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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Research

JoVE Journal

Cancer Research

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

A Murine Orthotopic Bladder Tumor Model and Tumor Detection System

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified to secrete human Prostate Specific Antigen (PSA), and the procedure for the confirmation of tumor implantation. In brief, mice are anesthetized using injectable drugs and are made to lay in the dorsal position. Urine is vacated from the bladder and 50 &#181;L of poly-L-lysine (PLL) is slowly instilled at a rate of 10 &#181;L/20 s using a 24 G IV catheter. It is left in the bladder for 20 min by stoppering the catheter. The catheter is removed and PLL is vacated by gentle pressure on the bladder. This is followed by instillation of the murine bladder cancer cell line (1 x 105 cells/50 &#181;L) at a rate of 10 &#181;L/20 s. The catheter is stoppered to prevent premature evacuation. After 1 h, the mice are revived with a reversal drug, and the bladder is vacated. The slow instillation rate is important, as it reduces vesico-ureteral reflux, which can cause tumors to occur in the upper urinary tract and in the kidneys. The cell line should be well re-suspended to reduce clumping of cells, as this can lead to uneven tumor sizes after implantation.
This technique induces tumors with high efficiency. Tumor growth is monitored by urinary PSA secretion. PSA marker monitoring is more reliable than ultrasound or fluorescence imaging for the detection of the presence of tumors in the bladder. Tumors in mice generally reach a maximum size that negatively impacts health by about 3 - 4 weeks if left untreated. By monitoring tumor growth, it is possible to differentiate mice that were cured from those that were not successfully implanted with tumors. With only end-point analysis, the latter may be mistakenly assumed to have been cured by therapy.

This protocol describes the generation of murine orthotopic bladder tumors in female C57BL/6J mice and the monitoring of tumor growth.

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified ...

Transplantation of Zebrafish Pediatric Brain Tumors into Immune-competent Hosts for Long-term Study of Tumor Cell Behavior and Drug Response

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to assess potential patient response to therapy. Current methods largely depend on using syngeneic or immune-compromised animals to avoid rejection of the tumor graft. Such methods require the use of specific genetic strains that often prevent the analysis of immune-tumor cell interactions and/or are limited to specific genetic backgrounds. An alternative method in zebrafish takes advantage of an incompletely developed immune system in the embryonic brain before 3 days, where tumor cells are transplanted for use in short-term assays (i.e., 3 to 10 days). However, these methods cause host lethality, which prevents the long-term study of tumor cell behavior and drug response. This protocol describes a simple and efficient method for the long-term orthotopic transplantation of zebrafish brain tumor tissue into the fourth ventricle of a 2-day-old immune-competent zebrafish. This method allows: 1) long-term study of tumor cell behaviors, such as invasion and dissemination; 2) durable tumor response to drugs; and 3) re-transplantation of tumors for the study of tumor evolution and/or the impact of different host genetic backgrounds. In summary, this technique allows cancer researchers to assess engraftment, invasion, and growth at distant sites, as well as to perform chemical screens and cell competition assays over many months. This protocol can be extended to studies of other tumor types and can be used to elucidate mechanisms of chemoresistance and metastasis.

The transplantation of cancer cells is an important tool for the identification of cancer mechanisms and therapeutic responses. Current techniques depend on immune-incompetent animals. Here, we describe a method to transplant zebrafish tumor cells into immune-competent embryos for the long-term analysis of tumor cell behavior and in vivo drug responses.

Tumor cell transplantation is an important technique to define the mechanisms controlling cancer cell growth, migration, and host response, as well as to ...

Target Cell Pre-enrichment and Whole Genome Amplification for Single Cell Downstream Characterization

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently developed method uses a medical wire functionalized with anti-epithelial cell adhesion molecule (EpCAM) antibodies for in vivo isolation of circulating tumor cells (CTCs)1. A patient-matched cohort in non-metastatic prostate cancer showed that the in vivo isolation technique resulted in a higher percentage of patients positive for CTCs as well as higher CTC counts as compared to the current gold standard in CTC enumeration. As cells cannot be recovered from current medical devices, a new functionalized wire (referred to as Device) was manufactured allowing capture and subsequent detachment of cells by enzymatic treatment. Cells are allowed to attach to the Device, visualized on a microscope and detached using enzymatic treatment. Recovered cells are cytocentrifuged onto membrane-coated slides and harvested individually by means of laser microdissection or micromanipulation. Single-cell samples are then subjected to single-cell whole genome amplification allowing multiple downstream analysis including screening and target-specific approaches. The procedure of isolation and recovery yields high quality DNA from single cells and does not impair subsequent whole genome amplification (WGA). A single cell&#39;s amplified DNA can be forwarded to screening and/or targeted analysis such as array comparative genome hybridization (array-CGH) or sequencing. The device allows ex vivo isolation from artificial rare cell samples (i.e. 500 target cells spiked into 5 mL of peripheral blood). Whereas detachment rates of cells are acceptable (50 - 90%), the recovery rate of detached cells onto slides spans a wide range dependent on the cell line used (&#60;10 - &#62;50%) and needs some further attention. This device is not cleared for the use in patients.

This protocol is to recover and prepare rare target cells from a mixture with non-target background cells for molecular genetic characterization at the single-cell level. DNA quality is equal to non-treated single cells and allows for single-cell application (both screening based and targeted analysis).

Rare target cells can be isolated from a high background of non-target cells using antibodies specific for surface proteins of target cells. A recently ...

A Method to Define the Effects of Environmental Enrichment on Colon Microbiome Biodiversity in a Mouse Colon Tumor Model

Several recent studies have illustrated the beneficial effects of living in an enriched environment on improving human disease. In mice, environmental enrichment (EE) reduces tumorigenesis by activating the mouse immune system, or affects tumor bearing animal survival by stimulating the wound repair response, including improved microbiome diversity, in the tumor microenvironment. Provided here is a detailed procedure to assess the effects of environmental enrichment on the biodiversity of the microbiome in a mouse colon tumor model. Precautions regarding animal breeding and considerations for animal genotype and mouse colony integration are described, all of which ultimately affect microbial biodiversity. Heeding these precautions may allow more uniform microbiome transmission, and consequently will alleviate non-treatment dependent effects that can confound study findings. Further, in this procedure, microbiota changes are characterized using 16S rDNA sequencing of DNA isolated from stool collected from the distal colon following long-term environmental enrichment. Gut microbiota imbalance is associated with the pathogenesis of inflammatory bowel disease and colon cancer, but also of obesity and diabetes among others. Importantly, this protocol for EE and microbiome analysis can be utilized to study the role of microbiome pathogenesis across a variety of diseases where robust mouse models exist that can recapitulate human disease.

Environmental Enrichment (EE) is an animal housing environment that is used to reveal mechanisms that underlie the connections between lifestyle, stress, and disease. This protocol describes a procedure that uses a mouse model of colon tumorigenesis and EE to specifically define alterations in microbiota biodiversity that may impact animal mortality.

Several recent studies have illustrated the beneficial effects of living in an enriched environment on improving human disease. In mice, environmental ...

Murine Model of Metastatic Liver Tumors in the Setting of Ischemia Reperfusion Injury

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce multiple tissue and organ damage. Despite recent advances and therapeutic approaches, the overall morbidity has remained unsatisfactory especially in patients with underlying parenchymal abnormalities. In the context of aggressive cancer growth and metastasis, surgical I/R is suspected to be the promoter regulating tumor recurrence. This article aims to describe a clinically relevant murine model of liver I/R and colorectal liver metastasis. In doing so, we aim to assist other investigators in establishing and perfecting this model for their routine research practice to better understand the effects of liver I/R on promoting liver metastases.

We describe in detail a clinically relevant colorectal cancer liver metastases (CRLM) tumor model and the influence of liver ischemia reperfusion (I/R) in tumor growth and metastasis. This model can help to better understand the mechanisms underlying surgery-induced promotion of liver metastatic growth.

Liver ischemia and reperfusion (I/R) injury, a common clinical challenge, remains an inevitable pathophysiological process that has been shown to induce ...

Establishment and Characterization of Small Bowel Neuroendocrine Tumor Spheroids

Small bowel neuroendocrine tumors (SBNETs) are rare cancers originating from enterochromaffin cells of the gut. Research in this field has been limited because very few patient derived SBNET cell lines have been generated. Well-differentiated SBNET cells are slow growing and are hard to propagate. The few cell lines that have been established are not readily available, and after time in culture may not continue to express characteristics of NET cells. Generating new cell lines could take many years since SBNET cells have a long doubling time and many enrichment steps are needed in order to eliminate the rapidly dividing cancer-associated fibroblasts. To overcome these limitations, we have developed a protocol to culture SBNET cells from surgically removed tumors as spheroids in extracellular matrix (ECM). The ECM forms a 3-dimensional matrix that encapsulates SBNET cells and mimics the tumor micro-environment for allowing SBNET cells to grow. Here, we characterized the growth rate of SBNET spheroids and described methods to identify SBNET markers using immunofluorescence microscopy and immunohistochemistry to confirm that the spheroids are neuroendocrine tumor cells. In addition, we used SBNET spheroids for testing the cytotoxicity of rapamycin.

Neuroendocrine tumors (NETs) originate from neuroendocrine cells of the neural crest. They are slow growing and challenging to culture. We present an alternative strategy to grow NETs from the small bowel by culturing them as spheroids. These spheroids have small bowel NET markers and can be used for drug testing.

Small bowel neuroendocrine tumors (SBNETs) are rare cancers originating from enterochromaffin cells of the gut. Research in this field has been limited ...

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 ...

Modeling Primary Bone Tumors and Bone Metastasis with Solid Tumor Graft Implantation into Bone

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These lesions compromise bone structure, increase the risk of pathologic fracture, and leave patients with limited treatment options. Primary bone tumors metastasize to distant organs, with some types capable of spreading to other skeletal sites. However, recent evidence suggests that with many solid tumors, cancer cells that have spread to bone may be the primary source of cells that ultimately metastasize to other organ systems. Most syngeneic or xenograft mouse models of primary bone tumors involve intra-osseous (orthotopic) injection of tumor cell suspensions. Some animal models of skeletal metastasis from solid tumors also depend on direct bone injection, while others attempt to recapitulate additional steps of the bone metastatic cascade by injecting cells intravascularly or into the organ of the primary tumor. However, none of these models develop bone metastasis reliably or with an incidence of 100%. In addition, direct intra-osseous injection of tumor cells has been shown to be associated with potential tumor embolization of the lung. These embolic tumor cells engraft but do not recapitulate the metastatic cascade. We reported a mouse model of osteosarcoma in which fresh or cryopreserved tumor fragments (consisting of tumor cells plus stroma) are implanted directly into the proximal tibia using a minimally invasive surgical technique. These animals developed reproducible engraftment, growth, and, over time, osteolysis and lung metastasis. This technique has the versatility to be used to model solid tumor bone metastasis and can readily employ grafts consisting of one or multiple cell types, genetically-modified cells, patient-derived xenografts, and/or labeled cells that can be tracked by optical or advanced imaging. Here, we demonstrate this technique, modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone.

Bone metastasis models do not develop metastasis uniformly or with a 100% incidence. Direct intra-osseous tumor cell injection can result in embolization of the lung. We present our technique modeling primary bone tumors and bone metastasis using solid tumor graft implantation into bone, leading to reproducible engraftment and growth.

Primary bone tumors or bone metastasis from solid tumors result in painful osteolytic, osteoblastic, or mixed osteolytic/osteoblastic lesions. These ...

Automated Dissection Protocol for Tumor Enrichment in Low Tumor Content Tissues

Tumor enrichment in low tumor content tissues, those below 20% tumor content depending on the method, is required to generate quality data reproducibly with many downstream assays such as next generation sequencing. Automated tissue dissection is a new methodology that automates and improves tumor enrichment in these common, low tumor content tissues by decreasing the user-dependent imprecision of traditional macro-dissection and time, cost, and expertise limitations of laser capture microdissection by using digital image annotation overlay onto unstained slides. Here, digital hematoxylin and eosin (H&#38;E) annotations are used to target small tumor areas using a blade that is 250 &#181;m2 in diameter in unstained formalin fixed paraffin embedded (FFPE) or fresh frozen sections up to 20 &#181;m in thickness for automated tumor enrichment prior to nucleic acid extraction and whole exome sequencing (WES). Automated dissection can harvest annotated regions in low tumor content tissues from single or multiple sections for nucleic acid extraction. It also allows for capture of extensive pre- and post-harvest collection metrics while improving accuracy, reproducibility, and increasing throughput with utilization of fewer slides. The described protocol enables digital annotation with automated dissection on animal and/or human FFPE or fresh frozen tissues with low tumor content and could also be used for any region of interest enrichment to boost adequacy for downstream sequencing applications in clinical or research workflows.

Digital annotation with automated tissue dissection provides an innovative approach to enriching tumor in low tumor content cases and is adaptable to both paraffin and frozen tissue types. The described workflow improves accuracy, reproducibility and throughput and could be applied to both research and clinical settings.

Tumor enrichment in low tumor content tissues, those below 20% tumor content depending on the method, is required to generate quality data reproducibly ...