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| Status |
Public on Oct 29, 2020 |
| Title |
CNS-Native Myeloid Cells Drive Immune Suppression in the Brain Metastatic Niche through Cxcl10 [R08_MgliaDep] |
| Organism |
Mus musculus |
| Experiment type |
Other
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| Summary |
We performed CITE-seq (10x Genomics-based) to profile and compare the transcriptomes and cell surface expression of immune epitopes in the brains and blood of different transgenic mice (Cx3cr1CreERT; Cx3cr1CreERT-ROSA-iDTR) during homeostasis or brain metastasis with or without DT-based microglia depletion. We sequenced a total of eight different samples. We created an antibody pool consisting of 35 different antibodies and stained each sample individually with this antibody pool. Then, we stained each sample with it's own unique hashing antibody so that we could subsequently pool the samples for loading onto the 10x Chromium and later prepare one library consisting of all sight samples and finally separate each sample in silico by it's unique hashing antibody. The samples are as follows: (1) HTO1: brain of naïve Cx3cr1CreERT mouse (non-microglia depleted); (2) HTO2: brain of metastasis-burdened Cx3cr1CreERT mouse (non-microglia depleted); (3) HTO3: brain of metastasis-burdened Cx3cr1CreERT mouse (non-microglia depleted); (4) HTO4: brain of naïve Cx3cr1CreERT-ROSA-iDTR mouse (microglia depleted); (5) HTO5: brain of metastasis-burdened Cx3cr1CreERT-ROSA-iDTR mouse (microglia depleted); (6) HTO6: brain of metastasis-burdened Cx3cr1CreERT-ROSA-iDTR mouse (microglia depleted); (7) HTO7: blood of metastasis-burdened Cx3cr1CreERT mouse (non-microglia depleted); (8) HTO8: blood of metastasis-burdened Cx3cr1CreERT-ROSA-iDTR mouse (microglia depleted). The following sample comparisons were made: HTO1 versus HTO4; HTO2 and HTO3 versus HTO5 and HTO6; HTO7 versus HTO8.
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| Overall design |
Naïve brains were obtained from 2-3 month old female Cx3cr1CreERT or Cx3cr1CreERT-ROSA-iDTR mice without brain metastases. These mice were administered tamoxifen followed by a one month cessation before being administered diptheria toxin, which induced microglia depletion in Cx3cr1CreERT-ROSA-iDTR, but not control Cx3cr1CreERT mice. Brains from brain metastasis-bearing mice initiated by carotid injection of E0771 cells were obtained from 2-3 month old female mice of the aforementioned genotypes with established brain metastases (~2 weeks post injection). These mice also underwent the aforementioned tamoxifen and diptheria toxin treatment to deplete microglia during brain metastasis. Cell suspensions were prepared by percoll gradient centrifugation enrichment to obtain suspensions enriched for immune cells from the brain. We created an antibody pool consisting of 35 different antibodies and stained each sample individually with this antibody pool. Then, we stained each sample with it's own unique hashing antibody so that we could subsequently pool the samples for loading onto the 10x Chromium and later prepare one library consisting of all eight samples and finally separate each sample in silico by it's unique hashing antibody. NOTE: Brain immune cells were enriched through percoll gradient before 10X sequencing. Thus, there are remanent microglia cells in the depleted samples.
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| Contributor(s) |
Zhang S, Guldner IH |
| Citation(s) |
33113353 |
| NIH grant(s) |
| Grant ID |
Grant title |
Affiliation |
Name |
| R01 CA194697 |
(PQD-3) Spatiotemporal Molecular Interrogation of Early Metastatic Evolution In Situ |
UNIVERSITY OF NOTRE DAME |
Siyuan Zhang |
| R01 CA222405 |
Mechanisms of neuroinflammation in brain metastasis progression |
UNIVERSITY OF NOTRE DAME |
Siyuan Zhang |
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| Submission date |
Nov 05, 2019 |
| Last update date |
Jan 30, 2021 |
| Contact name |
Siyuan Zhang |
| E-mail(s) |
szhang8@nd.edu
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| Phone |
574-631-4635
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| Organization name |
University of Notre Dame
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| Department |
Biological Sciences
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| Street address |
1234 N. Notre Dame Ave
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| City |
South Bend |
| ZIP/Postal code |
46617 |
| Country |
USA |
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| Platforms (1) |
| GPL24247 |
Illumina NovaSeq 6000 (Mus musculus) |
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| Samples (3) |
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| This SubSeries is part of SuperSeries: |
| GSE134285 |
CNS-Native Myeloid Cells Drive Immune Suppression in the Brain Metastatic Niche through Cxcl10 |
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| Relations |
| BioProject |
PRJNA587758 |
| SRA |
SRP228493 |
