2024-09-01
ENA first public
ERC000011
ENA-CHECKLIST
Unit of Molecular Oncology, Centro di Riferimento Oncologico di Aviano (CRO), IRCCS, National Cancer Institute
INSDC center name
public
INSDC status
ERS19932288
SRA accession
E-MTAB-14085:Sample 1
Submitter Id
ArrayExpress
broker name
not collected
collection date
human
common name
Protocols: Tumor samples were collected from CDK4/6 inhibitor sensitive and resistant PDX at necroscopy. Tumors were processed in ice and fragments flash frozen for subsequent nucleic acid extraction. Stable third-passage PDX derived from luminal BCRO#28 were implanted in NSG mice (10 mice/PDX). When tumor volume reached about 60 mm3, mice were randomly divided in two cohorts and continuously treated via oral gavage with vehicle or Palbociclib (PD-0332991 hydrochloride, Clinisciences, Nanterre, France) resuspended in ringer lactate (100 mg/kg, 5 days/week). Treatments were prolonged up to 8 months with the same dosing regimen.  After emergence of acquired-resistance tumors (volume >400mm3), mice were sacrificed, tumors and distant organs collected and stored for further molecular analysis. To confirm the resistant phenotype, tumor pieces derived from excised masses were implanted in 10 recipient NSG mice. As described above, when the tumors reached 60 mm3, mice were separated into two different groups (vehicle- and Palbociclib-treated) and the treatments followed as described. In all experiments, tumor dimensions were recorded weekly with a caliper and tumor volume was calculated based on the formula: length x width2/2. Total RNA was isolated from fresh solid tumor samples using TRIzol solution (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA), according to manufacturer protocol. GentleMACS™ Dissociator (Miltenyi Biotec, Bergisch Gladbach, Germany) was used to disrupt tumors, and lysates were passed at least five times through a 23‐gauge needle fitted to an RNase‐free syringe. RNA quantification was performed using NanoDrop (Thermo Fisher Scientific, Waltham, MA, USA) and Tape Station RNA Screen Tape kit (Agilent Technologies, Inc., Santa Clara, CA, USA) according to manufacturer protocol. An input of 500 ng of total RNA per sample was used for subsequent RNA Sequencing analysis. RNA-Seq libraries were generated using the Illumina Stranded total RNA prep library kit (Illumina, San Diego, CA, USA) according to protocol’s instructions. Pools of 28 purified libraries were quantified using High Sensitivity D1000 Screen Tape System (Agilent Technologies, Inc., Santa Clara, CA, USA) and normalized to 2nΜ.
description
adult
developmental stage
breast cancer
disease
not collected
geographic location (country and/or sea)
1118-1DX
individual
not applicable
isolate
Homo sapiens
organism
Homo sapiens
organism
breast
organism part
Resistant
response to treatment
Homo sapiens
scientific_name
female
sex
Sample 1
title