Modelling lymphangioleiomyomatosis (LAM) using two-dimensional and three-dimensional in vitro co-culture systems

Background: Lymphangioleiomyomatosis (LAM) is a rare progressive neoplastic cystic lung disease that primarily affects women of child bearing age leading to lung destruction, respiratory failure and death. Thought to be a consequence of dysregulated protease expression, cells of unknown origin accumulate in the lung, often forming clusters or nodules of cells with both melanocytic and smooth muscle properties. Some of these cells, known as LAM cells, have bi-allelic mutations in TSC2 resulting in constitutive mTOR activation. However LAM nodules are heterogeneous structures and genotyping analyses suggest that cells without LOH for TSC2 including wild-type fibroblasts are also common within LAM nodules. Hypotheses and aims: We hypothesise that LAM cells recruit wild-type fibroblasts and modify their properties to generate a permissive microenvironment, akin to a tumour stroma including the production and activation of matrix-degrading proteases which contribute to the destruction of the lung parenchyma. This study has therefore deigned in vitro co-culture models with an aim to study the expression patterns and activation of proteases in a LAM lung leading to matrix destruction. Another aim was also to characterise transcriptional differences normal human lung fibroblasts (NHLFs) and LAM-associated fibroblasts (LAFs) and to investigate changes in their gene expression when cultured together with a model LAM cell line, 621-101 angiomyolipoma cells which were derived from a LAM patient and have bi-allelic loss of TSC2. Methods: In vitro 2-dimensional (2D) and 3-dimensionalD (3D) co-culture models were designed and validated using fibroblasts characterised and isolated from 4 LAM lung donors, now termed LAFs, and 621-101 cells. The 3D extracellular matrix (ECM) incorporated the two cell types in a 10:1 ratio embedded in a basement membrane extract (BME) mimicking the lung matrix. An organotypic spheroid model was also developed incorporating both cell types thereby mimicking a LAM nodule. 6 LAM lung and 3 normal lung tissue donors were screened for candidate proteases in LAM pathology using qRT-PCR and identified upregulated proteases which may contribute to a role in LAM pathology. These findings were verified in the 2D and 3D in vitro models as well as ex vivo tissue using a variety of immunostaining techniques, activity assays and ELISA. Lastly, commercially bought NHLF (n=3) and LAF (n=3) were cultured in the presence or absence of 621-101 cells in the 2D Boyden chamber co-culture model. LAF and NHLF RNA was analysed using Affymetrix Human Genome U133 Plus 2.0 Arrays and Genomics Suite and Pathway (Partek). Findings were validated by multiplex assay and immunohistochemistry in 2D and 3D in vitro models and tissue respectively. Inflammatory cell migration and function was examined in co-culture model and LAM tissue. Results: The 3D BME model showed that TSC2-/- 621-101 cells and fibroblasts spontaneously form aggregates and clump together akin to a LAM nodule. The two cell types exhibited strong heterotypic cell-cell adhesive forces and resulted in strictly spherical spheroids. The 3D models designed all showed expression of markers of LAM nodules thereby representing LAM nodules in a dish. Of 30 proteases screened, cathepsin K gene expression was increased almost 15-fold in LAM lung compared to normal tissue and was also found to be elevated in 3D BME model. Cathepsin K in LAM tissue was expressed in the LAM nodules associated with cysts and was expressed exclusively by fibroblasts in the 3D spheroid model. As cathepsin K requires low pH for activity it was determined if LAFs and TSC2-/- cells can acidify the extracellular space. TSC2-/- cells but not LAFs decreased extracellular pH, over 24 hours and pH values <7 were associated with increased cathepsin K activity in co-cultures. TSC2-/- cells expressed membrane transporters associated with extra-cellular acidification and inhibitors of the sodium bicarbonate co-transporters, carbonic anhydrases and mTOR reduced the pH gradient and decreased CTSK activity in co-cultures. Transcriptomic analysis using the 2D co-culture model showed 148 genes were significantly altered in both NHLF and LAF by 621-101 cells. Soluble factors from 621-101 cells induce pro-inflammatory transcriptional changes in both NHLFs and LAFs and pathway analysis showed enhanced chemokine signalling which highlighted stimulation of mainly the C-X-C motif chemokines and chemokine receptor signalling. The analysis identified 6 C-X-C motif chemokines all possessing a cognate receptor. The gene and protein expression of these chemokines was validated in the in vitro models and in ex vivo LAM lung tissue. Conclusions: The in vitro models are versatile and mimic the LAM lung nodule and environment. A potent matrix degrading protease possibly playing a role in LAM has been identified and using the in vitro models a possible mechanism of activation of CTSK resulting from a synergistic relationship between TSC2-/- cells and LAFs has been demonstrated. Also, soluble factors from the TSC2-/- LAM cell line elicit changes in gene expression in co-cultured fibroblasts. Chemokine signalling is associated with cell migration; elevated chemokine expression may be associated with the recruitment of inflammatory cells to the LAM nodule. The identification of these mechanisms and pathways opens up new avenues for therapeutic interventions in LAM

Cathepsin K in lymphangioleiomyomatosis: LAM cell-fibroblast Interactions enhance protease activity by extracellular acidification

Lymphangioleiomyomatosis (LAM) is a rare disease in which clonal ‘LAM’ cells infiltrate the lungs and lymphatics. In association with recruited fibroblasts, LAM cells form nodules adjacent to lung cysts. It is assumed LAM nodule derived proteases lead to cyst formation although, this is uncertain. We profiled protease gene expression in whole lung tissue and observed cathepsin K was 40 fold over-expressed in LAM compared with control lungs (p≤0.0001). Immunohistochemistry confirmed cathepsin K protein in LAM nodules but not control lungs. Cathepsin K gene expression, protein and protease activity was detected in LAM associated fibroblasts but not the LAM cell line 621-101. In lung nodules, cathepsin K immune reactivity was predominantly co-localised with LAM associated fibroblasts. In vitro, extra-cellular cathepsin K activity was minimal at pH 7.5 but significantly enhanced in fibroblast cultures at pH 7 and 6. 621-101 cells reduced extracellular pH by 0.5 units over 24 hours. Acidification was dependent upon 621-101 cell mTOR activity and net hydrogen ion transporters, particularly sodium/bicarbonate co-transporters and carbonic anhydrases which were also expressed in LAM lung tissue. In LAM cell/fibroblast co-cultures, acidification paralleled cathepsin K activity and both were inhibited by sodium bicarbonate co-transporter (p≤0.0001) and carbonic anhydrase inhibitors (p=0.0021). Our findings suggest cathepsin K activity is dependent on LAM cell/fibroblast interactions and inhibitors of extracellular acidification may be potential therapies for LAM

Extra-cellular matrix proteins induce matrix metalloproteinase-1 (MMP-1) activity and increase airway smooth muscle contraction in asthma

Airway remodelling describes the histopathological changes leading to fixed airway obstruction in patients with asthma and includes extra-cellular matrix (ECM) deposition. Matrix metalloproteinase-1 (MMP-1) is present in remodelled airways but its relationship with ECM proteins and the resulting functional consequences are unknown. We used airway smooth muscle cells (ASM) and bronchial biopsies from control donors and patients with asthma to examine the regulation of MMP-1 by ECM in ASM cells and the effect of MMP-1 on ASM contraction. Collagen-I and tenascin-C induced MMP-1 protein expression, which for tenascin-C, was greater in asthma derived ASM cells. Tenascin-C induced MMP-1 expression was dependent on ERK1/2, JNK and p38 MAPK activation and attenuated by function blocking antibodies against the β1 and β3 integrin subunits. Tenascin-C and MMP-1 were not expressed in normal airways but co-localised in the ASM bundles and reticular basement membrane of patients with asthma. Further, ECM from asthma derived ASM cells stimulated MMP-1 expression to a greater degree than ECM from normal ASM. Bradykinin induced contraction of ASM cells seeded in 3D collagen gels was reduced by the MMP inhibitor ilomastat and by siRNA knockdown of MMP-1. In summary, the induction of MMP-1 in ASM cells by tenascin-C occurs in part via integrin mediated MAPK signalling. MMP-1 and tenascin-C are co-localised in the smooth muscle bundles of patients with asthma where this interaction may contribute to enhanced airway contraction. Our findings suggest that ECM changes in airway remodelling via MMP-1 could contribute to an environment promoting greater airway narrowing in response to broncho-constrictor stimuli and worsening asthma symptoms

Modelling lymphangioleiomyomatosis (LAM) using two-dimensional and three-dimensional in vitro co-culture systems

Background: Lymphangioleiomyomatosis (LAM) is a rare progressive neoplastic cystic lung disease that primarily affects women of child bearing age leading to lung destruction, respiratory failure and death. Thought to be a consequence of dysregulated protease expression, cells of unknown origin accumulate in the lung, often forming clusters or nodules of cells with both melanocytic and smooth muscle properties. Some of these cells, known as LAM cells, have bi-allelic mutations in TSC2 resulting in constitutive mTOR activation. However LAM nodules are heterogeneous structures and genotyping analyses suggest that cells without LOH for TSC2 including wild-type fibroblasts are also common within LAM nodules. Hypotheses and aims: We hypothesise that LAM cells recruit wild-type fibroblasts and modify their properties to generate a permissive microenvironment, akin to a tumour stroma including the production and activation of matrix-degrading proteases which contribute to the destruction of the lung parenchyma. This study has therefore deigned in vitro co-culture models with an aim to study the expression patterns and activation of proteases in a LAM lung leading to matrix destruction. Another aim was also to characterise transcriptional differences normal human lung fibroblasts (NHLFs) and LAM-associated fibroblasts (LAFs) and to investigate changes in their gene expression when cultured together with a model LAM cell line, 621-101 angiomyolipoma cells which were derived from a LAM patient and have bi-allelic loss of TSC2. Methods: In vitro 2-dimensional (2D) and 3-dimensionalD (3D) co-culture models were designed and validated using fibroblasts characterised and isolated from 4 LAM lung donors, now termed LAFs, and 621-101 cells. The 3D extracellular matrix (ECM) incorporated the two cell types in a 10:1 ratio embedded in a basement membrane extract (BME) mimicking the lung matrix. An organotypic spheroid model was also developed incorporating both cell types thereby mimicking a LAM nodule. 6 LAM lung and 3 normal lung tissue donors were screened for candidate proteases in LAM pathology using qRT-PCR and identified upregulated proteases which may contribute to a role in LAM pathology. These findings were verified in the 2D and 3D in vitro models as well as ex vivo tissue using a variety of immunostaining techniques, activity assays and ELISA. Lastly, commercially bought NHLF (n=3) and LAF (n=3) were cultured in the presence or absence of 621-101 cells in the 2D Boyden chamber co-culture model. LAF and NHLF RNA was analysed using Affymetrix Human Genome U133 Plus 2.0 Arrays and Genomics Suite and Pathway (Partek). Findings were validated by multiplex assay and immunohistochemistry in 2D and 3D in vitro models and tissue respectively. Inflammatory cell migration and function was examined in co-culture model and LAM tissue. Results: The 3D BME model showed that TSC2-/- 621-101 cells and fibroblasts spontaneously form aggregates and clump together akin to a LAM nodule. The two cell types exhibited strong heterotypic cell-cell adhesive forces and resulted in strictly spherical spheroids. The 3D models designed all showed expression of markers of LAM nodules thereby representing LAM nodules in a dish. Of 30 proteases screened, cathepsin K gene expression was increased almost 15-fold in LAM lung compared to normal tissue and was also found to be elevated in 3D BME model. Cathepsin K in LAM tissue was expressed in the LAM nodules associated with cysts and was expressed exclusively by fibroblasts in the 3D spheroid model. As cathepsin K requires low pH for activity it was determined if LAFs and TSC2-/- cells can acidify the extracellular space. TSC2-/- cells but not LAFs decreased extracellular pH, over 24 hours and pH values <7 were associated with increased cathepsin K activity in co-cultures. TSC2-/- cells expressed membrane transporters associated with extra-cellular acidification and inhibitors of the sodium bicarbonate co-transporters, carbonic anhydrases and mTOR reduced the pH gradient and decreased CTSK activity in co-cultures. Transcriptomic analysis using the 2D co-culture model showed 148 genes were significantly altered in both NHLF and LAF by 621-101 cells. Soluble factors from 621-101 cells induce pro-inflammatory transcriptional changes in both NHLFs and LAFs and pathway analysis showed enhanced chemokine signalling which highlighted stimulation of mainly the C-X-C motif chemokines and chemokine receptor signalling. The analysis identified 6 C-X-C motif chemokines all possessing a cognate receptor. The gene and protein expression of these chemokines was validated in the in vitro models and in ex vivo LAM lung tissue. Conclusions: The in vitro models are versatile and mimic the LAM lung nodule and environment. A potent matrix degrading protease possibly playing a role in LAM has been identified and using the in vitro models a possible mechanism of activation of CTSK resulting from a synergistic relationship between TSC2-/- cells and LAFs has been demonstrated. Also, soluble factors from the TSC2-/- LAM cell line elicit changes in gene expression in co-cultured fibroblasts. Chemokine signalling is associated with cell migration; elevated chemokine expression may be associated with the recruitment of inflammatory cells to the LAM nodule. The identification of these mechanisms and pathways opens up new avenues for therapeutic interventions in LAM

Wild type mesenchymal cells contribute to the lung pathology of lymphangioleiomyomatosis.

Lymphangioleiomyomatosis (LAM) is a rare disease leading to lungs cysts and progressive respiratory failure. Cells of unknown origin accumulate in the lungs forming nodules and eventually resulting in lung cysts. These LAM cells are described as clonal with bi-allelic mutations in TSC-2 resulting in constitutive mTOR activation. However LAM nodules are heterogeneous structures containing cells of different phenotypes; we investigated whether recruited wild type cells were also present alongside mutation bearing cells. Cells were isolated from LAM lung tissue, cultured and characterised using microscopy, immunocytochemistry and western blotting. Fibroblast-like cells were identified in lung tissue using immunohistochemical markers. Fibroblast chemotaxis toward LAM cells was examined using migration assays and 3D cell culture. Fibroblast-like cells were obtained from LAM lungs: these cells had fibroblast-like morphology, actin stress fibres, full length tuberin protein and suppressible ribosomal protein S6 activity suggesting functional TSC-1/2 protein. Fibroblast Activation Protein, Fibroblast Specific Protein/S100A4 and Fibroblast Surface Protein all stained subsets of cells within LAM nodules from multiple donors. In a mouse model of LAM, tuberin positive host derived cells were also present within lung nodules of xenografted TSC-2 null cells. In vitro, LAM 621-101 cells and fibroblasts formed spontaneous aggregates over three days in 3D co-cultures. Fibroblast chemotaxis was enhanced two fold by LAM 621-101 conditioned medium (p=0.05), which was partially dependent upon LAM cell derived CXCL12. Further, LAM cell conditioned medium also halved fibroblast apoptosis under serum free conditions (p=0.03). Our findings suggest that LAM nodules contain a significant population of fibroblast-like cells. Analogous to cancer associated fibroblasts, these cells may provide a permissive environment for LAM cell growth and contribute to the lung pathology of LAM lung disease

FSP is expressed by variable numbers of cells within LAM nodules.

<p>There is heterogeneity between different LAM lung donors in the number of anti-FSP immunoreactive cells in LAM nodules. Panels (a) and (b) at x10 magnification, (c) at x20, (d) at x40. (e) and (f) show anti-tuberin reactive and non-reactive cells (arrowed) within nodules from two donors, x20.</p

Fibroblasts protect 621–101 cells against apoptosis.

<p>(a) Culture of either 621–101 cells or fibroblasts on glass in serum free conditions results in cell death. Co-culture of the two cell types under the same conditions results in formation of viable cell colonies. (b) Under serum-free conditions, 621–101 cells have a basal rate of apoptosis of 8% assessed by TUNEL positive nuclear staining (arrowed). TUNEL positivity is reduced by half in the presence of fibroblast conditioned medium (*p = 0.032).</p

LAM nodules express fibroblast markers.

<p>LAM nodules react with antibodies against alpha-Smooth Muscle Actin, Fibroblast Surface Protein (FSP), Fibroblast Activation Protein (FAP) and S100A4. Left panels are x20 magnification and right are inset area at x40 taken from serial sections of a representative donor. Fibroblast markers display different expression patterns within LAM tissue, with anti-FSP reacting with 30–70% of cells within a nodule, anti-FAP detecting the majority of cells and S100A4 detecting only small nests of cells within nodules.</p

Characteristics of wild-type fibroblast-like cells obtained from LAM lungs.

<p>(a) Explant culture of lung tissue from patients with LAM yields a fibroblast-like population with spindle shaped morphology under phase contrast microscopy (phase) and immunoreactivity to anti-alpha Smooth Muscle Actin, and fibroblast markers FSP, S100A4 and TE-7 (these cells are also DAPI stained for clarity). (b) Western blot of lysates of fibroblast like-cells from two donors with LAM compared with TSC-2 mutation bearing 621–101 cells. Unlike 621–101 cells fibroblast-like cells have full length tuberin, and suppression of phospho-S6 in the absence of serum.</p