Daniel L. Farkas

Isolation of cancer stem cells from adult glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most common adult primary brain tumor and is comprised of a heterogeneous population of cells. It is unclear which cells within the tumor mass are responsible for tumor initiation and maintenance. In this study, we report that brain tumor stem cells can be identified from adult GBMs. These tumor stem cells form neurospheres, possess the capacity for self-renewal, express genes associated with neural stem cells (NSCs), generate daughter cells of different phenotypes from one mother cell, and differentiate into the phenotypically diverse populations of cells similar to those present in the initial GBM. Having a distinguishing feature from normal NSCs, these tumor stem cells can reform spheres even after the induction of differentiation. Furthermore, only these tumor stem cells were able to form tumors and generate both neurons and glial cells after in vivo implantation into nude mice. The identification of tumor stem cells within adult GBM may represent a major step forward in understanding the origin and maintenance of GBM and lead to the identification and testing of new therapeutic targets.

Identification of amyloid plaques in retinas from Alzheimer's patients and noninvasive in vivo optical imaging of retinal plaques in a mouse model.

Noninvasive monitoring of β-amyloid (Aβ) plaques, the neuropathological hallmarks of Alzheimers disease (AD), is critical for AD diagnosis and prognosis. Current visualization of Aβ plaques in brains of live patients and animal models is limited in specificity and resolution. The retina as an extension of the brain presents an appealing target for a live, noninvasive optical imaging of AD if disease pathology is manifested there. We identified retinal Aβ plaques in postmortem eyes from AD patients (n=8) and in suspected early stage cases (n=5), consistent with brain pathology and clinical reports; plaques were undetectable in age-matched non-AD individuals (n=5). In APP(SWE)/PS1(∆E9) transgenic mice (AD-Tg; n=18) but not in non-Tg wt mice (n=10), retinal Aβ plaques were detected following systemic administration of curcumin, a safe plaque-labeling fluorochrome. Moreover, retinal plaques were detectable earlier than in the brain and accumulated with disease progression. An immune-based therapy effective in reducing brain plaques, significantly reduced retinal Aβ plaque burden in immunized versus non-immunized AD mice (n=4 mice per group). In live AD-Tg mice (n=24), systemic administration of curcumin allowed noninvasive optical imaging of retinal Aβ plaques in vivo with high resolution and specificity; plaques were undetectable in non-Tg wt mice (n=11). Our discovery of Aβ specific plaques in retinas from AD patients, and the ability to noninvasively detect individual retinal plaques in live AD mice establish the basis for developing high-resolution optical imaging for early AD diagnosis, prognosis assessment and response to therapies.

Tumor labeling in vivo using cyanine-conjugated monoclonal antibodies

Far-red-emitting cyanine fluorochromes have many properties desirable for in vivo imaging: absorption and emission at wavelengths where blood and tissue are relatively transparent, high quantum yields, and good solubility even at high molar ratios of fluorochrome to antibody. Potentially, conjugation by multiple linkages should minimize hydrolysis in vivo. We conjugated two tumor-targeting monoclonal antibodies: anti-SSEA-1 (IgM, κ) at ratios of 1.2–35 mol dye/mol antibody and 9.2.27 (IgG2a, κ) at 0.6–6 mol dye/mol antibody, using the cyanine fluorochromes Cy3.18, Cy5.18, and Cy5.5.18. Nude mice were inoculated using the SSEA-1-expressing MH-15 teratocarcinoma or the 9.2.27 antigen-expressing SK-MEL-2 melanoma to give tumors at several sites. Conjugated antibody was injected, and mice were imaged immediately after injection and at appropriate intervals thereafter using a standard camera lens, dissecting microscope, or endoscopes. Images were acquired using either an image-intensified video camera or cooled CCD cameras. Immediately after injection, major blood vessels and the heart, liver, and kidneys were readily visualized. After 1 day, tumor-targeting antibody conjugates were concentrated in tumors and there was little circulating conjugate; however, the bladder and kidneys were still visible. Tumors labeled by specific antibody were the most fluorescent tissues at 2 days after injection, but non-specific antibody conjugates did not concentrate in the tumors. The small intestine was weakly visualized by both specific and non-specific antibody conjugates. These data support the possibility of visualizing tumor metastasis by optical means, including currently available endoscopes.

Tumor detection and elimination by a targeted gallium corrole

Sulfonated gallium(III) corroles are intensely fluorescent macrocyclic compounds that spontaneously assemble with carrier proteins to undergo cell entry. We report in vivo imaging and therapeutic efficacy of a tumor-targeted corrole noncovalently assembled with a heregulin-modified protein directed at the human epidermal growth factor receptor (HER). Systemic delivery of this protein-corrole complex results in tumor accumulation, which can be visualized in vivo owing to intensely red corrole fluorescence. Targeted delivery in vivo leads to tumor cell death while normal tissue is spared. These findings contrast with the effects of doxorubicin, which can elicit cardiac damage during therapy and required direct intratumoral injection to yield similar levels of tumor shrinkage compared with the systemically delivered corrole. The targeted complex ablated tumors at >5 times a lower dose than untargeted systemic doxorubicin, and the corrole did not damage heart tissue. Complexes remained intact in serum and the carrier protein elicited no detectable immunogenicity. The sulfonated gallium(III) corrole functions both for tumor detection and intervention with safety and targeting advantages over standard chemotherapeutic agents.

Non-invasive image acquisition and advanced processing in optical bioimaging

Light is a most versatile tool for investigating biological systems and phenomena; the range, non-destructiveness, spatial discrimination and speed of optical imaging are all important for investigating structure and function at the cellular, tissue or even whole organism level. In live biological imaging, where the technological requirements are heightened, other features of light, such as coherence and wavelength, are used to generate the additional contrast and resolution needed. We report here recent improvements in our ability to image biological specimens optically, focusing on (a) spectral resolution and the related image processing issues, and (b) tomographic three-dimensional fluorescence imaging in vivo.

Near-simultaneous hemoglobin saturation and oxygen tension maps in mouse brain using an AOTF microscope

A newly developed microscope using acousto-optic tunable filters (AOTFs) was used to generate in vivo hemoglobin saturation (SO2) and oxygen tension (PO2) maps in the cerebral cortex of mice. SO2 maps were generated from the spectral analysis of reflected absorbance images collected at different wavelengths, and PO2 maps were generated from the phosphorescence lifetimes of an injected palladium-porphyrin compound using a frequency-domain measurement. As the inspiratory O2 was stepped from hypoxia (10% O2), through normoxia (21% O2), to hyperoxia (60% O2), measured SO2 and PO2 levels rose accordingly and predictably throughout. A plot of SO2 versus PO2 in different arterial and venous regions of the pial vessels conformed to the sigmoidal shape of the oxygen-hemoglobin dissociation curve, providing further validation of the two mapping procedures. The study demonstrates the versatility of the AOTF microscope for in vivo physiologic investigation, allowing for the generation of nearly simultaneous SO2 and PO2 maps in the cerebral cortex, and the frequency-domain detection of phosphorescence lifetimes. This class of study opens up exciting new possibilities for investigating the dynamics of hemoglobin and O2 binding during functional activation of neuronal tissues.

NONINVASIVE IMAGING OF LIVING HUMAN SKIN WITH DUAL-WAVELENGTH OPTICAL COHERENCE TOMOGRAPHY IN TWO AND THREE DIMENSIONS

We demonstrate the potential of optical coherence-domain tomography (OCT) for noninvasive imaging of living skin simultaneously at two wavelengths in the near infrared range (830 and 1285 nm). The technical details of a prototype monomode fiber-optic coherence tomographic scanner providing rapid two-dimensional (2D) and three-dimensional (3D) imaging of biological tissues are described. The effects of both instrumentation parameters and the dynamic characteristics of living tissue on image contrast and resolution and on speckle reduction are discussed. The impact of imaging speed on OCT image quality is studied by a comparison between a single scan and the corresponding frame-averaged OCT images, with the latter resulting in decreased speckle noise as well as loss of some subtle structures. Both theoretical predictions and experimental results in human skin imaging show that longer wavelength can minimize the influence of multiple scattering on image contrast and resolution and thus increase the effective penetration depth of OCT imaging to about 2 mm. Some high-resolution 2D and 3D images of microscopic anatomic structures of living human skin are presented and analyzed, illustrating the unique capability of OCT for in depth, noninvasive visualization of living skin microscopic morphology in vivo.

Tumor Detection and Visualization Using Cyanine Fluorochrome‐Labeled Antibodies

Tumor localization using fluorescence has been made practical by current improvements in tumor targeting molecules, especially monoclonal antibodies and their derivatives, by the development of convenient near‐infrared emitting fluorochromes and by the availability of digital cameras having high sensitivity in this spectral region. Recent studies in animals have demonstrated that fluorochrome labeling of monoclonal antibodies confers adequate sensitivity and improved resolution. Distribution and catabolism of fluorochrome‐labeled and radiolabeled antibodies are similar. Simultaneous localization of multiple reagents is made possible by labeling with several different near‐infrared emitting fluorochromes; thus background subtraction and differential labeling of multiple tumor‐associated components can be performed. Difficulties in using the fluorochrome labels are mainly related to light scattering and absorption in tissues, but detection of small tumors at depths of several millimeters is feasible. The major medical use of this new technology is likely to be endoscopic location of tumors. Scientific uses include studies of tumor metastasis, uptake and distribution of drugs and tumor‐targeting molecules by tumors, and migration patterns of near‐infrared labeled cells in vivo.

Induction of potent antitumor immunity by intratumoral injection of interleukin 23-transduced dendritic cells

Dendritic cells (DCs) are potent antigen-presenting cells that play a critical role in priming immune responses to tumor. Interleukin (IL)-23 can act directly on DC to promote immunogenic presentation of tumor peptide in vitro. Here, we evaluated the combination of bone marrow-derived DC and IL-23 on the induction of antitumor immunity in a mouse intracranial glioma model. DCs can be transduced by an adenoviral vector coding single-chain mouse IL-23 to express high levels of bioactive IL-23. Intratumoral implantation of IL-23-expressing DCs produced a protective effect on intracranial tumor-bearing mice. The mice consequently gained systemic immunity against the same tumor rechallenge. The protective effect of IL-23-expressing DCs was comparable with or even better than that of IL-12-expressing DCs. IL-23-transduced DC (DC-IL-23) treatment resulted in robust intratumoral CD8(+) and CD4(+) T-cell infiltration and induced a specific TH1-type response to the tumor in regional lymph nodes and spleen at levels greater than those of nontransduced DCs. Moreover, splenocytes from animals treated with DC-IL-23 showed heightened levels of specific CTL activity. In vivo lymphocyte depletion experiments showed that the antitumor immunity induced by DC-IL-23 was mainly dependent on CD8(+) T cells and that CD4(+) T cells and natural killer cells were also involved. In summary, i.t. injection of DC-IL-23 resulted in significant and effective systemic antitumor immunity in intracranial tumor-bearing mice. These findings suggest a new approach to induce potent tumor-specific immunity to intracranial tumors. This approach may have therapeutic potential for treating human glioma.

Isolation of tumour stem-like cells from benign tumours.

Background:Cancerous stem-like cells (CSCs) have been implicated as cancer-initiating cells in a range of malignant tumours. Diverse genetic programs regulate CSC behaviours, and CSCs from glioblastoma patients are qualitatively distinct from each other. The intrinsic connection between the presence of CSCs and malignancy is unclear. We set out to test whether tumour stem-like cells can be identified from benign tumours.Methods:Tumour sphere cultures were derived from hormone-positive and -negative pituitary adenomas. Characterisation of tumour stem-like cells in vitro was performed using self-renewal assays, stem cell-associated marker expression analysis, differentiation, and stimulated hormone production assays. The tumour-initiating capability of these tumour stem-like cells was tested in serial brain tumour transplantation experiments using SCID mice.Results:In this study, we isolated sphere-forming, self-renewable, and multipotent stem-like cells from pituitary adenomas, which are benign tumours. We found that pituitary adenoma stem-like cells (PASCs), compared with their differentiated daughter cells, expressed increased levels of stem cell-associated gene products, antiapoptotic proteins, and pituitary progenitor cell markers. Similar to CSCs isolated from glioblastomas, PASCs are more resistant to chemotherapeutics than their differentiated daughter cells. Furthermore, differentiated PASCs responded to stimulation with hypothalamic hormones and produced corresponding pituitary hormones that are reflective of the phenotypes of the primary pituitary tumours. Finally, we demonstrated that PASCs are pituitary tumour-initiating cells in serial transplantation animal experiments.Conclusion:This study for the first time indicates that stem-like cells are present in benign tumours. The conclusions from this study may have applications to understanding pituitary tumour biology and therapies, as well as implications for the notion of tumour-initiating cells in general.