GoKawiilStudy participant details from the ARTEMIS Trial
The clinical trial protocol was reviewed and approved by The University of Texas MD Anderson Cancer Center Institutional Review Board and all participants provided written informed consent (NCT02276443; MDA no. 2014-0185). All study procedures performed were in accordance with ethical standards of the Institutional Review Board and with the 1964 Helsinki declaration and its later amendments or comparable ethical standards. TNBC was defined as breast cancer that was oestrogen receptor-negative (lower than 1%) or low oestrogen receptor-positive (1% to less than 10%), progesterone receptor-negative (less than 1%) or low progesterone receptor-positive (1% to less than 10%) and HER2-negative by immunohistochemistry and fluorescence in situ hybridization. Residual cancer burden (RCB) status after chemotherapy treatment was assessed by histopathology from the resected surgical specimen61. Chemotherapy response was classified into pCR (RCB-0) or RD (RCB-I, II or III) by RCB status. A subset of patients (N = 19) with suboptimal mid-treatment response were changed to targeted therapies before surgery and had no NAC response data available. The gene-expression data from patients with TNBC without NAC response data were used for the biological analysis (for example, cell type and cell state clustering) but were excluded from the analyses comparing the response groups. For each patient, 1–4 fresh tumour breast core needle biopsies were collected and transported to the laboratory in media for cell dissociation and frozen for spatial transcriptomic experiments.
Experimental method details
scRNA-seq
The biopsies were placed in 10-cm sterile tissue culture dishes and were cut into pieces around 1 mm3, then digested in 1 ml of dissociation buffer (collagenase A (1 mg ml−1 working solution, Sigma no. 11088793001) dissolved in DMEM F12/HEPES media (Gibco no. 113300) and BSA fraction V solutions (Gibco no. 15260037) mixed at a 3:1 ratio, respectively). Cell suspensions were transferred into a 1.5-ml tube and rotated in a hybridization oven for 30 min at 37 °C. The cell pellet was collected by centrifuging at 500g for 5 min and then resuspended in 1 ml of trypsin (Corning no. 25053CI) before incubating in a rotating hybridization oven at 37 °C for 5 min. Next, 2 ml of DMEM containing 10% fetal bovine serum (Sigma no. F0926) was used for trypsin neutralization. The solution was then filtered through a 70-μm strainer (Falcon no. 352350) and then centrifuged at 500g for 5 min to collect the cell pellet. To remove red blood cells, the cell pellet was nutated at room temperature for 10 min in 10 ml of 1× MACS red blood cell lysis buffer (MACS no. 130-094-183). Then 10 ml of DMEM was added to stop red blood cell lysis and then centrifuged at 500g for 5 min. The resulting pellet was washed in 1 ml of cold DMEM and then resuspended in 100 µl of cold PBS (Sigma no. D8537) + 0.04% BSA solution (Ambion no. AM2616) and filtered through a 40-μm flowmi (Bel-Art no. h13680-0040) on ice until use. Before loading, cell viability and cell counts were quantified by Trypan blue staining with the Countless II FL automated cell counter (Thermo Fisher) and their concentration was adjusted to 300–1,000 cells per microlitre. Single-cell capture, barcoding and library preparation were performed by following the 10X Genomics Single Cell Chromium 3′ protocols (V2, CG00052; V3, CG000183; V3.1, CG000204). A detailed step-by-step description of this protocol is also publicly available at Protocols.IO https://doi.org/10.17504/protocols.io.t3geqjw.
Visium spatial transcriptomics experiments
Visium spatial transcriptomics experiments were performed using the Visium Platform (10X Genomics) following the manufacturer’s protocol with a minor modification based on the recommendations in the Tissue Optimization protocol (10X protocol no. CG000238). Specifically, fresh tumour biopsies were embedded in cryogenic (cryo) moulds with optimal cutting temperature (OCT) compound (Fisher nos. NC9542860, 1437365) on dry ice and stored at −80 °C until cryo-sectioning. Next 12-μm sections were prepared on a cryomicrotome (Cryostar NX70, Thermo Scientific) with chuck and blade temperatures set at −17 °C and −15 °C, respectively. Two core sections were placed within the capture area of the Visium spatial slide (10X Genomics PN-1000184). According to the manufacturer’s tissue optimization protocol, we determined that the optimal permeabilization time for breast tissue was 12 min. Sectioned slides were fixed and stained with H&E as described by the manufacturer (10X protocol no. CG000160). Images of H&E staining were captured on the Nikon Eclipse Ti2 system following imaging guidelines (10X protocol no. CG000241). The final libraries were constructed by following the user guide (10X protocol no. CG000239) and were sequenced on the Illumina next-generation sequencing system (NovoSeq6000) using the S2-100 flow cell.
Xenium in situ RNA experiments
Fresh frozen breast core biopsy tissues were embedded in cryomolds using OCT compound (Thermo Fisher Scientific no. 1437365) over dry ice and cryo-sectioned at 12 μm using a cryomicrotome (Leica, CM1950). Sections were placed within the capture area of the Xenium slides (10X Genomics, PN3000941). Tissue fixation and permeabilization were performed according to the 10X Genomics user guide (CG000581, revision D), with a modification extending the 37 °C incubation to 3 min for breast tissue samples. FFPE tissues were sectioned at 5 μm and placed on xenium slides by the pathology laboratory. Deparaffinization and decrosslinking were performed following the 10X Genomics user guide (CG000580, revision E), with a modification extending the nuclease-free water incubation time to 1 min to remove residual eosin from previously stained tissue before embedding. Both OCT and FFPE slides were subsequently proceeded according to the Xenium prime in situ gene expression with cell segmentation staining User Guide (CG000760, revision E). A custom 100-gene panel was also designed (Supplementary Table 4) and incorporated as an add-on to the Xenium Human 5K Pan Tissue & Pathways Panel. Post-Xenium H&E staining was conducted following the manufacturer’s protocol (10X Genomics user guide CG000613 revision A).
VisiumHD spatial transcriptomics experiments
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