PS-LIBR-g013
Homo sapiens
Protocols: PC9 CDX animal studies were conducted in accordance with U.K. Home Office legislation, the Animal Scientific Procedures Act 1986, and AstraZeneca Global Bioethics policy. All experimental work is outlined in project license PP3292652, which has gone through the AstraZeneca Ethical Review Process. Female severe combined immunodeficiency disease (SCID) mice were purchased from Envigo UK. All mice were older than 6 weeks at the time of implant. Cells were implanted under isoflurane anaesthesia with microchipping performed at same time. Approximately 5 million PC9 cells in a total volume of 0.1 mL containing 50% Matrigel were injected subcutaneously in the right flank of female mice. PC9 xenografts were established in female SCID mice. Mice were marked with a subcutaneous microchip to follow tumour growth and tumours were measured with an electronic calliper twice weekly. Tumour volume was calculated as 0.5 x tumour length x tumour width x 2. Tumour volume was monitored twice weekly by bilateral calliper measurements until the end of study or until tumours reached the ethical limit of 10% of the mouse's bodyweight.  Mice were randomized into vehicle or treatment groups with approximate mean tumour volume of 0.2 to 0.4 cm3. Randomization for animal studies was based on initial tumour volumes to ensure equal distribution across groups. Mice were dosed once in the morning daily by oral gavage for the duration of the treatment period. All compounds were synthesised by AstraZeneca. TKI (osimertinib) was prepared fresh every 7 days in 0.5% HPMC. Mice were sacrificed at indicated time point and the collected tumours were passed through a McIlwain tissue chopper and stored in 1mL RPMI1640 until subsequent processing. Minced PC9 CDX tissue samples were dissociated using the Miltenyi human tumour dissociation kit (130-095-929) (40 minutes, 37C), filtered through 30uM Miltenyi pre-separation filters, counted using AO/PI double stain (Nexcelom) and processed as per 10x Flex protocol for fixation of dissociated tumour cells.  Cells were processed according to the 10x protocol for the fixation of cells and nuclei. Briefly, suspensions of up to 10 million cells were spun-down at 350 rcf for 5 min at 4C and cells were resuspended in 1 mL of Fixation Buffer (4% Formaldehyde, 1X Conc. Fix and Perm Buffer – 10x Genomics PN-2000517). Cells were fixed for 16-24h at 4C. To stop the fixation, cells were spun-down at 850 rcf for 5 min at room temperature and quenched with 1 mL of Quenching Buffer (1X Conc. Quench Buffer – 10x Genomics PN-2000516). Preparations were then processed by adding 0.1 volumes of Enhancer (10x Genomics PN-2000482) and 10% glycerol for long-term storage and shipment to Single-cell Discoveries B.V..  Samples were then thawed at room temperature, centrifuged at 850 rcf for 5 min and resuspended in 1 mL 0.5X PBS - 0,02% BSA. Cell concentration and viability was then assessed by AO-PI staining with a LUNA-FX7™ Automated Cell Counter (Logos Biosystems).  Up to 2 million cells were processed per hybridization following 10x recommendations. Hybridizations were set up in 80 uL of hybridization mix with 20 uL of Human or Mouse WTA probes (10x Genomics PN-2000510 or PN-2000718). For experiments with probe mixing 10 uL of each probe set were used per hybridization. Hybridizations were performed at 42C for 16-24h.  After hybridization samples were diluted in Post-Hyb Wash Buffer and measured by AO-PI staining in a LUNA-FX7™ Automated Cell Counter (Logos Biosystems). For each experiment, we pooled an equal number of cells from each hybridization to have an equal contribution per sample. Cell pools were then washed 3 times in Post-Hyb Wash Buffer for 10 min at 42C. After the washes, cells were resuspended in Post-Hyb Resuspension Buffer, filtered through a Miltenyi Biotec 30 um filter and measured with the cell counter to determine the amount needed for the Chromium X run.   For the GEM encapsulation we followed the 10x Genomics protocol and their guidelines on the volume of cells and reagents required per well according to the targeted cell recovery. After loading the Chip Q and running it on the Chromium X, GEMs were recovered and processed as indicated by 10x Genomics. After processing the GEMs, the product is pre-amplified and indexed to construct the sequencing library according t the 10x Genomics Gene Expression Flex User Guide.
strain
SCID
organism
Homo sapiens
organism
Homo sapiens
collection date
not collected
sex
female
scientific_name
Homo sapiens
common name
human
organism part
tumor
isolate
not applicable
xenograft
PC9 lung cancer cell
growth condition
xenograft PC9 cells grown in mice
cell_type
lung cancer cell
age
6
geographic location (country and/or sea)
not collected