There are limited methods that are currently available for longitudinal and non-invasive in vivo ... more There are limited methods that are currently available for longitudinal and non-invasive in vivo assessment of the transport kinetics of carrier-based therapeutics, such as those relying on liposomes. A viable strategy to noninvasively track liposomes in vivo is to load them with contrast-enhancing agents that are well retained within the carrier. The encapsulation of contrast agents such as iodine and gadolinium allows for the liposomes to be tracked by computed tomography (CT) and magnetic resonance (MR) imaging systems, respectively. This study evaluated the feasibility of using CT and MR imaging as a means to gain a quantitative assessment of the pathway and fate of liposomes in vivo in rabbits. The liposome formulation examined was composed of DPPC/Cholesterol/DSPEPEG2000 in a 55/40/5 molar ratio and the vesicles had an average diameter of 80 nm. The commercially available agents iohexol (CT agent) and gadoteridol (MR agent) were encapsulated within the liposomes and found to b...
An integrated augmented reality (AR) surgical navigation system that potentially improves intra-o... more An integrated augmented reality (AR) surgical navigation system that potentially improves intra-operative visualization of concealed anatomical structures. Integration of real-time tracking technology with a laser pico-projector allows the surgical surface to be augmented by projecting virtual images of lesions and critical structures created by multimodality imaging. We aim to quantitatively and qualitatively evaluate the performance of a prototype interactive AR surgical navigation system through a series of pre-clinical studies. Four pre-clinical animal studies using xenograft mouse models were conducted to investigate system performance. A combination of CT, PET, SPECT, and MRI images were used to augment the mouse body during image-guided procedures to assess feasibility. A phantom with machined features was employed to quantitatively estimate the system accuracy. All the image-guided procedures were successfully performed. The tracked pico-projector correctly and reliably depi...
Nanoscale perfluorocarbon (PFC) droplets have enormous potential as clinical theranostic agents. ... more Nanoscale perfluorocarbon (PFC) droplets have enormous potential as clinical theranostic agents. They are biocompatible and are currently used in vivo as contrast agents for a variety of medical imaging modalities, including ultrasound, computed tomography, photoacoustic and 19 F-magnetic resonance imaging. PFC nanodroplets can also carry molecular and nanoparticulate drugs and be activated in situ by ultrasound or light for targeted therapy. Recently, there has been renewed interest in using PFC nanodroplets for hypoxic tumor reoxygenation towards radiosensitization based on the high oxygen solubility of PFCs. Previous studies showed that tumor oxygenation using PFC agents only occurs in combination with enhanced oxygen breathing. However, recent studies suggest that PFC agents that accumulate in solid tumors can contribute to radiosensitization, presumably due to tumor reoxygenation without enhanced oxygen breathing. In this study, we quantify the impact of oxygenation due to PFC nanodroplet accumulation in tumors alone in comparison with other reoxygenation methodologies, in particular, carbogen breathing. Methods: Lipid-stabilized, PFC (i.e., perfluorooctyl bromide, CF3(CF2)7Br, PFOB) nanoscale droplets were synthesized and evaluated in xenograft prostate (DU145) tumors in male mice. Biodistribution assessment of the nanodroplets was achieved using a fluorescent lipophilic indocarbocyanine dye label (i.e., DiI dye) on the lipid shell in combination with fluorescence imaging in mice (n≥3 per group). Hypoxia reduction in tumors was measured using PET imaging and a known hypoxia radiotracer, [ 18 F]FAZA (n≥ 3 per group). Results: Lipid-stabilized nanoscale PFOB emulsions (mean diameter of ~250 nm), accumulated in the xenograft prostate tumors in mice 24 hours post-injection. In vivo PET imaging with [ 18 F]FAZA showed that the accumulation of the PFOB nanodroplets in the tumor tissues alone significantly reduced tumor hypoxia, without enhanced oxygen (i.e., carbogen) breathing. This reoxygenation effect was found to be comparable with carbogen breathing alone. Conclusion: Accumulation of nanoscale PFOB agents in solid tumors alone successfully reoxygenated hypoxic tumors to levels comparable with carbogen breathing alone, an established tumor oxygenation method. This study confirms that PFC agents can be used to reoxygenate hypoxic tumors in addition to their current applications as multifunctional theranostic agents.
Purpose: Hypoxia is linked to aggressiveness, resistance to therapy, and poor prognosis of pancre... more Purpose: Hypoxia is linked to aggressiveness, resistance to therapy, and poor prognosis of pancreatic tumors. Liposomal irinotecan (nal-IRI, ONIVYDE®) has shown potential in reducing hypoxia in the HT29 colorectal cancer model, and here, we investigate its therapeutic activity and ability to modulate hypoxia in patient-derived orthotopic tumor models of pancreatic cancer. Procedures: Mice were randomized into nal-IRI treated and untreated controls. Magnetic resonance imaging was used for monitoring treatment efficacy, positron emission tomography (PET) imaging with F-18-labelled fluoroazomycinarabinoside ([ 18 F]FAZA) for tumor hypoxia quantification, and F-18-labelled fluorothymidine ([ 18 F]FLT) for tumor cell proliferation. Results: The highly hypoxic OCIP51 tumors showed significant response following nal-IRI treatment compared with the less hypoxic OCIP19 tumors. [ 18 F]FAZA-PET detected significant hypoxia reduction in treated OCIP51 tumors, 8 days before significant changes in tumor volume. OCIP19 tumors also responded to therapy, although tumor volume control was not accompanied by any reduction in [ 18 F]FAZA uptake. In both models, no differences were observable in [ 18 F]FLT uptake in treated tumors compared with control mice. Conclusions: Hypoxia modulation may play a role in nal-IRI's mechanism of action. Nal-IRI demonstrated greater anti-tumor activity in the more aggressive and hypoxic tumor model. Furthermore, hypoxia imaging provided early prediction of treatment response.
To evaluate CF800, a novel lipid-based liposomal nanoparticle that co-encapsulates indocyanine gr... more To evaluate CF800, a novel lipid-based liposomal nanoparticle that co-encapsulates indocyanine green (ICG) and iohexol, for CT imaging of pulmonary vasculature in minimallyinvasive thoracic surgery planning. Methods CF800 was intravenously administered to 7 healthy rabbits. In vivo CT imaging was performed 15 min post-injection, with a subset of animals imaged at 24h, 48h, and 72h post injection. Signal-to-background ratios (SBR) were calculated at the inferior vena cava and compared across time-points. A similar protocol was applied to 2 healthy pigs to evaluate the feasibility and efficacy in a large animal model. To evaluate the feasibility of clinical application, a survey was completed by 7 surgical trainees to assess pre-and post-injection CT images of rabbits and pigs. Responses on the discernibility of pulmonary vasculature subbranches and comfort level to use the images for pre-operative planning were collected and analyzed. Results CF800 injection improved visualization of pulmonary vessels in both rabbit and pig models. The SBR of rabbit pulmonary vasculature was significantly higher after CF800 injection (range 3.7-4.4) compared to pre-injection (range 3.3-3.8, n = 7; p<0.05). SBR remained significantly different up to 24 hours after injection (range 3.7-4.3, n = 4; p<0.05). Trainees' evaluation found the post-injection CT images had significantly higher discernibility at the second vessel branch generation in both rabbit and pig models. Trainees identified smaller vasculature branch generations in the post-injection images compared to the pre-treatment images in both rabbit (mean 6.7±1.8 vs 5.4±2.1; p<0.05) and pig (mean 6.7±1.8 vs 5.4±2.1; p<0.05). Trainees were significantly more comfortable using post-injection images for
A novel liposomal nanoparticle, CF800, that co-encapsulates indocyanine green for near-infrared (... more A novel liposomal nanoparticle, CF800, that co-encapsulates indocyanine green for near-infrared (NIR) imaging and iohexol for CT imaging has shown preferential tumor accumulation after intravenous injection via the enhanced permeability and retention (EPR) effect. We hypothesized that CF800-enhanced NIR imaging would facilitate intraoperative localization of small lung nodules. A rabbit VX2 lung tumor model was implemented. CF800 was injected intravenously followed by sequential CT acquisitions to track the biodistribution of CF800. Eleven rabbits were employed for NIR fluorescence evaluation after thoracotomy at timepoints until 7 days post injection using a NIR fluorescence thoracoscope in vivo. Organs of interests were removed for ex vivo analysis using NIR imaging. Tumor-to-background (inflated lung) ratio was calculated and compared among the timepoints. Both CT and NIR imaging demonstrated enhanced accumulation of CF800 within the VX2 tumor. NIR image analysis revealed the hig...
Triple negative breast cancer (TNBC) represents a significant therapeutic challenge due to its hi... more Triple negative breast cancer (TNBC) represents a significant therapeutic challenge due to its highly aggressive nature and lack of effective treatment options. Liposomal irinotecan (nal-IRI, ONIVYDE) was approved in 2015 (by the Food and Drug Administration, European Medicines Agency, and Therapeutic Goods Administration) and is a topoisomerase inhibitor indicated, in combination with fluorouracil and leucovorin, for the treatment of patients with metastatic adenocarcinoma of the pancreas after disease progression following gemcitabine-based therapy. This study investigates the potential therapeutic benefit of nal-IRI for the treatment of advanced TNBC in a clinically relevant mouse model of spontaneous metastasis (LM2-4). Female SCID mice were orthotopically inoculated with TNBC LM2-4-luc cells in the lower mammary fat pad. Following primary tumor resection, bioluminescence imaging (BLI) was used to monitor both metastasis formation and spread as well as response to treatment with...
Deposition of liposomal drugs into solid tumors is a potentially rate-limiting step for drug deli... more Deposition of liposomal drugs into solid tumors is a potentially rate-limiting step for drug delivery and has substantial variability that may influence probability of response. Tumor deposition is a shared mechanism for liposomal therapeutics such that a single companion diagnostic agent may have utility in predicting response to multiple nanomedicines. We describe the development, characterization and preclinical proof-of-concept of the positron emission tomography (PET) agent, MM-DX-929, a drug-free untargeted 100 nm PEGylated liposome stably entrapping a chelated complex of 4-DEAP-ATSC and Cu (copper-64). MM-DX-929 is designed to mimic the biodistribution of similarly sized therapeutic agents and enable quantification of deposition in solid tumors. MM-DX-929 demonstrated sufficient and stability with PET images accurately reflecting the disposition of liposome nanoparticles over the time scale of imaging. MM-DX-929 is also representative of the tumor deposition and intratumoral ...
Preclinical breast cancer models recapitulating the clinical course of metastatic disease are cru... more Preclinical breast cancer models recapitulating the clinical course of metastatic disease are crucial for drug development. Highly metastatic cell lines forming spontaneous metastasis following orthotopic implantation were previously developed and characterized regarding their biological and histological characteristics. This study aimed to non-invasively and longitudinally characterize the spatiotemporal pattern of metastasis formation and progression in the MDA-MB-231-derived triple negative LM2-4 and HER2+ LM2-4H2N cell lines, using bioluminescence imaging (BLI), contrast enhanced computed tomography (CT), fluorescence imaging, and 2-deoxy-2-[fluorine-18]fluoro-D-glucose positron emission tomography ([18F]FDG-PET). LM2-4, LM2-4H2N, and MDA-MB-231 tumors were established in the right inguinal mammary fat pad (MFP) of female SCID mice and resected 14-16 days later. Metastasis formation was monitored using BLI. Metabolic activity of primary and metastatic lesions in mice bearing LM2...
Paneth cell dysfunction causes deficiencies in intestinal C-type lectins and antimicrobial peptid... more Paneth cell dysfunction causes deficiencies in intestinal C-type lectins and antimicrobial peptides, which leads to dysbiosis of the intestinal microbiota, alters the mucosal barrier, and promotes development of inflammatory bowel diseases. We investigated whether transgenic expression of the human regenerating family member 3 alpha gene (REG3A) alters the fecal microbiota and affects development of colitis in mice. We performed studies with C57BL/6 mice that express human regenerating family member 3 alpha (hREG3A) in hepatocytes, via the albumin gene promoter. In these mice, hREG3A travels via the bile to the intestinal lumen. Some mice were given dextran sodium sulphate (DSS) to induce colitis. Feces were collected from mice and the composition of the microbiota was analyzed by 16S rRNA sequencing. The fecal microbiome was also analyzed from mice that express only 1 copy of human REG3A transgene but were fed feces from control mice (not expressing hREG3A) as newborns. Mice expres...
Tumor hypoxia is strongly linked to aggressive disease progression and resistance to therapy. Pos... more Tumor hypoxia is strongly linked to aggressive disease progression and resistance to therapy. Positron emission tomography (PET) imaging with hypoxia tracers such as [18F]fluoroazomycin arabinoside (FAZA) allows for non-invasive quantification of tumor hypoxia during treatment. We and others have previously shown by immunohistochemical methods that treatments with longer lasting camptothecin formulations reduce tumor hypoxia after either single or multiple treatment cycles. Here we evaluated the kinetics and magnitude of hypoxia changes in tumors after treatment with irinotecan sucrosofate liposome injection (MM-398) which has shown an extended plasma half-life and higher intratumoral deposition in animal models relative to free pro-drug and compare it to the effects of free irinotecan at equivalent exposure levels. FAZA-PET/CT was used for longitudinal monitoring of tumor hypoxia changes in the HT29 mouse colon cancer xenograft model over a 21-day period following weekly chemotherapy administrations of either MM-398 (5 & 10mg/kg) or free irinotecan (50mg/kg). These dosages were predicted to result in comparable SN-38 exposure in either plasma or tumor based on a mechanistic pharmacokinetic model of MM-398 and free irinotecan that was developed using a systems pharmacology approach. Baseline levels of FAZA uptake in tumors were similar across treatment groups. Significant differences in tumor FAZA uptake were observed between these groups as early as Day 7 post treatment initiation, with increased FAZA uptake seen in tumors treated with free irinotecan. In contrast, differences in tumor volume only became statistically significant on Day 16. MM-398 at 10mg/kg was the most effective treatment for control of tumor volume and also minimized changes in FAZA uptake at all time-points. Background FAZA levels in the muscle were consistent over time across all treatment groups (0.78±0.18 %ID/g, 0.81±0.11 %ID/g and 0.71±0.20 %ID/g). However, normalization with muscle signal did not improve quantification of FAZA uptake differences in tumors. Tumor-specific hypoxia status at the study end point was confirmed by co-staining for CA9 and EF5 levels, which were, as expected, highly correlated. Average EF5 intensity/tumor area was lowest in the MM-398 (10mg/kg) treatment group, while being highest in the irinotecan (50mg/kg) treatment group. This study demonstrated the feasibility of performing longitudinal and repeated tumor hypoxia assessment using FAZA-PET imaging. Treatment with MM-398, but not free irinotecan, led to significant changes in the tumor microenvironment as measured by reduced hypoxia levels that occurred far earlier than anatomical changes assessed by tumor volume. Imaging of hypoxia levels after anti-cancer therapy with MM-398 has the potential to allow early assessment of treatment activity. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C293. Citation Format: Stephan G. Klinz, Jinzi Zheng, Raquel De Souza, Michael Dunne, Jason Cain, Jaeyeon Kim, Nancy Paz, Ashish Kalra, David Jaffray, Jonathan Fitzgerald. Irinotecan sucrosofate liposome injection, MM-398, demonstrates superior activity and control of hypoxia as measured through longitudinal imaging using [18F]FAZA PET compared to free irinotecan in a colon adenocarcinoma xenograft model. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C293.
Imaging technologies are being increasingly employed to guide the delivery of cancer therapies wi... more Imaging technologies are being increasingly employed to guide the delivery of cancer therapies with the intent to increase their performance and efficacy. To date, many patients have benefited from image-guided treatments through prolonged survival and improvements in quality of life. Advances in nanomedicine have enabled the development of multifunctional imaging agents that can further increase the performance of image-guided cancer therapy. Specifically, this talk will focus on examples that demonstrate the benefits and application of nanomedicine in the context of image-guide surgery, personalized drug delivery, tracking of cell therapies and high precision radiotherapy delivery.
Identification of necrosis in tumors is of prognostic value in treatment planning, as necrosis is... more Identification of necrosis in tumors is of prognostic value in treatment planning, as necrosis is associated with aggressive forms of cancer and unfavourable outcomes. To facilitate rapid detection of necrosis with Mass Spectrometry (MS), we report the lipid MS profile of necrotic breast cancer with Desorption Electrospray Ionization Mass Spectrometry (DESI-MS) imaging validated with statistical analysis and correlating pathology. This MS profile is characterized by (1) the presence of the ion of m/z 572.48 [Cer(d34:1) + Cl] − which is a ceramide absent from the viable cancer subregions; (2) the absence of the ion of m/z 391.25 which is present in small abundance only in viable cancer subregions; and (3) a slight increase in the relative intensity of known breast cancer biomarker ions of m/z 281.25 [FA(18:1)-H] − and 303.23 [FA(20:4)-H] −. Necrosis is accompanied by alterations in the tissue optical depolarization rate, allowing tissue polarimetry to guide DESI-MS analysis for rapid MS profiling or targeted MS imaging. This workflow, in combination with the MS profile of necrosis, may permit rapid characterization of necrotic tumors from tissue slices. Further, necrosis-specific biomarker ions are detected in seconds with single MS scans of necrotic tumor tissue smears, which further accelerates the identification workflow by avoiding tissue sectioning and slide preparation. Necrosis is a form of cell death caused by factors external to the cell, such as hypoxia, and is often associated with rapidly growing, aggressive forms of cancer in the breast, colon, brain, lung, kidney, and pancreas. Identification of tumor necrosis in breast cancer can provide prognostic information indicating early recurrence or death, and is used in treatment planning 1. Therefore, necrosis in tumors must be detected during surgery to allow immediate treatment planning once the tumor is encountered. Currently, intraoperative histology can be used to identify necrosis in resected lumpectomy samples. The method uses either a snap frozen slice of the biopsied tissue or a tissue smear prepared on a microscope slide. Morphological signatures of necrosis, such as alterations in the cellular and nuclear membranes, are then used to identify necrotic centers through microscopy. The process can take anywhere between 10-30 min, and requires feedback from a trained pathologist. Due to lengthy processing times, only small samples of tissue are examined during surgery and regional sampling error can lead to necrotic areas being overlooked. Intraoperative assessment of necrosis is often completed while the patient is under general
Investigation of CF800, a novel PEGylated nano-liposomal imaging agent containing indocyanine gre... more Investigation of CF800, a novel PEGylated nano-liposomal imaging agent containing indocyanine green (ICG) and iohexol, for real-time near infrared (NIR) fluorescence and computed tomography (CT) image-guided surgery in an orthotopic lung cancer model in nude mice. CF800 was intravenously administered into 13 mice bearing the H460 orthotopic human lung cancer. At 48 h post-injection (peak imaging agent accumulation time point), ex vivo NIR and CT imaging was performed. A clinical NIR imaging system (SPY®, Novadaq) was used to measure fluorescence intensity of tumor and lung. Tumor-to-background-ratios (TBR) were calculated in inflated and deflated states. The mean Hounsfield unit (HU) of lung tumor was quantified using the CT data set and a semi-automated threshold-based method. Histological evaluation using H&E, the macrophage marker F4/80 and the endothelial cell marker CD31, was performed, and compared to the liposomal fluorescence signal obtained from adjacent tissue sections. Th...
Journal of visualized experiments : JoVE, Jan 18, 2016
The heterogeneous intra-tumoral accumulation of liposomes is a critical determinant of their effi... more The heterogeneous intra-tumoral accumulation of liposomes is a critical determinant of their efficacy. Both the chaotic tumor microcirculation and elevated IFP are linked to the heterogeneous intra-tumoral distribution of nanotechnology-based drug delivery systems such as liposomes. In the present study, the relationship between tumor microcirculation, elevated IFP, and accumulation of nanoparticles was investigated through in vivo experimentation. This was accomplished by evaluation of the tumor microcirculation using dynamic contrast enhanced computed tomography (DCE-CT) and measurement of tumor IFP using a novel image-guided robotic needle placement system connected to the micro-CT scanner. The intra-tumoral accumulation of liposomes was determined by CT image-based assessment of a nanoparticle liposomal formulation that stably encapsulate the contrast agent iohexol (CT-liposomes). CT imaging allowed for co-localization of the spatial distribution of tumor hemodynamics, IFP and C...
Journal of visualized experiments : JoVE, Jan 13, 2015
Liposomes have been employed as drug delivery systems to target solid tumors through exploitation... more Liposomes have been employed as drug delivery systems to target solid tumors through exploitation of the enhanced permeability and retention (EPR) effect resulting in significant reductions in systemic toxicity. Nonetheless, insufficient release of encapsulated drug from liposomes has limited their clinical efficacy. Temperature-sensitive liposomes have been engineered to provide site-specific release of drug in order to overcome the problem of limited tumor drug bioavailability. Our lab has designed and developed a heat-activated thermosensitive liposome formulation of cisplatin (CDDP), known as HTLC, to provide triggered release of CDDP at solid tumors. Heat-activated delivery in vivo was achieved in murine models using a custom-built laser-based heating apparatus that provides a conformal heating pattern at the tumor site as confirmed by MR thermometry (MRT). A fiber optic temperature monitoring device was used to measure the temperature in real-time during the entire heating per...
Picosecond InfraRed Laser (PIRL) is capable of cutting through biological tissues in the absence ... more Picosecond InfraRed Laser (PIRL) is capable of cutting through biological tissues in the absence of significant thermal damage. As such, PIRL is a standalone surgical scalpel with the added bonus of minimal post-operative scar tissue formation. In this work, a tandem of PIRL Ablation with ElectroSpray Ionization (PIR-LAESI) mass spectrometry is demonstrated and characterized for tissue molecular imaging, with a limit of detection in the range of 100nM for Reserpine or better than 5 nM for Verapamil in aqueous solution. We characterized PIRL crater size using agar films containing Rhodamine. PIR-LAESI offers a 20-30 μm vertical resolution (~3 μm removal per pulse) and a lateral resolution of ~100μm. We were able to detect 25 fmoles of Rhodamine in agar ablation experiments. PIR-LAESI was used to map the distribution of endogenous methoxykaempferol glucoronide in zebra plant (Aphelandra squarrosa) leaves producing a localization map that is corroborated by the literature. PIRL-LAESI w...
Each medical imaging modality has unique strengths and limitations and it is often through the us... more Each medical imaging modality has unique strengths and limitations and it is often through the use of mul-tiple modalities that a complete assessment of a patient is achieved. The use of CT and MR for radiation treatment planning, PET/CT in oncological diagnoses and PET/MRI in neurosurgery are a few instances of the role of multimodal imaging in treatment planning and diagnosis. However, very few attempts have been made to develop a contrast agent that can be used across multiple imaging modalities. Due to its stability, prolonged imaging window, and modular nature; the liposome-based contrast agent platform de-scribed here is an effective system to integrate the complementary information gained from the use of currently available non-invasive imaging techniques (CT, MR, SPECT, PET and optical).
Given the bulky nature of nanotherapeutics relative to small molecules, it is hypothesized that e... more Given the bulky nature of nanotherapeutics relative to small molecules, it is hypothesized that effective tumor delivery and penetration are critical barriers to their clinical activity. HER2-targeted PEGylated liposomal doxorubicin (MM-302, HER2-tPLD) is an antibody-liposomal drug conjugate designed to deliver doxorubicin to HER2-overexpressing cancer cells while limiting uptake into non-target cells. In this work we demonstrate that the administration and appropriate dose sequencing of cyclophosphamide can improve subsequent MM-302 delivery and enhance anti-tumor activity in preclinical models without negatively affecting non-target tissues, such as the heart and skin. We demonstrate that this effect is critically dependent on the timing of cyclophosphamide administration. Further, the effect was found to be unique to cyclophosphamide and related analogues, and not shared by other agents, such as taxanes or eribulin, under the conditions examined. Analysis of the cyclophosphamide-...
There are limited methods that are currently available for longitudinal and non-invasive in vivo ... more There are limited methods that are currently available for longitudinal and non-invasive in vivo assessment of the transport kinetics of carrier-based therapeutics, such as those relying on liposomes. A viable strategy to noninvasively track liposomes in vivo is to load them with contrast-enhancing agents that are well retained within the carrier. The encapsulation of contrast agents such as iodine and gadolinium allows for the liposomes to be tracked by computed tomography (CT) and magnetic resonance (MR) imaging systems, respectively. This study evaluated the feasibility of using CT and MR imaging as a means to gain a quantitative assessment of the pathway and fate of liposomes in vivo in rabbits. The liposome formulation examined was composed of DPPC/Cholesterol/DSPEPEG2000 in a 55/40/5 molar ratio and the vesicles had an average diameter of 80 nm. The commercially available agents iohexol (CT agent) and gadoteridol (MR agent) were encapsulated within the liposomes and found to b...
An integrated augmented reality (AR) surgical navigation system that potentially improves intra-o... more An integrated augmented reality (AR) surgical navigation system that potentially improves intra-operative visualization of concealed anatomical structures. Integration of real-time tracking technology with a laser pico-projector allows the surgical surface to be augmented by projecting virtual images of lesions and critical structures created by multimodality imaging. We aim to quantitatively and qualitatively evaluate the performance of a prototype interactive AR surgical navigation system through a series of pre-clinical studies. Four pre-clinical animal studies using xenograft mouse models were conducted to investigate system performance. A combination of CT, PET, SPECT, and MRI images were used to augment the mouse body during image-guided procedures to assess feasibility. A phantom with machined features was employed to quantitatively estimate the system accuracy. All the image-guided procedures were successfully performed. The tracked pico-projector correctly and reliably depi...
Nanoscale perfluorocarbon (PFC) droplets have enormous potential as clinical theranostic agents. ... more Nanoscale perfluorocarbon (PFC) droplets have enormous potential as clinical theranostic agents. They are biocompatible and are currently used in vivo as contrast agents for a variety of medical imaging modalities, including ultrasound, computed tomography, photoacoustic and 19 F-magnetic resonance imaging. PFC nanodroplets can also carry molecular and nanoparticulate drugs and be activated in situ by ultrasound or light for targeted therapy. Recently, there has been renewed interest in using PFC nanodroplets for hypoxic tumor reoxygenation towards radiosensitization based on the high oxygen solubility of PFCs. Previous studies showed that tumor oxygenation using PFC agents only occurs in combination with enhanced oxygen breathing. However, recent studies suggest that PFC agents that accumulate in solid tumors can contribute to radiosensitization, presumably due to tumor reoxygenation without enhanced oxygen breathing. In this study, we quantify the impact of oxygenation due to PFC nanodroplet accumulation in tumors alone in comparison with other reoxygenation methodologies, in particular, carbogen breathing. Methods: Lipid-stabilized, PFC (i.e., perfluorooctyl bromide, CF3(CF2)7Br, PFOB) nanoscale droplets were synthesized and evaluated in xenograft prostate (DU145) tumors in male mice. Biodistribution assessment of the nanodroplets was achieved using a fluorescent lipophilic indocarbocyanine dye label (i.e., DiI dye) on the lipid shell in combination with fluorescence imaging in mice (n≥3 per group). Hypoxia reduction in tumors was measured using PET imaging and a known hypoxia radiotracer, [ 18 F]FAZA (n≥ 3 per group). Results: Lipid-stabilized nanoscale PFOB emulsions (mean diameter of ~250 nm), accumulated in the xenograft prostate tumors in mice 24 hours post-injection. In vivo PET imaging with [ 18 F]FAZA showed that the accumulation of the PFOB nanodroplets in the tumor tissues alone significantly reduced tumor hypoxia, without enhanced oxygen (i.e., carbogen) breathing. This reoxygenation effect was found to be comparable with carbogen breathing alone. Conclusion: Accumulation of nanoscale PFOB agents in solid tumors alone successfully reoxygenated hypoxic tumors to levels comparable with carbogen breathing alone, an established tumor oxygenation method. This study confirms that PFC agents can be used to reoxygenate hypoxic tumors in addition to their current applications as multifunctional theranostic agents.
Purpose: Hypoxia is linked to aggressiveness, resistance to therapy, and poor prognosis of pancre... more Purpose: Hypoxia is linked to aggressiveness, resistance to therapy, and poor prognosis of pancreatic tumors. Liposomal irinotecan (nal-IRI, ONIVYDE®) has shown potential in reducing hypoxia in the HT29 colorectal cancer model, and here, we investigate its therapeutic activity and ability to modulate hypoxia in patient-derived orthotopic tumor models of pancreatic cancer. Procedures: Mice were randomized into nal-IRI treated and untreated controls. Magnetic resonance imaging was used for monitoring treatment efficacy, positron emission tomography (PET) imaging with F-18-labelled fluoroazomycinarabinoside ([ 18 F]FAZA) for tumor hypoxia quantification, and F-18-labelled fluorothymidine ([ 18 F]FLT) for tumor cell proliferation. Results: The highly hypoxic OCIP51 tumors showed significant response following nal-IRI treatment compared with the less hypoxic OCIP19 tumors. [ 18 F]FAZA-PET detected significant hypoxia reduction in treated OCIP51 tumors, 8 days before significant changes in tumor volume. OCIP19 tumors also responded to therapy, although tumor volume control was not accompanied by any reduction in [ 18 F]FAZA uptake. In both models, no differences were observable in [ 18 F]FLT uptake in treated tumors compared with control mice. Conclusions: Hypoxia modulation may play a role in nal-IRI's mechanism of action. Nal-IRI demonstrated greater anti-tumor activity in the more aggressive and hypoxic tumor model. Furthermore, hypoxia imaging provided early prediction of treatment response.
To evaluate CF800, a novel lipid-based liposomal nanoparticle that co-encapsulates indocyanine gr... more To evaluate CF800, a novel lipid-based liposomal nanoparticle that co-encapsulates indocyanine green (ICG) and iohexol, for CT imaging of pulmonary vasculature in minimallyinvasive thoracic surgery planning. Methods CF800 was intravenously administered to 7 healthy rabbits. In vivo CT imaging was performed 15 min post-injection, with a subset of animals imaged at 24h, 48h, and 72h post injection. Signal-to-background ratios (SBR) were calculated at the inferior vena cava and compared across time-points. A similar protocol was applied to 2 healthy pigs to evaluate the feasibility and efficacy in a large animal model. To evaluate the feasibility of clinical application, a survey was completed by 7 surgical trainees to assess pre-and post-injection CT images of rabbits and pigs. Responses on the discernibility of pulmonary vasculature subbranches and comfort level to use the images for pre-operative planning were collected and analyzed. Results CF800 injection improved visualization of pulmonary vessels in both rabbit and pig models. The SBR of rabbit pulmonary vasculature was significantly higher after CF800 injection (range 3.7-4.4) compared to pre-injection (range 3.3-3.8, n = 7; p<0.05). SBR remained significantly different up to 24 hours after injection (range 3.7-4.3, n = 4; p<0.05). Trainees' evaluation found the post-injection CT images had significantly higher discernibility at the second vessel branch generation in both rabbit and pig models. Trainees identified smaller vasculature branch generations in the post-injection images compared to the pre-treatment images in both rabbit (mean 6.7±1.8 vs 5.4±2.1; p<0.05) and pig (mean 6.7±1.8 vs 5.4±2.1; p<0.05). Trainees were significantly more comfortable using post-injection images for
A novel liposomal nanoparticle, CF800, that co-encapsulates indocyanine green for near-infrared (... more A novel liposomal nanoparticle, CF800, that co-encapsulates indocyanine green for near-infrared (NIR) imaging and iohexol for CT imaging has shown preferential tumor accumulation after intravenous injection via the enhanced permeability and retention (EPR) effect. We hypothesized that CF800-enhanced NIR imaging would facilitate intraoperative localization of small lung nodules. A rabbit VX2 lung tumor model was implemented. CF800 was injected intravenously followed by sequential CT acquisitions to track the biodistribution of CF800. Eleven rabbits were employed for NIR fluorescence evaluation after thoracotomy at timepoints until 7 days post injection using a NIR fluorescence thoracoscope in vivo. Organs of interests were removed for ex vivo analysis using NIR imaging. Tumor-to-background (inflated lung) ratio was calculated and compared among the timepoints. Both CT and NIR imaging demonstrated enhanced accumulation of CF800 within the VX2 tumor. NIR image analysis revealed the hig...
Triple negative breast cancer (TNBC) represents a significant therapeutic challenge due to its hi... more Triple negative breast cancer (TNBC) represents a significant therapeutic challenge due to its highly aggressive nature and lack of effective treatment options. Liposomal irinotecan (nal-IRI, ONIVYDE) was approved in 2015 (by the Food and Drug Administration, European Medicines Agency, and Therapeutic Goods Administration) and is a topoisomerase inhibitor indicated, in combination with fluorouracil and leucovorin, for the treatment of patients with metastatic adenocarcinoma of the pancreas after disease progression following gemcitabine-based therapy. This study investigates the potential therapeutic benefit of nal-IRI for the treatment of advanced TNBC in a clinically relevant mouse model of spontaneous metastasis (LM2-4). Female SCID mice were orthotopically inoculated with TNBC LM2-4-luc cells in the lower mammary fat pad. Following primary tumor resection, bioluminescence imaging (BLI) was used to monitor both metastasis formation and spread as well as response to treatment with...
Deposition of liposomal drugs into solid tumors is a potentially rate-limiting step for drug deli... more Deposition of liposomal drugs into solid tumors is a potentially rate-limiting step for drug delivery and has substantial variability that may influence probability of response. Tumor deposition is a shared mechanism for liposomal therapeutics such that a single companion diagnostic agent may have utility in predicting response to multiple nanomedicines. We describe the development, characterization and preclinical proof-of-concept of the positron emission tomography (PET) agent, MM-DX-929, a drug-free untargeted 100 nm PEGylated liposome stably entrapping a chelated complex of 4-DEAP-ATSC and Cu (copper-64). MM-DX-929 is designed to mimic the biodistribution of similarly sized therapeutic agents and enable quantification of deposition in solid tumors. MM-DX-929 demonstrated sufficient and stability with PET images accurately reflecting the disposition of liposome nanoparticles over the time scale of imaging. MM-DX-929 is also representative of the tumor deposition and intratumoral ...
Preclinical breast cancer models recapitulating the clinical course of metastatic disease are cru... more Preclinical breast cancer models recapitulating the clinical course of metastatic disease are crucial for drug development. Highly metastatic cell lines forming spontaneous metastasis following orthotopic implantation were previously developed and characterized regarding their biological and histological characteristics. This study aimed to non-invasively and longitudinally characterize the spatiotemporal pattern of metastasis formation and progression in the MDA-MB-231-derived triple negative LM2-4 and HER2+ LM2-4H2N cell lines, using bioluminescence imaging (BLI), contrast enhanced computed tomography (CT), fluorescence imaging, and 2-deoxy-2-[fluorine-18]fluoro-D-glucose positron emission tomography ([18F]FDG-PET). LM2-4, LM2-4H2N, and MDA-MB-231 tumors were established in the right inguinal mammary fat pad (MFP) of female SCID mice and resected 14-16 days later. Metastasis formation was monitored using BLI. Metabolic activity of primary and metastatic lesions in mice bearing LM2...
Paneth cell dysfunction causes deficiencies in intestinal C-type lectins and antimicrobial peptid... more Paneth cell dysfunction causes deficiencies in intestinal C-type lectins and antimicrobial peptides, which leads to dysbiosis of the intestinal microbiota, alters the mucosal barrier, and promotes development of inflammatory bowel diseases. We investigated whether transgenic expression of the human regenerating family member 3 alpha gene (REG3A) alters the fecal microbiota and affects development of colitis in mice. We performed studies with C57BL/6 mice that express human regenerating family member 3 alpha (hREG3A) in hepatocytes, via the albumin gene promoter. In these mice, hREG3A travels via the bile to the intestinal lumen. Some mice were given dextran sodium sulphate (DSS) to induce colitis. Feces were collected from mice and the composition of the microbiota was analyzed by 16S rRNA sequencing. The fecal microbiome was also analyzed from mice that express only 1 copy of human REG3A transgene but were fed feces from control mice (not expressing hREG3A) as newborns. Mice expres...
Tumor hypoxia is strongly linked to aggressive disease progression and resistance to therapy. Pos... more Tumor hypoxia is strongly linked to aggressive disease progression and resistance to therapy. Positron emission tomography (PET) imaging with hypoxia tracers such as [18F]fluoroazomycin arabinoside (FAZA) allows for non-invasive quantification of tumor hypoxia during treatment. We and others have previously shown by immunohistochemical methods that treatments with longer lasting camptothecin formulations reduce tumor hypoxia after either single or multiple treatment cycles. Here we evaluated the kinetics and magnitude of hypoxia changes in tumors after treatment with irinotecan sucrosofate liposome injection (MM-398) which has shown an extended plasma half-life and higher intratumoral deposition in animal models relative to free pro-drug and compare it to the effects of free irinotecan at equivalent exposure levels. FAZA-PET/CT was used for longitudinal monitoring of tumor hypoxia changes in the HT29 mouse colon cancer xenograft model over a 21-day period following weekly chemotherapy administrations of either MM-398 (5 & 10mg/kg) or free irinotecan (50mg/kg). These dosages were predicted to result in comparable SN-38 exposure in either plasma or tumor based on a mechanistic pharmacokinetic model of MM-398 and free irinotecan that was developed using a systems pharmacology approach. Baseline levels of FAZA uptake in tumors were similar across treatment groups. Significant differences in tumor FAZA uptake were observed between these groups as early as Day 7 post treatment initiation, with increased FAZA uptake seen in tumors treated with free irinotecan. In contrast, differences in tumor volume only became statistically significant on Day 16. MM-398 at 10mg/kg was the most effective treatment for control of tumor volume and also minimized changes in FAZA uptake at all time-points. Background FAZA levels in the muscle were consistent over time across all treatment groups (0.78±0.18 %ID/g, 0.81±0.11 %ID/g and 0.71±0.20 %ID/g). However, normalization with muscle signal did not improve quantification of FAZA uptake differences in tumors. Tumor-specific hypoxia status at the study end point was confirmed by co-staining for CA9 and EF5 levels, which were, as expected, highly correlated. Average EF5 intensity/tumor area was lowest in the MM-398 (10mg/kg) treatment group, while being highest in the irinotecan (50mg/kg) treatment group. This study demonstrated the feasibility of performing longitudinal and repeated tumor hypoxia assessment using FAZA-PET imaging. Treatment with MM-398, but not free irinotecan, led to significant changes in the tumor microenvironment as measured by reduced hypoxia levels that occurred far earlier than anatomical changes assessed by tumor volume. Imaging of hypoxia levels after anti-cancer therapy with MM-398 has the potential to allow early assessment of treatment activity. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C293. Citation Format: Stephan G. Klinz, Jinzi Zheng, Raquel De Souza, Michael Dunne, Jason Cain, Jaeyeon Kim, Nancy Paz, Ashish Kalra, David Jaffray, Jonathan Fitzgerald. Irinotecan sucrosofate liposome injection, MM-398, demonstrates superior activity and control of hypoxia as measured through longitudinal imaging using [18F]FAZA PET compared to free irinotecan in a colon adenocarcinoma xenograft model. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C293.
Imaging technologies are being increasingly employed to guide the delivery of cancer therapies wi... more Imaging technologies are being increasingly employed to guide the delivery of cancer therapies with the intent to increase their performance and efficacy. To date, many patients have benefited from image-guided treatments through prolonged survival and improvements in quality of life. Advances in nanomedicine have enabled the development of multifunctional imaging agents that can further increase the performance of image-guided cancer therapy. Specifically, this talk will focus on examples that demonstrate the benefits and application of nanomedicine in the context of image-guide surgery, personalized drug delivery, tracking of cell therapies and high precision radiotherapy delivery.
Identification of necrosis in tumors is of prognostic value in treatment planning, as necrosis is... more Identification of necrosis in tumors is of prognostic value in treatment planning, as necrosis is associated with aggressive forms of cancer and unfavourable outcomes. To facilitate rapid detection of necrosis with Mass Spectrometry (MS), we report the lipid MS profile of necrotic breast cancer with Desorption Electrospray Ionization Mass Spectrometry (DESI-MS) imaging validated with statistical analysis and correlating pathology. This MS profile is characterized by (1) the presence of the ion of m/z 572.48 [Cer(d34:1) + Cl] − which is a ceramide absent from the viable cancer subregions; (2) the absence of the ion of m/z 391.25 which is present in small abundance only in viable cancer subregions; and (3) a slight increase in the relative intensity of known breast cancer biomarker ions of m/z 281.25 [FA(18:1)-H] − and 303.23 [FA(20:4)-H] −. Necrosis is accompanied by alterations in the tissue optical depolarization rate, allowing tissue polarimetry to guide DESI-MS analysis for rapid MS profiling or targeted MS imaging. This workflow, in combination with the MS profile of necrosis, may permit rapid characterization of necrotic tumors from tissue slices. Further, necrosis-specific biomarker ions are detected in seconds with single MS scans of necrotic tumor tissue smears, which further accelerates the identification workflow by avoiding tissue sectioning and slide preparation. Necrosis is a form of cell death caused by factors external to the cell, such as hypoxia, and is often associated with rapidly growing, aggressive forms of cancer in the breast, colon, brain, lung, kidney, and pancreas. Identification of tumor necrosis in breast cancer can provide prognostic information indicating early recurrence or death, and is used in treatment planning 1. Therefore, necrosis in tumors must be detected during surgery to allow immediate treatment planning once the tumor is encountered. Currently, intraoperative histology can be used to identify necrosis in resected lumpectomy samples. The method uses either a snap frozen slice of the biopsied tissue or a tissue smear prepared on a microscope slide. Morphological signatures of necrosis, such as alterations in the cellular and nuclear membranes, are then used to identify necrotic centers through microscopy. The process can take anywhere between 10-30 min, and requires feedback from a trained pathologist. Due to lengthy processing times, only small samples of tissue are examined during surgery and regional sampling error can lead to necrotic areas being overlooked. Intraoperative assessment of necrosis is often completed while the patient is under general
Investigation of CF800, a novel PEGylated nano-liposomal imaging agent containing indocyanine gre... more Investigation of CF800, a novel PEGylated nano-liposomal imaging agent containing indocyanine green (ICG) and iohexol, for real-time near infrared (NIR) fluorescence and computed tomography (CT) image-guided surgery in an orthotopic lung cancer model in nude mice. CF800 was intravenously administered into 13 mice bearing the H460 orthotopic human lung cancer. At 48 h post-injection (peak imaging agent accumulation time point), ex vivo NIR and CT imaging was performed. A clinical NIR imaging system (SPY®, Novadaq) was used to measure fluorescence intensity of tumor and lung. Tumor-to-background-ratios (TBR) were calculated in inflated and deflated states. The mean Hounsfield unit (HU) of lung tumor was quantified using the CT data set and a semi-automated threshold-based method. Histological evaluation using H&E, the macrophage marker F4/80 and the endothelial cell marker CD31, was performed, and compared to the liposomal fluorescence signal obtained from adjacent tissue sections. Th...
Journal of visualized experiments : JoVE, Jan 18, 2016
The heterogeneous intra-tumoral accumulation of liposomes is a critical determinant of their effi... more The heterogeneous intra-tumoral accumulation of liposomes is a critical determinant of their efficacy. Both the chaotic tumor microcirculation and elevated IFP are linked to the heterogeneous intra-tumoral distribution of nanotechnology-based drug delivery systems such as liposomes. In the present study, the relationship between tumor microcirculation, elevated IFP, and accumulation of nanoparticles was investigated through in vivo experimentation. This was accomplished by evaluation of the tumor microcirculation using dynamic contrast enhanced computed tomography (DCE-CT) and measurement of tumor IFP using a novel image-guided robotic needle placement system connected to the micro-CT scanner. The intra-tumoral accumulation of liposomes was determined by CT image-based assessment of a nanoparticle liposomal formulation that stably encapsulate the contrast agent iohexol (CT-liposomes). CT imaging allowed for co-localization of the spatial distribution of tumor hemodynamics, IFP and C...
Journal of visualized experiments : JoVE, Jan 13, 2015
Liposomes have been employed as drug delivery systems to target solid tumors through exploitation... more Liposomes have been employed as drug delivery systems to target solid tumors through exploitation of the enhanced permeability and retention (EPR) effect resulting in significant reductions in systemic toxicity. Nonetheless, insufficient release of encapsulated drug from liposomes has limited their clinical efficacy. Temperature-sensitive liposomes have been engineered to provide site-specific release of drug in order to overcome the problem of limited tumor drug bioavailability. Our lab has designed and developed a heat-activated thermosensitive liposome formulation of cisplatin (CDDP), known as HTLC, to provide triggered release of CDDP at solid tumors. Heat-activated delivery in vivo was achieved in murine models using a custom-built laser-based heating apparatus that provides a conformal heating pattern at the tumor site as confirmed by MR thermometry (MRT). A fiber optic temperature monitoring device was used to measure the temperature in real-time during the entire heating per...
Picosecond InfraRed Laser (PIRL) is capable of cutting through biological tissues in the absence ... more Picosecond InfraRed Laser (PIRL) is capable of cutting through biological tissues in the absence of significant thermal damage. As such, PIRL is a standalone surgical scalpel with the added bonus of minimal post-operative scar tissue formation. In this work, a tandem of PIRL Ablation with ElectroSpray Ionization (PIR-LAESI) mass spectrometry is demonstrated and characterized for tissue molecular imaging, with a limit of detection in the range of 100nM for Reserpine or better than 5 nM for Verapamil in aqueous solution. We characterized PIRL crater size using agar films containing Rhodamine. PIR-LAESI offers a 20-30 μm vertical resolution (~3 μm removal per pulse) and a lateral resolution of ~100μm. We were able to detect 25 fmoles of Rhodamine in agar ablation experiments. PIR-LAESI was used to map the distribution of endogenous methoxykaempferol glucoronide in zebra plant (Aphelandra squarrosa) leaves producing a localization map that is corroborated by the literature. PIRL-LAESI w...
Each medical imaging modality has unique strengths and limitations and it is often through the us... more Each medical imaging modality has unique strengths and limitations and it is often through the use of mul-tiple modalities that a complete assessment of a patient is achieved. The use of CT and MR for radiation treatment planning, PET/CT in oncological diagnoses and PET/MRI in neurosurgery are a few instances of the role of multimodal imaging in treatment planning and diagnosis. However, very few attempts have been made to develop a contrast agent that can be used across multiple imaging modalities. Due to its stability, prolonged imaging window, and modular nature; the liposome-based contrast agent platform de-scribed here is an effective system to integrate the complementary information gained from the use of currently available non-invasive imaging techniques (CT, MR, SPECT, PET and optical).
Given the bulky nature of nanotherapeutics relative to small molecules, it is hypothesized that e... more Given the bulky nature of nanotherapeutics relative to small molecules, it is hypothesized that effective tumor delivery and penetration are critical barriers to their clinical activity. HER2-targeted PEGylated liposomal doxorubicin (MM-302, HER2-tPLD) is an antibody-liposomal drug conjugate designed to deliver doxorubicin to HER2-overexpressing cancer cells while limiting uptake into non-target cells. In this work we demonstrate that the administration and appropriate dose sequencing of cyclophosphamide can improve subsequent MM-302 delivery and enhance anti-tumor activity in preclinical models without negatively affecting non-target tissues, such as the heart and skin. We demonstrate that this effect is critically dependent on the timing of cyclophosphamide administration. Further, the effect was found to be unique to cyclophosphamide and related analogues, and not shared by other agents, such as taxanes or eribulin, under the conditions examined. Analysis of the cyclophosphamide-...
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