library 9
Protocols: Tissue digestion for generation of single-cell suspensions To generate a niche-labeled scRNA-seq dataset of multi-organ synchronous metastasis, single cell suspensions were generated from brain, lung, liver and bones from lower limbs tissues of FVB mice injected with VO-PyMT cells. One mouse was processed each time, and a total of 3 mice were used for this dataset. To generate single cell suspensions, tissue dissociation protocols were optimized to ensure the best possible representation of cellular diversity for each organ, cell yield and viability, considering technical feasibility as well. For each iteration of these experiments, a cohort of 3-5 mice were injected i.c. with VO-PyMT cells, and on days 10-11 post-i.c. injections, systemic metastatic burden was monitored in vivo by injecting 200 µl of 15 mg/ml D-Luciferin in PBS, i.p., allowing luciferase reaction for 10 minutes, and imaging mice under isoflurane anesthesia using an IVIS Lumina imager (PerkinElmer). To model high burden systemic metastasis and ensure the highest possible cancer cell and proximal stroma cell yield, one mouse with robust BLI signal from head (brain metastasis), thorax (lung metastasis), abdomen (liver metastasis) and lower limbs (bone metastasis) was selected to be processed for multi-organ scRNA-seq preparations. Liver dissociation was first performed by direct 2-step perfusion through the portal vein to ensure hepatocyte viability44,45. At endpoint, mice were first anesthetized using 100 mg/kg pentobarbital solution by intraperitoneal (i.p.) injection. An incision in the abdomen was made to expose the portal vein and inferior vena cava (IVC), a suture was placed around the IVC, and the portal vein was severed. Using a needle connected to a peristaltic pump by a cannula, the liver was first perfused with  pre-warmed chelating buffer (25 mM HEPES, 115 mM NaCl, 5 mM KCl, 1 mM KH2PO4, 2.5 mM MgSO4, 0.5 mM EGTA pH 7.4) for 5 minutes, followed by perfusion with pre-warmed digestion buffer (25 mM HEPES, 115 mM NaCl, 5 mM KCl, 1 mM KH2PO4, 2 mM CaCl2, 0.1 mg/ml Collagenase Type IV, pH 7.4)  for 15 minutes. After perfusion, gall bladder was removed and liver was collected and placed on a petri dish on ice containing liver resuspension buffer (25 mM HEPES, 115 mM NaCl, 5 mM KCl, 1 mM KH2PO4, 2.5 mM MgSO4, 4 mM CaCl2, pH 7.4) supplemented with 10 mg/ml bovine serum albumin (BSA, Fisher Scientific, cat. no. BP-9705). Livers were then peeled and gently shaken to release cells into resuspension buffer. Liver homogenates were then filtered through a 70 µm strainer and pelleted by centrifugation at 30 g for 1 minute at 4C. Supernatants contained non-hepatocyte cells (NHCs) and cell pellets were mostly hepatocytes. Supernatants were then separated, centrifuged at 300 g at 4C, and both NHCs and hepatocytes were resuspended in liver resuspension buffer supplemented with 10 mg/ml BSA. NHCs and hepatocyte suspensions were counted, mixed on a 1:1 ratio. NHCs and hepatocyte mixtures were kept on ice until cell multiplexing oligo (CMO) labeling. For generating metastatic brain single cell suspensions, brains were first chopped into 6 pieces using scalpels and simultaneous enzymatic and mechanic digestion with 2 ml 1 mg/ml Collagenase D (Millipore Sigma, cat. no. 11088858001) and 10 µg/mL Deoxyribonuclease I (Worthington Biochemical Corporation, cat. no. LS006342) in HBSS supplemented with Ca2+ and Mg2+ (HBSS++, Corning, cat. no. 21-023-CV) using GentleMACS C tubes (Miltenyi Biotec, cat. no. 130-093-237) and a GentleMACS Octo Dissociator with heaters (Miltenyi Biotec), for 30 minutes at 37°C using the built-in program 37C_ABDK_01. Upon enzymatic and mechanical digestion, brain homogenates were resuspended in 5 ml ice cold DMEM/F-12 medium supplemented with 10% vol/vol FBS and filtered through a 70 µm strainer (Fisherbrand cat. no. 22363548). Brain homogenates were pelleted by centrifugation at 300 g for 5 minutes at 4°C and debris was removed using 900 µl Debris Removal Solution (Miltenyi Biotec, cat. no. 130-109-398) according to the manufacturer's instructions. 4 ml of cold HBSS++ was gently overlayed onto brain suspensions in Debris Removal Solution and then centrifuged at 3,000 g for 10 minutes at 4°C, yielding a triphasic solution. Two top layers (supernatant and debris) were discarded by aspiration, and bottom layer was gently resuspended in 10 ml cold HBSS++ by inverting three times. Brain cells were then collected by centrifugation at 1,000 g at 4C for 10 minutes and resuspended and incubated in ACK Lysing Buffer (ThemorFisher Scientific, cat. no. A1049201) for red blood cell (RBC) lysis for 30 seconds at room temperature. RBC lysis was quenched by adding 12 ml cold HBSS++, and brain single cell suspensions were pelleted at 300 g at 4°C for 10 minutes, resuspended in HBSS++ supplemented with 10% vol/vol FBS and maintained on ice until CMO labeling. To obtain single cell suspensions from lungs affected by metastases, lungs were first mechanically digested using scalpels and lung homogenates were incubated in DMEM/F-12 medium supplemented with 10% vol/vol FBS, 2 mg/ml Collagenase Type IV (Millipore Sigma, cat. no. C5138), 1% (w/v) and 30 µg/ml Deoxyribonuclease I at 37°C for 20 minutes in constant shaking at 200 rpm. After enzymatic digestion, lung homogenates were filtered through a 70 µm cell strainer, pelleted at 300 g centrifugation for 5 minutes at 4°C and resuspended in 1 ml ACK Lysing buffer for RBC lysis for 30 seconds at room temperature. RBC lysis was quenched by adding cold PBS, and single cell suspensions from metastatic lungs were resuspended in PBS supplemented with 10% vol/vol FBS and maintained on ice until CMO labeling. For obtaining single cell suspensions from metastatic bones, tibiae and femora from both lower limbs were harvested, cleaned from surrounding soft tissues, and epiphysis were severed and placed on a sterilized mortar containing 1 ml bone digestion buffer, consisting of DMEM/F12 supplemented with 10% FBS vol/vol and 1 mg/ml Collagenase Type II (ThermoFisher Scientific, cat. no. 17101015). Bone marrows were flushed with ice cold DMEM/F12 supplemented with 10% FBS vol/vol using a 5 ml syringe coupled to a 27G needle, yielding the bone marrow fraction (BMF), which was maintained on ice. Bone diaphysis were placed in the same mortar with epiphysis containing bone digestion buffer, and using a pestle, mineral bone tissues were crushed until homogenization. Mineral bone homogenates were incubated for 30 min at 37°C, constantly shaking at 200 rpm, and then filtered through a 70 µm cell strainer to remove debris. Both bone marrow and mineral bone fractions were pelleted by centrifugation at 300 g for 5 min at 4°C, and pellets were resuspended and incubated in 1 ml ACK RBC lysis buffer at room temperature for 30 seconds to remove red blood cells. Bone-derived single-cell suspensions were resuspended in PBS supplemented with 10% FBS vol/vol, mixed, and maintained on ice until CMO labeling. CMO labeling Single-cell suspensions resulting from brain, lung, liver and bones were counted using a Countess II automated cell counter (ThermoFisher Scientific), and cells from each organ were labeled with one CMO of the 3' CellPlex Kit Set A (10X Genomics, cat. no. 1000261) according to the manufacturer's instructions. Briefly, 2 x 106 cells in ice cold PBS supplemented with 10% FBS were transferred to a 1.5 ml microcentrifuge tube, pelleted at 300 g for 5 min at 4C, resuspended in corresponding 100 µl Cell Multiplexing Oligos and incubated for 5 minutes at room temperature. Cells and CMO mixtures were then washed by adding 1.9 ml chilled PBS + 10% FBS vol/vol, pelleted by centrifugation at 300 g for 5 minutes at 4C, resuspended in 0.5 ml PBS + 10% FBS vol/vol and filtered through a 30 µm strainers into 5 ml conical tubes (Corning, cat. no. 352235) prior to FACS sorting. Cells were constantly kept at 4°C to preserve viability. Cell sorting for separation of cancer cells, proximal stroma, and distal stroma fractions CMO-labeled, single-cell suspensions were labeled with Sytox Blue (1:1,000, ThermoFisher Scientific, cat. no. S34857) to detect dead cells and sorted according to GFP and mCherry signal into PBS supplemented with 10% FBS vol/vol using either a FACS Aria II or an Aria Fusion cell sorter (both from Becton Dickinson). For brain, lung, and bone tissues, the entirety of the cell suspension resulting from organ dissociations were sorted. For liver tissues, recovery of >1x106 cells per cellular fraction (cancer cells, proximal and distal stroma fractions) was ensured. Cell sorting resulted in 12 samples, corresponding to cancer cell, proximal and distal stroma fractions from brain, lung, liver and bone cell suspensions. These 12 samples were centrifuged at 300 g for 5 min at 4°C, resuspended in chilled PBS + 10% FBS vol/vol, and counted using a Countess II automated counter. Cells from the same cellular fractions but from different organs were pooled at equal cell numbers into one sample to subsequently generate a scRNA-seq library. Droplet generation and scRNA-seq library preparation Cell pools were processed following the Chromium Next GEM Single Cell 3' user guide instructions (10X Genomics, CG000388 Rev B). In brief, cells were resuspended in chilled PBS + 10% FBS at a concentration of approximately 1,000 cells/µl and loaded onto the 10X Genomics Chromium Controller for generating single-cell droplets. Single-cell RNA-seq gene expression and cell multiplexing libraries were generated following the 10X Genomics Chromium v3.1 guideline.
ENA first public
2026-07-31
INSDC center name
University of California, Irvine
INSDC status
public
Submitter Id
E-MTAB-16621:library 9
age
7-8
broker name
ArrayExpress
cell type
mouse cells
collection date
not collected
common name
house mouse
developmental stage
adult
disease
breast cancer metastasis
genotype
wild type genotype
geographic location (country and/or sea)
not collected
individual
mouse02
isolate
not applicable
organism part
multiple organs
sample name
E-MTAB-16621:library 9
scientific_name
Mus musculus
sex
female
strain
FVB