This work was supported by grants R01-CA169354&#160;and R01-047822&#160;from the National Institutes of Health and a Merit Award from the Department of Veterans Affairs (ECB). RKP was supported by NIH T32 CA009287-30A1, an ASCO Young Investigator Award, California Breast Cancer Research Project Fellowship, and an American Cancer Society Mentored Research Scholar Grant; BAZ was supported by NIH grant AI079320. JM was supported by fellowships from NIH T-32 training grant T32-AI07290- 25, T32-AI07290-24 and American Cancer Society post-doctoral fellowship PF-12-052-01-CSM.

NOTE: All animal experiments were conducted in accordance with approved Stanford, Palo Alto VA HCS, and National Institutes of Health Institutional AnimalCare and Use Committee guidelines.
1. Preparing for Sample Collection and Processing (Time Required: ~10-15 min)
<ol>
	<li>Inoculate murine B16F0 melanoma cells (0.5-1 x 106) subcutaneously at or near the midline of the abdomen in female C57BL/6 mice as described previously6. Alternatively, inoculate tumor cells at add...

<ol>
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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changes+in+fluorescence+intensity+of+selected+leukocyte+surface+markers+following+fixation.">Changes in fluorescence intensity of selected leukocyte surface markers following fixation.</a> Cytometry A. 71 (6), 379-385 (2007).</li><li>Hackstein, H., 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=Heterogeneity+of+respiratory+dendritic+cell+subsets+and+lymphocyte+populations+in+inbred+mouse+strains.">Heterogeneity of respiratory dendritic cell subsets and lymphocyte populations in inbred mouse strains.</a> Respir Res. 13, 94 (2012).</li><li>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=Myeloid-derived+suppressor+cells:+more+mechanisms+for+inhibiting+antitumor+immunity.">Myeloid-derived suppressor cells: more mechanisms for inhibiting antitumor immunity.</a> Cancer Immunol Immunother. 59 (10), 1593-1600 (2010).</li><li>Curran, M. 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The authors have nothing to disclose.

Performing detailed analysis of the tumor microenvironment is critical in determining the mechanisms and effects of immunomodulation. With the increasing presence of immunotherapeutics in the human clinical realm, understanding the impact of these agents on the tumor infiltrating leukocytes is becoming requisite in defining their mechanism of action. In humans, there often exist clinical and/or logistic difficulties in obtaining and analyzing tumor tissue for leukocyte subset analysis, and thus analysis of the peripheral...

The balance between tumor growth and promotion and regression is, in part, dependent on the balance of pro- and anti- tumor infiltrating leukocytes present in the microenvironment1,2. In order to study the tumor microenvironment (TME) and specifically identify the infiltrating leukocyte subpopulations, we developed a method for evaluation of subcutaneous tumors in a murine tumor model. The importance of studying the tumor microenvironment is well known and supported in the literature. Numerous studies have shown that the balance of pro- and anti-tumor infiltrating immune cells can impact the outcomes of tumor growth, not only in mouse but also human studies (reviewed in3,4). For example, Curiel et al. showed that worsened clinical outcomes in ovarian cancer patients were correlated with the presence of increasing percentages of tumor-infiltrating regulatory CD4+ T cells (Tregs)5. Our own work also showed the effect of a novel leukocyte chemoattractant on the ratio of leukocyte subsets in a mouse melanoma model6, which also correlated with decreased tumor growth. Thus, the detailed analyses of the leukocyte subsets within a tumor is now more broadly recognized and increasingly important.
There are many ways to evaluate the tumor microenvironment for infiltrating leukocytes; for example groups have engineered transgenic mice to express various fluorescent proteins in order to image the TME7, classical immunohistochemistry and immunofluorescence of preserved sections8, including various imaging modalities such as MRI, PET, confocal microscopy9-11 - some with the ability to monitor intravitally10,12. These can be used with various molecular imaging agents, such as nanoparticles13 or novel contrast agents14 that label immune cells. Our method is a flow cytometry-based approach and has several advantages. First, the entire tumor microenvironment can be sampled; at the time of analysis, the entire subcutaneous tumor and surrounding periphery is surgically resected for processing. This eliminates any potentially sampling bias within a single tumor and gives a more &#8220;global&#8221; analysis of the tumor as a whole. Secondly, using multicolor flow cytometry to analyze the leukocyte subsets allows us to more specifically gauge the phenotype of infiltrating leukocytes. Depending on the number of colors used, very specific subsets can be identified. This is important as there are several leukocyte subsets within a particular cell type - or even in a general subtype classification &#8211; that have disparate functions that are potentially significant in determining the fate of the tumor. For example, plasmacytoid dendritic cells (pDC) have been implicated in anti-tumor immunity15. However, the CCR9+ subset of pDCs has been shown to be tolerogenic16, and shifting the balance of such a subset may have an impact on tumor growth.
Our method is appropriate for subcutaneous or other tumors that can be resected en bloc. In our hands, tumors were uniformly resected at the time of euthanasia. However, it is conceivable, as has been done in some studies, that a subcutaneous tumor could be fully resected with closure of the surrounding skin in a survival surgery17, thus allowing additional evaluation of the animal. The analysis is then performed on the resected tumor. Thus, the results represent a single timepoint in the tumor&#8217;s development. While this allows a detailed look into the microenvironment, it is also a static picture of what is no doubt a dynamic process. However, isolated leukocytes (e.g. via magnetic separation or density gradient) can then be analyzed separately from the tumor epithelia and stroma, or used in other, functional assays to further define their phenotype, as has been previously described18. This method, then, would be useful for any investigators interested in understanding the composition of the leukocytes within the tumor microenvironment at a given time point, whether in the setting of the natural disease course, or after a specific therapeutic perturbation. Although not done by us, variations of this procedure could also potentially be used to analyze specific portions of a tumor in isolation. For example, given the size of the tumor, the peripheral zone(s) could be dissected away from the central, possibly necrotic core of the tumor to give the researcher a more spatially segregated view of the tumor microenvironment.
In the burgeoning field of tumor immunology, there will no doubt be an exponentially growing number of studies evaluating novel immunomodulatory agents in murine tumor models. Several reports have highlighted the differences in specific leukocyte function within the tumor versus the peripheral environment. For example, Shafer-Weaver et al. showed in a mouse model that antigen-specific T effector CD8+ cells, while active in the periphery, were transformed into CD8+ T suppressor cells once they trafficked into the tumor microenvironment19. This was in part due to TGF&#946;, but other factors are likely involved as well. Thus, evaluating leukocyte subsets &#8211; numbers and ratios, as well as functional status &#8211; within the tumor itself will give a more accurate representation of the effect of a particular immunomodulation on tumor fate.
Our technique allows detailed analysis of the tumor and provides the researcher an opportunity to more closely identify changes in leukocyte populations than previous approaches.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:229px;">Name of the Material/Equipment</td>
			<td style="width:229px;">Company</td>
			<td style="width:229px;">Catalog Number</td>
			<td style="width:229px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RPMI</td>
			<td>Cellgro</td>
			<td>10-040</td>
			<td>http://cellgro.com; keep on ice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FBS</td>
			<td>Cellgro</td>
			<td>35-011-CV</td>
			<td>http://cellgro.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 ml conical tubes</td>
			<td>Falcon</td>
			<td>14-432-22</td>
			<td>fischersci.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">40 micron filter</td>
			<td>Falcon</td>
			<td>08-771-1</td>
			<td>fischersci.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5 ml syringe</td>
			<td>BD</td>
			<td>14-823-35</td>
			<td>fischersci.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">surgical scissors/forceps</td>
			<td>Roboz</td>
			<td>RS-5910</td>
			<td>roboz.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PBS</td>
			<td>Cellgro</td>
			<td>MT-21-030-CM</td>
			<td>http://cellgro.com; keep on ice</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">trypan blue</td>
			<td>Cellgro</td>
			<td>MT-25-900-CI</td>
			<td>fischersci.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">hemacytometer</td>
			<td>Hausser Scientifice&#160;</td>
			<td>02-671-54&#160;</td>
			<td>fischersci.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Live/Dead stain</td>
			<td>Life Technologies</td>
			<td>L34957</td>
			<td>lieftechnologies.com</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">FlowJo software</td>
			<td>TreeStar, Inc</td>
			<td></td>
			<td>flowjo.com</td>
		</tr>
	</tbody>
</table>

evaluation of tumor-infiltrating leukocyte subsets in a subcutaneous tumor model

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or subvert anti-tumor immune responses in order to survive and flourish. Tumors take advantage of a number of different mechanisms of immune &#8220;escape,&#8221; including the recruitment of tolerogenic DC, immunosuppressive regulatory T cells (Tregs), and myeloid-derived suppressor cells (MDSC) that inhibit cytotoxic anti-tumor responses. Conversely, anti-tumor effector immune cells can slow the growth and expansion of malignancies: immunostimulatory dendritic cells, natural killer cells which harbor innate anti-tumor immunity, and cytotoxic T cells all can participate in tumor suppression. The balance between pro- and anti-tumor leukocytes ultimately determines the behavior and fate of transformed cells; a multitude of human clinical studies have borne this out. Thus, detailed analysis of leukocyte subsets within the tumor microenvironment has become increasingly important. Here, we describe a method for analyzing infiltrating leukocyte subsets present in the tumor microenvironment in a mouse tumor model. Mouse B16 melanoma tumor cells were inoculated subcutaneously in C57BL/6 mice. At a specified time, tumors and surrounding skin were resected en bloc and processed into single cell suspensions, which were then stained for multi-color flow cytometry. Using a variety of leukocyte subset markers, we were able to compare the relative percentages of infiltrating leukocyte subsets between control and chemerin-expressing tumors. Investigators may use such a tool to study the immune presence in the tumor microenvironment and when combined with traditional caliper size measurements of tumor growth, will potentially allow them to elucidate the impact of changes in immune composition on tumor growth. Such a technique can be applied to any tumor model in which the tumor and its microenvironment can be resected and processed.

Our results showed that forced over-expression of chemerin in murine B16 tumors augmented the percentage of tumor infiltrating leukocytes (TILs). Additionally, changes to the relative ratio of leukocyte subsets represented within the tumor microenvironment associated with chemerin expression were identified. Re-print with permission from Pachynski et al.6.
Figure 1 shows that there was a significant increase in total CD45+ cells (TILs) within in the tumors th...

Watch this Scientific Journal Video about Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model at JoVE.com

Evaluation of Tumor-infiltrating Leukocyte Subsets in a Subcutaneous Tumor Model

This protocol describes a method for the detailed evaluation of leukocyte subsets within the tumor microenvironment in a mouse tumor model. Chemerin-expressing B16 melanoma cells were implanted subcutaneously into syngeneic mice. Cells from the tumor microenvironment were then stained and analyzed by flow cytometry, allowing for detailed leukocyte subset analyses.

Specialized immune cells that infiltrate the tumor microenvironment regulate the growth and survival of neoplasia.&#160; Malignant cells must elude or ...

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19.9K Views.  Washington University School of Medicine. This protocol describes a method for the detailed evaluation of leukocyte subsets within the tumor microenvironment in a mouse tumor model. Chemerin-expressing B16 melanoma cells were implanted subcutaneously into syngeneic mice. Cells from the tumor microenvironment were then stained and analyzed by flow cytometry, allowing for detailed leukocyte subset analyses. 