimize the perturbation to the tissue site.

Noninvasive optical spectroscopy for identification of non-melanoma skin cancer: Pilot study.

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.

XIAP Regulation by MNK Links MAPK and NFκB Signaling to Determine an Aggressive Breast Cancer Phenotype.

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.

NIR-emissive PEG-b-TCL micelles for breast tumor imaging and minimally invasive pharmacokinetic analysis.

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.

Synergistic Immuno Photothermal Nanotherapy (SYMPHONY) for the Treatment of Unresectable and Metastatic Cancers.

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.