There are a few kits that can be used to isolate MDSCs from mouse tumors such as the EasySep system (Stem Cell). I want to know if MDSCs are present in high abundance in tumors in order to plan tumor-derived MDSC characterization studies.
Could you recommend any tumor dissociation systems to use to increase the yield of MDSCs from dissociated tumors?
I previously tried dissociating 4T1 tumors (1500 mm3) from BALB/c mice using collagenase/dispase/DNase. The tumors were manually dissociated using a blades and a syringe plunger. After incubation for 1.5 hours in the dissociation solution at 37 C.
I used flow cytometry to determine Gr1+/CD11b+ cells from the tumor. There were virtually no MDSCs present in the tumor, so I am wondering if anyone is familiar with tissue dissociation protocols that are useful for isolating MDSCs from tumors.
I will be using the GentleMACS dissociator to enhance cell viability in my next iteration. I have had success in recovering over 50 million MDSCs from tumor-bearing mouse spleens. If anyone has experience or information regarding MDSC accumulation in tumors during tumorigenesis, I would appreciate the advice.
Many membrane proteins are sensitive to proteases. Thus, type IV collogenase is needed as it has low levels of proteases. In addition, dispase is a collection of proteases. The DNAse is to reduce cell clumping from the DNA of apoptotic/dead cells.
Gentle stirring, using magnetic stirrers in a water bath to maintain temp is good. Also note that collogenase is an enzyme that require Ca to be active so Ca Mg free HBSS is not your friend.
See any of the numerous papers by Talmadge, or Wiltrout or Fidler on dissociation. If memory serves there are several SOP like publications. I will also note there are a few papers that discuss the sensitivity of different CDs to different enzymes.
Thanks again for the advice, James E Talmadge . My main concern is regarding the abundance of MDSC during mid-to-late stage tumorigenesis - ie, "are the MDSCs a dominant population of stromal cells in highly proliferative tumor models such as 4T1?" That was my first and only attempt at isolating MDSCs from a highly necrotic tumor, so I'm sure those conditions should be taken into account.
Using more gentle conditions (and a more viable tumor) should improve the yield of tumor MDSCs in comparison to splenic MDSCs that can be seamlessly isolated without the use of any enzyme incubation.
I have published a number of papers regarding isolation of MDSCs from tumors including 4T1 tumor bearing mice. Lots of other people have published this as well. The infiltration is tumor burden dependent. Try searching on mammary and MDSC.
Mammary tumor heterogeneity in the expansion of myeloid-derived suppressor cells. Donkor MK, Lahue E, Hoke TA, Shafer LR, Coskun U, Solheim JC, Gulen D, Bishay J, Talmadge JE. Int Immunopharmacol. 2009 Jul;9(7-8):937-48. doi: 10.1016/j.intimp.2009.03.021. Epub 2009 Apr 9. PMID: 19362167
Myeloid-derived suppressor cells in mammary tumor progression in FVB Neu transgenic mice. Abe F, Dafferner AJ, Donkor M, Westphal SN, Scholar EM, Solheim JC, Singh RK, Hoke TA, Talmadge JE. Cancer Immunol Immunother. 2010 Jan;59(1):47-62. doi: 10.1007/s00262-009-0719-2. Epub 2009 May 18.
Therapeutic activity of sunitinib for Her2/neu induced mammary cancer in FVB mice.
Abe F, Younos I, Westphal S, Samson H, Scholar E, Dafferner A, Hoke TA, Talmadge JE. Int Immunopharmacol. 2010 Jan;10(1):140-5. doi: 10.1016/j.intimp.2009.09.023. Epub 2009 Oct 12. PMID: 19833232
Tumor- and organ-dependent infiltration by myeloid-derived suppressor cells.
Younos I, Donkor M, Hoke T, Dafferner A, Samson H, Westphal S, Talmadge J. Int Immunopharmacol. 2011 Jul;11(7):816-26. doi: 10.1016/j.intimp.2011.02.021. Epub 2011 Mar 2. PMID: 21376153
Tumor regulation of myeloid-derived suppressor cell proliferation and trafficking. Younos IH, Dafferner AJ, Gulen D, Britton HC, Talmadge JE. Int Immunopharmacol. 2012 Jul;13(3):245-56. doi: 10.1016/j.intimp.2012.05.002. Epub 2012 May 17. PMID: 22609473
Long-chain omega-3 polyunsaturated fatty acids decrease mammary tumor growth, multiorgan metastasis and enhance survival.
Khadge S, Thiele GM, Sharp JG, McGuire TR, Klassen LW, Black PN, DiRusso CC, Cook L, Talmadge JE. Clin Exp Metastasis. 2018 Dec;35(8):797-818. doi: 10.1007/s10585-018-9941-7. Epub 2018 Oct 16.
Chemokine-mediated rapid turnover of myeloid-derived suppressor cells in tumor-bearing mice. Sawanobori Y, Ueha S, Kurachi M, Shimaoka T, Talmadge JE, Abe J, Shono Y, Kitabatake M, Kakimi K, Mukaida N, Matsushima K. Blood. 2008 Jun 15;111(12):5457-66. doi: 10.1182/blood-2008-01-136895. Epub 2008 Mar 28. PMID: 18375791
Reduced inflammation in the tumor microenvironment delays the accumulation of myeloid-derived suppressor cells and limits tumor progression.
Bunt SK, Yang L, Sinha P, Clements VK, Leips J, Ostrand-Rosenberg S. Cancer Res. 2007 Oct 15;67(20):10019-26. doi: 10.1158/0008-5472.CAN-07-2354.
Gemcitabine directly inhibits myeloid derived suppressor cells in BALB/c mice bearing 4T1 mammary carcinoma and augments expansion of T cells from tumor-bearing mice. Le HK, Graham L, Cha E, Morales JK, Manjili MH, Bear HD. Int Immunopharmacol. 2009 Jul;9(7-8):900-9. doi: 10.1016/j.intimp.2009.03.015. Epub 2009 Mar 29. PMID: 19336265
Thank you, Dr. Talmadge. I will look through these papers and others that I have not read. Deeply appreciated.
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