Browsing by Subject "Tumor"

Three-dimensional (3D) tumor models aim to reduce the need for animal models for drug and gene delivery studies. However, many models are not conducive to environmental manipulation and may not be easily adapted for in situ microscopic analysis of transport phenomena. One goal of this study was to develop a 3D tumor model that can mimic 3D cell-cell interactions to mimic native tumor tissues.

To this end, a novel 3D microfluidics-based tumor model was created which allowed the overnight culture of a high density of tumor cells and could be used for small molecule penetration studies. This microfluidic device facilitated the loading of B16.F10 tumor cells in a densely-packed three-dimensional arrangement in a micro-channel which was accessible for nutrient supply via channels on either side through which culture media was continuously infused. Cell volume fraction in the micro-channel was determined via nuclear staining and counting of cells immediately after loading and after a 12-hr culture period. The average volume fraction of cells in this model was 0.32 immediately after loading and 0.26 after 12-hr culture. The values are comparable to cell volume fractions of the in vivo B16.F10 tumor previously measured in our lab. The reduction in cell volume fraction after overnight culture was due to the change in cell morphology to become more elongated after time in culture. Cell-cell adhesions appeared to have formed during culture, resulting in more uniform packing.

Sodium fluorescein dye was used as a drug analog and the extent of penetration of this fluorescent molecule through the cell compartment was assessed through microscopy. The dye was introduced on one side of the cell micro-channel and fluorescence images were captured for generation of concentration profiles in the cell compartment. Results showed that dye penetration through the cell chamber was greatly limited by the presence of the 3D cell culture and a linear concentration profile was achieved across the cell compartment. Also, the concentration of sodium fluorescein in the cell compartment of the 12-hr microfluidic cell culture was appreciably lower than the concentration in the cell compartment when the dye was introduced immediately after loading cells. These results suggest that the proposed tumor model shows significant resistance to dye penetration and could prove to be extremely useful for mimicking tumor tissue resistance to drug penetration via diffusion.

There are many barriers to gene delivery to tumors which highlight the importance of selecting an effective gene carrier system. Some pathogenic bacteria have been investigated as gene delivery vectors because of their innate ability to selectively proliferate in tumor environments. However, pathogenicity concerns arise when trying to achieve therapeutic levels of gene expression. It has been shown that non-pathogenic bacteria such as E. coli can be engineered to invade mammalian cells and participate as gene delivery vehicles. Hence, the second part of this research project involved the use of the newly developed microfluidic 3D tumor model previously described to visualize the transport behavior of invasive (inv+) and non-invasive (inv-) E. coli. The inv+ bacteria harbored a plasmid containing the inv gene encoding the protein invasin that binds to &beta₁ integrin receptors on the surface of mammalian cells resulting in the phagocytosis of invasin-expressing bacteria by normally non-phaogcytotic cells. Two tumor cells lines were used: B16.F10 and EMT6, which have been shown to differ in expression of &beta₁ integrins. The bacteria were also engineered to express mCherry for fluorescent detection.

A suspension of tumor cells and bacteria was loaded into the microfluidic device and cultured for 12 hrs before imaging bacteria distribution throughout the cell culture. Proliferation of inv+ bacteria was generally uniform throughout the cell compartment in the B16.F10 model and bacterial cells were primarily concentrated outside of cells. Bacteria that were internalized did not appear to migrate far from the plasma membrane of the tumor cell. The non-invasive bacteria proliferated to a much greater extent than the invasive form and this proliferation was also generally uniform throughout the cell compartment. Proliferation of both invasive and non-invasive bacteria in the EMT6 model was less uniform than in the B16.F10 model. Overall bacterial concentration appeared to be lower in the EMT6 model. Viability staining after bacterial infection showed that tumor cells in the 3D model were able to maintain viability despite bacterial cell proliferation.

An additional assay was conducted in culture plate wells to determine the effect of chemical factors secreted by tumor cells on bacterial cell proliferation. The results of this assay revealed that tumor cells may be secreting anti-microbial factors that inhibit the proliferation of bacteria and that the binding of invasin-expressing E. coli to tumor cells may further promote the release of these factors.

The results of this study suggest that tumor cell type plays a major role in the distribution and proliferation of bacteria in a 3D environment. The ability to visualize bacterial spread throughout a 3D tumor model will prove to be useful for observing the effect of various genetic modifications on the transport and gene delivery efficiency of E. coli.

Purpose: Accurately quantifying the extent vessel coverage in the pancreas is essential for determining the feasibility of surgery. The aim of this study is to train a segmentation model specialized for Pancreatic Ductal Adenocarcinoma (PDAC) imaging, focusing on delineating pancreas, tumor, arteries (celiac artery, superior mesenteric artery, common hepatic artery), and veins (portal vein, superior mesenteric vein) using an improved Attention Unet CNN approach.Methods: Data from 100 PDAC patients treated at the Ruijin Hospital between 2020 and 2022 were utilized. Using Synapse 3D software, masks of the tumor, arteries (including the celiac artery, superior mesenteric artery, and hepatic artery), and veins (including the superior mesenteric vein and portal vein) were generated semi-automatically and reviewed by radiologists. Standard image processing techniques, including adjustment of window level to 60 and width to 350 and histogram equalization were subsequently applied. Two types of CNN-based Attention Unet segmentation models were developed: (1) Unified Unet Model that segments all four components simultaneously, and (2) four Individual Unet Models that segments pancreas, tumor, veins, and arteries separately. The train-validation-test data assignment was set to 7:2:1. The segmentation efficacy was assessed using Dice similarity coefficient, with the Adam optimizer utilized for optimization. Results: The individual segmentation models achieve notable performance: pancreas (Accuracy: 0.84, IoU: 0.81, Dice: 0.76), tumor (Accuracy: 0.78, IoU: 0.77, Dice: 0.68), vein (Accuracy: 0.88, IoU: 0.86, Dice: 0.80), and artery (Accuracy: 0.91, IoU: 0.93, Dice: 0.93). However, the unified model demonstrates inferior performance with accuracy, IoU, and Dice coefficient scores of 0.61, 0.50, and 0.45, respectively. Conclusion: Accurate segmentation models have been developed for pancreas, pancreatic tumors, arteries, and veins in PDAC patients. This will enable the efficient quantification of vessel coverage in the pancreas, thereby enhancing the decision-making process regarding the feasibility of surgery for PDAC patients. The findings also demonstrate that Individual Models outperform the Unified Model in segmentation accuracy, highlighting the importance of tailored segmentation strategies for different anatomical structures in PDAC imaging.

There is a critical need to target tumor hypoxia as patients with hypoxic tumors have worse prognosis due to aggressive phenotypes and resistance to radiotherapy and chemotherapy. The overall goal of this work is to improve response to conventional cancer therapies by targeting tumor hypoxia. This has been carried out and evaluated through the use of polymersome-encapsulated myoglobin (PEMs) with the hypothesis that O2-releasing PEMs will increase tumor oxygenation, and thereby improve response to radiotherapy. Mb was chosen as an O2 carrying protein to deliver to tumors because it has a strong association to O2, providing a mechanism to deliver O2 only within the hypoxic regions of the tumor. Mb was loaded within nanoscale polymeric vesicles that were expected to accumulate within solid tumors due to the enhanced permeability and retention (EPR) effect. This hypothesis has been tested through the following aims:

1. Develop NIR imaging techniques for studying the biodistribution and pharmacokinetics of polymersomes

2. Establish the effects of Mb-containing polymersomes on tumor physiology

3. Modify tumor growth through delivery of Mb polymersomes in combination with a cytotoxic therapy specific to aerobic tumors

These aims have been evaluated through numerous in vivo studies. First, polymersomes of various polymer formulations and diameters ranging from 110-550 nm were prepared with a near-infrared (NIR) -emissive fluorophore. Using live animal fluorescence imaging, I was able to study the biodistribution of the polymersomes following i.v. administration, demonstrating significant polymersome accumulation in orthotopic 4T1 mammary carcinomas. In addition, a novel method for measuring pharmacokinetics was developed, using serial small volume blood draws from individual mice. The plasma fluorescence in microcapillary tubes was used to quantify polymersome concentrations, demonstrating long circulation half-lives that varied from 6-23 h. Toxicity of various polymersome formulations were also studied in vitro and in vivo, revealing negligible toxicities.

For the second aim, PEMs were administered i.v. in tumor-bearing mice. Unexpectedly, we observed a dramatic gross tumor effect within hours of treatment in both orthotopic 4T1 tumors and flank Renca renal cell carcinomas. Histological analysis revealed endothelial cell apoptosis as early as 1 h following treatment, with scattered tumor cell death throughout the tumor by 4 h. Hematoxylin and eosin staining showed significant necrosis 24 h following PEM treatment. Vascular effects and polymersome distribution were studied in 4T1 window chamber tumors. Following i.v. treatment with PEMs, intravital microscopy was used to image polymersome fluorescence, brightfield transmission was imaged for vessel morphology and blood flow, and a tunable filter was used for determining hemoglobin (Hb) oxygen saturation. Tumor hemorrhaging was observed within hours of PEM treatment, which was not seen with empty polymersomes. This was consistent with the gross tumor effects observed initially. Hb saturation decreased in both the PEM and empty polymersome groups, but not in saline-treated mice. While we expected to observe an increase in tumor oxygenation by using Mb as an oxygen carrier, we actually observed hemorrhage, decreased oxygenation, and central tumor necrosis. In vitro studies using human endothelial cells demonstrated dramatic changes in cell morphology and increased permeability due to Mb and PEM treatments, which appear to be enhanced in an oxidative environment. These in vitro and in vivo observations are similar to what is seen with tumor vascular disrupting agents.

For the third aim, I combined radiotherapy (RT) and PEM treatment with a new hypothesis. I originally expected the PEMs to increase tumor oxygenation, thus making the tumor more susceptible to RT. However, considering the results from the second aim, this hypothesis was modified: the PEMs would result in necrosis of the tumor core, while RT would target the more oxygenated rim of the tumor, thus leading to improved tumor growth delay compared with PEM or RT alone. This hypothesis was tested in both orthotopic, syngeneic 4T1 tumors as well as flank FaDu xenografts. 4T1 tumor cells were surgically implanted within the dorsal mammary fat pad of mice and grown until ~200 mm3. A CT microirradiator with a square collimator was used in order to locate and specifically irradiate the tumor. Within 1 h following RT, the PEMs were administered i.v.. Mice receiving PEMs with no RT showed a significant decrease in tumor growth compared with saline-treated mice (p = 0.0001 for time to 3x original tumor volume). In addition, the combination of RT plus PEMs reduced tumor growth compared with RT alone (p = 0.0144 for time to 3x original tumor volume). However, this effect was not seen with FaDu tumors. This may have been due to excessive radiation dose or other compounding factors: the timing between RT and PEM treatment was not optimized, and the number of mice per group was small (3-4).

Thus, the conclusions for each aim are as follows:

1. Develop NIR imaging techniques for studying the biodistribution and pharmacokinetics of polymersomes

NIR imaging techniques were optimized for studying polymersomes, demonstrating long plasma circulation times and accumulation within tumors.

2. Establish the effects of Mb-containing polymersomes on tumor physiology

While the hypothesis was that PEMs would accumulate within hypoxic tumors and subsequently increase O2 tension, we observed a rapid decrease in tumor oxygenation followed by a dramatic hemorrhagic effect of Mb polymersomes, which appear to be due to both endothelial cell apoptosis and morphological changes, resulting in central tumor necrosis.

3. Modify tumor growth through delivery of Mb polymersomes in combination with a cytotoxic therapy specific to aerobic tumors

Combination therapy of PEMs with RT results in enhanced tumor growth delay in aggressive 4T1 mammary carcinomas compared with RT or PEMs alone.

These studies have led to a proposed mechanism for the PEM anti-tumor effect in combination with RT. Prior to PEM administration, RT is administered, resulting in tumor cell kill of the well-oxygenated tumor periphery. Mb polymersomes are then injected i.v. and begin to accumulate within tumors due to the EPR effect. As shown in Aim 1, this accumulation occurs over a short time scale. Within 30 min of PEM treatment, the Mb is believed to act on tumor vessels, resulting in morphological changes and apoptosis of endothelial cells. These effects are expected to increase permeability of the vessels and expose the basement membrane, which leads to clotting and decreased blood flow. Both decreased perfusion and increased permeability are believed to have a catastrophic effect on interior tumor vessels. Hemorrhage results as the endothelial cells die, resulting in tumor core necrosis. Therefore, the result is tumor cell kill at the periphery due to RT and central tumor necrosis due to PEM treatment.

PEMs have potential in cancer therapy as a new class of VDAs. While the mechanism requires further investigation, this work has demonstrated that PEM treatment results in tumor vessel destruction and central necrosis. PEMs accumulate within tumors, thus minimizing the systemic toxicity of treatment commonly seen with VDAs. By combining PEMs with a therapy that kills the better perfused tumor periphery, PEMs show promise in improving tumor response. Future mechanistic studies will be needed in order to maximize vessel damage and optimize combination dosing schedules to improve outcome.

In this thesis, we present an overview of the development and application of surface-enhanced Raman scattering (SERS) and plasmonic nanoplatforms developed in our laboratory for sensing applications. Plasmonic nanomaterials, such as gold nanostars (AuNS) a hallmark particle pioneered by the Vo-Dinh group, increase inherently weak Raman signals from molecules providing an intense and unique SERS spectrum allowing for targets to be sensitively detected and easily identified. For this work, we investigated biosensing in plants and murine models using plasmonic nanoplatforms. In the first part of the work, we developed a wide variety of plasmonics-active substrates and nanoparticle-based sensing systems including inverse molecular sentinels (iMS) utilizing two platforms silver-coated gold nanostars (AuNS@Ag) and nanorods (AuNR@Ag). The AuNS@Ag were decorated onto fiber-optrodes for chemical fiber sensing and monitoring of genomic biomarkers in plants for renewable bioenergy research. Rapid chemical sensing of illegal food additives proves to be a challenge at the site of exposure thus requiring the need for in situ fiber detection. Also, the fiber sensing approach is necessary for facilitating field analyses of microRNAs since the gold standard methods can only be performed in laboratory settings on the timescale of days. The fiber-optrodes were capable of functioning as chemical and biological sensors for analytes such as illegal food additives and plant microRNAs. The work is also aimed at microRNA sensing but directly inside of plant tissue with spatial and temporal resolutions that PCR cannot achieve. Thus, the silver-coated nanorod (AuNR@Ag) was developed with the purpose of infiltrating plant cells. We designed the plasmonic nanorod to have a dimension smaller than the plant cell wall exclusion limit to permit cellular uptake, while improving SERS properties through a silver coating on the particle. We confirmed particle uptake in plant cells using a multi-modal approach consisting of confocal microscopy, transmission electron microscopy, and x-ray fluorescence microscopy. Dye coated AuNR@Ag served as a strong contrast agent for two-photon imaging, photoacoustic imaging, and Raman mapping during in vivo experiments in Tobacco leaves. The AuNR@Ag was further functionalized with iMS technology and was applied for sensing for microRNA targets in leaf tissue. To our knowledge, this is the first demonstration of intracellular SERS sensing in vivo of leaf tissue treated with the AuNR@Ag nanoprobes. In another chapter of the work, we used standoff shifted-excitation difference spectroscopy (SERDS) for remote detection of biomarkers in plants under ambient light conditions. Monitoring plant molecular targets in field conditions remain an elusive task for standard optical methods such as fluorescence and Raman spectroscopy. However, recent developments in nanoprobe technology and remote optical techniques have ushered in a novel mechanism for highly specific molecular monitoring of living organisms at different stages of growth and other phenotypic cues. We have successfully demonstrated nanoprobe detection in a live plant leaf at a minimal distance of 2 meters. This work brings the remote monitoring of plant genetic biomarkers closer to in vivo tracking and analysis without the need of a dark laboratory as required by traditional optical sensing. Applications of plasmonic AuNS for bioimaging of tumors in combination with SERDS is also presented. During surgery accurately removing the entire tumor without harming surrounding healthy tissue is critical; however, due to the lack of intraoperative imaging techniques, surgeons rely on visual and physical inspection to identify tumors. To address this problem, we established the first use of SERDS for in vivo tumor detection in a murine model under ambient light conditions, mimicking an intraoperative environment.  

Imbalance between oxygen consumption and supply often makes tumors hypoxic (Bristow and Hill 2008). Tumor hypoxia is significantly correlated with aggressive tumor growth, ineffective response to radiation and chemotherapy, and as a result, poor patient prognosis. Hyperthermia (HT) is a strong adjuvant treatment to overcome these challenges of tumor hypoxia because it causes tumor reoxygenation at temperatures lower than 43ºC (Song, Park, and Griffin 2001). However, the detailed molecular mechanisms of how HT enhances tumor oxygenation have not been elucidated. Here we determine that 1 hour HT activates hypoxia-inducible factor-1 (HIF-1) and its downstream targets, vascular endothelial growth factor (VEGF), lactate dehydrogenase A (LDHA), and pyruvate dehydrogenase kinase 1 (PDK1) in tumors. Consistent with HIF-1 activation and upregulation of its downstream genes, HT also enhances tumor perfusion/vascularization and decreases oxygen consumption rates. As a result, tumor hypoxia is reduced after HT suggesting that these physiological changes contribute to HT-induced tumor reoxygenation. Since HIF-1 is a potent regulator of tumor vascularization and metabolism, our findings suggest that HIF-1 plays a role in HT-induced tumor reoxygenation by transactivating its downstream targets. Mechanistically, we demonstrate that NADPH oxidase-mediated reactive oxygen species (ROS) production upregulates HIF-1 after HT. Further, we determine that this pathway is initiated by increased transcription of NADPH oxidase-1 (NOX1) through the ERK pathway.

A major research effort at Duke focuses on combinations of HT and doxorubicin in the treatment of locally advanced breast and other cancers. Thus, we investigated whether there are HIF-1 responses to doxorubicin treatment. We reveal that doxorubicin also activates HIF-1. Unlike HT, doxorubicin-induced HIF-1 promotes persistent tumor angiogenesis. We also reveal that the signal transducer and activator of transcription 1 (STAT1)/inducible nitric oxide synthase (iNOS) pathway causes HIF-1α accumulation in an oxygen-independent manner. We show that activated STAT1 upregulates iNOS expression and promotes nitric oxide (NO) production in tumor cells resulting in HIF-1α stabilization. We further determine that both iNOS inhibitor, 1400W and STAT1 inhibitor, epigallocatechin-3-gallate (EGCG) significantly decrease intracellular NO production and suppress doxorubicin-induced normoxic HIF-1α accumulation.

HIF-1 is often considered a promising therapeutic target because of its role in tumor progression (Semenza 2003) and therapy resistance (Moeller et al. 2004). However, our findings suggest that HIF-1 plays a pleiotropic role in response to HT and chemotherapy. Therefore, to preferentially take advantage of HT-induced HIF-1 activation and also to suppress its deleterious effects induced by chemotherapy or as we have previously reported, by radiation (Moeller et al. 2004), HIF-1 inhibition needs to be carefully regulated in a time-sensitive manner to achieve optimal therapeutic effects.