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Dissociation of human melanoma

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Posts: 1

Joined: Fri Mar 29, 2019 1:06 pm

Post Fri Mar 29, 2019 1:17 pm

Dissociation of human melanoma

I am dissociating human melanomas for Cytof analysis, but my cells are dying and the recovery rate is very low. Has someone a protocol to advice please?



Posts: 4

Joined: Mon Jul 13, 2015 9:07 pm

Post Sat Mar 30, 2019 1:01 pm

Re: Dissociation of human melanoma

Regarding dissociation of melanoma and other solid tumors for suspension mass cytometry, I would highlight the following publications from our group (Irish lab @ Vanderbilt).

1) Finding the right time and enzymes to prepare viable single cells from solid human tissue.

Leelatian N*, Doxie DB*, Greenplate AR, Lehman JM, Sinnaeve J, Mobley BC, Kauffmann RM, Werkhaven JA, Mistry AM, Weaver KD, Thompson RC, Massion PP, Hooks MA, Kelley MC, Chambless LB, Ihrie RA, Irish JM. Single cell analysis of human tissues and solid tumors with mass cytometry. Cytometry B. PMC5459378. DOI. *equal contribution
- Peer-reviewed methods research led by Nalin Leelatian and Deon Doxie and conducted in collaboration with Rebecca Ihrie's lab.
- Validated mass cytometry for human solid tumors, including glioblastoma, melanoma, and patient derived xenografts (PDXs) of small cell lung cancer.
- Selected as “Editor’s Best” 2016-2017.
- First use of suspension mass cytometry in human solid tumors and tissues (online 6 Sept 2016).

Cytometry B: https://onlinelibrary.wiley.com/doi/ful ... to.b.21481
Full text at PMC: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5459378/

2) Protocol for tumor and solid tissue disaggregation and tips sheet for troubleshooting.

Leelatian N, Doxie DB, Greenplate AR, Sinnaeve J, Ihrie RA*, Irish JM*. Preparing Viable Single Cells from Human Tissue and Tumors for Cytomic Analysis. Current Protocols in Molecular Biology 2017 Apr 3;118:25C.1.1-25C.1.23. PMC5518778. *co-corresponding
- Protocol co-authored with Rebecca Ihrie's lab.
- Detailed instructions for methods in Leelatian and Doxie et al., Cytometry B 2017.
- Tips sheet for troubleshooting issues like poor viability in Table 25C.1.2

Current Protocols: https://currentprotocols.onlinelibrary. ... 02/cpmb.37
Full text at PMC: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5518778/

3) Uses of these protocols in suspension mass cytometry solid tumor research with a melanoma cell focus.

Doxie DB, Greenplate AR, Gandelman JS, Diggins KE, Roe CE, Dahlman KB, Sosman JA, Kelley MC, Irish JM. BRAF and MEK inhibitor therapy eliminates nestin expressing melanoma cells in human tumors. Pigment Cell & Melanoma Research 2018 May 19. PMC6188784 [Available on 2019-11-01]

- Peer-reviewed research led by Irish lab graduate student Deon Doxie and initiated in collaboration with surgeon Mark Kelley.
- Revealed dramatic changes in the cellular composition of melanoma tumors that occur in vivo in patients.
- Nestin+ cells are eliminated and a new, Nestin- melanoma cell type remains after BRAF and MEK inhibitor treatment.
- Loss (or maintenance of low levels) of MHC I and MHC II proteins was also apparent on melanoma cells.
- First use of mass cytometry to study matched solid tumor serial biopsies over time during treatment (online 19 May 2018).

PCMR: https://onlinelibrary.wiley.com/doi/abs ... pcmr.12712
Full text at PMC [Available on 2019-11-01] : https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6188784/
(Full text here: https://www.researchgate.net/profile/Jonathan_Irish3)

4) Uses of these protocols in suspension mass cytometry solid tumor research with a melanoma tumor infiltrating immune cell focus.

Greenplate AR, McClanahan DD, Oberholtzer BK, Doxie DB, Roe CE, Diggins KE, Leelatian N, Rasmussen ML, Kelley MC, Gama V, Ferrell PB, Johnson DB, Irish JM. Computational immune monitoring reveals abnormal double negative T cells present across human tumor types. Cancer Immunology Research 2018 Nov 9. PMID: 30413431. PMC6318034 [Available on 2020-01-01].

Cancer Immunology Research: http://cancerimmunolres.aacrjournals.or ... /1/86.long
Full text at PMC [Available 2020-01-01]: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6318034/
(Full text here: https://www.researchgate.net/profile/Jonathan_Irish3)

- Peer-reviewed research led by Irish lab graduate student Allison Greenplate and initiated in collaboration with Doug Johnson.
- Developed a comprehensive computational biology workflow for time series / longitudinal mass cytometry data.
- Characterized cells in samples collected over time from melanoma blood or melanoma tumor (different studies).
- Revealed a novel population of CD3+ CD4- CD8- double negative T cells present in human tumor types, including brain, kidney, and melanoma skin tumors.
- The cancer associated double negative T cells increased over time following kinase inhibitor treatment in melanoma tumors.

5) While I'm here, I'll plug our recent review focused on mass cytometry for solid tumors...

Mistry AM*, Greenplate AR, Ihrie RA, Irish JM*. Beyond the Message: Advantages of Snapshot Proteomics with Single-Cell Mass Cytometry in Solid Tumors. FEBS J 2018 Dec 13. doi: 10.1111/febs.14730. * co-corresponding

Full text at FEBS J: https://febs.onlinelibrary.wiley.com/do ... febs.14730

- Review led by neurosurgeon Akshitkumar Mistry.
- Emphasizes use of single cell mass cytometry for solid tumor and tissue research.
- Includes a table comparing complementary single cell technologies, including mass, fluorescence, and RNA-seq.
- Suspension analysis emphasis, although imaging is discussed.

6) And if you still want more suspension mass cytometry of solid tumors...

Vanderbilt researchers & collaborators will be at CYTO 2019 in Vancouver this summer and speaking on solid tumor mass cytometry at the pre-meeting and CYTO. My CYTO presentation will be on our glioblastoma mass cytometry collaboration with the Ihrie lab (scheduled for the 10:30 AM - 12:00 PM session on Monday, June 24, 2019 in Parallel 05: Application of Multimodal Analysis to Disease Biology). We'll have a few other posters and talks about this and related work, as well.

Hope to see you at CYTO!


Jonathan M. Irish, Ph.D.
Assistant Professor of Cell & Developmental Biology
Assistant Professor of Pathology, Microbiology, & Immunology
Scientific Director, Cancer & Immunology Core (CIC),
Mass Cytometry Center of Excellence (MCCE)
Vanderbilt University

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