Friday, April 20, 2012

Nanoparticles Light Up Brain Tumors


Introduction & Background
Sanjiv (Sam) Gambhir (1)
            Last week in class, Dr. Islas mentioned that there is an “art” to surgical oncology.  Dr. Islas explained how surgeons will often excise the tumor as well as additional healthy cells surrounding it, to ensure they are removing the entire tumor and no residual cancer cells are left.  Over the weekend, I came across a study conducted by a group of Stanford scientist who have developed nanoparticles that can home in on brain tumors, thereby increasing the accuracy of surgical removal.
The senior author of the study is Sanjiv (Sam) Gambhir, MD, PhD, professor and chair of radiology at the Stanford University School of Medicine.  The research was published online on April 15, 2012 in Nature Medicine.
            Each year in the United States approximately 14,000 people are diagnosed with brain cancer.  Of these cases about 3,000 are glioblastomas (1).  Glioblastoma multiforme (GBM) is a tumor of the central nervous system, which originates from glial (supportive) tissue of the brain and spinal cord (2).  Without treatment, the median survival time is three months, making GBM the most aggressive form of brain tumor (1).  In most cases resection of the tumor is required.  Surgery, however, only prolongs the average patient’s survival time by less than a year (1).  GBM’s grim prognosis is due to the fact that even the most skilled neurosurgeons are unable to remove the entire tumor while sparing normal brain tissue.
Two common features of glioblastomas make them especially difficult for neurosurgeons to remove.  Glioblastomas are characteristically described as rough-edged tumors, because small “finger-like” projections often extend from the central mass and protrude into healthy tissue along paths of blood vessels and nerve fibers (1).  In addition to these “finger-like” extensions, micrometastases further complicate the process of glioblastoma resection.  Micrometastases are miniscule tumor patches that have spread from the primary tumor to other sites (3).  Scattered amongst healthy tissue, micrometastases are invisible to the surgeon’s naked eye and if left unnoticed they can readily grow into new tumors (1).
As Dr. Islas said when surgeons tackle tumors in the body they can usually afford to remove some healthy tissue surround the cancerous mass in hopes of getting rid of every harmful cell.  But, in the brain, each cell is important and too critical to sacrifice.  As Dr. Gambhir says, “With brain tumors, surgeons don’t have the luxury of removing large amounts of surrounding normal brain tissue to be sure no cancer cells are left.  You clearly have to leave as much of the healthy brain intact as you possibly can.” (1)

In this study, Gambhir and his team developed minuscule nanoparticles that highlighted brain tumors, precisely delineating their boundaries and increasing the ease of the tumor’s complete removal (1).  The nanoparticles, engineered by Gambhir, are tiny gold balls coated with various layers of imaging reagents.  When injected intravenously into mice with brain tumors, the nanoparticles diffused out of damaged blood vessels feeing the tumors and attached to cancerous cells therein.  The nanoparticle’s gold core along with their specialized coatings are visible to three distinct methods of imaging – magnetic resonance imaging (MRI), photoacoustic imaging, and Raman imaging.  Using this tri-modality imaging method, Gambhir obtained high resolution images of the tumors before and during resection in tumor-bearing mice.  Capitalizing on the strengths of each imaging technique to guide each craniotomy, Gambhir successfully removed the bulk of the glioblastomas, along with its “finger-like” projections, and micrometastases.  Before delving into exactly how the scientists went about their study, let’s take a step back and talk about the nanoparticles, MRI, photoacoustic imaging, and Raman imaging.

The nanoparticles
Each nanoparticle is composed of a 60-nm gold core surrounded by a thin Raman-active outer layer that is further protected by a 30-nm silica coating.  Maleimide-DOTA-Gd3+ was then attached to thiol (SH) groups on the silica (5) (Fig. 1a).  The diameter of each nanoparticle measures less than five one-millionths of an inch, which is approximately one-sixteenth the size of a human erythrocyte (1).  Upon intravenous injection, the nanoparticles work by capitalizing on the damage caused by brain tumors to the blood-brain barrier.  In doing so, they diffuse through the “leaky” vessels surrounding the tumor, enter the extravascular space, and accumulate in cells that comprise the tumor.  In other, non-cancerous regions of the brain where the blood-brain barrier is intact, the nanoparticles are too big to cross and are therefore unable to accumulate in healthy brain tissue (5).  Unlike other agents used for imaging that have a short blood half-life (i.e. Gadolinium for MRIs), a single injection of Gambhir’s nanoparticles are retained in tumor brain tissue long enough (~ seven days) to allow for preoperative planning and intraoperative resection of the tumor (5).

MRI
Magnetic resonance imaging (MRI) uses radio waves and a powerful magnet linked to a computer to generate images of areas inside the body (4).  Currently, MRIs are used to give surgeons a macroscopic outline of the tumor before surgery (5).  Use of MRIs during surgery is possible, but as stated earlier, requires repeated administration and high dosages of contrast agents.  In addition, surgeons often experience discrepancies between preoperative and intraoperative MRI scans.  Before surgery, MRIs are well-equipped to generate images that outline a tumor’s boundaries, but given the rapidly-growing nature of brain cancers and the constantly changing dynamics of the brain, preoperative MRI’s are often not representative of the tumor during surgery.  MRIs are also limited in the spatial resolution they can provide (5).

Photoacoustic imaging
In photoacoustic imaging, red shifted light pulses are sent into the body, which interact with image-enhancing nanoparticles.  Excitation of the nanoparticles cause them to heat slightly (without damaging nearby tissue) (6).  Thermal expansion of the nanoparticles creates pressure in the form of ultrasound waves.  The sound waves are then recorded by an ultrasound transducer that generates a three-dimensional image of the nanoparticles distribution in the tissue.  In this study, the nanoparticle’s gold-core served as a good material for absorbing light, and subsequently a good material at producing sound (6). 

Raman imaging
            Raman imaging generates images based on a given material’s Raman spectrum.  The Raman spectrum is a signature pattern consisting of several wavelengths unique to the specific material (1).  A compete spectrum is acquired at each and every pixel of the image and then analyzed to create colored images based on the material’s composition and structure (7).  In this study, the nanoparticle’s silica and maleimide-DOTA-Gd3+ outer coating served to generate the initial Raman signal, while the nanoparticle’s gold-core amplified the signal to be captured and recorded by a microscope (1).
           
Histological validation of nanoparticle sequestration by brain tumors
            Gambhir and his team used immunohistochemistry to determine their image-enhancing nanoparticles targeted tumor tissue, and tumor tissue only (5).  The researchers implanted different types of human glioblastoma cells (transduced with GFP) into the brains of mice.  Once the tumors were well established, they treated the mice with the nanoparticles via the mice’s tail vein.  They then stained with antibodies against GFP and CD11b (microglia marker antibody) to visualize tumor cells and glial cells, respectively (5).  Gambhir then took samples of tissue at sites where tumor cells intersected with healthy cells.  Using Raman imaging in conjunction with scanning transmission electron microscopy (STEM) Gambhir confirmed the nanoparticles accumulated within tumors, but not in healthy brain tissue (Fig. 2).  In addition, Gambhir’s lab found that the nanoparticles highlighted the glioblastoma’s “finger-like” protrusions as well as isolated microscopic tumor foci (5).
            The results indicate that the nanoparticles are able to diffuse through the damaged blood-brain barrier surrounding brain tumors and accumulate in cancer cells therein, without needing a specific target mechanism.

Nanoparticles guide brain tumor resection in vivo
            Gambhir and his team then demonstrated that their nanoparticles along with their three-pronged imaging proposal, could indeed facilitate tumor resection in glioblastoma-bearing mice.  To do this, the researchers once again implanted different types of human glioblastoma cells (transduced with GFP) into the brains of mice.  Twenty-four hours after injection of the image-enhancing nanoparticles into the mice’s tail vein, they used all three imaging techniques to visualize the tumors the human glioblastoma cells had spawned (5).  The researchers then placed the mice under anesthesia and performed craniotomies.  Gambhir used visual inspection only to sequentially remove quarters of the brain tumor (Fig. 3a).  Following each resection step, MRI, photoacoustic, and Raman imaging were performed.  After the tumor resection seemed to be complete by visual inspection, Gambhir obtained another set of images using all three methods.  MRI scans and photoacoustic imaging indicated the tumor had been removed.  But, Raman imaging showed several remnant micrometastases (Fig. 3b).  On further histological analysis of the tumor (using antibody staining against GFP (tumor) and CD11b (microglia)), Gambhir found tiny “finger-like” extensions of the tumor protruding into the surrounding brain tissue (Fig. 3c).  A Raman image taken of this same section also confirmed the presence of these microscopic cancerous growths, due to the retention of the nanoparticles therein (5).
Overall, the results suggest that neither MRI nor photoacoustic imaging alone can distinguish healthy from cancerous tissue at a microscopic level to highlight every last bit of a tumor (1).  Raman imaging, however, can.  With its high sensitivity, Raman imaging was able to flag residual microscopic tumor deposits that were otherwise not visible to the naked eye, MRI, or photoacoustic imaging.  Impart to its high accuracy, using real-time Raman images, Gambhir was able to go back in and remove leftover cancer cells that otherwise would have been left untreated.

The engineering of functionalized nanoparticles in conjunction with a tri-modality imaging strategy has great promise for enabling more accurate brain tumor imaging and resection.  Gambhir and his team’s proposed strategy entails 1) learning of the tumor’s general position and character before surgery using MRI, 2) excise of the bulk of the tumor mass using photoacoustic imaging, and 3) removal of once-invisible residual tumor deposits using Raman imaging (Fig. 4).  Ultimately, Gambhir’s triple-modality-nanoparticle approach will help radiologists and neurosurgeons better visualize the intricate outline of tumors before and during surgery, thereby allowing for more accurate and successful brain tumor resection.


MRI
Photoacoustic Imaging
Raman Imaging
How it works?
Radiowaves and magnet to generate image of body
Light heats up the nanoparticle’s gold-core causing them to emit sound.  The sound is measured by an ultrasound transducer to generate real-time images of the body
The nanoparticle’s silica & Gd outer coating gives off a weak Raman signal that is amplified by the nanoparticle’s gold-core.  The amplification is measured by a spectroscope to generate real-times images of the body
Proposed clinical use
Preoperative
- initial detection of the tumor
- surgical planning
Intraoperative
- guiding resection of the bulk of the tumor
Intraoperative
- guiding resection of residual micrometastases
Strengths
Detectability
- High spatial resolution
- 3D imaging
- Deep tissue penetration
- High spatial resolution
- High sensitivity

Questions & Thoughts:
After reading the paper, a few questions and thoughts came to mind:
1)       What are nanoparticles?  How do they differ from the fluorescent-antibodies Gambhir used to locate tumor cells and microglia?
Answer: There are four major advantages of imaging using nanoparticles versus imaging using molecules:
a.   Signaling power.  In molecular imaging, molecules are injected into the body and locate other molecules (i.e. molecules indicative of cancer).  But, in order for those imaging-molecules to be detected outside the body they need to produce a very large signal.  Nanoparticles are very good at producing signals because they are very good amplifiers.  Therefore, nanoparticles are able to generate signals that are much stronger than your typical imaging-molecules.
b.       Versatility.  Nanoparticles can be customized with different molecules based on their proposed used.  (For example, in this study Gambhir used a gold-core along with silica and Gd outer layers.)
c.       Multi-modality competence.  Nanoparticles can be made so that they produce a signal for positron emission tomography (PET) scanning, computed tomography (CT), and even a third modality such as single photon emission computed tomography (SPECT) scanning.  (For example, in this study Gambhir created nanoparticles for MRI, photoacoustic imaging, and Raman imaging.)  In molecular imaging, the multi-modality feature is harder to create.
d.       Diagnostic and therapeutic abilities.  Nanoparticles themselves can serve as both a diagnostic tool and a therapeutic agent.  Gambhir believes that if you are going to go through the trouble to create nanoparticles that can home in on abnormal tumors just to get an image, then that particle should be able to act as a diagnostic and therapeutic as well.  Therefore, nanoparticles could essentially act as an imaging agent, a diagnostic, and a therapeutic.  In molecular imaging, the molecules are strictly used for identification. (6)


2) Can the nanoparticles be used to light up other tumors elsewhere in the body?
Answer: Yes, the nanoparticles can be used to highlight cancer cells in the colon.
In fact, in one of Gambhir’s original studies with nanoparticles (prior to the one I blogged about) he engineered particles to go into the bowel and detect colorectal cancer.
The use of colonoscopy has significantly lowered colon cancer mortality rates, but this method of detection has its drawbacks.  Dr. Gambhir explains, “Colonoscopy relies on the human eye.  So, this screening too, while extremely useful, still misses many cancer lesions such as those that are too tiny, obscure, or flat to be noticed.” (10)  Through his nanoparticles, Gambhir has created a way to catch hard-to-see colon cancer cells early.
The nanoparticles used in the colon cancer study are very similar to the ones I described in the brain cancer paper.  They have a gold core, surrounded by a Raman-active layer, which is further protected by a silica coating.  The only difference is that the colon cancer-nanoparticles are further functionalized with molecules that target colon cancer cells, rather than the malemide-DOTA-Gd that functionalized the brain cancer-nanoparticles.  This makes sense, because while the brain tumor-nanoparticles worked by diffusing through the damaged blood-brain barrier (and did not have a specific target mechanism), the colon cancer-nanoparticles obviously cannot do this.  So, Gambhir needed to attach molecules, specific to colorectal cancer cells, on the outside of the colon cancer-nanoparticles.  Using the colon cancer-nanoparticle’s gold core to amplify the Raman signal, Gambhir demonstrated that his nanoparticles latched onto colon cancer cells.
His findings have huge implications for improving detection of colon cancer.  Dr. Gambhir says, “Right now, flat lesions might be entirely missed by the endoscopist who might be doing screening for colorectal cancer, but by having the gold nanoparticles light up where the cancer might be hiding in the bowel, now the hope is that the endoscopist will be able to act on a lesion they would have otherwise missed.” (10)
One can then imagine, that just by functionalizing the outer layer of the nanoparticles with molecules specific to certain types of cancer, the particles can hopefully be used to detect a wide variety of tumors.

3) Are the nanoparticles toxic?
Answer: No, the nanoparticles are nontoxic.
In a previous study, Gambhir showed that nanoparticles similar to the ones used in this study (except without gadolinium, Gd3+) were non-toxic in mice, suggesting that they will also behave well in humans (11).  Researchers administered nanoparticles to two groups of mice, each containing 30 female and 30 male animals.  The dose was 1,000-fold larger than would be required to get a clear signal from the nanoparticles (10).
The first group of mice received the nanoparticles rectally.  Gambhir then monitored the mice at five different time points, ranging from five minutes to two weeks.  At each time point, Gambhir and his team took a variety of measurements.  They followed the mices’ blood pressure, electrocardiograms, and white-blood-cell counts.  They examined several tissues for increased expression of antioxidant enzymes or pro-inflammatory signals, which would indicate physiological stress on the test mices’ cells.  Gambhir also stained tissues with dyes that tag dying cells.  By the end of two weeks, none of the tests resulted in signs of stress to any tissues.  To determine where in the body the nanoparticles had accumulated, Gambhir scanned the mices’ tissue using electron microscopy.  He found the nanoparticles remained confined to the bowel, thereby posing no systemic threat to other organs.  The nanoparticles were quickly excreted.
The second group of mice received the nanoparticles intravenously.  Once again, Gambhir and his colleagues found no significant signs of inflammation or other signs of toxicity.   Scavenger cells present in organs such as the spleen and liver quickly sequestered the nanoparticles.
With strong evidence that the nanoparticles are safe in mice, Gambhir is now filing for FDA approval to proceed to human clinical studies of the nanoparticles for diagnosis of cancer.

4)       Overall, Gambhir’s study seems very promising.  I would have liked to seen Dr. Gambhir monitor the mice for sometime after their craniotomies to see whether or not the brain tumors recurred.  If he had done this and found that remission rates are low, then Gambhir’s findings – that image-enhancing nanoparticles selectively targets all cancer cells, including finger-like projections and micrometastases, and therefore aids in complete tumor removal become much more powerful.  To relate this back to my first blog, CD47 Antibody: A Promising Breakthrough (8), the scientists in this study monitored the mice for an additional four months after administering the anti-CD47 antibody treatment (9).  Only after finding no recurrence of breast cancer were they able to make the claim that their treatment may be effective at eliminating all cancer cells, even cancer stem cells.  In this study, Gambhir claims his tri-modality-nanoparticle approach could enable more accurate surgical resection of brain tumors, which can subsequently help to improve the current poor prognosis for GBM.  He should then compare recurrence or mortality rates of tumor bearing-mice that undergo tumor resection guided by nanoparticle-imaging with that of mice that undergo tumor resection guided by visual inspection only.  It could very well be that the nanoparticles do not accumulate in all the “finger-like” projections or micrometastases, and if this were the case then we would expect to see no difference in the long-term surgical outcome between nanoparticle-image treated mice and visually inspected mice.

5)       This may be useful information for groups interested in doing their cancer project on new, cutting-edge cancer therapies.

Thanks for reading!