Morphometry and Proteomics of Lung Cancer Invasion

Desmoplastic stroma of lung squamous cell carcinoma – localized molecular analysis of tumor heterogeneity
Lung squamous cell carcinoma (SCC) is a highly fibrotic malignancy, elaborating a prominent desmoplastic stroma. SCC cells infiltrate into this stroma as either detached small cohorts, also called buds, or larger sheets maintaining contact to the tumor centre. Tumor epithelia display biochemical epithelial-mesenchymal transition (EMT) at the invasion fronts. Thereby, cells acquire a more motile phenotype via upregulation of mesenchymal proteins like vimentin and concomitant downregulation of cell adhesion molecules like E-cadherin. Epithelial invasion fronts are immediately surrounded by a particular form of stroma, called the matricellular space, in which heterotopic and reciprocal signaling occurs. This oedematous space is enriched in specialized non-structure matricellular proteins like periostin or tenascin. Periostin is a recognized EMT marker and directly binds to epithelial integrin receptors. Farther away from the invasion front, a denser, more collagenous stroma is elaborated.
 
We originally detected the secreted N-glycoprotein periostin in malignant pleural effusions of lung adenocarcinomas by shot gun mass spectrometry in the lab of Prof. Aebersold, ETHZ. In a non-small cell lung carcinoma (NSCLC) patient cohort (n = 532), assembled on a tissue microarray (TMA), the periostin protein was found to be most closely associated with higher pT, larger size, pM1, higher stage and vascular invasion. Further, it correlated with the squamous cell histotype and was a significant prognosticator for decreased patient survival. It was found to be upregulated in both tumor epithelia and the matricellular space. A high content of intratumoral CD68+ macrophages correlated with high cytosolic periostin and vimentin but low membranous E-cadherin of tumor cells. Invasion fronts also presented increased expression of EMT proteins Slug and L1CAM, a neuronal cell adhesion molecule. In frozen tissues, 8 periostin isoforms were detected in fetal lung but only 5 in NSCLC and matched normal lung, suggesting splice-specific regulation during embryogenesis. Laser capture microdissection proved that both tumor epithelia as well as stromal cells produce periostin mRNA.

 In a next step, we aim to perform a more detailed analysis of the lung SCC stroma. We will compare chemotherapy-naïve versus -treated and pT1 versus pT3 tumors in order to assess the prognostic impact of stromal features and to define the topographic region with the highest drug resistance. The tumor-stroma ratio is morphometrically measured using cytokeratin immunohistochemistry (IHC) on both TMA cores and tissue whole sections. Lung SCC will be compared with head neck SCC. Architectural parameters will be correlated with next generation sequencing data, including oncogenic alterations such as discoidin domain receptor 2 (DDR2) mutations. DDR2 mutant cases are of particular interest, since this proteins serves as a direct collagen receptor. Topographic expression of relevant EMT markers at the tumor-stroma interface will be assessed by micro-IHC and localized DNA extraction, using the microfluidic probe technology (MFP, IBM Research) and laser capture microdissection. This may allow for spatial genetic profiling of oncofetal isoforms of matricellular proteins.

Clinical relevance: Tumor heterogeneity and microarchitecture, respectively, is of high oncologic significance. Tumor epithelia bordering the matricellular space are most resistant to platinum chemotherapy, whereas the more differentiated, keratinized and partially necrotic centers are more vulnerable. These bordering epithelia may express the so-called cancer stem cell (CSC) phenotype, requiring special targeting. A future lung SCC anti-tumor microenvironment therapy may combine inhibitors against genomic alterations of tumor epithelia such as fibroblast growth factor receptor 1 (FGFR1) or DDR2 with antibodies directed against oncofetal isoforms of matricellular proteins.

Main Fields of Research, Keywords
Lung cancer, head neck cancer, epithelial-mesenchymal transition, tumor heterogeneity, immunohistochemistry, localized molecular analysis.

Special Techniques and Equipment
 Immunohistochemistry (IHC), fluorescent in-situ hybridization (FISH), chromogenic/silver in-situ hybridization (CISH/SISH), laser capture microdissection (LCM), microfluidic probe (MFP), tissue microarray (TMA).
 
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