Membrane slides

Five-µm-thick FFPE sections were mounted on membrane slides (Leica, Germany). The sections were deparaffinized for 30 s in Xylene, rehydrated in graded ethanol, stained for 2 s in Mayers hematoxylin and washed in sterile water. After drying, five consecutive sections were microdissected using a microdissection system (LMD 630, Leica Germany). Cancer cells and tumor stromal cells were collected in separate tubes.

Microdissection of bronchi, lung vessels, and adjacent tissue was performed with PFA-fixed and paraffin-embedded lung tissue sections (13 µm) on MembraneSlides (Leica #11600288, Wetzlar, Germany). Tissues were deparaffinized in isopropanol (Roth, #6752, Karlsruhe, Germany) and rehydrated in a graded ethanol series (100%, 96%, 80%, and 70% for one minute each) to DEPC-H₂O. Sections of bronchi and vessels were dissected with PALMProbe software (Zeiss, Oberkochen, Germany) at PALMMicroBeam microscope (Zeiss) through laser-microdissection with laser settings at speed 73, energy 56 µJ, and focus 88. Laser-microdissected bronchi and vessels were collected in AdhesiveCap 500 opaque tubes (Zeiss, #415190-9201-000). After collecting the dissectates, samples were incubated with protein kinase K (Fermentas, Thermo Fisher Scientific, #EO0491, Waltham, Massachusetts, USA) overnight to dissolve the lung tissue out of the adhesive cap. Afterward, RNA was extracted according to an established protocol with TRI reagent (Sigma-Aldrich, #T9424-200ml, St. Louis, MO, USA).

Fresh frozen tissues were placed in OCT and sections were cut at 7 μm thickness and mounted onto membrane slides (Leica, 11600288). LCM of regions of interest was performed using Leica LMD 7000 Microscope in which study pathologists were careful to exclude areas with overt inflammatory infiltrates, carcinoma, and high-grade PIN when isolating normal-appearing regions. Tissue digestion and DNA/RNA extraction was performed using Allprep DNA/RNA Kits following the manufacturer’s recommendations (Qiagen, 802804). Sequencing was performed using Illumina HiSeqX and NovaSeq6000 with Paired End 150 bp × 150 bp read configuration. Trim galore v0.6.3 was used to trim the reads. Bwa v0.7.7 (mem) was used to align to the hg19 and hg38 human genome builds. Piccard tools v1.119 and GATK v3.6.0 were used to create a recalibrated bam file. Bedtools v2.27.1 (genomecov) was used to determine the nuclear coverage (i.e., all chromosomes except mtDNA) and the mitochondrial coverage. mtDNAcn was computed using the following formula: mitochondrial coverage/nuclear coverage. RNA-Seq and gene expression measures on mtDNA replication related genes were performed as previously described (102 (link)). The analysis of the WGS and RNA-Seq data here is limited to the reported mtDNAcn assessment. Full details and reporting of the WGS data will be reported as part of another study.

3 µm MCF-7 FFPE sections were adhered to positively charged slides and membrane slides (Leica; coated with 0.01% Poly-L-Lysine) by incubating at 37 °C overnight. Following deparaffinization, the sections were stained using the standard hematoxylin and eosin staining and immunocytochemically for PR, ER and HER2 expression. The immunocytochemical detection of the protein targets was performed using the primary antibodies, ER (Abcam; AB108398 rabbit monoclonal; clone EPR4097; 1:250), PR (Abcam; AB2765 mouse monoclonal; clone Alpha PR6; 1:20) and HER2 (Abcam; AB214275 rabbit monoclonal; clone EPR19547-12; 1:4000). Antigen retrieval was done in pH 9.0 Tris-EDTA solution for ER and Her2 and pH 6.0 sodium citrate solution for PR in microwave oven at 95 °C for 15 min. The primed proteins were chromogenically detected using Ventana’s Optiview DAB IHC Detection kit.