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 tumors from the mice and ensure that the tumors are free of any contaminating tissue (i.e., hair, skin, peritoneum, etc.). Place each tumor in 1.8 mL of base RPMI-1640 media without fetal bovine serum (FBS) in a 24-well plate. For larger harvests, keep plates cold on ice to preserve cellular viability. 
	NOTE: If sterility is required, all tumor dissections will need to be performed in a biosafety cabinet with sterile surgical tools (i.e., scissors, forceps, etc.), as well as any further steps. Tumors with the longest diameter between 0.5 -&#160;1 cm are optimal, and larger or smaller tumors will require scaling of reagents.</li>
	<li>Use scissors to cut the tumors into less than 1 mm3 pieces within each well.</li>
	<li>Prepare a 10x digestion cocktail by dissolving collagenase I at 10 mg/mL, collagenase IV at 2,500 U/mL, and DNase I at 200 U/mL in base RPMI-1640 media (without FBS). 
	NOTE: The digestion cocktail can be prepared a day in advance and stored at -20 &#176;C; however, prolonged storage is not recommended as enzymes will lose activity over time once dissolved.</li>
	<li>Add 200 &#181;L of 10x dissociation cocktail containing collagenase I, collagenase IV, and DNase I to each well with the 1 mm3 pieces.</li>
	<li>Allow the samples to digest for 1 h at 37 &#176;C while lightly shaking at 60 - 100 rpm using an orbital plate shaker. 
	NOTE: The elevation of temperature to 37 &#176;C during the enzymatic digestion may cause immune cell activation.</li>
	<li>After 1 h, neutralize the reaction by adding 1 mL of RPMI-1640 media supplemented with 5% FBS and 2 mM EDTA to each well.</li>
	<li>Pipette the tumor digestion and the remaining tumor pieces into a 40 &#181;m cell strainer seated on top of a 50 mL centrifuge tube. 
	NOTE: Cut the tip off of a 1 mL pipette tip for easier transfer of the tumor digestion to the strainer.</li>
	<li>Use a 10 mL syringe plunger to mechanically disaggregate the tumor pieces through the strainer. Periodically rinse the strainer with 2 mL of supplemented RPMI-1640 media (containing 5% FBS) and repeat until the cell strainer is clear of tumor. 
	NOTE: Approximately 4 - 10 mL of total media should be sufficient to adequately rinse the strainer of tumor. 
	NOTE: Place samples on ice until this step is completed for all samples to preserve cellular viability.</li>
	<li>Adjust each 50 mL centrifuge tube to the same volume using the supplemented RPMI-1640 media. 
	NOTE: The adjustment of volume is critical for centrifuge balancing. Skipping this step could result in centrifuge damage or personal injury.</li>
	<li>Centrifuge the cell suspensions (5 min, 805 x g, 4 &#176;C), discard the supernatants, and re-suspend in 2 mL of supplemented RPMI-1640 media. 
	NOTE: A cell aggregate may form following the centrifugation that is difficult to resuspend. Use a 5 mL serological pipette and mix rapidly to break it up before proceeding.</li>
</ol>
2. Separation of Immune and Tumor Cellular Fractions
NOTE: The time required for this step is approximately 1 h.
<ol>
	<li>Add 3 mL of density gradient medium to the bottom of a 15 mL centrifuge tube. Prepare and label enough tubes for each tumor.</li>
	<li>Layer the 2 mL of tumor cell suspension on top of the density gradient medium by pipetting slowly down the side of the tube to prevent mixing of the two layers.</li>
	<li>Carefully place the layered tubes in the centrifuge and spin for 20 min at 805 x g, 20 &#176;C, with no brake. 
	NOTE: It is extremely important to verify proper balancing and ensure that no brake is used during centrifugation. Any disruption of this process will result in layer mixing and the full sample recovery will be difficult.</li>
	<li>Carefully transfer the tumor infiltrating leukocyte (TIL) layer and the top media layer to a new 15 mL tube using a transfer pipette. Discard all remaining density gradient medium and resuspend the tumor pellet at the bottom of the tube in 2 mL of supplemented RPMI-1640. 
	NOTE: After centrifugation there will be four visible layers from the top to bottom which respectively correspond to: media, TILs, density gradient medium, and tumor cell pellet. See Figure 2A for an example of the various layers.</li>
	<li>Centrifuge both the TIL and tumor samples in separate tubes (5 min, 805 x g, 4 &#176;C) and discard the supernatant.</li>
	<li>Resuspend the TIL sample in 200 &#181;L and the tumor pellet in 2 mL of supplemented RPMI-1640 media. Keep on ice to preserve viability.</li>
</ol>
3. Plating and Staining
Note: The time required for plating is 30 s/tumor; the time required for surface staining is 1 h; the time required for intracellular staining is 40 min; the time required for flow cytometry analysis is 1 - 4 min/tumor.
<ol>
	<li>Prepare and label a 96-well U-bottom plate for each immune staining panel and an additional plate for cell counts. 
	NOTE: Typically, three plates are prepared in total: a lymphocyte-focused panel plate, a myeloid-focused panel plate, and a cell count plate.</li>
	<li>Aliquot the single-cell suspensions into each of the staining panel plates and a set volume into the count plate. 
	NOTE: The count plate is used to calculate the total number of cells added to each staining panel, thereby allowing accurate cell population counts. A flow cytometer with 96-well plate sampling and volumetric analysis capabilities can provide the number of cells per &#181;L in each sample, and therefore, calculate the number of cells added to each staining panel. Count plates can be acquired the following day after fixation overnight at 4 &#176;C, rinsing with FACs buffer (phosphate buffered saline (PBS) with 2% FBS), and resuspending in a set volume of FACs buffer.</li>
	<li>Wash the cells twice with 200 &#181;L of Dulbecco&#39;s phosphate buffered saline (DPBS) per sample by centrifuging (5 min, 805 x g, 4 &#176;C) and discarding the supernatant between each rinse to remove any free FBS.</li>
	<li>Add 50 &#181;L of Fc block, mix the samples using a multi-channel pipette, and incubate for 20 min at 4 &#176;C.</li>
	<li>Add 50 &#181;L of 2x concentrated extracellular targeting antibody and fixable viability stain mixture, mix using a multi-channel pipette, and incubate for 30 min in the dark at RT or 4 &#176;C. 
	NOTE: The exact staining conditions for the antibody depend on the manufacturer&#39;s instructions. All staining dilutions must be in DPBS, with no added FBS to prevent saturation of the fixable viability dye. Please see the Table of Materials/Equipment for the optimal dilutions of the staining panel used in this manuscript. Antibody and fixable viability staining solution is prepared at a 2x concentration to provide a final 1x staining concentration in 100 &#181;L of total staining volume when added to the Fc block.</li>
	<li>Wash the samples twice with FACs buffer by centrifuging (5 min, 805 x g, 4 &#176;C) and discarding the supernatants between each rinse.</li>
	<li>Resuspend in 200 &#181;L of 1x fixation/permeabilization solution and incubate overnight at 4 &#176;C protected from light. 
	NOTE: The experiment can be paused overnight at this point: keep samples at 4 &#176;C protected from light.</li>
	<li>The following day, centrifuge the plates (5 min, 805 x g, 4 &#176;C) and discard the supernatant.</li>
	<li>Rinse once with 200 &#181;L of 1x permeabilization buffer, centrifuge (5 min, 805 x g, 4 &#176;C), and discard the supernatant.</li>
	<li>Add 100 &#181;L of 1x concentrated intracellular antibody staining panel prepared in 1x permeabilization buffer and incubate for 30 min in the dark at RT or 4 &#176;C (this depends on staining recommendations from the manufacturer).</li>
	<li>Rinse once with 1x permeabilization buffer (5 min, 805 x g, 4 &#176;C), discard the supernatant, and rinse a second time with FACs buffer (PBS with 2% FBS).</li>
	<li>Resuspend the samples in 300 &#181;L of FACs buffer (PBS with 2% FBS), transfer the samples to flow cytometry tubes, and perform flow cytometry analysis.</li>
</ol>

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The authors have nothing to disclose.

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.

<table><tbody><tr><td>6 to 12 week old male C57BL/6 mice</td><td>Jackson Laboratory&amp;nbsp;</td><td>N/A</td><td>Mice used in our tumor studies</td></tr><tr><td>MEER Murine Syngeneic Cancer Cell line</td><td>Acquired from collaborator&amp;nbsp;</td><td>N/A</td><td>E6/E7 HPV antigen expressing murine tonsillar epithelial cancer cell line&amp;nbsp;</td></tr><tr><td>70% isopropyl alcohol&amp;nbsp;</td><td>EMD Milipore&amp;nbsp;</td><td>PX1840</td><td></td></tr><tr><td>Murine surgical tool kit</td><td>World Precision Instruments</td><td>MOUSEKIT</td><td>Primarily only need scissors, tweezers, and a scalpel.&amp;nbsp;</td></tr><tr><td>24-well plate&amp;nbsp;</td><td>Denville Scientific Inc.</td><td>T1024</td><td>Or any 24-well flat bottom plate.&amp;nbsp;</td></tr><tr><td>RPMI-1640 media</td><td>Sigma-Aldrich</td><td>R0883</td><td></td></tr><tr><td>Collagenase I&amp;nbsp;</td><td>EMD Milipore</td><td>234153</td><td></td></tr><tr><td>Collagenase IV</td><td>Worthington Biochemical Corporation</td><td>LS004189</td><td></td></tr><tr><td>DNase I&amp;nbsp;</td><td>Sigma-Aldrich</td><td>11284932001</td><td></td></tr><tr><td>Low Speed Orbital Shaker</td><td>BioExpress (supplier: Genemate)&amp;nbsp;</td><td>S-3200-LS</td><td>Or any orbital shaker.&amp;nbsp;</td></tr><tr><td>Thermoregulating incubator</td><td>Fisher Scientific</td><td>13-255-27</td><td>Or any other thermo-regulated incubator</td></tr><tr><td>FBS</td><td>Sigma-Aldrich</td><td>F8192</td><td></td></tr><tr><td>EDTA</td><td>AMRESCO</td><td>E177</td><td></td></tr><tr><td>1 mL Pipette and tips</td><td>Eppendorf</td><td>13-690-032</td><td>Or any other pipette and tips</td></tr><tr><td>40 um Cell Strainers</td><td>Fisher Scientific</td><td>22363547</td><td></td></tr><tr><td>50 mL Centrifuge Tubes</td><td>Denville Scientific Inc.</td><td>C1062-P</td><td></td></tr><tr><td>15 mL Conical Tubes</td><td>Denville Scientific Inc.</td><td>C1012</td><td></td></tr><tr><td>10 mL Luer-Lok Syringe without needles</td><td>BD</td><td>309604</td><td></td></tr><tr><td>Lymphoprep Density Gradient Medium</td><td>STEMCELL Technologies</td><td>7811</td><td></td></tr><tr><td>Transfer Pipettes</td><td>Denville Scientific Inc.</td><td>P7212</td><td></td></tr><tr><td>96-well U-bottom plates</td><td>Denville Scientific Inc.</td><td></td><td></td></tr><tr><td>DPBS</td><td>Sigma Aldrich&amp;nbsp;</td><td>D8573</td><td></td></tr><tr><td>FoxP3 Transcription Factor Staining Buffer Set&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>00-5523-00</td><td>Fix/Permeabilization Buffer and Permeabilization Buffer</td></tr><tr><td>Thermoregulated Centrifuge</td><td>Eppendorf&amp;nbsp;</td><td>5810 R</td><td></td></tr><tr><td>Attune NxT Flow Cytometer</td><td>ThermoFisher Scientific</td><td>N/A</td><td>For 96-well cell volumetric counting&amp;nbsp;</td></tr><tr><td>Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block)&amp;nbsp;Clone 2.4G2&amp;nbsp;</td><td>ThermoFisher Scientific</td><td>553141</td><td>Fc Block</td></tr><tr><td>LIVE/DEAD Fixable Blue Dead Cell Stain Kit, for UV excitation</td><td>ThermoFisher Scientific</td><td>L34962</td><td>Fixable viability stain</td></tr><tr><td>CD45 Monoclonal Antibody (30-F11), APC-eFluor 780</td><td>ThermoFisher Scientific</td><td>47-0451-80</td><td></td></tr><tr><td>TCR beta Monoclonal Antibody (H57-597), APC</td><td>ThermoFisher Scientific</td><td>17-5961-82</td><td></td></tr><tr><td>CD8-&amp;alpha; Antibody (KT15), PE</td><td>Santa Cruz Biotechnology</td><td>sc-53473&amp;nbsp;</td><td></td></tr><tr><td>CD4 Monoclonal Antibody (GK1.5), PE-Cyanine5</td><td>ThermoFisher Scientific</td><td>15-0041-81</td><td></td></tr><tr><td>MHC Class II (I-A/I-E) Monoclonal Antibody (M5/114.15.2), Alexa Fluor 700</td><td>ThermoFisher Scientific</td><td>56-5321-82</td><td></td></tr><tr><td>CD11c Monoclonal Antibody (N418), PE-Cyanine5</td><td>ThermoFisher Scientific</td><td>15-0114-82</td><td></td></tr><tr><td>F4/80 Monoclonal Antibody (BM8), eFluor 450</td><td>ThermoFisher Scientific</td><td>48-4801-80</td><td></td></tr><tr><td>CD11b Monoclonal Antibody (M1/70), APC</td><td>ThermoFisher Scientific</td><td>17-0112-81</td><td></td></tr><tr><td>Ly-6G (Gr-1) Monoclonal Antibody (RB6-8C5), PE</td><td>ThermoFisher Scientific</td><td>12-5931-82</td><td></td></tr><tr><td>CD274 (PD-L1, B7-H1) Monoclonal Antibody (MIH5), PE-Cyanine7</td><td>ThermoFisher Scientific</td><td>25-5982-80</td><td></td></tr><tr><td>CD273 (B7-DC) Monoclonal Antibody (122), PerCP-eFluor 710</td><td>ThermoFisher Scientific</td><td>46-9972-82</td><td></td></tr><tr><td>Ki-67 Monoclonal Antibody (B56), BV711</td><td>BD Biosciences</td><td>563755</td><td></td></tr></tbody></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...

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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 ...

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Cancer Research

9.5K Views.  Baylor College of Medicine. Hier beschrijven we een methode om te scheiden en verrijken van de componenten van de tumor immuun en niet-immuun communicatie in gevestigde subcutane tumoren. Deze techniek maakt het mogelijk voor de afzonderlijke analyse van tumor immuun infiltreren en niet-immuun tumor breuken die uitgebreide karakterisering van de tumor immuun communicatie kunnen toestaan.

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

A Mimic of the Tumor Microenvironment: A Simple Method for Generating Enriched Cell Populations and Investigating Intercellular Communication

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events leading to stromal activation and establishment of the tumor microenvironment (TME). Several in vitro and in vivo models of the TME have been developed; however, in general these models do not readily permit isolation of individual cell populations, under non-perturbing conditions, for further study. To circumvent this difficulty, we have employed an in vitro TME model using a cell growth substrate consisting of a permeable microporous membrane insert that permits simple generation of highly enriched cell populations grown intimately, yet separately, on either side of the insert&#39;s membrane for extended co-culture times. Through use of this model, we are capable of generating greatly enriched cancer-associated fibroblast (CAF) populations from normal diploid human fibroblasts following co-culture (120 hr) with highly metastatic human breast carcinoma cells, without the use of fluorescent tagging and/or cell sorting. Additionally, by modulating the pore-size of the insert, we can control for the mode of intercellular communication (e.g., gap-junction communication, secreted factors) between the two heterotypic cell populations, which permits investigation of the mechanisms underlying the development of the TME, including the role of gap-junction permeability. This model serves as a valuable tool in enhancing our understanding of the initial events leading to cancer-stroma initiation, the early evolution of the TME, and the modulating effect of the stroma on the responses of cancer cells to therapeutic agents.

We adapted a permeable microporous membrane insert to mimic the tumor microenvironment (TME). The model consists of a mixed cell culture, allows simplified generation of highly enriched individual cell populations without using fluorescent tagging or cell sorting, and permits studying intercellular communication within the TME under normal or stress conditions.

Understanding the early heterotypic interactions between cancer cells and the surrounding non-cancerous stroma is important in elucidating the events ...

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 ...

Live Imaging of Drug Responses in the Tumor Microenvironment in Mouse Models of Breast Cancer

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional co-culture systems are frequently used to explicate tumor-stroma interactions, including their role in drug responses. However, many of the interactions that occur in vivo in the intact microenvironment cannot be completely replicated in these in vitro settings. Thus, direct visualization of these processes in real-time has become an important tool in understanding tumor responses to therapies and identifying the interactions between cancer cells and the stroma that can influence these responses. Here we provide a method for using spinning disk confocal microscopy of live, anesthetized mice to directly observe drug distribution, cancer cell responses and changes in tumor-stroma interactions following administration of systemic therapy in breast cancer models. We describe procedures for labeling different tumor components, treatment of animals for observing therapeutic responses, and the surgical procedure for exposing tumor tissues for imaging up to 40 hours. The results obtained from this protocol are time-lapse movies, in which such processes as drug infiltration, cancer cell death and stromal cell migration can be evaluated using image analysis software.

We describe a method for imaging response to anti-cancer treatment in vivo and at single cell resolution.

The tumor microenvironment plays a pivotal role in tumor initiation, progression, metastasis, and the response to anti-cancer therapies. Three-dimensional ...

Medicine

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 ...

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 ...

Testing Cancer Immunotherapeutics in a Humanized Mouse Model Bearing Human Tumors

Reversing the immunosuppressive nature of the tumor microenvironment is critical for the successful treatment of cancers with immunotherapy drugs. Murine cancer models are extremely limited in their diversity and suffer from poor translation to the clinic. To serve as a more physiological preclinical model for immunotherapy studies, this protocol has been developed to evaluate the treatment of human tumors in a mouse reconstituted with a human immune system. This unique protocol demonstrates the development of human immune system (HIS, &#34;humanized&#34;) mice, followed by implantation of a human tumor, either a cell-line derived xenograft (CDX) or a patient derived xenograft (PDX). HIS mice are generated by injecting CD34+ human hematopoietic stem cells isolated from umbilical cord blood into neonatal BRGS (BALB/c Rag2-/- IL2R&#947;C-/- NODSIRP&#945;) highly immunodeficient mice that are also capable of accepting a xenogeneic tumor. The importance of the kinetics and characteristics of the human immune system development and tumor implantation is emphasized. Finally, an in-depth evaluation of the tumor microenvironment using flow cytometry is described. In numerous studies using this protocol, it was found that the tumor microenvironment of individual tumors is recapitulated in HIS-PDX mice; &#34;hot&#34; tumors exhibit large immune infiltration while &#34;cold&#34; tumors do not. This model serves as a testing ground for combination immunotherapies for a wide range of human tumors and represents an important tool in the quest for personalized medicine.

This protocol outlines the generation of human immune system (HIS) mice for immuno-oncology studies. Instructions and considerations in the use of this model for testing human immunotherapeutics on human tumors implanted in this model are presented with an emphasis on characterizing the response of the human immune system to the tumor.

Reversing the immunosuppressive nature of the tumor microenvironment is critical for the successful treatment of cancers with immunotherapy drugs. Murine ...

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.

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 ...

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

Microfluidic Co-Culture Models for Dissecting the Immune Response in in vitro Tumor Microenvironments

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise invisible processes. Advanced cell assays closely mimicking in vivo scenery are establishing themselves as novel ways to visualize and measure the bidirectional tumor-host interplay influencing the progression of cancer. Here we describe two versatile protocols to recreate highly controllable 2D and 3D co-cultures in microdevices, mimicking the complexity of the tumor microenvironment (TME), under natural and therapy-induced immunosurveillance. In section 1, an experimental setting is provided to monitor crosstalk between adherent tumor cells and floating immune populations, by bright field time-lapse microscopy. As an applicative scenario, we analyze the effects of anti-cancer treatments, such as the so-called immunogenic cancer cell death inducers on the recruitment and activation of immune cells. In section 2, 3D tumor-immune microenvironments are assembled in a competitive layout. Differential immune infiltration is monitored by fluorescence snapshots up to 72 h, to evaluate combination therapeutic strategies. In both settings, image processing steps are illustrated to extract a plethora of immune cell parameters (e.g., immune cell migration and interaction, response to therapeutic agents). These simple and powerful methods can be further tailored to simulate the complexity of the TME encompassing the heterogeneity and plasticity of cancer, stromal and immune cells subtypes, as well as their reciprocal interactions as drivers of cancer evolution. The compliance of these rapidly evolving technologies with live-cell high-content imaging can lead to the generation of large informative datasets, bringing forth new challenges. Indeed, the triangle &#39;&#39;co-cultures/microscopy/advanced data analysis&#34; sets the path towards a precise problem parametrization that may assist tailor-made therapeutic protocols. We expect that future integration of cancer-immune on-a-chip with artificial intelligence for high-throughput processing will synergize a large step forward in leveraging the capabilities as predictive and preclinical tools for precision and personalized oncology.

In the age of immunotherapy and single-cell genomic profiling, cancer biology requires novel in vitro and computational tools for investigating the tumor-immune interface in a proper spatiotemporal context. We describe protocols to exploit tumor-immune microfluidic co-cultures in 2D and 3D settings, compatible with dynamic, multiparametric monitoring of cellular functions.

Complex disease models demand cutting-edge tools able to deliver physiologically and pathologically relevant, actionable insights, and unveil otherwise ...

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

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