This work was supported by the Ann and Sol Schreiber Mentored Investigator Award (POE/DF/02.2011) awarded to SS.

All procedures were performed under the guidance of University of Texas at MD Anderson IACUC review board.
1. Reagent Preparation
<ol>
	<li>Preparation of medium for migration assay
	<ol>
		<li>Prepare 500 mL of complete RPMI media for the migration assay: RPMI medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1% pen-strep (penicillin 500 U/mL and streptomycin 500 &#181;g/mL).</li>
	</ol>
	</li>
	<li>Preparation of buffers for staining MDSCs<...

<ol>
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Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reversion+of+immune+tolerance+in+advanced+malignancy:+modulation+of+myeloid-derived+suppressor+cell+development+by+blockade+of+stem-cell+factor+function.">Reversion of immune tolerance in advanced malignancy: modulation of myeloid-derived suppressor cell development by blockade of stem-cell factor function.</a> Blood. 111 (1), 219-228 (2008).</li><li>Serafini, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-dose+granulocyte-macrophage+colony-stimulating+factor-producing+vaccines+impair+the+immune+response+through+the+recruitment+of+myeloid+suppressor+cells.">High-dose granulocyte-macrophage colony-stimulating factor-producing vaccines impair the immune response through the recruitment of myeloid suppressor cells.</a> Cancer Res. 64 (17), 6337-6343 (2004).</li><li>Gabrilovich, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vascular+endothelial+growth+factor+inhibits+the+development+of+dendritic+cells+and+dramatically+affects+the+differentiation+of+multiple+hematopoietic+lineages+in+vivo.">Vascular endothelial growth factor inhibits the development of dendritic cells and dramatically affects the differentiation of multiple hematopoietic lineages in vivo.</a> Blood. 92 (11), 4150-4166 (1998).</li><li>Movahedi, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+discrete+tumor-induced+myeloid-derived+suppressor+cell+subpopulations+with+distinct+T+cell-suppressive+activity.">Identification of discrete tumor-induced myeloid-derived suppressor cell subpopulations with distinct T cell-suppressive activity.</a> Blood. 111 (8), 4233-4244 (2008).</li><li>Delano, M. 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C., Ochoa, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Arginine+regulation+by+myeloid+derived+suppressor+cells+and+tolerance+in+cancer:+mechanisms+and+therapeutic+perspectives.">Arginine regulation by myeloid derived suppressor cells and tolerance in cancer: mechanisms and therapeutic perspectives.</a> Immunol Rev. , 180-191 (2008).</li><li>Bingisser, R. M., Tilbrook, P. A., Holt, P. G., Kees, U. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Macrophage-derived+nitric+oxide+regulates+T+cell+activation+via+reversible+disruption+of+the+Jak3/STAT5+signaling+pathway.">Macrophage-derived nitric oxide regulates T cell activation via reversible disruption of the Jak3/STAT5 signaling pathway.</a> J Immunol. 160 (12), 5729-5734 (1998).</li><li>Kusmartsev, S., Nefedova, Y., Yoder, D., Gabrilovich, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antigen-specific+inhibition+of+CD8++T+cell+response+by+immature+myeloid+cells+in+cancer+is+mediated+by+reactive+oxygen+species.">Antigen-specific inhibition of CD8+ T cell response by immature myeloid cells in cancer is mediated by reactive oxygen species.</a> J Immunol. 172 (2), 989-999 (2004).</li><li>Waris, G., Ahsan, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+species:+role+in+the+development+of+cancer+and+various+chronic+conditions.">Reactive oxygen species: role in the development of cancer and various chronic conditions.</a> J Carcinog. 5, 14 (2006).</li><li>Schmielau, J., Finn, O. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activated+granulocytes+and+granulocyte-derived+hydrogen+peroxide+are+the+underlying+mechanism+of+suppression+of+t-cell+function+in+advanced+cancer+patients.">Activated granulocytes and granulocyte-derived hydrogen peroxide are the underlying mechanism of suppression of t-cell function in advanced cancer patients.</a> Cancer Res. 61 (12), 4756-4760 (2001).</li><li>Szuster-Ciesielska, A., Hryciuk-Umer, E., Stepulak, A., Kupisz, K., Kandefer-Szerszen, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+species+production+by+blood+neutrophils+of+patients+with+laryngeal+carcinoma+and+antioxidative+enzyme+activity+in+their+blood.">Reactive oxygen species production by blood neutrophils of patients with laryngeal carcinoma and antioxidative enzyme activity in their blood.</a> Acta Oncol. 43 (3), 252-258 (2004).</li><li>Youn, J. I., Nagaraj, S., Collazo, M., Gabrilovich, D. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Subsets+of+myeloid-derived+suppressor+cells+in+tumor-bearing+mice.">Subsets of myeloid-derived suppressor cells in tumor-bearing mice.</a> J Immunol. 181 (8), 5791-5802 (2008).</li><li>Sinha, P., Clements, V. K., Bunt, S. K., Albelda, S. M., Ostrand-Rosenberg, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cross-talk+between+myeloid-derived+suppressor+cells+and+macrophages+subverts+tumor+immunity+toward+a+type+2+response.">Cross-talk between myeloid-derived suppressor cells and macrophages subverts tumor immunity toward a type 2 response.</a> J Immunol. 179 (2), 977-983 (2007).</li><li>Li, H., Han, Y., Guo, Q., Zhang, M., Cao, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cancer-expanded+myeloid-derived+suppressor+cells+induce+anergy+of+NK+cells+through+membrane-bound+TGF-beta+1.">Cancer-expanded myeloid-derived suppressor cells induce anergy of NK cells through membrane-bound TGF-beta 1.</a> J Immunol. 182 (1), 240-249 (2009).</li><li>Sarkar, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PRKCI+promotes+immune+suppression+in+ovarian+cancer.">PRKCI promotes immune suppression in ovarian cancer.</a> Genes Dev. 31 (11), 1109-1121 (2017).</li><li>Cribbs, A. 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We have described the methodology to isolate MDSCs from mouse ovarian tumor. The same method can be utilized for isolating MDSCs or other immune cells from any solid normal tissue or solid tumor using cell-specific markers. Additionally, depending on the nature of the tissue the incubation time with the dissociation buffer will need to be optimized.
The isolation of viable immune cells from tumor tissue depends on performing the different isolation steps from dissecting tumors to obtain the so...

In recent years a number of studies have focused on understanding the role of immune cells in cancer development and progression. One way by which tumor cells evade the immune system is through the expression of immunosuppressive factors that activate, upregulate, and attract immune suppressive cells like MDSCs in the tumor microenvironment1.
MDSCs are a population of immature myeloid cells that are generated in the bone marrow. Under normal conditions, these immature cells differentiate into mature myeloid cells like macrophages, monocytes, or dendritic cells2. Under pathologic condition, these cells fail to differentiate completely into mature myeloid cells. Instead, they are expanded and activated by factors secreted by activated T cells, tumor cells, and stromal cells. Secretory proteins like stem-cell factor3, IL-6, granulocyte/macrophage CSF (G-CSF;M-CSF)4, vascular endothelial growth factor5,IFN&#947;6, ligands for Toll-like receptors (TLRs)7, IL-48, IL-139, and transforming growth factor &#946; (TGF&#946;)10 promote MDSC proliferation and activation. Upon activation, these cells upregulate expression of factors like arginase 111, inducible nitric oxide synthase12, and reactive oxygen species13,14,15,16,17, resulting in their remarkable potential to suppress the T-cell response. In addition to inhibiting the adaptive immune response, MDSCs have been reported to negatively influence the innate immune response through macrophages18 and NK cells19.
These cells lack markers of mature myeloid cells. In mice, MDSCs are characterized by the expression of the cell surface markers GR1 and CD11b. Based on morphology, they are further characterized as granulocytic (CD11b+LY6G+LY6Clow) ormonocytic (CD11b+LY6G&#8722;LY6Chi) MDSCs. Both subtypes have immunosuppressive properties, but in mouse tumors, the granulocytic MDSCs are the major population17. In humans, MDSC markers are not well characterized. Often, the monocytic MDSCs are identified as CD11b+ CD14+ CD33+high HLA-DRneg/Low and CD66bneg, while granulocytic MDSCs are identified as CD11b+ CD14neg CD33+low HLA-DRneg CD66b.
A number of functional studies in mouse tumor models have established the importance of MDSCs in tumor development and T cell suppression. Hence, they are potential targets for therapeutic intervention and it is critical to study this subset of immune cells to understand immune suppression in cancer. We need to identify new factors that may promote or inhibit MDSC expansion and activation, which can be utilized for therapy. To perform such tests, we need to isolate MDSCs in a simple and timely manner to obtain live, non-contaminated cells. Here, we outline the methods to isolate a viable MDSC population from mouse tumor. This method can be used to isolate MDSC from any other tissue following the same method. Cell types other than MDSCs can also be isolated by this method using the appropriate cell-specific markers. We also outline methods to determine MDSC migration potential towards a cytokine gradient. Here we use tumor necrosis factor &#945; (TNF&#945;) as an example. Other cytokines can also be tested. Instead of conditioned media from cell lines, regular media supplemented with specific cytokines in varying concentrations can be used to test the role of those cytokines in MDSC migration from a particular tumor type.

<table border="0" cellpadding="0" cellspacing="0" width="707">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Collagenase IV</td>
			<td style="width:115px;">Thermo Fisher</td>
			<td style="width:133px;">17104019</td>
			<td style="width:243px;">Tumor dissociation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cell strainer</td>
			<td style="width:115px;">Falcon</td>
			<td style="width:133px;">352350</td>
			<td style="width:243px;">Cell strain</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RBC lysis buffer</td>
			<td style="width:115px;">Biolegend</td>
			<td style="width:133px;">420301</td>
			<td style="width:243px;">Cell culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DMEM+Glutamax</td>
			<td style="width:115px;">Gibco</td>
			<td style="width:133px;">10569010</td>
			<td style="width:243px;">Cell culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RPMI</td>
			<td style="width:115px;">Gibco</td>
			<td style="width:133px;">11875093</td>
			<td style="width:243px;">Cell culture medium</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FBS</td>
			<td style="width:115px;">Gibco</td>
			<td style="width:133px;">10082147</td>
			<td style="width:243px;">Serum for Cell culture</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:216px;">Phosphate Buffered Saline (PBS)</td>
			<td style="width:115px;">Sigma</td>
			<td style="width:133px;">D8537-500ML</td>
			<td style="width:243px;">PBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trypsin</td>
			<td style="width:115px;">Invitrogen</td>
			<td style="width:133px;">25200-056</td>
			<td style="width:243px;">Cell dissociation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Penstrep</td>
			<td style="width:115px;">HyClone</td>
			<td style="width:133px;">SV30010</td>
			<td style="width:243px;">Antibiotic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sterile water&#160;</td>
			<td style="width:115px;">Invitrogen</td>
			<td style="width:133px;">10977-015</td>
			<td style="width:243px;">For dilution of buffers</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">BV605-CD11b</td>
			<td>Biolegend</td>
			<td>101237</td>
			<td style="width:243px;">Antibody for MDSC labeling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">FITC-GR1</td>
			<td>Biolegend</td>
			<td>108405</td>
			<td style="width:243px;">Antibody for MDSC labeling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">APC-Cy7-CD45</td>
			<td style="width:115px;">Biolegend</td>
			<td>103115</td>
			<td style="width:243px;">Antibody for lymphocyte labeling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">PE-Cy7-Ly6C</td>
			<td style="width:115px;">Biolegend</td>
			<td>128017</td>
			<td style="width:243px;">Antibody for MDSC labeling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">APC-Ly6G</td>
			<td style="width:115px;">Biolegend</td>
			<td>127613</td>
			<td style="width:243px;">Antibody for MDSC labeling</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TNF&#945;</td>
			<td style="width:115px;">Sigma</td>
			<td>T7539</td>
			<td style="width:243px;">Cytokine</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TNF&#945; neutralizing antibody</td>
			<td style="width:115px;">Biolegend</td>
			<td>506309</td>
			<td style="width:243px;">neutralizing antibody</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:216px;">Ghost Dye Violet 450</td>
			<td style="width:115px;">Tonbo Biosciences</td>
			<td style="width:133px;">13-0863</td>
			<td style="width:243px;">Cell viability dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">UltraComp Beads</td>
			<td style="width:115px;">Invitrogen</td>
			<td style="width:133px;">01-2222-42</td>
			<td style="width:243px;">Compensation beads</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cell culture inserts</td>
			<td style="width:115px;">Corning</td>
			<td style="width:133px;">353097</td>
			<td style="width:243px;">Migration chamber</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:216px;">LSR Fortessa X-20</td>
			<td style="width:115px;">BD Biosciences</td>
			<td style="width:133px;">LSR Fortessa X-20</td>
			<td style="width:243px;">Fluorescent Cell analyzer</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:216px;">BD FACSAria Fusion</td>
			<td style="width:115px;">BD Biosciences</td>
			<td style="width:133px;">BD FACSAria Fusion</td>
			<td style="width:243px;">Fluorescent Cell sorter</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Cell countess</td>
			<td style="width:115px;">Cell countess</td>
			<td style="width:133px;">Cell countess</td>
			<td style="width:243px;">Cell countess</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Flow Jo</td>
			<td style="width:115px;"></td>
			<td style="width:133px;"></td>
			<td style="width:243px;">Software for Analysis of flow data</td>
		</tr>
		<tr height="39">
			<td height="39" style="height:39px;width:216px;">Prism</td>
			<td style="width:115px;"></td>
			<td style="width:133px;"></td>
			<td style="width:243px;">Plotting of graph and statistical analysis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">C57BL/6 mice</td>
			<td style="width:115px;">Taconic</td>
			<td style="width:133px;">B6-F</td>
			<td style="width:243px;">mice for tumor generation</td>
		</tr>
	</tbody>
</table>

isolation of myeloid-derived suppressor cells from mouse tumor and determining their migration potential in vitro

The importance of the immune response in cancer and other diseases (like diabetes mellitus, alzheimers, cystic fibrosis) is now known, and the manipulation of the immune system as a therapy to treat cancer is gaining attention. The immune system regulates tumorigenesis both negatively and positively. The myeloid-derived suppressor cells (MDSCs) are a population of immune cells that are increased during cancer, inflammation, and infection. These cells influence the immune response and effectively suppresses the anti-tumor T cell response. They serve as potential targets for therapeutic intervention to effectively use the immune system to inhibit tumorigenesis. To better understand how such intervention can be applied it is important to study these cell types. Using mouse ovarian tumors, we describe the isolation of MDSCs from solid tissue using gentle dissociation techniques. We further describe how MDSCs are isolated from such dissociated tissue based on the expression of cell surface markers with the help of flow cytometry. Additionally, we describe the procedure to perform an in vitro MDSC migration assay to determine the migration potential of these cells in response to soluble factors like cytokines and chemokines.

Here, we present results obtained from the isolation of MDSCs from mouse ovarian tumors20. Following the procedure described above, we isolated single cells and stained them for MDSCs. MDSCs in the tumors were labeled with APC-Cy7-CD45, FITC-GR1, PE-CD11b. To elucidate the MDSC population, these cells can be further stained with Ly6C and Ly6G, as shown in the gating strategy in Figure 1. Labeled cells were sorted by flow cytometry. Lab...

This article outlines the procedures to isolate myeloid-derived suppressor cells from mouse solid tumors and perform an in vitro assay with the cells to determine their response migration potential to certain soluble factors like cytokines and chemokines.

Isolation of Myeloid-derived Suppressor Cells from Mouse Tumor and Determining Their Migration Potential <em>In Vitro</em>

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