Name: enrichment and characterization of the tumor immune and non-immune microenvironments in established subcutaneous murine tumors
Uploaded: 2018-06-07T14:45:00
Description: Watch this Scientific Journal Video about Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors at JoVE.com

JMN acknowledges financial support from National Institute of General Medical Sciences (T32GM088129) and the National Institute of Dental &amp; Craniofacial Research (F31DE026682) both of the National Institutes of Health. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. This project was also supported by the Cytometry and Cell Sorting Core at Baylor College of Medicine with funding from the NIH (P30 AI036211, P30 CA125123, and S10 RR024574) and the expert assistance of Joel M. Sederstrom.

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Baylor College of Medicine.
1. Tumor Harvest and Digestion
NOTE: The time required for harvest is ~3 - 5 min/tumor, and the time required for processing is ~1 h + 2 - 3 min/tumor.
<ol>
	<li>Euthanize mice by carbon dioxide inhalation and spray each mouse down with 70% ethanol before harvesting to prevent hair contamination. Surgically remove subcutaneous tum...

<ol>
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The TIME is composed of diverse and complex cellular components and molecules. Recent evidence suggests that accurate characterization of this environment can provide a better understanding of treatment success or failure, and can even help identify mechanisms of therapeutic resistance. For example, increasing intratumoral levels of various immunosuppressive cells (i.e., MDSCs, T regulatory cells, etc.) can mitigate effector immune responses and abrogate immunotherapeutic effects. On the other hand, the...

The suppressive TIME has recently been recognized as a hallmark of cancer, and is known to play a significant role in the development, progression, and protection of solid tumor cancers as well as mitigate their susceptibility to immunotherapy1. The TIME is composed of numerous cellular subsets and phenotypes, all of which provide critical insight into the immunologic state of the tumor. These immunologic subsets can be further stratified into cells of lymphocyte or myeloid origin, which together constitute the majority of the innate and adaptive immune responses2,3. Recent advances in the field of cancer immunotherapy have demonstrated that immunotherapeutic strategies (i.e., immune checkpoint inhibitors, chimeric antigen receptor T-cells, etc.) have the potential to induce durable cancer regressions; however, they remain relatively ineffective in solid tumor cancers4,5. Numerous groups have shown the critical hurdle that the TIME can play on treatment success6,7, and thus, there remains a need to accurately evaluate new immunotherapies in the pre-clinical setting specifically focusing on their ability to modulate or overcome the TIME8.
Current efforts to characterize the TIME typically utilize either microscopy or flow cytometry along with antibody labeling strategies to identify immune cellular subsets and their features9,10. These two strategies provide uniquely different information, as microscopy allows spatial appreciation of cellular subsets and flow cytometry provides high throughput and broader quantification of cellular changes. Despite the recent improvements in fluorescent multiplex immunohistochemistry optimizing spectral imaging systems, which now can support up to 7 parameters, limited panel size makes broad level immune profiling difficult and thus this technique is often reserved for more focused analyses. As a result, flow cytometry remains one of the most widely used immune profiling techniques. Despite its widespread use in TIME characterization, the methods used for processing and staining are quite variable. Most often protocols utilize a tumor dissociating enzyme (i.e., collagenases, DNase, etc.) and manual dissociation methods to achieve single-cell suspensions, followed by antibody staining and analysis7. Despite the benefits of each method, numerous groups have shown the extensive variability that can be induced through these techniques11. This makes cross-study comparisons of tumor microenvironment profiling extremely difficult, even when assessing the same murine tumor model. Furthermore, these methods provide limited potential to assess tumor cellular and tumor immune infiltrate components independently, since both components are interspersed after digestion. Sample quantity then limits multi-panel staining and analysis, which becomes a major issue when attempting to characterize rare immunologic subsets (i.e., tumor-specific T-cells)12. More recent techniques such as mass cytometry, or cytometry by time of flight (CyTOF), allow high-dimensional phenotypic analysis of cellular subsets with some systems supporting panel designs of greater than 42 independent parameters13. Despite the tremendous power of CyTOF technology in immune profiling, it remains limited because of the expense, analysis expertise, and the access to the equipment. In addition, many CyTOF protocols recommend purification of immune subsets to improve signal-to-noise ratios14, and thus we suggest that our enrichment method could be used upstream of CyTOF analysis to improve data quality.
Herein we describe a tumor microenvironment digestion and analysis method that incorporates tumor immune infiltrate separation. The purpose of this method is to allow independent high-throughput profiling of the tumor immune infiltrate and tumor cellular fractions for broader characterization of the tumor microenvironment. Using this method, we further demonstrate the importance of performing whole tumor analysis, as a subcutaneous solid tumor model was found to have significant spatial immunologic heterogeneity. Overall, this method can more accurately and consistently compare between samples because it enriches for immune cellular subsets within the tumor and allows for independent profiling of tumor cell and immune fractions of a tumor.

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			<td height="41" style="height:41px;width:216px;">6 to 12 week old male C57BL/6 mice</td>
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			<td height="81" style="height:81px;width:216px;">MEER Murine Syngeneic Cancer Cell line</td>
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			<td style="width:113px;">ThermoFisher Scientific</td>
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			<td style="width:113px;">BD Biosciences</td>
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</table>

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.

Our results demonstrate the significant benefit of TIL separation from non-immune tumor components, as explained in the protocol. Additionally, using the described method we demonstrate the significant immunologic heterogeneity of established solid tumors.
A significant problem with many tumor dissociation techniques is the loss of sample viability, most often as a result of harsh digestion conditions (i.e., elevated te...

Watch this Scientific Journal Video about Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors at JoVE.com

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

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 ...

The overall goal of this technique is to provide a method for simultaneous profiling of both the immune and cancer cell fractions contributing to the immune microenvironment of solid tumors. This method can help answer key questions in the immune oncology fields, such as what immune and non-immune serial changes occur within the tumor following a given treatment. The main advantage of this technique is that it facilitates the effective enrichment of tumor immune and non-immune components, allowing a thorough profiling of the tumor immunologic state.The overall goal of this method is to efficiently profile the tumor immune microenvironment and identify treatment induced changes. After spraying the mouse with 70%ethanol to prevent hair contamination, surgically remove the subcutaneous tumors, taking care that the tumors are free of any contaminating tissue. Then, place each tumor in 1.8 milliliters of base RPMI 1640 medium without fetal bovine serum in individual wells of a 24-well plate on ice.When all of the tumors have been collected, use scissors to cut the samples into less than one cubic millimeter pieces and add 200 microliters of freshly prepared 10X dissociation cocktail containing collagenase-1, collagenase-4, and DNase one to each well. After one hour at 37 degrees Celsius, in an orbital plate shaker at 60 to 100 RPM, neutralize the reaction with one milliliter of RPMI 1640 medium supplemented with 5%FBS and two millimolar EDTA, and use a modified one milliliter pipette tip to transfer each tumor slurry sample into individual 50 milliliter conical tubes topped with individual 40 micrometer strainers. Using a 10 milliliter syringe plunger, mechanically disaggregate the tumor pieces through the strainers, periodically rinsing each strainer with two milliliters of RPMI 1640 medium plus 5%FBS until the strainers are clear of tumor.When all of the tumors have been dissociated, add medium to each tube as necessary to bring all of the samples up to the same volume and pellet the cells by centrifugation. Then, use a five milliliter serological pipette to resuspend the pellets in two milliliters of FBS supplemented medium per tube. To separate the immune and tumor cell fractions, add three milliliters of density gradient medium to the bottom of one 15 milliliter conical tube per sample and slowly layer two milliliters of tumor cell suspension down the side of each tube onto each gradient.Take care to ensure that the tumor cell suspensions are mixed well before layering, that the cell suspensions are slowly pipette down the side of the tubes, and that the tubes are handled carefully to prevent mixing of the layers. Separate the cells by density gradient centrifugation and carefully collect the top medium and tumor infiltrating leukocyte layers into one new 15 milliliter tube per sample. Discard the remaining density gradient media and resuspend the tumor pellets in two milliliters of medium plus FBS per tube.Then, centrifuge all of the samples and resuspend the tumor infiltrating leukocytes in 200 microliters and the tumor cells in two milliliters of medium plus FBS per tube on ice. To plate the cells, label one 96-well U-bottom plate for each immune staining panel plus an additional plate for cell counts and aliquot the single cell suspensions into each of the staining panel plates and the calc plate. Wash the cells two times with 200 microliters of Dulbecco's PBS per sample and block the cells with 50 microliters of FC block per well for 20 minutes at four degrees Celsius.At the end of the blocking incubation, add 50 microliters of 2X concentrated extracellular targeting antibody and fixable viability stain mixture to each well for a 30 minute incubation in the dark. At the end of the staining incubation, wash the samples two times with FACS buffer and resuspend the pellets in 200 microliters of fixation and permeabilization solution overnight at four degrees Celsius, protected from light. The next day, collect the cells by centrifugation, followed by a wash with 200 microliters of permeabilization buffer per well.Label the cells with 100 microliters of intracellular antibody staining panel in permeabilization buffer for a 30 minute incubation in the dark, followed by another permeabilization buffer wash. Then, wash the cells again with FACS buffer and resuspend the samples in 300 microliters of fresh FACS buffer for their analysis by flow cytometry. Tumor digestion, as demonstrated, with or without density gradient medium enrichment, results in an approximately 70 to 90%viable whole tumor cell population as assessed by flow cytometry.Density gradient medium enrichment promotes a greater than two-fold increase in the CD45 positive tumor infiltrating leukocyte fraction, compared to non-enriched tumor digestions, as well as numerous immune cell subsets commonly observed in immune microenvironment studies. The CD45 negative tumor cell fraction can also be profiled for numerous tumor features, such as overall tumor viability or apoptosis, proliferation capacity, and the expression of various immunosuppressive markers. To determine how myeloid and lymphocyte immunologic subsets vary throughout a single solid tumor, the tumor can be divided into four equal parts in such a way that each section contains approximately equal intratumoral regions, as well as equal dermal and peritoneal facing portions.Each piece can then be digested separately and analyzed, as demonstrated, for the presence of various immune cell populations. Once mastered, this technique can be completed in four to 8 hours depending on how many samples you have. While attempting this procedure, it's important to work quickly but efficiently to preserve cell viability.Visual demonstration of this method is critical, as the dissociation and separation steps involve techniques that are difficult to learn by written text alone. Following this procedure, flow cytometry can be performed to answer additional questions about how cellular populations change within a tumor, both in terms of abundance and phenotype. After watching this video, you should have a good understanding of how to separate and analyze both the immune and non-immune components of an established subcutaneous murine tumor using the demonstrated digestion and density gradient separation techniques.

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