Steven G. Allen

Nanoroughened Surfaces for Efficient Capture of Circulating Tumor Cells without Using Capture Antibodies

Circulating tumor cells (CTCs) detached from both primary and metastatic lesions represent a potential alternative to invasive biopsies as a source of tumor tissue for the detection, characterization and monitoring of cancers. Here we report a simple yet effective strategy for capturing CTCs without using capture antibodies. Our method uniquely utilized the differential adhesion preference of cancer cells to nanorough surfaces when compared to normal blood cells and thus did not depend on their physical size or surface protein expression, a significant advantage as compared to other existing CTC capture techniques.

Single-cell Migration Chip for Chemotaxis-based Microfluidic Selection of Heterogeneous Cell Populations

Tumor cell migration toward and intravasation into capillaries is an early and key event in cancer metastasis, yet not all cancer cells are imbued with the same capability to do so. This heterogeneity within a tumor is a fundamental property of cancer. Tools to help us understand what molecular characteristics allow a certain subpopulation of cells to spread from the primary tumor are thus critical for overcoming metastasis. Conventional in vitro migration platforms treat populations in aggregate, which leads to a masking of intrinsic differences among cells. Some migration assays reported recently have single-cell resolution, but these platforms do not provide for selective retrieval of the distinct migrating and non-migrating cell populations for further analysis. Thus, to study the intrinsic differences in cells responsible for chemotactic heterogeneity, we developed a single-cell migration platform so that individual cells’ migration behavior can be studied and the heterogeneous population sorted based upon chemotactic phenotype. Furthermore, after migration, the highly chemotactic and non-chemotactic cells were retrieved and proved viable for later molecular analysis of their differences. Moreover, we modified the migration channel to resemble lymphatic capillaries to better understand how certain cancer cells are able to move through geometrically confining spaces.

Image-Guided Non-Invasive Ultrasound Liver Ablation Using Histotripsy: Feasibility Study in an In Vivo Porcine Model

Hepatocellular carcinoma is one of the fastest growing cancers worldwide. Histotripsy is a non-invasive ablation method that fractionates soft tissue through the control of acoustic cavitation. In this study, we demonstrate the feasibility of using histotripsy for non-invasive liver ablation. Fourteen ~1cm3 lesions were created in the livers of eight pigs through the intact chest in vivo without using aberration correction. Complete fractionation of liver parenchyma was observed with <;500 μm sharp boundaries. In addition, two larger volumes of 18 cm3 and 60 cm3 were generated within 60 minutes. Histotripsy liver fractionation was self-limited at the boundaries of critical structures including the gallbladder and major vessels. The liver surrounding major vessels was completely fractionated while the vessels remained intact. This work demonstrates that histotripsy is capable of noninvasively fractionating liver tissue while preserving critical anatomical structures within the liver. Results suggest histotripsy has potential for the non-invasive ablation of liver tumors.

Macrophages Enhance Migration in Inflammatory Breast Cancer Cells via RhoC GTPase Signaling

Inflammatory breast cancer (IBC) is the most lethal form of breast cancer. All IBC patients have lymph node involvement and one-third of patients already have distant metastasis at diagnosis. This propensity for metastasis is a hallmark of IBC distinguishing it from less lethal non-inflammatory breast cancers (nIBC). Genetic profiling studies have been conducted to differentiate IBC from nIBC, but no IBC cancer-cell-specific gene signature has been identified. We hypothesized that a tumor-extrinsic factor, notably tumor-associated macrophages, promotes and contributes to IBC’s extreme metastatic phenotype. To this end, we studied the effect of macrophage-conditioned media (MCM) on IBC. We show that two IBC cell lines are hyper-responsive to MCM as compared to normal-like breast and aggressive nIBC cell lines. We further interrogated IBC’s hyper-responsiveness to MCM using a microfluidic migration device, which permits individual cell migration path tracing. We found the MCM “primes” the IBC cells’ cellular machinery to become extremely migratory in response to a chemoattractant. We determined that interleukins −6, −8, and −10 within the MCM are sufficient to stimulate this enhanced IBC migration effect, and that the known metastatic oncogene, RhoC GTPase, is necessary for the enhanced migration response.

Modified Nanoemulsions with Iron Oxide for Magnetic Resonance Imaging

A nanoemulsion (NE) is a surfactant-based, oil-in-water, nanoscale, high-energy emulsion with a mean droplet diameter of 400–600 nm. When mixed with antigen and applied nasally, a NE acts as a mucosal adjuvant and induces mucosal immune responses. One possible mechanism for the adjuvant effect of this material is that it augments antigen uptake and distribution to lymphoid tissues, where the immune response is generated. Biocompatible iron oxide nanoparticles have been used as a unique imaging approach to study the dynamics of cells or molecular migration. To study the uptake of NEs and track them in vivo, iron oxide nanoparticles were synthesized and dispersed in soybean oil to make iron oxide-modified NEs. Our results show that iron oxide nanoparticles can be stabilized in the oil phase of the nanoemulsion at a concentration of 30 µg/μL and the iron oxide-modified NEs have a mean diameter of 521 nm. In vitro experiments demonstrated that iron oxide-modified NEs can affect uptake by TC-1 cells (a murine epithelial cell line) and reduce the intensity of magnetic resonance (MR) images by shortening the T2 time. Most importantly, in vivo studies demonstrated that iron oxide-modified NE could be detected in mouse nasal septum by both transmission electron microscopy and MR imaging. Altogether these experiments demonstrate that iron oxide-modified NE is a unique tool that can be used to study uptake and distribution of NEs after nasal application.

Nanoroughened adhesion-based capture of circulating tumor cells with heterogeneous expression and metastatic characteristics

BackgroundCirculating tumor cells (CTCs) have shown prognostic relevance in many cancer types. However, the majority of current CTC capture methods rely on positive selection techniques that require a priori knowledge about the surface protein expression of disseminated CTCs, which are known to be a dynamic population.MethodsWe developed a microfluidic CTC capture chip that incorporated a nanoroughened glass substrate for capturing CTCs from blood samples. Our CTC capture chip utilized the differential adhesion preference of cancer cells to nanoroughened etched glass surfaces as compared to normal blood cells and thus did not depend on the physical size or surface protein expression of CTCs.ResultsThe microfluidic CTC capture chip was able to achieve a superior capture yield for both epithelial cell adhesion molecule positive (EpCAM+) and EpCAM- cancer cells in blood samples. Additionally, the microfluidic CTC chip captured CTCs undergoing transforming growth factor beta-induced epithelial-to-mesenchymal transition (TGF-β-induced EMT) with dynamically down-regulated EpCAM expression. In a mouse model of human breast cancer using EpCAM positive and negative cell lines, the number of CTCs captured correlated positively with the size of the primary tumor and was independent of their EpCAM expression. Furthermore, in a syngeneic mouse model of lung cancer using cell lines with differential metastasis capability, CTCs were captured from all mice with detectable primary tumors independent of the cell lines’ metastatic ability.ConclusionsThe microfluidic CTC capture chip using a novel nanoroughened glass substrate is broadly applicable to capturing heterogeneous CTC populations of clinical interest independent of their surface marker expression and metastatic propensity. We were able to capture CTCs from a non-metastatic lung cancer model, demonstrating the potential of the chip to collect the entirety of CTC populations including subgroups of distinct biological and phenotypical properties. Further exploration of the biological potential of metastatic and presumably non-metastatic CTCs captured using the microfluidic chip will yield insights into their relevant differences and their effects on tumor progression and cancer outcomes.

Intermediate Endpoints After Postprostatectomy Radiotherapy: 5-Year Distant Metastasis to Predict Overall Survival

BACKGROUND Intermediate clinical endpoints (ICEs) prognostic for overall survival (OS) are needed for men receiving postprostatectomy radiation therapy (PORT) to improve clinical trial design. OBJECTIVE To identify a potential ICE for men receiving PORT. DESIGN, SETTING, AND PARTICIPANTS We performed an institutional review board-approved multi-institutional retrospective study of 566 men consecutively treated with PORT at tertiary care centers from 1986 to 2013. The median follow-up was 8.2 yr. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS Biochemical failure (BF), distant metastases (DM), and castrate-resistant prostate cancer (CRPC) were evaluated for correlation with OS and assessed as time-dependent variables in a multivariable Cox proportional hazards model and in landmark analyses at 1, 3, 5, and 7 yr after PORT. Cross-validated concordance (c) indices were used to assess model discrimination. RESULTS AND LIMITATIONS OS at 1, 3, 5, and 7 yr after PORT was 98%, 95%, 90%, and 82%, respectively. In a time-varying model controlling for clinical and pathologic variables, BF (hazard ratio [HR] 2.32, 95% confidence interval [CI] 1.45-3.71; p<0.001), DM (HR 6.52, 95% CI 4.20-10.1; p<0.001), and CRPC (HR 2.47, 95% CI 1.56-3.92; p<0.001) were associated with OS. In landmark analyses, 5-yr DM had the highest c index when adjusting for baseline covariates (0.78), with 5-yr DM also providing the greatest increase in discriminatory power over a model only including baseline covariates. These findings require validation in prospective randomized data. CONCLUSIONS While limited by the retrospective nature of the data, 5-yr DM is associated with lower OS following PORT, outperforming the prognostic capability of BF and CRPC at 1, 3, 5, or 7 yr after treatment. Confirmation of this ICE as a surrogate for OS is needed from randomized trial data so that it can be incorporated into future clinical trial design. PATIENT SUMMARY We assessed potential intermediate clinical endpoints prognostic for overall survival in a cohort of men receiving radiotherapy after prostatectomy. We identified the development of metastatic disease within 5 yr after treatment as the strongest predictor of overall survival.

Real-time MRI feedback of cavitation ablation therapy (histotripsy).

Histotripsy ablation of liver tumors is a non-invasive surgery that uses high-intensity acoustic pulses to control an inertial cavitation cloud. Repeated exposure to the cavitation cloud renders a target tissue into a homogenous slurry which is then gradually absorbed by the body. Like other non-invasive surgeries, histotripsy requires a feedback system that can estimate therapy location and dose in real-time. Because the time-average power output of histotripsy is very small, the treatment region does not express a significant rise in temperature, making MRI thermometry an ineffective method to actively monitor therapy. Previous work has shown that MRI pulse sequences can be made sensitive to the chaotic water flow present during inertial cavitation. This is done by synchronizing the timing of the histotripsy pulses with the timing of the sequence’s gradient waveforms. Incoherent water flow caused by cavitation attenuates the MR signal through a process similar to that used in diffusion-weighted MRI. It was shown that these sequences can give localized contrast specific to histotripsy bubble clouds. However, the MR images could only be acquired in a piecemeal fashion over several minutes such that a single image represented the influence of many cavitation events. Here, we introduce a single-shot MR acquisition sequence that is able to rapidly acquire a complete MR image and remain sensitive to histotripsy cavitation. This is done by synchronizing each histotripsy pulse with incoherent motion-weighting gradients placed just before the readout portion of the sequence. When repeated at the same rate as the histotripsy pulses, the resulting MR images can give feedback on the location of every cavitation cloud applied to the target tissue.

Chronic Mastitis in Egypt and Morocco: Differentiating between Idiopathic Granulomatous Mastitis and IgG4-Related Disease

Idiopathic granulomatous mastitis (IGM) is a benign, frequently severe chronic inflammatory lesion of the breast. Its etiology remains unknown and reported cases vary in their presentation and histologic findings with an optimal treatment algorithm yet to be described owing mainly to the diseases heterogeneity. IgG4‐related disease (IgG4‐RD) is a newly recognized systemic fibroinflammatory condition characterized by a dense lymphoplasmacytic infiltrate with many IgG4‐positive plasma cells, storiform fibrosis, and obliterative phlebitis. Immunosuppressive therapy is considered to be an effective first‐line therapy for IgG4‐RD. We sought to clarify and classify chronic mastitis according to the histologic findings of IgG4‐RD mastitis with respect to IGM and to develop a robust diagnostic framework to help select patients for optimal treatment strategies. Using the largest collection to date (43 cases from Egypt and Morocco), we show that despite sharing many features, IGM and IgG4‐RD mastitis are separate diseases. To diagnostically separate the diseases, we created a classification schema—termed the Michigan Classification—based upon our large series of cases, the consensus statement on IgG4‐RD, and the histologic description of IGM in the literature. Using our classification, we discerned 17 cases of IgG4‐RD and 8 cases of IGM among the 43 chronic mastitis cases, with 18 indeterminate cases. Thus, our Michigan Classification can form the basis of rational stratification of chronic mastitis patients between these two clinically and histopathologically heterogeneous diseases.

Abstract 4977: Chronic mastitis in North Africa: Geographic overlap and a potential precursor comorbidity of inflammatory breast cancer

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA North African countries, such as Egypt and Morocco, have a high prevalence of non-infectious, chronic mastitis (up to 10% of patients presenting with breast problems) compared to less than 1% worldwide. This same geographic region also suffers from an unusually high prevalence of inflammatory breast cancer (IBC) - 10-15% of breast cancer cases - versus just 1-2% of cases in the U.S. IBC is a serious public health issue as it is the most lethal form of breast cancer because of the propensity for rapid onset of disseminated metastases, which are present in one-third of cases at diagnosis. It is our hypothesis that this geographic overlap is not coincidental, but that the high level of background chronic breast inflammation changes the mammary microenvironment making it more auspicious for the development of IBC. Here we report our work aimed at determining molecular and histologic criteria to classify mastitis into pathogenic groups in order to rationally guide treatment for this serious inflammatory illness, as well as study its possible role in the development of IBC. We identified 44 cases of chronic mastitis of unknown etiology. Cases were defined as any female patient with histopathological diagnosis of chronic mastitis seen at the 5 study hospitals in Egypt and Morocco from 2008-2011. Exclusion criterion was previous diagnosis of malignancy. Biopsy slides were analyzed by a pathologist and stained for IgG4 and IgG. Out of the 44 cases of chronic mastitis using consensus guidelines and our molecular IgG4 profiling, we identified 10 as idiopathic granulomatous mastitis (IGM) and 17 as breast manifestations of IgG4-related disease. IGM is a rare non-neoplastic, chronic, often severe inflammatory lesion of the breast that mimics carcinoma clinically and radiologically. Treatment strategies include watchful waiting, immunosuppressive therapy, wide local or other excisions, or combinations of the above. IgG4-related disease is a newly recognized fibro-inflammatory condition. It is characterized by the formation of tumefactive lesions, a dense lymphoplasmacytic infiltrate with many IgG4-positive plasma cells, storiform fibrosis, obliterative phlebitis and, frequently, elevated serum IgG4 concentrations. While no randomized clinical trials have been conducted, glucocorticoid treatment is the standard first line therapy and appears to be quite effective in the majority of patients. This series is the largest reported and is the first step toward changing the chronic breast inflammatory landscape in North Africa by classifying the chronic mastitis into pathogenic groups that can guide personalized treatment protocols. Further studies are needed to discern whether effective and timely treatment of these chronic inflammatory conditions may lower the incidence of IBC. Citation Format: Steven G. Allen, Hanna Oltean, Kathy Toy, Omar S. Omar, Tamer Youssef, Mehdi Karkouri, Azza Abdel-Aziz, Ahmad Hablas, Ali Tahri, Celina Kleer, Amr Soliman, Sofia D. Merajver. Chronic mastitis in North Africa: Geographic overlap and a potential precursor comorbidity of inflammatory breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4977. doi:10.1158/1538-7445.AM2014-4977