Tumor hypoxia is an important factor in assessment of both cancer progression and cancer treatment efficacy. This has driven a substantial effort toward development of imaging modalities that can directly measure oxygen distribution and therefore hypoxia in tissue. Although several approaches to measure hypoxia exist, direct measurement of tissue oxygen through an imaging approach is still an unmet need. To address this, we present a new approach based on in vivo application of photoacoustic lifetime imaging (PALI) to map the distribution of oxygen partial pressure (pO 2 ) in tissue. This method utilizes methylene blue, a dye widely used in clinical applications, as an oxygen-sensitive imaging agent. PALI measurement of oxygen relies upon pO 2 -dependent excitation lifetime of the dye. A multimodal imaging system was designed and built to achieve ultrasound (US), photoacoustic, and PALI imaging within the same system. Nude mice bearing LNCaP xenograft hindlimb tumors were used as the target tissue. Hypoxic regions were identified within the tumor in a combined US/PALI image. Finally, the statistical distributions of pO 2 in tumor, normal, and control tissues were compared with measurements by a needle-mounted oxygen probe. A statistically significant drop in mean pO 2 was consistently detected by both methods in tumors.
Oxygen plays a key role in the energy metabolism of living organisms. Any imbalance in the oxygen levels will affect
the metabolic homeostasis and lead to pathophysiological diseases. Hypoxia, a status of low tissue oxygen, is a key
factor in tumor biology as it is highly prominent in tumor tissues. However, clinical tools for assessing tissue
oxygenation are limited. The gold standard is polarographic needle electrode which is invasive and not capable of
mapping (imaging) the oxygen content in tissue.
We applied the method of photoacoustic lifetime imaging (PALI) of oxygen-sensitive dye to small animal tissue hypoxia
research. PALI is new technology for direct, non-invasive imaging of oxygen. The technique is based on mapping the
oxygen-dependent transient optical absorption of Methylene Blue (MB) by pump-probe photoacoustic imaging. Our
studies show the feasibility of imaging of dissolved oxygen distribution in phantoms. In vivo experiments demonstrate
that the hypoxia region is consistent with the site of subcutaneously xenografted prostate tumor in mice with adequate
spatial resolution and penetration depth.
Recent advances in nanotechnology have allowed for the effective use of iron oxide nanoparticles (IONPs) for cancer
imaging and therapy. When activated by an alternating magnetic field (AMF), intra-tumoral IONPs have been
effective at controlling tumor growth in rodent models. To accurately plan and assess IONP-based therapies in clinical
patients, noninvasive and quantitative imaging technique for the assessment of IONP uptake and biodistribution will
Proven techniques such as confocal, light and electron microscopy, histochemical iron staining, ICP-MS, fluorescent
labeled mNPs and magnetic spectroscopy of Brownian motion (MSB), are being used to assess and quantify IONPs in
vitro and in ex vivo tissues. However, a proven noninvasive in vivo IONP imaging technique has not yet been
developed. In this study we have demonstrated the shortcomings of computed tomography (CT) and magnetic
resonance imaging (MRI) for effectively observing and quantifying iron /IONP concentrations in the clinical setting.
Despite the poor outcomes of CT and standard MR sequences in the therapeutic concentration range, ultra-short T2
MRI methods such as, Sweep Imaging With Fourier Transformation (SWIFT), provide a positive iron contrast
enhancement and a reduced signal to noise ratio. Ongoing software development and phantom and in vivo studies,
will further optimize this technique, providing accurate, clinically-relevant IONP biodistribution information.
Essential developments in the reliable and effective use of heat in medicine include: 1) the ability to model energy
deposition and the resulting thermal distribution and tissue damage (Arrhenius models) over time in 3D, 2) the
development of non-invasive thermometry and imaging for tissue damage monitoring, and 3) the development of
clinically relevant algorithms for accurate prediction of the biological effect resulting from a delivered thermal dose in
mammalian cells, tissues, and organs. The accuracy and usefulness of this information varies with the type of thermal
treatment, sensitivity and accuracy of tissue assessment, and volume, shape, and heterogeneity of the tumor target and
normal tissue. That said, without the development of an algorithm that has allowed the comparison and prediction of the
effects of hyperthermia in a wide variety of tumor and normal tissues and settings (cumulative equivalent minutes/
CEM), hyperthermia would never have achieved clinical relevance. A new hyperthermia technology, magnetic
nanoparticle-based hyperthermia (mNPH), has distinct advantages over the previous techniques: the ability to target the
heat to individual cancer cells (with a nontoxic nanoparticle), and to excite the nanoparticles noninvasively with a noninjurious
magnetic field, thus sparing associated normal cells and greatly improving the therapeutic ratio. As such, this
modality has great potential as a primary and adjuvant cancer therapy. Although the targeted and safe nature of the
noninvasive external activation (hysteretic heating) are a tremendous asset, the large number of therapy based variables
and the lack of an accurate and useful method for predicting, assessing and quantifying mNP dose and treatment effect is
a major obstacle to moving the technology into routine clinical practice. Among other parameters, mNPH will require
the accurate determination of specific nanoparticle heating capability, the total nanoparticle content and biodistribution
in the target cells/tissue, and an effective and matching alternating magnetic field (AMF) for optimal and safe excitation
of the nanoparticles. Our initial studies have shown that appropriately delivered and targeted nanoparticles are capable of
achieving effective tumor cytotoxicity at measured thermal doses significantly less than the understood thermal dose
values necessary to achieve equivalent treatment effects using conventional heat delivery techniques. Therefore
conventional CEM based thermal dose - tissues effect relationships will not hold for mNPH. The goal of this effort is to
provide a platform for determining the biological and physical parameters that will be necessary for accurately planning
and performing safe and effective mNPH, creating a new, viable primary or adjuvant cancer therapy.
Biomedical applications of nanoparticle heating range in scale from molecular activation (i.e. molecular beacons, protein
denaturation, lipid melting and drug release), cellular heating (i.e. nanophotolysis and membrane permeability control
and rupture) to whole tumor heating (deep and superficial). This work will present a review on the heating of two
classes of biologically compatible metallic nanoparticles: iron oxide and gold with particular focus on spatial and
temporal scales of the heating event. The size range of nanoparticles under discussion will focus predominantly in the 10
- 200 nm diameter size range. Mechanisms of heating range from Néelian and Brownian relaxation due to magnetic
susceptibility at 100s of kHz, optical absorption due to VIS and NIR lasers and "Joule" heating at higher frequency RF
(13.56 MHz). The heat generation of individual nanoparticles and the thermal responses at nano-, micro-, and macroscales
are presented. This review will also discuss how to estimate a specific absorption rate (SAR, W/g) based on
individual nanoparticles heating in bulk samples. Experimental setups are designed to measure the SAR and the results
are compared with theoretical predictions.
Local recurrence of cancer after cryosurgery is related to the inability to monitor and predict destruction of cancer
(temperatures > -40°C) within an iceball. We previously reported that a cytokine adjuvant TNF-α could be used to
achieve complete cancer destruction at the periphery of an iceball (0 to -40°C). This study is a further development of
that work in which cryosurgery was performed using cryoprobes operating at temperatures > -40°C. LNCaP Pro 5 tumor
grown in a dorsal skin fold chamber (DSFC) was frozen at -6°C after TNF-α incubation for 4 or 24 hours. Tumors
grown in the hind limb were frozen with a probe tip temperature of -40°C, 4 or 24 hours after systemic injection with
TNF-α. Both cryosurgery alone or TNF-α treatment alone caused only a minimal damage to the tumor tissue at the
conditions used in the study. The combination of TNF-α and cryosurgery produced a significant damage to the tumor
tissue in both the DSFC and the hind limb model system. This augmentation in cryoinjury was found to be time-dependent
with 4-hour time period between the two treatments being more effective than 24-hour. These results suggests
the possibility of cryotreatment at temperatures > -40°C with the administration of TNF-α.
Magnetic iron oxide nanoparticles have intrinsic advantages over other nanoparticles for various biomedical
applications. These advantages include visualization, heating, and movement properties. There are now numerous efforts
underway to expand the applications of these particles for non-invasive magnetic targeting/localization, drug/adjuvant
delivery and release, cellular imaging and cellular therapies. In order to move these applications forward it is necessary
to define new assays and methods to visualize, move and heat these particles and define their interactions with cellular
systems. Our studies of the movement and heating of these nanoparticles in solutions and gels suggest a strong response
of these properties to the size and coating of the particles, the suspending medium and the field parameters. Additionally,
cellular association is a strong function of the coating and concentration of the nanoparticles and the time of incubation.
X-ray computed tomography (CT) can be used to image at least two orders of concentration (1-40 mg Fe/ml) higher than
that by 1.5 T Magnetic Resonance (MR) (0.01-0.4 mg Fe/ml) and could prove to be useful for image-guided treatments
Minimally invasive cryothermic and hyperthermic therapies are being increasingly used to destroy dysfunctional and neoplastic tissues in several organ systems. This report morphologically compares the acute tissue response that follow cryothermic and microwave therapy in porcine kidneys. Three cryothermic and hyperthermic groups of treated kidneys were pooled from other studies for evaluation: 1) in vitro treated non-perfused, 2) in situ treated with 2-hour post in vivo perfusion, and 3) in situ treated with 3-day or 7-day post in vivo perfusion. The cryolesions showed uniform central coagulative-type necrosis and interstitial hemorrhage. The hyperthermic lesions showed central thermal fixation and a rim of coagulative necrosis. The cryothermic and hyperthermic lesions both had a similar narrow transition zone of partial cell injury. The cryothermic lesions developed a wound healing response that advanced into the central lesion. In contrast, the heat-treated tissues lacked a prominent wound healing response and appeared to resist breakdown/repair by the body. Thus, the tissue effects of and response to cryothermic and heat injury appear to be different.
An in vitro study was performed to investigate a more effective method of destroying malignant tissue during cyrosurgery, which is based on eutectic crystallization. Eutectic formation is a solidification process through which water and solutes form a hydrate and can be recognized by a secondary heat release in differential scanning calorimetry (DSC). We investigated whether it is possible to induce eutectic crystallization by infusing concentrated salt solutions into cell suspension and tissue systems. These systems included AT-1 rat prostate tumor and normal rat liver tissues. In cell suspensions, the post-thaw viability significantly drops at or below the temperatures where eutectic crystallization occurred. When eutectic crystallization is induced in tissues, histological analysis shows significantly enhanced freezing injury. These results imply that this method may be of benefit in cryosurgical applications particularly at the edge of the iceball where tumor cell survival is in question. The possible advantages of inducing eutectic crystallization are i) enhancement of direct cell injury; ii) enlargement of effective cryosurgical cell/tissue destruction zone by selecting a salt with a high eutectic temperature; and iii) improvement of the efficacy of monitoring during cryosurgery.
Uterine leiomyomata are the most common pelvic tumor in women. Minimally invasive cryosurgery is being investigated as a therapeutic option for symptomatic women. Direct cryothermic cell injury thresholds for leiomyomata and the adjacent myometrium are not well quantified. Using a directional solidification stage to simulate in-vivo cryothermic cooling, tissue sections (3 mm) from ten leiomyomata and six portions of myometrium were cooled (5°C/min) to -20°C, -40°C, -60°C, and - 80°C, held for 15 minutes, and then rapidly thawed to 21°C. In conjunction with tissue culturing and appropriate controls, cell death was assessed using a viability dye (ethidium homodimer/Hoechst) and routine histology. After normalizing to controls, leiomyomata cell death (LCD) increased from -20 to -80°C by histology (12 to 27 percent LCD) and dye assay (26 to 38 percent LCD). Myometrial cell death (MCD) from -20 to -80°C was 10 to 12 percent by histology and 4 to 20 percent by dye assay. In contrast to LCD from -40 to -80°C, MCD was significantly less and plateaued over this range (p<0.05). The dye assay appears to be more sensitive for detecting cell death than histology. This study suggests that both leiomyomata and myometrium are moderately resistant to direct cryothermic injury, with leiomyomata somewhat more susceptible.