Abstract 1571: Novel microfluidic technology and biology based approach for isolation and characterization of circulating cells from the peripheral blood of patients with Lymphangioleiomyomatosis
Authors: Nagrath Sunitha , Moore Alessandra L. , Zenali Mahnaz , Chekaluk Yvonne I. , Peters Elizabeth M. , Kwiatowski David J. , Haber Daniel A. , Henske Elizabeth P. , Toner Mehmet 
Published: June 2012

Abstract
The rare multisystem disease Lymphangioleiomyomatosis (LAM) that effects almost exclusively women is characterized by the aberrant proliferation of smooth muscle-like cells (LAM cells). Although the specific molecular determinants are unknown, metastasis seems to be the mechanism by which the LAM cells are disseminated. In fact the multisystem manifestations of LAM have been correlated with a metastatic dissemination of the LAM cells, which were identified in donor lungs transplanted to LAM patients. Furthermore, LAM cells have been detected in blood, chyle, and urine. However, there is still a significant gap between the detection of circulating cells and their use as a biomarker for diagnosis, monitoring of response to a therapy, and prognosis. It is well established that the LAM is characterized by cystic lung destruction caused by LAM cells (smooth-muscle-like-cells) that have mutations in tumor suppressor genes, tuberous sclerosis complex (TSC) 1 or 2, and have the capacity to metastasize. Given the limitations of the sensitivity of the current technologies, the genotyping of the circulating LAM cells (CLCs) has not been established. Hence we applied a novel microfluidic technology (CTC-chip) for the reliable detection of circulating LAM cells from blood. An integrated technology and biology based approach is employed to address the efficient and specific isolation and applications of CLCs. Since LAM cells isolated from LAM patient lungs expresses CD44 and CD44v6 and additionally CD44v6 positive cells show loss heterozygosity (LOH) at the TSC2 locus, we evaluated CD44 as a capture antibody and Cd44v6 together with Cd45 as a negative marker as detection antibodies. Furthermore, to enhance the sensitivity and specificity of isolation and identification of CLCs, we extensively tested various LAM specific, neural crest and melanocytic markers. Specifically, to increase the specificity of the capture we explored several neural crest markers. Since, stem cell precursors that can give rise to smooth muscle and melanocytes have shown to express neural stem cell marker Ng2, we optimized the antibodies against Ng2 as a capture moiety. To enhance the specificity of the detection, we stained CLCs for gp100, a melanocyte-related protein immunorecognized by antibody HMB45. Using this strategy, blood samples from 10 healthy controls and 25 samples from LAM patients are analyzed. The results indicate a clear difference in the levels of CLCs between healthy and patient population. Using this novel biology and technology approach, we were able to detect and identify CLCs was with high specificity and sensitivity. We hypothesize that with the presented strategies, it is feasible to use CLCs as a blood “biopsy”, which may lead not only to elucidating cellular and molecular mechanisms, but also may pave the way to finding cure for LAM.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1571. doi:10.1158/1538-7445.AM2011-1571