Abstract A64: The development of novel cell culture models and preclinical models of lymphangioleiomyomatosis
Authors: Morrison Tasha A. , Yu Jane , Henske Elizabeth P. 
Published: June 2012

Abstract
Introduction: Lymphangioleiomyomatosis (LAM) is a rare lung disease, with an incidence rate of approximately 2.6 per 1 million women. LAM is the result of benign metastasis of TSC2-null cells causing cystic lung destruction, chylous pleural effusions and renal angiomyolipomas. Establishing pure LAM cell cultures from explanted lungs, chylous pleural fluid and renal angiomyolipomas from women with LAM has been a challenging endeavor. The first barrier is that LAM cells represent a small portion of the resected lung tissue. Less than 20% of cells cultured from an explanted LAM lung represent LAM cells, which was confirmed by the presence of somatic inactivating TSC2 gene mutations. Histologically, LAM cells are smooth muscle-like in appearance. Other cell types grow in LAM culture, making it hard to select for and use pure LAM cells for experiments. The second barrier is that the TSC2-deficient LAM cells become almost non-existent in later passages, which can be determined by comparing tuberin expression of later passages to earlier passages. Therefore, culturing cells from tissue of women with LAM alone does not work in establishing a pure LAM cell line. A multifaceted approach, which includes looking at cell morphology, genetic analysis, and selection using in vivo and in vitro experiments, will be implemented to obtain pure LAM culture models. Developing primary TSC2-null cells into immunodeficient mice in pre-clinical models of LAM has also been a challenge. No tumor or substantial phenotype developed. Inoculating triple immune-deficient NOD/SCIDII2rg-/-(NOD/SCID) mice has been successful in developing tumors using primary LAM patient-derived cells that would not otherwise become tumorigenic.
Methods: 621-101 cells are an immortalized TSC2-deficient cell line derived from an angiomyolipoma of a LAM patient. NOD/SCID mice (Jackson Laboratory) were subcutaneously injected with 621-101 cells (2.5 × 106 in 200 L medium with 25% matrigel). Tumors developed from 621-101 cells inoculation was minced, incubated in 0.2% collagenase, washed and cultured. These cells are labeled as 621NST cells. 621NST cells were subcutaneously injected with SCID mice (2.5 × 106 in 200ul medium with 25% matrigel). Tuberin protein levels were determined by immunoblot analysis. Chylous pleural fluid and explanted lung was received 24 hours after thoracentesis or resection. Pleural fluid was filtered through a 40 m Nylon Mesh. Cells caught in filter were harvested, and cultured in IIA-Complete medium. Fresh tissue received was minced, incubated in 0.2% collagenase, washed and cultured.
Results: One hundred percent of NOD/SCID mice subcutaneously injected with 621-101 cells developed tumors. Lack of tuberin expression was confirmed in 621NST cells using western blot analysis. Next, we hypothesized that 621NST cells are more tumorigenic in regular SCID mice. One of the reasons why we are more interested in using SCID mice rather than NOD/SCID is the fact that SCID are three times less expensive. Seventy-five percent of SCID mice subcutaneously injected with 621NST cells developed tumors. Ongoing experiments are being done to determine how biologically different 621NST cells are from 621-101 cells.
Ongoing experiments include the establishment of xenograft tumors in NOD/SCID using LAM tissue from explanted lung and cells from chylous pleural fluid. We anticipate that these NOD/SCID mice will develop tumors that will mostly compose of LAM cells. We will resect the tumor, and grow cells from that tumor in an attempt to develop a pure population of LAM cells. Estrogen treatment and targeted drug therapy will also be implemented to determine the pathogenesis of LAM.
Citation Information: Cancer Epidemiol Biomarkers Prev 2011;20(10 Suppl):A64.