Lymphangioleiomyomatosis and TSC2-/- cells

Abstract

The cells comprising pulmonary lymphangioleiomyomatosis (LAM) and renal angiomyolipomas (AMLs) are heterogeneous, with variable mixtures of cells exhibiting differentiation towards smooth muscle, fat, and vessels. Cells grown from LAM and AMLs have likewise tended to be heterogeneous. The discovery that LAM and AMLs contain cells with mutations in the TSC1 or TSC2 genes is allowing investigators to discriminate between "two-hit" cells and neighboring cells, providing insights into disease pathogenesis. In rare cases, it has been possible to derive cells from human tumors, including AMLs and TSC skin tumors that are highly enriched for TSC2(-/-) cells. Cells derived from an Eker rat uterine leiomyoma (ELT3 cells) are Tsc2-null and these have been used in a rodent cell models for LAM. Further improvements in the ability to reliably grow well-characterized TSC2(-/-) cells from human tumors are critical to developing in vitro and in vivo model systems for studies of LAM pathogenesis and treatment.

FIG. 1.

Phenotypic and genotypic heterogeneity in LAM cell cultures. Reaction of cells cultured from LAM lung (A) or pulmonary artery smooth muscle cells (PASM) (B) with monoclonal antibody against SMA. Reaction of cultured LAM cells (C) and MALME-3M melanoma cells (D) with monoclonal antibody HMB-45. Fluorescence in situ hybridization (FISH) for TSC1 (green) and TSC2 (red) on cells grown from LAM lungs (E–G), reveals two populations of cells (E) with some nuclei showing the expected presence of two signals for each allele, TSC1 (arrows) and TSC2 (arrowheads) (F) and other nuclei exhibiting a single signal for TSC2 (G). Bar, 20 μm. (Reference 14, reproduced with permission. Copyright 2007 American Association for Cancer Research).

FIG. 2.

Enrichment of LAM cells showing loss of heterozygosity (LOH) at the TSC2 locus. Cells were incubated with CD44-R-phycoerythin and CD44v6-fluorescein antibodies for fluorescence-activated cell sorting. (A) Side (SSC) and forward (FSC) scatter; cells within the R1 gate were selected for sorting. (B) Four populations (CD44–CD44v6–, CD44 + CD44v6–, CD44–CD44v6 + , and CD44 + CD44v6+) of cells defined by reaction with CD44-RPE and/or CD44v6-FITC antibodies. (C) LOH analysis of sorted cells. Chromatograms show profiles for the microsatellite marker Kg8. Controls are samples from cells before sorting. Arrows show the position of the missing allele. (Reference 14, reproduced with permission. Copyright 2007 American Association for Cancer Research).

FIG. 3.

(A) Comparison of the proliferation of TSC2^-/- (solid line) and TSC2^-/meth (dashed line) ASM cells in presence or absence of EGF, and with anti-EGFR or anti-IGF-1R antibodies. Cell number at the beginning of the treatment was considered 100%. The growth rates of the two cell lines were similar. (B) Constitutive phosphorylation of S6 (Ser235/236) was markedly decreased by TSC2 transfection in TSC2^-/- ASM cells and by trichostatin A (TSA) in TSC2^-/meth ASM cells. (C) Tuberin was undetectable by immunocytochemistry in TSC2^-/-ASM cells, but it was expressed in these cells following TSC2 transfection. (Reference 60, Copyright 2008 Lesma et al.). (D) Following incubation with TSA (3.3 μM) for 72 h or 5-azacytidine (5-AZA) (1 μM) for 96 ho, tuberin expression was increased in TSC2^-/meth ASM cells. (Reference 19, reproduced with permission. Copyright 2009 American Society for Investigative Pathology).

FIG. 4.

Heterogeneity of mTORC1 activation in TSC skin tumors and cells grown from TSC skin tumors. Sections of normal-appearing skin (A) or periungual fibromas (B) were stained for phosphorylated ribosomal protein S6 (red). Fibroblast-like cells in the dermis of tumors, but not fibroblasts in a paired sample of normal-appearing skin, are immunoreactive for phospho-S6. The epidermis of the TSC skin tumor also shows immunoreactivity for phospho-S6, due to release of paracrine factors by tumor fibroblast-like cells. (C) Staining for phospho-S6 in cells with allelic deletion of TSC2. Cells grown from a periungual fibroma were analyzed by combined immunocytochemistry for phospho-S6 (green) and fluorescence in situ hybridization for TSC2 (red signals, indicated by arrowheads), using DAPI to stain nuclei (blue). The nucleus on top with two signals for TSC2 shows undetectable phospho-S6, and the nucleus below with allelic deletion of TSC2 shows strong staining for phospho-S6.