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 additional anatomical sites (e.g. flank, mammary fat pad, etc). 
	NOTE: Any number of tumors (implanted or spontaneous) can be used and evaluated using this protocol. For example, other commonly used syngeneic tumors in C57BL/6 include the colon line MC38, Lewis Lung Carcinoma (LLC), and the prostate TRAMP-C2 line. While human xenografts inoculated into immunodeficient mice can also be evaluated in this way, it should be noted that the profiles of the tumor-infiltrating leukocytes will necessarily be altered in accordance with the immunodeficient state of the host mouse.
	<ol>
		<li>Use complete media (e.g. RPMI containing 10% FBS and additives) or another appropriate protein-containing buffer (e.g. HBSS or PBS +1% FBS or 1% BCS). Chill media or buffer to 4 &#176;C prior to euthanization.</li>
	</ol>
	</li>
	<li>Set up collection tubes and filters for resected tumors; place these on ice.
	<ol>
		<li>Use one 50 ml conical tube per tumor to be resected. Insert a 40-70 &#181;m cell suspension filter in each one. Alternatively, use 15 ml conical tubes with metal filter mesh. Use a 5 ml syringe plunger or thick pen to create a cup-shaped filter at the top of the 15 ml tube.</li>
	</ol>
	</li>
	<li>Prepare instruments for resection and tumor processing; use surgical forceps and fine scissors to resect and process tumors. Use 70% ethanol to clean instruments between resections/tumor samples. For larger number of samples (e.g. &#62;10), we recommend simultaneous processing of samples by multiple researchers to avoid significant differences in processing time between tumor samples.</li>
</ol>
2. Tumor Resection (Time Required: ~5 min/tumor)
<ol>
	<li>Euthanize B16 tumor-bearing mice individually using carbon dioxide or other approved method.</li>
	<li>Secure the mouse on a surgical stage. Spray the tumor area with 70% ethanol and wipe excess off; this helps prevent spreading of any hair present. For non-nude mice, shave the skin area around the tumor prior to resection as needed.</li>
	<li>Using forceps and scissors, resect the subcutaneous tumor en bloc, including overlying and surrounding skin. The amount of &#8220;margin&#8221; skin taken can be varied, but typically resect 2-3 mm of skin around the tumor.
	<ol>
		<li>Weigh resected tumors (+/- surrounding skin) at this point. Place the resected skin/tumor section in the filter secured within the plastic conical tube.</li>
	</ol>
	</li>
</ol>
3. Preparing a Single Cell Tumor Suspension (Time Required: ~5-7 min/tumor)
<ol>
	<li>Use forceps and scissors to mince tumor and overlying/surrounding skin. Mince the sample using scissors until all large sections of tissue are processed into 1-2 mm sections. 
	NOTE: Typically, whole tumors are processed; alternatively, for larger tumors, representative sections of tumors could be minced and processed.</li>
	<li>Using a plunger from a disposable 5 or 10 ml syringe, mechanically disaggregate the minced tumor tissue against the secured filter.</li>
	<li>Using a pipettor, wash the processed tumor tissue with 5-10 mls of cold media or buffer. This will flush the single cells through the filter.</li>
	<li>Repeat the above two steps several times, ensuring that the total number and volumes of washings are approximately the same for similarly sized tumors.
	<ol>
		<li>After washing the processed tumor tissue, observe small fragments of skin and/or other connective tissue should be left behind in the filter.</li>
		<li>Alternatively, for tumors where mechanical disaggregation alone may not be sufficient, digestion with collagenase and/or other enzymes can be performed prior to the mincing procedure. This will allow for a more thorough single cell suspension. However, it should be noted that such digestion protocols can affect surface antigen expression20, so caution should be taken in interpreting these results.</li>
	</ol>
	</li>
	<li>Wash and pellet the cells by adding media/buffer and centrifuging at 4 &#176;C 500 x g for 5 min.</li>
</ol>
4. FACS Staining of Single Cell Suspension (Time Required: ~30-60 min)
<ol>
	<li>Resuspend cells in ice cold FACS buffer (PBS + 1% FBS).</li>
	<li>Count viable cells using trypan blue and a hemacytometer, or automated cell counter.</li>
	<li>Determine total viable cell yield per volume. This data can be used to extrapolate absolute numbers of tumor infiltrating leukocytes in combination with FACS data.</li>
	<li>Determine the number of cells to be stained and analyzed by FACS. This will be influenced by several factors (e.g. tumor histology type, tumor age and size, average percentage of infiltrating leukocytes, etc.). 
	NOTE: In B16 melanoma, we found a low percentage of tumor infiltrating leukocytes (TIL; typically &#60;5%) compared to tumor cells. Thus, at least 1 x 106 total tumor (tumor + infiltrating leukocytes) cells were typically collected via FACS for analysis.
	<ol>
		<li>Using the trypan-stained counting data, calculated volume of cells to be stained for FACS. For example, if TIL typically constitute 5% of total tumor cells (many of the tumor cells will be dead on trypan stain), factor this into the total number of tumor single cell suspension to be stained.</li>
		<li>For less common leukocyte subsets e.g. CCR9+ pDC) use a larger number of total tumor cells (e.g. 2-3 x 106) for FACS staining, given the relative low frequency of these cells in the single cell suspension.</li>
	</ol>
	</li>
	<li>Stain the tumor single cell suspension for FACS.
	<ol>
		<li>For live/dead discrimination, use a fixable stain or non-fixable stain (e.g. propidium iodide) can be used. If cell are stained without live/dead discrimination, caution must be advised given the potential for non-specific binding of antibodies.</li>
		<li>Stain the cell suspension for specific leukocyte subsets using a standard FACS staining protocol. Block cells to reduce non-specific antibody binding with PBS/FBS containing 1% rat serum and Fc block (anti-CD16/32) for 10 min prior to staining with antigen-specific antibodies. Include the correct isotype control antibodies to ensure staining is specific.</li>
		<li>At this point, after the FACS staining is complete, fix samples with 4% formaldehyde or other similar fixative agent. Store samples at 4 &#176;C in the dark (as to avoid quenching of fluorophores) until time of collection and analysis. 
		NOTE: Storing fixed samples for long periods of time may result in alteration in signal and intensity of the fluorophores21.</li>
		<li>Collect the samples on a flow cytometer. Optimal flow cytometer settings will vary depending on type of instrument and laser/detector setup. 
		NOTE: Additionally, there can be variability between experiments even if using the same machine. Thus, perform appropriate machine setup (e.g. using calibration beads) to ensure appropriate compensation setting prior to FACS collection and analysis of each experiment.</li>
	</ol>
	</li>
	<li>Analysis of leukoctye subsets
	<ol>
		<li>Using FACS data analysis software, to analyze the data. For a live/dead stain, start by gating out dead cells. Use antibody against a leukocyte specific marker (e.g. anti-CD45) to identify leukocytes within the live cells.
		<ol>
			<li>After this, use various gating schemes to identify specific subsets, depending on the staining protocol. For example, to identify CD4+ T cells, select the SSClo region from the CD45+ population and select the CD19-CD3+ cells; from this CD4+ T cells can be identified22.</li>
		</ol>
		</li>
		<li>Analyze data and present it in a number of different ways to ensure consistency and identify patterns or trends (i.e. use total numbers, percentages, or ratios to quantify subsets). 
		NOTE: For example, we looked at total live CD45+ cells as a percentage of total tumor cells collected (Figure 1). Additionally, we then calculated the percentage of each leukocyte subset (eg CD4+ T cells) within the tumor and compared these as ratios between the control and test groups (Figure 3).</li>
	</ol>
	</li>
</ol>

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X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Innate+and+adaptive+immune+cells+in+the+tumor+microenvironment.">Innate and adaptive immune cells in the tumor microenvironment.</a> Nat Immunol. 14 (10), 1014-1022 (2013).</li><li>Fridman, W. H., Pages, F., Sautes-Fridman, C., Galon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+immune+contexture+in+human+tumours:+impact+on+clinical+outcome.">The immune contexture in human tumours: impact on clinical outcome.</a> Nat Rev Cancer. 12 (4), 298-306 (2012).</li><li>Curiel, T. 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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.

<table><tbody><tr><td>RPMI</td><td>Cellgro</td><td>10-040</td><td>http://cellgro.com; keep on ice</td></tr><tr><td>FBS</td><td>Cellgro</td><td>35-011-CV</td><td>http://cellgro.com</td></tr><tr><td>50 ml conical tubes</td><td>Falcon</td><td>14-432-22</td><td>fischersci.com</td></tr><tr><td>40 &amp;#956;m filter</td><td>Falcon</td><td>08-771-1</td><td>fischersci.com</td></tr><tr><td>5 ml syringe</td><td>BD</td><td>14-823-35</td><td>fischersci.com</td></tr><tr><td>surgical scissors/forceps</td><td>Roboz</td><td>RS-5910</td><td>roboz.com</td></tr><tr><td>PBS</td><td>Cellgro</td><td>MT-21-030-CM</td><td>http://cellgro.com; keep on ice</td></tr><tr><td>trypan blue</td><td>Cellgro</td><td>MT-25-900-CI</td><td>fischersci.com</td></tr><tr><td>hemacytometer</td><td>Hausser Scientifice&amp;nbsp;</td><td>02-671-54&amp;nbsp;</td><td>fischersci.com</td></tr><tr><td>Live/Dead stain</td><td>Life Technologies</td><td>L34957</td><td>lieftechnologies.com</td></tr><tr><td>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...

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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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Research

JoVE Journal

Medicine

20.1K 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. 

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

Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for immunotherapies. Recent clinical advances in immunotherapy highlight the need for reproducible methods to accurately and thoroughly characterize the tumor and its associated immune infiltrate. Tumor enzymatic digestion and flow cytometric analysis allow broad characterization of numerous immune cell subsets and phenotypes; however, depth of analysis is often limited by fluorophore restrictions on panel design and the need to acquire large tumor samples to observe rare immune populations of interest. Thus, we have developed an effective and high throughput method for separating and enriching the tumor immune infiltrate from the non-immune tumor components. The described tumor digestion and centrifugal density-based separation technique allows separate characterization of tumor and tumor immune infiltrate fractions and preserves cellular viability, and thus, provides a broad characterization of the tumor immunologic state. This method was used to characterize the extensive spatial immune heterogeneity in solid tumors, which further demonstrates the need for consistent whole tumor immunologic profiling techniques. Overall, this method provides an effective and adaptable technique for the immunologic characterization of subcutaneous solid murine tumors; as such, this tool can be used to better characterize the tumoral immunologic features and in the preclinical evaluation of novel immunotherapeutic strategies.

Here we describe a method to separate and enrich components of the tumor immune and non-immune microenvironment in established subcutaneous tumors. This technique allows for the separate analysis of tumor immune infiltrate and non-immune tumor fractions which can permit comprehensive characterization of the tumor immune microenvironment.

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for ...

Cancer Research

Intramucosal Inoculation of Squamous Cell Carcinoma Cells in Mice for Tumor Immune Profiling and Treatment Response Assessment

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop better therapeutic strategies, understanding tumor microenvironmental factors that contribute to the treatment resistance is important. A major impediment to understanding disease mechanisms and improving therapy has been a lack of murine cell lines that resemble the aggressive and metastatic nature of human HNSCCs. Furthermore, a majority of murine models employ subcutaneous implantations of tumors which lack important physiological features of the head and neck region, including high vascular density, extensive lymphatic vasculature, and resident mucosal flora. The purpose of this study is to develop and characterize an orthotopic model of HNSCC. We employ two genetically distinct murine cell lines and established tumors in the buccal mucosa of mice. We optimize collagenase-based tumor digestion methods for the optimal recovery of single cells from established tumors. The data presented here show that mice develop highly vascularized tumors that metastasize to regional lymph nodes. Single-cell multiparametric mass cytometry analysis shows the presence of diverse immune populations with myeloid cells representing the majority of all immune cells. The model proposed in this study has applications in cancer biology, tumor immunology, and preclinical development of novel therapeutics. The resemblance of the orthotopic model to clinical features of human disease will provide a tool for enhanced translation and improved patient outcomes.

Here we present a reproducible method for developing an orthotopic murine model of head and neck squamous cell carcinoma. Tumors demonstrate clinically relevant histopathological features of the disease, including necrosis, poor differentiation, nodal metastases, and immune infiltration. Tumor-bearing mice develop clinically relevant symptoms including dysphagia, jaw displacement, and weight loss.

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop ...

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The orthotopic murine model can be performed on large cohorts of immunocompetent mice simultaneously, is relatively inexpensive and preserves the cognate tissue microenvironment. The quantification of T cell infiltration and cytotoxic activity within orthotopic tumors provides a useful indicator of an antitumoral response.
This protocol describes the methodology for surgical generation of orthotopic pancreatic tumors by injection of a low number of syngeneic tumor cells resuspended in 5 &#181;L basement membrane directly into the pancreas. Mice bearing orthotopic tumors take approximately 30 days to reach endpoint, at which point tumors can be harvested and processed for characterization of tumor-infiltrating T cell activity. Rapid enzymatic digestion using collagenase and DNase allows a single-cell suspension to be extracted from tumors. The viability and cell surface markers of immune cells extracted from the tumor are preserved; therefore, it is appropriate for multiple downstream applications, including flow-assisted cell sorting of immune cells for culture or RNA extraction, flow cytometry analysis of immune cell populations. Here, we describe the ex vivo stimulation of T cell populations for intracellular cytokine quantification (IFN&#947; and TNF&#945;) and degranulation activity (CD107a) as a measure of overall cytotoxicity. Whole-tumor digests were stimulated with phorbol myristate acetate and ionomycin for 5 h, in the presence of anti-CD107a antibody in order to upregulate cytokine production and degranulation. The addition of brefeldin A and monensin for the final 4 h was performed to block extracellular transport and maximize cytokine detection. Extra- and intra-cellular staining of cells was then performed for flow cytometry analysis, where the proportion of IFN&#947;+, TNF&#945;+ and CD107a+ CD4+ and CD8+ T cells was quantified.
This method provides a starting base to perform comprehensive analysis of the tumor microenvironment.

Generation of Orthotopic Pancreatic Tumors and Ex vivo Characterization of Tumor-Infiltrating T Cell Cytotoxicity

This protocol describes the surgical generation of orthotopic pancreatic tumors and the rapid digestion of freshly isolated murine pancreatic tumors. Following digestion, viable immune cell populations can be used for further downstream analysis, including ex vivo stimulation of T cells for intracellular cytokine detection by flow cytometry.

In vivo models of pancreatic cancer provide invaluable tools for studying disease dynamics, immune infiltration and new therapeutic strategies. The ...

Functional Analysis of Tumor-Infiltrating Myeloid Cells by Flow Cytometry and Adoptive Transfer

The tumor-infiltrating myeloid cell compartment represents a heterogeneous population of broadly immunosuppressive cells that have been exploited by the tumor to support its growth. Their accumulation in tumor and secondary lymphoid tissue leads to the suppression of antitumor immune responses and is thus a target for therapeutic intervention. As it is known that the local cytokine milieu can dictate the functional programming of tumor-infiltrating myeloid cells, strategies have been devised to manipulate the tumor microenvironment (TME) to express a cytokine landscape more conducive to antitumor myeloid cell activity. To evaluate therapy-induced changes in tumor-infiltrating myeloid cells, this paper will outline the procedure to dissociate intradermal/subcutaneous tumor tissue from solid tumor-bearing mice in preparation for leukocyte recovery. Strategies for flow cytometric analysis will be provided to enable the identification of heterogeneous myeloid populations within isolated leukocytes and the characterization of unique myeloid phenotypes. Lastly, this paper will describe a means of purifying viable myeloid cells for functional assays and determining their therapeutic value in the context of adoptive transfer.

This protocol provides reliable methods of solid tumor dissociation and myeloid cell isolation in murine intradermal or subcutaneous tumor models. Flow cytometry allows for phenotypic characterization of heterogeneous myeloid populations within the tumor microenvironment and sorting will demonstrate their functionality in the context of adoptive transfer.

The tumor-infiltrating myeloid cell compartment represents a heterogeneous population of broadly immunosuppressive cells that have been exploited by the ...

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in eradicating cancerous cells. However, the origins and replenishment of tumor antigen-specific CD8+ T cells within the tumor microenvironment (TME) remain obscure. This protocol employs the B16F10-OVA melanoma cell line, which stably expresses the surrogate neoantigen, ovalbumin (OVA), and TCR transgenic OT-I mice, in which over 90% of CD8+ T cells specifically recognize the OVA-derived peptide OVA257-264 (SIINFEKL) bound to the class I major histocompatibility complex (MHC) molecule H2-Kb. These features enable the study of antigen-specific T cell responses during tumorigenesis.
Combining this model with tumor transplantation surgery, tumor tissues from donors were transplanted into tumor-matched syngeneic recipient mice to precisely trace the influx of recipient-derived immune cells into transplanted donor tissues, allowing the analysis of the immune responses of tumor-inherent and periphery-originated antigen-specific CD8+ T cells. A dynamic transition was found to occur between these two populations. Collectively, this experimental design has provided another approach to precisely investigate the immune responses of CD8+ T cells in TME, which will shed new light on tumor immunology.

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

Here, we present a tumor transplantation protocol for the characterization of tumor-inherent and periphery-derived tumor-infiltrated lymphocytes in a mouse tumor model. Specific tracing of the influx of recipient-derived immune cells with flow cytometry reveals the dynamics of the phenotypic and functional changes of these cells during antitumor immune responses.

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in ...

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1&#945; Nanoparticles

Cytokine therapy is a promising immunotherapeutic strategy that can produce robust antitumor immune responses in cancer patients. The proinflammatory cytokine interleukin-1 alpha (IL-1&#945;) has been evaluated as an anticancer agent in several preclinical and clinical studies. However, dose-limiting toxicities, including flu-like symptoms and hypotension, have dampened the enthusiasm for this therapeutic strategy. Polyanhydride nanoparticle (NP)-based delivery of IL-1&#945; would represent an effective approach in this context since this may allow for a slow and controlled release of IL-1&#945; systemically while reducing toxic side effects. Here an analysis of the antitumor activity of IL-1&#945;-loaded polyanhydride NPs in a head and neck squamous cell carcinoma (HNSCC) syngeneic mouse model is described. Murine oropharyngeal epithelial cells stably expressing HPV16 E6/E7 together with hRAS and luciferase (mEERL) cells were injected subcutaneously into the right flank of C57BL/6J mice. Once tumors reached 3-4 mm in any direction, a 1.5% IL-1a - loaded 20:80 1,8-bis(p-carboxyphenoxy)-3,6-dioxaoctane:1,6-bis(p-carboxyphenoxy)hexane (CPTEG: CPH) nanoparticle (IL-1&#945;-NP) formulation was administered to mice intraperitoneally. Tumor size and body weight were continuously measured until tumor size or weight loss reached euthanasia criteria. Blood samples were taken to evaluate antitumor immune responses by submandibular venipuncture, and inflammatory cytokines were measured through cytokine multiplex assays. Tumor and inguinal lymph nodes were resected and homogenized into a single-cell suspension to analyze various immune cells through multicolor flow cytometry. These standard methods will allow investigators to study the antitumor immune response and potential mechanism of immunostimulatory NPs and other immunotherapy agents for cancer treatment.

Evaluation of the In vivo Antitumor Activity of Polyanhydride IL-1&#945; Nanoparticles

A standard protocol is described to study the antitumor activity and associated toxicity of IL-1&#945; in a syngeneic mouse model of HNSCC.

Cytokine therapy is a promising immunotherapeutic strategy that can produce robust antitumor immune responses in cancer patients. The proinflammatory ...

A Simple and Rapid Protocol to Non-enzymatically Dissociate Fresh Human Tissues for the Analysis of Infiltrating Lymphocytes

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted as an important hallmark of cancer. Our research on breast cancer focuses on the active role that tumor infiltrating lymphocytes play in tumor progression and patient outcome. Toward this goal, we developed a methodology for the rapid isolation of intact lymphoid cells from normal and abnormal tissues in an effort to evaluate them proximate to their native state. Homogenates prepared using a mechanical dissociator show both increased viability and cell recovery while preserving surface receptor expression compared to enzyme-digested tissues. Furthermore, enzymatic digestion of the remaining insoluble material did not recover additional CD45+ cells indicating that quantitative and qualitative measurements in the primary homogenate likely genuinely reflect infiltrating subpopulations in the tissue fragment. The lymphoid cells in these homogenates can be easily characterized using immunological (phenotype, proliferation, etc.) or molecular (DNA, RNA and/or protein) approaches. CD45+ cells can also be used for subpopulation purification, in vitro expansion or cryopreservation. An additional benefit of this approach is that the primary tissue supernatant from the homogenates can be used to characterize and compare cytokines, chemokines, immunoglobulins and antigens present in normal and malignant tissues. This protocol functions extremely well for human breast tissues and should be applicable to a wide variety of normal and abnormal tissues.

This protocol describes the rapid non-enzymatic dissociation of fresh human tissue fragments for qualitative and quantitative assessment of CD45+ cells (lymphocytes/leukocytes) present in various normal and malignant human tissues. Additionally, the supernatant obtained from the primary tissue homogenate can be collected and stored for further analysis or experimentation.

The ability of malignant cells to evade the immune system, characterized by tumor escape from both innate and adaptive immune responses, is now accepted ...

Immunology and Infection

Experimental Melanoma Immunotherapy Model Using Tumor Vaccination with a Hematopoietic Cytokine

Fms-like tyrosine kinase 3 ligand (Flt3L) is a hematopoietic cytokine that promotes the survival and differentiation of dendritic cells (DCs). It has been used in tumor vaccines to activate innate immunity and enhance antitumor responses. This protocol demonstrates a therapeutic model using cell-based tumor vaccine consisting of Flt3L-expressing B16-F10 melanoma cells along with phenotypic and functional analysis of immune cells in the tumor microenvironment (TME). Procedures for cultured tumor cell preparation, tumor implantation, cell irradiation, tumor size measurement, intratumoral immune cell isolation, and flow cytometry analysis are described. The overall goal of this protocol is to provide a preclinical solid tumor immunotherapy model, and a research platform to study the relationship between tumor cells and infiltrating immune cells. The immunotherapy protocol described here can be combined with other therapeutic modalities, such as immune checkpoint blockade (anti-CTLA-4, anti-PD-1, anti-PD-L1 antibodies) or chemotherapy in order to improve the cancer therapeutic effect of melanoma.

The protocol presents a cancer immunotherapy model using cell-based tumor vaccination with Flt3L-expressing B16-F10 melanoma. This protocol demonstrates the procedures, including preparation of cultured tumor cells, tumor implantation, cell irradiation, measurement of tumor growth, isolation of intratumoral immune cells, and flow cytometry analysis.

Fms-like tyrosine kinase 3 ligand (Flt3L) is a hematopoietic cytokine that promotes the survival and differentiation of dendritic cells (DCs). It has been ...

Isolation of Immune Cells from Primary Tumors

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various chemokines and growth factors 1,2. However, once in the TME, these cells lose the ability to promote anti-tumor immunity and begin to support tumor growth and down-regulate anti-tumor immune responses 3-4. Studies on tumor-associated leukocytes have mainly focused on cells isolated from tumor-draining lymph nodes or spleen due to the inherent difficulties in obtaining sufficient cell numbers and purity from the primary tumor. While identifying the mechanisms of cell activation and trafficking through the lymphatic system of tumor bearing mice is important and may give insight to the kinetics of immune responses to cancer, in our experience, many leukocytes, including dendritic cells (DCs), in tumor-draining lymph nodes have a different phenotype than those that infiltrate tumors 5,6 . Furthermore, we have previously demonstrated that adoptively-transferred T cells isolated from the tumor-draining lymph nodes are not tolerized and are capable of responding to secondary stimulation in vitro unlike T cells isolated from the TME, which are tolerized and incapable of proliferation or cytokine production 7,8. Interestingly, we have shown that changing the tumor microenvironment, such as providing CD4+ T helper cells via adoptive transfer, promotes CD8+ T cells to maintain pro-inflammatory effector functions 5. The results from each of the previously mentioned studies demonstrate the importance of measuring cellular responses from TME-infiltrating immune cells as opposed to cells that remain in the periphery. To study the function of immune cells which infiltrate tumors using the Miltenyi Biotech isolation system9, we have modified and optimized this antibody-based isolation procedure to obtain highly enriched populations of antigen presenting cells and tumor antigen-specific cytotoxic T lymphocytes. The protocol includes a detailed dissection of murine prostate tissue from a spontaneous prostate tumor model (TRansgenic Adenocarcinoma of the Mouse Prostate -TRAMP) 10 and a subcutaneous melanoma (B16) tumor model followed by subsequent purification of various leukocyte populations.

In this report, we describe a protocol for isolating highly purified populations of leukocytes that infiltrate tumors. This protocol is adapted from the Miltenyi Biotech protocol to enhance yield and purity for isolating cells from complex tumor tissue.

Tumors create a unique immunosuppressive microenvironment (tumor microenvironment, TME) whereby leukocytes are recruited into the tumor by various ...

Biology

Predictive Immune Modeling of Solid Tumors

Immunotherapies show promise in the treatment of oncology patients, but complex heterogeneity of the tumor microenvironment makes predicting treatment response challenging. The ability to resolve the relative populations of immune cells present in and around the tumor tissue has been shown to be clinically-relevant to understanding response, but is limited by traditional techniques such as flow cytometry and immunohistochemistry (IHC), due the large amount of tissue required, lack of accurate cell type markers, and many technical and logistical hurdles. One assay (e.g., the ImmunoPrism Immune Profiling Assay) overcomes these challenges by accommodating both small amounts of RNA and highly degraded RNA, common features of RNA extracted from clinically archived solid tumor tissue. The assay is accessed via a reagent kit and cloud-based informatics that provides an end-to-end quantitative, high-throughput immuno-profiling solution for Illumina sequencing platforms. Researchers start with as few as two sections of formalin-fixed paraffin-embedded (FFPE) tissue or 20-40 ng of total RNA (depending on sample quality), and the protocol generates an immune profile report quantifying eight immune cell types and ten immune escape genes, capturing a complete view of the tumor microenvironment. No additional bioinformatic analysis is required to make use of the resulting data. With the appropriate sample cohorts, the protocol may also be used to identify statistically significant biomarkers within a patient population of interest.

The use of an RNA-based approach to determine quantitative immune profiles of solid tumor tissues and leverage clinical cohorts for immune-oncology biomarker discovery is described through a molecular and informatics protocols.

Immunotherapies show promise in the treatment of oncology patients, but complex heterogeneity of the tumor microenvironment makes predicting treatment ...

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

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