Danielle E. Green

Single-walled carbon nanotubes as a multimodal-thermoacoustic and photoacoustic-contrast agent

We have developed a novel carbon nanotube-based contrast agent for both thermoacoustic and photoacoustic tomography. In comparison to deionized water, single-walled carbon nanotubes exhibited more than twofold signal enhancement for thermoacoustic tomography at 3 GHz. In comparison to blood, they exhibited more than sixfold signal enhancement for photoacoustic tomography at 1064 nm wavelength. The large contrast enhancement of single-walled carbon nanotubes was further corroborated by tissue phantom imaging studies.

In vivo carbon nanotube-enhanced non-invasive photoacoustic mapping of the sentinel lymph node

Sentinel lymph node biopsy (SLNB), a less invasive alternative to axillary lymph node dissection (ALND), has become the standard of care for patients with clinically node-negative breast cancer. In SLNB, lymphatic mapping with radio-labeled sulfur colloid and/or blue dye helps identify the sentinel lymph node (SLN), which is most likely to contain metastatic breast cancer. Even though SLNB, using both methylene blue and radioactive tracers, has a high identification rate, it still relies on an invasive surgical procedure, with associated morbidity. In this study, we have demonstrated a non-invasive single-walled carbon nanotube (SWNT)-enhanced photoacoustic (PA) identification of SLN in a rat model. We have successfully imaged the SLN in vivo by PA imaging (793 nm laser source, 5 MHz ultrasonic detector) with high contrast-to-noise ratio (=89) and good resolution ( approximately 500 microm). The SWNTs also show a wideband optical absorption, generating PA signals over an excitation wavelength range of 740-820 nm. Thus, by varying the incident light wavelength to the near infrared region, where biological tissues (hemoglobin, tissue pigments, lipids and water) show low light absorption, the imaging depth is maximized. In the future, functionalization of the SWNTs with targeting groups should allow the molecular imaging of breast cancer.

Devastation of adult stem cell pools by irradiation precedes collapse of trabecular bone quality and quantity.

Stem cell depletion and compromised bone marrow resulting from radiation exposure fosters long‐term deterioration of numerous physiologic systems, with the degradation of the skeletal system ultimately increasing the risk of fractures. To study the interrelationship of damaged bone marrow cell populations with trabecular microarchitecture, 8‐ and 16‐week‐old C57BL/6 male mice were sublethally irradiated with 5 Gy of 137Cs γ‐rays, and adult stem cells residing in the bone marrow, as well as bone quantity and quality, were evaluated in the proximal tibia after 2 days, 10 days, and 8 weeks compared with age‐matched controls. Total extracted bone marrow cells in the irradiated 8‐week, young adult mice, including the hematopoietic cell niches, collapsed by 65% ± 11% after 2 days, remaining at those levels through 10 days, only recovering to age‐matched control levels by 8 weeks. As early as 10 days, double‐labeled surface was undetectable in the irradiated group, paralleled by a 41% ± 12% and 33% ± 4% decline in bone volume fraction (BV/TV) and trabecular number (Tb.N), respectively, and a 50% ± 10% increase in trabecular separation (Tb.Sp) compared with the age‐matched controls, a compromised structure that persisted to 8 weeks postirradiation. Although the overall collapse of the bone marrow population and devastation of bone quality was similar between the “young adult” and “mature” mice, the impact of irradiation—and the speed of recovery—on specific hematopoietic subpopulations was dependent on age, with the older animals slower to restore key progenitor populations. These data indicate that, independent of animal age, complications arising from irradiation extend beyond the collapse of the stem cell population and extend toward damage to key organ systems. It is reasonable to presume that accelerating the recovery of these stem cell pools will enable the prompt repair of the skeletal system and ultimately reduce the susceptibility to fractures.

Bone structure and B-cell populations, crippled by obesity, are partially rescued by brief daily exposure to low-magnitude mechanical signals

Deterioration of the immune and skeletal systems, each of which parallel obesity, reflects a fragile interrelationship between adiposity and osteoimmunology. Using a murine model of diet‐induced obesity, this study investigated the ability of mechanical signals to protect the skeletal‐immune systems at the tissue, cellular, and molecular level. A long‐term (7 mo) high‐fat diet increased total adiposity (+62%), accelerated age‐related loss of trabecular bone (–61%), and markedly reduced B‐cell number in the marrow (–52%) and blood (–36%) compared to mice fed a regular diet. In the final 4 mo of the protocol, the application of low‐magnitude mechanical signals (0.2 g at 90 Hz, 15 min/d, 5 d/wk) restored both bone structure and B cells to those levels measured in control mice fed a regular diet. These phenotypic outcomes were achieved, in part, by reductions in osteoclastic activity and a biasing of hematopoietic stem cell differentiation toward the lymphoid B‐cell lineage and away from a myeloid fate. These results emphasize that obesity undermines both the skeletal and immune systems, yet brief exposure to mechanical signals, perhaps as a surrogate to the salutary influence of exercise, diminishes the consequences of diabetes and obesity, restoring bone structure and normalizing B‐cell populations by biasing of the fate of stem cells through mechanosensitive pathways.—Chan, M. E., Adler, B. J., Green, D. E., Rubin, C. T. Bone structure and B‐cell populations, crippled by obesity, are partially rescued by brief daily exposure to low‐magnitude mechanical signals. FASEB J. 26, 4855–4863 (2012). www.fasebj.org

High Fat Diet Rapidly Suppresses B Lymphopoiesis by Disrupting the Supportive Capacity of the Bone Marrow Niche

The bone marrow (BM) niche is the primary site of hematopoiesis, and cues from this microenvironment are critical to maintain hematopoiesis. Obesity increases lifetime susceptibility to a host of chronic diseases, and has been linked to defective leukogenesis. The pressures obesity exerts on hematopoietic tissues led us to study the effects of a high fat diet (HFD: 60% Kcal from fat) on B cell development in BM. Seven week old male C57Bl/6J mice were fed either a high fat (HFD) or regular chow (RD) diet for periods of 2 days, 1 week and 6 weeks. B-cell populations (B220+) were not altered after 2 d of HFD, within 1 w B-cell proportions were reduced by −10%, and by 6 w by −25% as compared to RD (p<0.05). BM RNA was extracted to track the expression of B-cell development markers Il-7, Ebf-1 and Pax-5. At 2 d, the expression of Il-7 and Ebf-1 were reduced by −20% (p = 0.08) and −11% (p = 0.06) whereas Pax-5 was not significantly impacted. At one week, however, the expressions of Il-7, Ebf-1, and Pax-5 in HFD mice fell by -19%, −20% and −16%, and by six weeks were further reduced to −23%, −29% and −34% as compared to RD (p<0.05 for all), a suppression paralleled by a +363% increase in adipose encroachment within the marrow space (p<0.01). Il-7 is a critical factor in the early B-cell lineage which is secreted by supportive cells in the BM niche, and is necessary for B-cell commitment. These data indicate that BM Il-7 expression, and by extension B-cell differentiation, are rapidly impaired by HFD. The trend towards suppressed expression of Il-7 following only 2 d of HFD demonstrates how susceptible the BM niche, and the cells which rely on it, are to diet, which ultimately could contribute to disease susceptibility in metabolic disorders such as obesity.

Consequences of irradiation on bone and marrow phenotypes, and its relation to disruption of hematopoietic precursors

The rising levels of radiation exposure, specifically for medical treatments and accidental exposures, have added great concern for the long term risks of bone fractures. Both the bone marrow and bone architecture are devastated following radiation exposure. Even sub-lethal doses cause a deficit to the bone marrow microenvironment, including a decline in hematopoietic cells, and this deficit occurs in a dose dependent fashion. Certain cell phenotypes though are more susceptible to radiation damage, with mesenchymal stem cells being more resilient than the hematopoietic stem cells. The decline in total bone marrow hematopoietic cells is accompanied with elevated adipocytes into the marrow cavity, thereby inhibiting hematopoiesis and recovery of the bone marrow microenvironment. Poor bone marrow is also associated with a decline in bone architectural quality. Therefore, the ability to maintain the bone marrow microenvironment would hinder much of the trabecular bone loss caused by radiation exposure, ultimately decreasing some comorbidities in patients exposed to radiation.

Low magnitude mechanical signals mitigate osteopenia without compromising longevity in an aged murine model of spontaneous granulosa cell ovarian cancer.

Cancer progression is often paralleled by a decline in bone mass, raising risk of fracture. Concerns persist regarding anabolic interventions for skeletal protection, as these may inadvertently exacerbate neoplastic tissue expansion. Given bones inherent mechanosensitivity, low intensity vibration (LIV), a mechanical signal that encourages osteoblastogenesis, could possibly slow cancer-associated bone loss, but this goal must be achieved without fostering disease progression. Seventy 12w female F1-SWRxSWXJ-9 mice, a strain prone to developing granulosa cell tumors, were randomized into baseline control (BC: n=10), age-matched control (AC: n=30), and LIV (n=30), which received mechanical signals (90Hz @ 0.3g) for 15m/day, 5 day/w over the course of 1 year. Survival curves for AC (10 died) and LIV (8 died) followed similar trends (p=0.62), indicating longevity was unperturbed by LIV. At 1 year, bone volume of proximal tibiae in LIV mice was 25% greater than AC (p<0.02), while bone volume of L5 vertebrae was 16% higher in LIV over AC (p<0.02). Primary lesions and peripheral metastases were apparent in both LIV and AC; however, overall tumor incidence was approximately 30% less in LIV (p=0.27) and, when disease was evident, involved fewer organ systems (p=0.09). Marrow-derived mesenchymal stem cells (MSC) were 52% lower (p<0.01) in LIV, and 31% lower (p=0.08) in mice lacking pathology, suggesting higher MSC levels in this model of cancer susceptibility may have contributed to tumor progression. These experiments indicate that LIV helps protect bone mass in mice inherently susceptible to cancer without compromising life expectancy, perhaps through mechanical control of stem cell fate. Further, these data reflect the numerous system-level benefits of exercise in general, and mechanical signals in particular, in the preservation of bone density and the suppression of cancer progression.

Altered composition of bone as triggered by irradiation facilitates the rapid erosion of the matrix by both cellular and physicochemical processes.

Radiation rapidly undermines trabecular architecture, a destructive process which proceeds despite a devastated cell population. In addition to the ‘biologically orchestrated’ resorption of the matrix by osteoclasts, physicochemical processes enabled by a damaged matrix may contribute to the rapid erosion of bone quality. 8w male C57BL/6 mice exposed to 5 Gy of Cs137 γ-irradiation were compared to age-matched control at 2d, 10d, or 8w following exposure. By 10d, irradiation had led to significant loss of trabecular bone volume fraction. Assessed by reflection-based Fourier transform infrared imaging (FTIRI), chemical composition of the irradiated matrix indicated that mineralization had diminished at 2d by −4.3±4.8%, and at 10d by −5.8±3.2%. These data suggest that irradiation facilitates the dissolution of the matrix through a change in the material itself, a conclusion supported by a 13.7±4.5% increase in the elastic modulus as measured by nanoindentation. The decline in viable cells within the marrow of irradiated mice at 2d implies that the immediate collapse of bone quality and inherent increased risk of fracture is not solely a result of an overly-active biologic process, but one fostered by alterations in the material matrix that predisposes the material to erosion.

Novel breast cancer detection system combining both thermoacoustic (TA) and photoacoustic (PA) tomography using carbon nanotubes (CNTs) as a dual contrast agent

We report here a novel breast cancer scanner using microwave and light excitation and ultrasound detection. This combined thermoacoustic and photoacoustic tomography scanner is a nonionizing low cost system that can potentially provide high-resolution, dual contrast (microwave and light absorption) three dimensional images of the breast. Front breast compression will be used in this scanner to alleviate patient discomfort, experienced in side breast compression during traditional X-ray mammography. This scanner will use dry instead of gel ultrasonic coupling. We have also developed a carbon nanotube-based contrast agent for both thermoacoustic and photoacoustic imaging. In the future, targeted molecular photoacoustic and thermoacoustic imaging should be possible using this contrast agent.

In vivo photoacoustic (PA) mapping of sentinel lymph nodes (SLNs) using carbon nanotubes (CNTs) as a contrast agent

Sentinel lymph node biopsy (SLNB), a less invasive alternative to axillary lymph node dissection (ALND), is routinely used in clinic for staging breast cancer. In SLNB, lymphatic mapping with radio-labeled sulfur colloid and/or blue dye helps identify the sentinel lymph node (SLN), which is most likely to contain metastatic breast cancer. Even though SLNB, using both methylene blue and radioactive tracers, has a high identification rate, it still relies on an invasive surgical procedure, with associated morbidity. In this study, we have demonstrated a non-invasive single-walled carbon nanotube (SWNT)-enhanced photoacoustic (PA) identification of SLN in a rat model. We have used single-walled carbon nanotubes (SWNTs) as a photoacoustic contrast agent to map non-invasively the sentinel lymph nodes (SLNs) in a rat model in vivo. We were able to identify the SLN non-invasively with high contrast to noise ratio (~90) and high resolution (~500 μm). Due to the broad photoacoustic spectrum of these nanotubes in the near infrared wavelength window we could easily choose a suitable light wavelength to maximize the imaging depth. Our results suggest that this technology could be a useful clinical tool, allowing clinicians to identify SLNs non-invasively in vivo. In the future, these contrast agents could be functionalized to do molecular photoacoustic imaging.