As lmd

Formalin-fixed paraffin-embedded (FFPE) tissue samples from three independent tumors per genotype were sectioned (10 µm sections). We used tumors at three days post B16F1 injection corresponding to the earliest time point at which tumor size between WT and ADAM9^−/− was significantly different [15 (link)]. Proteins used for mass spectrometry were isolated from the tumor-stroma border by laser dissection using a Leica ASLMD instrument (Leica Microsystems Inc., Buffalo Grove, IL, USA). Processing of tissues and mass analysis was performed as elsewhere described [20 (link)]. Values obtained from the study of two mice tissue specimens per genotype were probabilistically validated by Scaffold Software Q+ (4.11.0) [20 (link)] and only proteins identified by two or more peptides and a minimum protein identification probability of >95% were listed. The ratios of averaged values were used here.

In total, 8–13 sequential paraffin-embedded slide sections with a thickness of 5 µm were prepared and mounted on pet (polyethylene terephthalate) slides (Leica AS LMD; Leica Microsystems Ltd., Wetzlar, Germany). Paraffin was removed by soaking in xylene twice for 10 min, and sections were stained with hematoxylin. One section was stained with CD3 antibody (NCL-L-CD3-565), which was used as a guide to differentiate between lymphocytic and epithelial cells (Supplementary Figure S1). Laser capture microdissection (LCM) was performed using a ZEISS PALM MicroBeam Microsdissection system (Carl Zeiss Microscopy GmbH, Oberkochen, Germany) and adhesive collection caps.
After microdissection, the samples were placed into collection vessels containing appropriate volumes of Depaffinization Solution (#19093, Qiagen GmbH, Hilden, Germany), and RNA was extracted using an MiRNeasy FFPE extraction kit (#217504, Qiagen GmbH, Hilden, Germany) generally according to the manufacturer’s instructions. However, after adding proteinase K solution, the samples were incubated overnight at 56 °C with gentle shaking for a better yield.

To examine the expression of miR-186 in ECs within tumor and non-tumor lung tissues, ECs were exclusively isolated using the LCM technique. For this purpose, formalin-fixed paraffin-embedded (FFPE) tissue sections at 5 μm were cut and mounted on membrane slides (Leica Microsystems, Wetzlar, Germany). After staining the sections with hematoxylin and eosin, around 2,000 ECs were collected from each sample by using an LCM microscope (Leica AS LMD; Leica Microsystems) for RNA isolation.