PI 200

Microscopy samples were prepared as follows (more detailed instructions are available for download from http://users.ox.ac.uk/~path0493/htiaot.html). T. brucei, L. mexicana and C. fasciculata were harvested from culture by centrifugation, washed with phosphate-buffered saline (PBS) and fixed with 2% paraformaldehyde in PBS. Cells were permeabilized with -20°C methanol and rehydrated with PBS. Many base pair intercalating stains also label RNA, therefore samples were incubated with 50 μg/ml RNaseA at room temperature for 1 h prior to staining. For analysis of a single sample (method 1) cells were stained with a combination of 1 μg/ml DAPI or bisbenzimide (Hoechst) 33342 and 40 μg/ml PI or 1:10,000 SYBR green (Sigma-Aldrich, S9430) for 2 min, washed with PBS and mounted in glycerol with 1% 1,4-diazabicyclo[2.2.2]octane and 10% 50 mM sodium phosphate, pH 8.0. Images were captured with an HCX PL APO 40 × NA 1.25 oil immersion lens (Leica Microsystems, Milton Keynes, UK) on a DM5500 B epifluorescence microscope (Leica Microsystems) with an Orca cooled CCD camera (Hamamatsu Photonics, Welwyn Garden City, UK). Images were only captured from regions of the sample not pre-exposed to the fluorescence light source; this avoids photobleaching and therefore allows accurate quantification of DNA from the fluorescent signal.
For comparative analysis of multiple samples (method 2) slides were stained by immersion in 40 ng/ml DAPI and 200 ng/ml PI in PBS for 2 min then mounted in the staining solution. This method aims to minimize variation, that is, give maximum precision, by keeping the DNA within the sample in equilibrium with the same concentration of the fluorescent DNA stains at all times. This is at the cost of photostability of the fluorescent stains as no photoprotective agent is present.

A panel targeting the exon of 53 lung cancer-associated genes (see Supplementary Table 1) was established to perform targeted sequencing. We searched the literature and selected these genes based on the following criteria: (a) genes often involved in lung cancer reported TCGA [22 (link), 23 (link)] and other projects [24 –28 (link)] or (b) genes frequently mutated in lung cancer from the COSMIC database (http://cancer.sanger.ac.uk/cancergenome/projects/cosmic). The primer design for the targeted sequencing was performed by Ion AmpliSeq designer software (Thermo Fisher Scientific), as we previously reported [29 (link), 30 (link)]. Sequencing libraries were prepared using Ion AmpliSeq Library kit (Thermo Fisher Scientific), according to the manufacturer's instruction. After barcode ligation using Ion Xpress Barcode Adapters kit (Thermo Fisher Scientific), library samples were purified using Agencourt AMPure XP reagent (Beckman Coulter, Tokyo, Japan) and subsequently quantified using Ion Library Quantitation Kit (Thermo Fisher Scientific). The libraries were templated with the Ion PI Template OT2 200 Kit v3 (Thermo Fisher Scientific). Sequencing was carried out on an Ion Proton (Ion Torrent) using the Ion PI Sequencing 200 Kit v3.
The sequence data were processed using standard Ion Torrent Suite Software running on the Torrent Server. Raw signal data were analyzed using Torrent Suite version 4.0. The pipeline included signaling processing, base calling, quality score assignment, read alignment to the human genome 19 reference (hg19), quality control of mapping and coverage analysis. Following data analysis, annotation of single nucleotide variants, insertions and deletions was performed by the Ion Reporter Server System (Thermo Fisher Scientific), and lymphocytes from peripheral blood DNA were used as a control to detect any variants (Tumor-Normal pair analysis). Sequence data were visually confirmed with the Integrative Genomics Viewer.
Unsupervised hierarchical clustering was used to identify potential distinct subgroups among the multi-clonal lesions based on the genetic profiling. The genetic informations were standardized, clustered and visualized with the CLUSTER and TREEVIEW programs.

A modified propidium iodide assay [90 (link)] was used to assess the compound’s activity in Oncotest’s panel of 74 tumor cell lines. Briefly, cells were harvested from exponential phase cultures by trypsinization, counted, and plated in 96-well flat-bottomed microtiter plates at a cell density depending on the cell line (4000–10,000 cells/well). After a 24 h recovery period to allow the cells to adhere and resume exponential growth, 10 µL of culture medium (six control wells/plate) or culture medium containing the test compounds was added to the cells. The compounds were applied in triplicates at five concentrations. Following four days of continuous drug exposure, the medium or medium with the test compound, including all dead cells suspended in the culture medium, was aspirated and replaced by 200 µL of an aqueous PI solution (7 µg/mL). To measure the fraction of living cells, cells were permeabilized by freezing the plates, resulting in the death of all cells that had remained attached to the bottom of the well after the incubation period. After thawing of the plates, fluorescence was measured using the Cytofluor 4000 micro-plate reader (excitation 530 nm, emission 620 nm), providing a direct relationship with the total viable cell number.

For cell-cycle analysis, cells were trypsinized and fixed with 70% ice-cold ethanol for 30 min at 4 °C. After washing twice with PBS (Gibco, Thermo Fisher Scientific Waltham, MA, USA), 5 µL RNAse A (20–40 mg/mL) (Thermo Fisher Scientific, Waltham, MA, USA) was added and incubated for 30 min at 37 °C. After the addition of 200 µL PI (from 50 µg/mL stock solution) (Abcam, Cambridge, UK), the stained cells were analyzed on a BD Accuri C6 Plus Flow Cytometer (BD Biosciences, Franklin Lakes, NJ, USA) using FlowJo software.

As the size of cfDNA fragments in cancer patients was recently reported to follow a narrow-range, unimodal distribution reaching a peak at 166 bp [39 (link)], the primers were designed to amplify 83 bp of the partial exon 7, covering codons 237 to 261 (hg19: ch17: 7,577,489–ch17: 7,577,571). The forward and reverse primer sequences were 5′-TGTGTAACAGTTCCTGCAT-3′ and 5′-GGCTCCTGACCTGGAGTCTT-3′, respectively. PCR amplification was performed using 5 ng of cfDNA, 5x AccuStart Buffer, 200 nM of forward and reverse primers and 0.04 U/mL of AccuStart HiFi Taq Polymerase (Quanta BioSciences, Beverly, MA, USA) with the following conditions: 2 min at 94 °C, 40 cycles of 30 s at 94 °C, 30 s at 58 °C and 40 s at 72 °C and a final elongation of 5 min at 72 °C. Approximately 20% of the PCR products were quantified by a QubitTM dsDNA HS Assay Kit and (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) and Qubit^® 2.0 fluorometer and 20 ng of the PCR product were purified with Serapure magnetic beads at a final concentration of 2.5x and 28% of isopropanol. The library preparation was performed using the NEB Next^® Fast DNA Library Prep Set for Ion Torrent™ kit (New England Biolabs, Ipswich, MA, USA) with some modifications, where each volume of reagent was reduced by a factor of 4. Briefly, 12.5 μL of the 20 μL purified products were end-repaired in 15 μL, and added to 8.6 μL of the ligation reaction mix, 0.7 μL of the Ion P1 Adapter and 0.7 μL of each Ion Barcode for the ligation step. The barcoded products were purified using the Serapure magnetic beads at a final concentration of 1.8x, amplified in 25 μL and quantified using the Qubit quantification system. A total of 40 ng of the amplified barcoded products were pooled into a single tube and the cleanup and size selection of the pooled libraries (~180 bp) was performed in a 2% agarose gel and MinElute Gel Extraction Kit (Qiagen, Courtaboeuf, France). The pool of purified barcoded libraries was quantified using the Qubit quantification system and the assessment of the library quality (molarity and size analysis) was performed using the Agilent^® High Sensitivity DNA Kit and the Agilent Technologies 2100 BioanalyzerTM (Agilent Technologies, Santa Clara, CA, USA). Emulsion amplification was performed on the Ion OneTouch 2 system (Thermo Fisher Scientific) using 7 μL of 100 pM library and the Ion PI Hi-Q OT2 200 Kit (Thermo Fisher Scientific), according to the manufacturer’s protocol. Quality control of Ion PI Ion Sphere Particles was performed using the Qubit 2.0 fluorometer, as described in the protocol. The sequencing reaction was performed on an Ion Proton System (Thermo Fisher Scientific) using Life Technologies’ Ion PI™ Chip Kit v3 and Ion PGM™ Hi-Q™ Sequencing Kit (Thermo Fisher Scientific), according to the manufacturer’s instructions. The library preparation and sequencing conditions were adapted from previous protocols [40 (link),41 (link),42 (link)].