This work was supported by National Institutes of Health/National Institute of Neurological Disorders &#38; Stroke (NIH/NINDS) grants R01-NS074387, R01-NS057711 and R21-NS091555 to M.G.C.; NIH/NINDS grants R01-NS061107, R01-NS076991, R01-NS082311 and R21-NS084275 to P.R.L.; grants from Leah&#8217;s Happy Hearts, University of Michigan Comprehensive Cancer Center awarded to M.G.C. and P.R.L; the Department of Neurosurgery, University of Michigan School of Medicine; the Michigan Institute for Clinical and Health Research, supported by NIH grant 2UL1-TR000433; the University of Michigan Cancer Biology Training Grant supported by NIH/NCI (National Cancer Institute) grant T32-CA009676; the University of Michigan Training in Clinical and Basic Neuroscience supported by NIH/NINDS grant T32-NS007222; and the University of Michigan Medical Scientist Training Program supported by NIH/NIGMS (National Institute of General Medicine Sciences) grant T32-GM007863. The authors are thankful for the academic leadership and support received from Dr. Karin Muraszko and the Department of Neurosurgery; to M. Dahlgren, D. Tomford, and S. Napolitan for superb administrative support; to M. Dzaman for outstanding technical assistance; and to Phil F. Jenkins for generous support toward the purchase of a Zeiss 3D Scanning Electron Microscope. We also acknowledge the Kuchroo laboratory at Harvard Medical School from which a modified version of the density centrifugation media-mediated strategy for isolation of brain mononuclear cells was drawn.

<ol>
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The authors have nothing to disclose.

This protocol describes a robust and reproducible method for the isolation and flow cytometric analysis of PBMCs that have infiltrated the early mouse brain tumor microenvironment. Glioma cell suspensions are generated at a concentration specified by the experimentalist that are stereotactically engrafted into the striatum of the mouse brain. Mice are then euthanized at predetermined time points specified by the experimental design and their brains are harvested and processed to isolate glioma-infiltrating PBMCs which ar...

Gliomas are a class of neuroepithelial brain cancers arising from transformed glia within the central nervous system (CNS). Of all the gliomas, World Health Organization (WHO) grade IV glioma, or glioblastoma (GBM), is the most common and lethal1. GBM is highly refractory to the current standard-of-care which consists of tumor resection to the extent possible followed by radiation plus concomitant and adjuvant chemotherapy with temozolomide2. These deadly cancers carry a dismal prognosis of only 15-18 months of survival from the time of initial diagnosis with only 5% of patients surviving the disease after 5 years3.
The presence of the blood brain barrier (BBB), lack of professional antigen presenting cells (APCs), and the previously unidentified existence of bona fide lymphatic structures within the brain4 have led to the notion of GBM as immune privileged. However, numerous studies now show that these brain cancers indeed engender the recruitment of peripheral immune cells that are predominantly myeloid in origin which include monocytes, macrophages, and myeloid-derived suppressor cells (MDSCs)5. GBM also influences the activity of brain-resident microglia to become pro-tumorigenic6,7. Lymphoid cells such as CD8+ T cells8 and CD56+&#160;natural killer&#160;cells9 are also present within the tumor microenvironment, but in much fewer numbers, a fact thought to be due to immunosuppressive function instigated by glioma-derived factors on tumor associated macrophages (TAMs)10. CD4+ T cells are also present in GBM, but much of this population also expresses CD25 and FoxP3, makers of immunosuppressive T regulatory (Treg) cells11. The overall immunosuppressive state of GBM culminates in the promotion of immunologic escape and tumor progression12.
A better understanding of the mechanisms of GBM immunosuppression is critical to the development of effective immunotherapeutic strategies designed to stimulate the immune system against the tumor. Over the last 15 years our lab has worked to overcome the mechanisms of brain tumor immunosuppresson in order to develop efficacious new anti-GBM immunotherapeutics13-19. The culmination of this work has now led to a clinical trial designed to evaluate a combined cytotoxic and immune-stimulatory therapeutic for patients with newly diagnosed GBM (ClinicalTrials.gov Identifier: NCT01811992).
Our most recent work shows that mouse GL26 and rat CNS-1 GBM cells block anti-tumor NK cell immune surveillance by producing large amounts of the &#946;-galactoside-binding lectin galectin-1 (gal-1)20. This was demonstrated by suppressing the expression of gal-1 in glioma cells using shRNA-mediated gene knockdown. In vitro experiments showed that gal-1-deficient glioma cells proliferated normally in culture, yet underwent rapid rejection soon after intracranial engraftment into syngeneic C57BL/6J or RAG1-/- mice, thus establishing the independence of T- or B- cells on this form of tumor rejection. NK cell immunodepletion with anti-asialo GM1 anti-serum or monoclonal NK1.1 antibodies led to the complete restoration of intracranial gal-1-deficient glioma growth, establishing the role of NK cells in gal-1-deficient glioma rejection. We now show that immunodepletion of Gr-1+/CD11b+ myeloid cells is sufficient to prevent gal-1-deficient glioma rejection despite the presence of NK cells, thus revealing a indispensible auxiliary role for myeloid cells in the aiding of NK-mediated gal-1-deficient tumor lysis (unpublished data). This unexpected result has led us to develop a comprehensive protocol for the isolation and analysis of peripheral blood mononuclear cells (PBMCs) that infiltrate the brain tumor microenvironment soon after intracranial engraftment so that we may better characterize the immune infiltration events that predicate gal-1-deficient glioma rejection.
The method is demonstrated here by using mouse GL26 glioma cells that constitutively express mCitrine fluorescent protein, called GL26-Cit, which permit direct tumor cell visualization by fluorescence microscopy21. These cells are stereotactically engrafted into the brain of syngeneic C57BL/6J mice and are allowed to grow for 24, 48, or 72 hr prior to mouse euthanasia. Glioma-infiltrating PBMCs are then isolated and immunolabeled using anti -CD45, -Gr-1, -CD11b and -NK1.1 cell surface antibodies together with intracellular immunolabeling for granzyme B (GzmB). This specific combination of antibodies allows for the identification of tumor-infiltrating Gr-1+/CD11b+ myeloid cells and NK1.1+, NK cells, cell types we&#160;have been implicated in gal-1-deficient tumor rejection. The immune infiltration profile of gal-1-deficient GL26-Cit glioma, referred to here as GL26-Cit-gal1i, is then compared to that of gliomas expressing normal levels of gal-1 called GL26-Cit-NT that contain a non-targeting control shRNA hairpin. The protocol begins with a description on how to culture GL26-Cit glioma cells in vitro, which is followed by an explanation on how to orthotopically engraft these cells into the striatum of syngeneic C57BL/6J mice. It then proceeds to enumerate the steps involved in the isolation and immunolabeling of glioma-infiltrating PBMCs for flow cytometric analysis. The protocol concludes with an explanation of standard data analysis and graphical representation.
The demonstration reveals that both Gr-1+/CD11b+ myeloid cells and NK1.1+ NK cells preferentially accumulate within the gal-1-deficient brain tumor microenvironment within 48 hr of tumor implantation, a result which helps explain why these tumors rapidly undergo complete tumor lysis approximately 1 week post-tumor engraftment20. The method is easily adaptable to a number of different in vivo experimental designs in which temporal data on immune infiltration into the brain is required. A single experimentalist can perform the protocol from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in about 4-6 hr depending on the number of samples to be analyzed. The method may also be combined with experiments aimed to characterize the profile of circulating PBMCs in tumor bearing mice for comparison with those that infiltrate the brain so to identify immunosuppression phenotypes specifically induced by the tumor microenvironment. Application of this and similar methods should facilitate a better understanding of the factors involved in the trafficking of peripheral immune cells into the brain tumor microenvironment.

<table><tbody><tr><td>Dulbecco&amp;rsquo;s Modification of Eagles Medium</td><td>Gibco</td><td>12430-054</td><td>with 4.5g/L D-glucose, L-glutamine, 25 mM HEPES and phenol red&amp;nbsp;</td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate-buffered Saline&amp;nbsp;</td><td>Gibco</td><td>14190-144&amp;nbsp;</td><td>without calcium chloride or Magnesium chloride</td></tr><tr><td>Fetal bovine serum</td><td>Omega Scientific Inc.</td><td>FB-11</td><td></td></tr><tr><td>Penicillin Streptomycin&amp;nbsp;</td><td>Gibco</td><td>15140-122</td><td>10,000 U/ml Penicillin; 10,000 &amp;mu;g/ml Streptomycin&amp;nbsp;</td></tr><tr><td>L-glutamine</td><td>Gibco</td><td>25030-081</td><td>200 mM</td></tr><tr><td>G418 sulfate&amp;nbsp;</td><td>Omega Scientific Inc.</td><td>GN-04</td><td></td></tr><tr><td>Puromycin dihydrochloride&amp;nbsp;&amp;nbsp;</td><td>Sigma-Aldrich</td><td>P8833</td><td>from Streptomyces alboniger</td></tr><tr><td>HyClone HyQTase non-mammalian trypsin alternative&amp;nbsp;</td><td>Thermo Scientific</td><td>SV30030.01</td><td>in DPBS with EDTA&amp;nbsp;</td></tr><tr><td>Phase contrast hemocytometer</td><td>Sigma-Aldrich</td><td>Z359629</td><td>0.1 mm deep</td></tr><tr><td>Trypan blue stain</td><td>Gibco</td><td>15250-061</td><td></td></tr><tr><td>Sterile 0.9% NaCl injection, USP</td><td>Hospira</td><td>NDC: 0409-4888</td><td>10 ml vials</td></tr><tr><td>0.6 ml conical polypropylenemi microtubes</td><td>Genesee Scientific</td><td>22-272</td><td></td></tr><tr><td>Atipamezole hydrochloride injection</td><td>Orion Pharma</td><td>NDC: 52483-6298</td><td>5 mg/ml solution</td></tr><tr><td>Ketamine hydrochloride injection</td><td>Fort Dodge</td><td>NDC: 0409-2051</td><td>100 mg/ml solution</td></tr><tr><td>Dexmedetomidine hydrochloride injection</td><td>Zoetis</td><td>NDC: 54771-2805</td><td>0.5 mg/ml solution</td></tr><tr><td>Xylazine hydrochloride injection</td><td>Lloyd</td><td>NDC: 61311-481</td><td>100 mg/ml solution</td></tr><tr><td>Carprofen injection</td><td>Pfizer</td><td>NDC: 61106-8501</td><td>50 mg/ml solution</td></tr><tr><td>Buprenorphine hydrochloride injection</td><td>Reckitt Benckiser</td><td>NDC: 12496-0757</td><td>0.3 mg/ml ampuls</td></tr><tr><td>1 ml tuberculin syringes&amp;nbsp;</td><td>Covidien</td><td>8881501400</td><td></td></tr><tr><td>26G x &amp;frac12; (0.45 mm x 13 mm) syringe needles&amp;nbsp;</td><td>BD</td><td>305111</td><td></td></tr><tr><td>Surgical clippers</td><td>3M</td><td>9661</td><td></td></tr><tr><td>Providone-iodine solution</td><td>Aplicare</td><td>82-217</td><td>NDC: 52380-1905</td></tr><tr><td>Sterile petrolatum ophthalmic ointment&amp;nbsp;</td><td>Dechra</td><td>NDC: 17033-211</td><td></td></tr><tr><td>70% isopropyl alcohol prep pads</td><td>Kendall</td><td>6818</td><td></td></tr><tr><td>Sterile gauze</td><td>Covidien</td><td>8044</td><td>non-woven, 4&quot; x 4&quot;, 4-ply&amp;nbsp;</td></tr><tr><td>1.7 ml conical polypropylene microtubes</td><td>Genesee Scientific</td><td>22-281</td><td></td></tr><tr><td>Mouse stereotactic frame</td><td>Stoelting</td><td>51730</td><td></td></tr><tr><td>Surgical lamp</td><td>Philips Burton</td><td>CS316W</td><td>Coolspot II Variable Spotlight</td></tr><tr><td>Curved dissecting forceps</td><td>Ted Pella Inc.</td><td>5431</td><td></td></tr><tr><td>Colibri retractors&amp;nbsp;</td><td>FST</td><td>17000-03</td><td></td></tr><tr><td>Cordless precision power drill</td><td>Dremel&amp;nbsp;</td><td>1100-01</td><td>Stylus model; 7.2-Volt Lithium-Ion with Docking Station</td></tr><tr><td>Engraving Cutter</td><td>Dremel</td><td>105</td><td>1/32&quot; or 0.8 mm bit diameter</td></tr><tr><td>Microliter syringe</td><td>Hamilton</td><td>75</td><td>Hamilton Microliter Syringes 700 series; 5 &amp;mu;l volume</td></tr><tr><td>Microliter syringe needles</td><td>Hamilton</td><td>7762-06</td><td>33 G, small hub RN NDL, 1.5 in, point style 3, 6/PK</td></tr><tr><td>Ethilon 3-0 black monofilament nylon sutures&amp;nbsp;</td><td>Ethicon</td><td>1663</td><td></td></tr><tr><td>Magnetic stir bar</td><td>VWR</td><td>74950-296</td><td>7.9 mm diameter x 50 mm length (5/16&quot; diameter x 2&quot; length)</td></tr><tr><td>1 L glass screw-cap storage bottle</td><td>Corning</td><td>1395</td><td>Type 1, Class A borosilicate glass</td></tr><tr><td>Heparin sodium&amp;nbsp;</td><td>Sagent</td><td>NDC: 25021-400</td><td>1,000 U/ml solution</td></tr><tr><td>Peristaltic pump</td><td>Cole Parmer Instruments Group</td><td>77200-60</td><td>Master Flex Easy Load II console drive</td></tr><tr><td>compressed carbogen (95% O&lt;sub&gt;2 &lt;/sub&gt;/ 5% CO&lt;sub&gt;2&lt;/sub&gt;)</td><td>available from local vendor</td><td>N/A</td><td>A-type, large cylinder&amp;nbsp;</td></tr><tr><td>20 G x 1 &amp;frac12;&quot; aluminum hub blunt needles</td><td>Kendall</td><td>8881202363</td><td>0.9 mm x 38.1 mm</td></tr><tr><td>Polyurethane ice bucket</td><td>Fisherbrand</td><td>02-591-45</td><td>Capacity: 0.152 oz. (4.5L)</td></tr><tr><td>Collagenase (Type I-S)</td><td>Sigma Aldrich</td><td>C1639</td><td>from Clostridium histolyticum&amp;nbsp;</td></tr><tr><td>Deoxyribonuclease I</td><td>Worthington Biochemical Corp.</td><td>LS002007</td><td>&gt;2,000 Kunitz units per mg dry weight</td></tr><tr><td>Antistatic polystyrene hexagonal weighing dishes</td><td>Ted Pella Inc.</td><td>20157-3</td><td>top I.D. 115 mm, base I.D. 85 mm, 203 ml volume&amp;nbsp;</td></tr><tr><td>Stainless steel single edged razor blades&amp;nbsp;</td><td>Garvey</td><td>40475</td><td></td></tr><tr><td>Bone rongeurs</td><td>FST</td><td>16001-15</td><td></td></tr><tr><td>Hemostat</td><td>FST</td><td>13014-14</td><td></td></tr><tr><td>Large dissection scissors</td><td>Ted Pella Inc.</td><td>1316</td><td></td></tr><tr><td>Small dissection scissors</td><td>FST</td><td>14094-11</td><td></td></tr><tr><td>Blunt end forceps</td><td>Ted Pella Inc.</td><td>13250</td><td></td></tr><tr><td>7 ml glass Dounce tissue grinder</td><td>Kontes</td><td>KT885300-0007</td><td></td></tr><tr><td>Percoll Density Centrifugation Media&amp;nbsp;</td><td>GE Healthcare</td><td>GE17-0891-01</td><td></td></tr><tr><td>10 ml serological pipettes</td><td>Genesee Scientific</td><td>12-104</td><td>single use; sterile</td></tr><tr><td>70 &amp;mu;m sterile nylon mesh cell strainers&amp;nbsp;</td><td>Fisherbrand</td><td>22363548</td><td></td></tr><tr><td>Alexa Fluor 700-conjugated rat anti-mouse CD45 (clone: 30-F11)</td><td>Biolegend</td><td>103128</td><td></td></tr><tr><td>APC-conjugated mouse anti-mouse NK1.1 (clone: PK136)</td><td>eBiosciences&amp;nbsp;</td><td>17-5941-82</td><td></td></tr><tr><td>PE-conjugated rat anti-mouse Gr-1 (clone:RB6-8C5)</td><td>BD Pharmigen</td><td>553128</td><td></td></tr><tr><td>PerCP/Cy5.5-conjugated rat anti-mouse CD11b (clone: M1/70)&amp;nbsp;</td><td>Biolegend</td><td>101228</td><td></td></tr><tr><td>Alexa Fluor 700-conjugated rat IgG2b, &amp;kappa; (clone: RTK4530) isotype control&amp;nbsp;</td><td>Biolegend</td><td>400628</td><td></td></tr><tr><td>APC-conjugated mouse IgG2a, &amp;kappa; (clone: eBM2a) isotype control&amp;nbsp;</td><td>eBioscience</td><td>17-4724</td><td></td></tr><tr><td>PE-conjugated rat IgG2b, &amp;kappa; (clone: eB149/10H5) isotype control&amp;nbsp;</td><td>eBioscience</td><td>12-4031-82</td><td></td></tr><tr><td>PerCP/Cy5.5-conjugated rat IgG2b, &amp;kappa; (clone: RTK4530) isotype control</td><td>Biolegend</td><td>400632</td><td></td></tr><tr><td>Pacific Blue-conjugated mouse anti-mouse granzyme B (Clone: GB11)&amp;nbsp;</td><td>Biolegend</td><td>515403</td><td></td></tr><tr><td>Pacific Blue-conjugated mouse IgG1, &amp;kappa; (Clone: MOPC-21) isotype control&amp;nbsp;</td><td>Biolegend</td><td>400151</td><td></td></tr><tr><td>Cytofix/Cytoperm</td><td>BD</td><td>554714</td><td></td></tr><tr><td>15 ml polypropylene centrifuge tubes</td><td>Genesee Scientific</td><td>21-103</td><td>conical bottom&amp;nbsp;</td></tr><tr><td>50 ml polypropylene centrifuge tubes</td><td>Genesee Scientific</td><td>21-108</td><td>conical bottom</td></tr><tr><td>12 mm x 75 mm round-bottom polypropylene FACS tubes&amp;nbsp;</td><td>Fisherbrand</td><td>14-956-1B</td><td></td></tr><tr><td>FACSAria Special Order Research Product flow cytometer/cell sorter</td><td>BD&amp;nbsp;</td><td>650033</td><td></td></tr><tr><td>Class II Biological Safety Cabinet</td><td>The Baker Company</td><td>SG603</td><td>model: SterilGARD III Advance</td></tr></tbody></table>

isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 malignant gliomas soon after intracranial engraftment if the cancer cells are rendered deficient in their expression of the &#946;-galactoside-binding lectin galectin-1 (gal-1). More recent work now shows that a population of Gr-1+/CD11b+ myeloid cells is critical to this effect. To better understand the mechanisms by which NK and myeloid cells cooperate to confer gal-1-deficient tumor rejection we have developed a comprehensive protocol for the isolation and analysis of glioma-infiltrating peripheral blood mononuclear cells (PBMC). The method is demonstrated here by comparing PBMC infiltration into the tumor microenvironment of gal-1-expressing GL26 gliomas with those rendered gal-1-deficient via shRNA knockdown. The protocol begins with a description of&#160;how to culture and prepare GL26 cells for inoculation into the syngeneic C57BL/6J mouse brain. It then explains the steps involved in the isolation and flow cytometric analysis of glioma-infiltrating PBMCs from the early brain tumor microenvironment. The method is adaptable to a number of in vivo experimental designs in which temporal data on immune infiltration into the brain is required. The method is sensitive and highly reproducible, as glioma-infiltrating PBMCs can be isolated from intracranial tumors as soon as 24 hr post-tumor engraftment with similar cell counts observed from time point matched tumors throughout independent experiments. A single experimentalist can perform the method from brain harvesting to flow cytometric analysis of glioma-infiltrating PBMCs in roughly 4-6 hr depending on the number of samples to be analyzed. Alternative glioma models and/or cell-specific detection antibodies may also be used at the experimentalists&#8217; discretion to assess the infiltration of several other immune cell types of interest without the need for alterations to the overall procedure.

The following gating strategy is used for a typical experiment: FSC-A vs. SSC-A &#8594; SSC-H vs. SSC-W &#8594; FSC-H vs. FSC-W &#8594; CD45 vs. count &#8594; Gr-1 vs. CD11b &#8594; NK1.1 vs. count. Gates placed on Gr-1+/CD11b+ myeloid cells and NK1.1+ NK cells are then stratified based on GzmB expression (Figure 5A). Backgating those cells identified as NK1.1+ NK cells and Gr-1+/CD11b+ myeloid cells in our experiments onto FSC-A vs. SSC-A...

Watch this Scientific Journal Video about Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells at JoVE.com

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Presented here is a straightforward method for the isolation and flow cytometric analysis of glioma-infiltrating peripheral blood mononuclear cells that yields time-dependent quantitative data on the number and activation status of immune cells entering the early brain tumor microenvironment.

Our laboratory has recently demonstrated that natural killer (NK) cells are capable of eradicating orthotopically implanted mouse GL26 and rat CNS-1 ...

isolation-flow-cytometric-analysis-glioma-infiltrating-peripheral

Research

JoVE Journal

Immunology and Infection

15.8K Views.  University of Michigan.  Hier voorgesteld is een eenvoudige methode voor het isoleren en flowcytometrische analyse van glioma infiltrerende perifere mononucleaire cellen die tijdsafhankelijk kwantitatieve gegevens over het aantal en activeringsstatus van immuuncellen die het begin hersentumor micro oplevert. 

Video: Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

Isolation of Brain and Spinal Cord Mononuclear Cells Using Percoll Gradients

Isolation of immune cells that infiltrate the central nervous system (CNS) during infection, trauma, autoimmunity or neurodegeneration, is often required to define their phenotype and effector functions. Histochemical approaches are instrumental to determine the location of the infiltrating cells and to analyze the associated CNS pathology. However, in-situ histochemistry and immunofluorescent staining techniques are limited by the number of antibodies that can be used at a single time to characterize immune cell subtypes in a particular tissue. Therefore, histological approaches in conjunction with immune-phenotyping by flow cytometry are critical to fully characterize the composition of local CNS infiltration. This protocol is based on the separation of CNS cellular suspensions over discontinous percoll gradients. The current article describes a rapid protocol to efficiently isolate mononuclear cells from brain and spinal cord tissues that can be effectively utilized for identification of various immune cell populations in a single sample by flow cytometry.

The current article describes a rapid protocol to efficiently isolate mononuclear cells from brain and spinal cord tissues that can be effectively utilized for flow cytometric analyses.

Isolation of immune cells that infiltrate the central nervous system (CNS) during infection, trauma, autoimmunity or neurodegeneration, is often required ...

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester (CFSE)

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic strategies1. To overcome the issue of tumor heterogeneity, it is essential to develop assays and tools enabling phenotypic, (epi)genetic and functional identification and characterization of tumor subpopulations that drive specific disease pathologies and represent clinically relevant targets. It is now well established that tumors exhibit distinct sub-fractions of cells with different frequencies of cell division, and that the functional criteria of being slow cycling is positively associated with tumor formation ability in several cancers including those of the brain, breast, skin and pancreas as well as leukemia2-8. The fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) has been used for tracking the division frequency of cells in vitro and in vivo in blood-borne tumors and solid tumors such as glioblastoma2,7,8. The cell-permeant non-fluorescent pro-drug of CFSE is converted by intracellular esterases into a fluorescent compound, which is retained within cells by covalently binding to proteins through reaction of its succinimidyl moiety with intracellular amine groups to form stable amide bonds9. The fluorescent dye is equally distributed between daughter cells upon divisions, leading to the halving of the fluorescence intensity with every cell division. This enables tracking of cell cycle frequency up to eight to ten rounds of division10. CFSE retention capacity was used with brain tumor cells to identify and isolate a slow cycling subpopulation (top 5% dye-retaining cells) demonstrated to be enriched in cancer stem cell activity2. 
This protocol describes the technique of staining cells with CFSE and the isolation of individual populations within a culture of human glioblastoma (GBM)-derived cells possessing differing division rates using flow cytometry2. The technique has served to identify and isolate a brain tumor slow-cycling population of cells by virtue of their ability to retain the CFSE labeling.

Identification and Isolation of Slow-Dividing Cells in Human Glioblastoma Using Carboxy Fluorescein Succinimidyl Ester CFSE

This video protocol demonstrates the application of the fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) for the identification and separation of different sub-populations of cells in human glioblastoma based on frequency of cell division.

Tumor heterogeneity represents a fundamental feature supporting tumor robustness and presents a central obstacle to the development of therapeutic ...

Medicine

Processing of Primary Brain Tumor Tissue for Stem Cell Assays and Flow Sorting

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction of cells in the tumor with stem cell properties, termed brain tumor initiating cells (BTICs), possess an ability to differentiate along multiple lineages, self-renew, and initiate tumors in vivo. We applied culture conditions originally used for normal neural stem cells (NSCs) to a variety of human brain tumors and found that this culture method specifically selects for stem-like populations. Serum-free medium (NSC) allows for the maintenance of an undifferentiated stem cell state, and the addition of bFGF and EGF allows for the proliferation of multi-potent, self-renewing, and expandable tumorspheres.
To further characterize each tumor's BTIC population, we evaluate cell surface markers by flow cytometry. We may also sort populations of interest for more specific characterization. Self-renewal assays are performed on single BTICs sorted into 96 well plates; the formation of tumorspheres following incubation at 37 &deg;C indicates the presence of a stem or progenitor cell. Multiple cell numbers of a particular population can also be sorted in different wells for limiting dilution analysis, to analyze self-renewal capacity. We can also study differential gene expression within a particular cell population by using single cell RT-PCR.
The following protocols describe our procedures for the dissociation and culturing of primary human samples to enrich for BTIC populations, as well as the dissociation of tumorspheres. Also included are protocols for staining for flow cytometry analysis or sorting, self-renewal assays, and single cell RT-PCR.

The identification of brain tumor initiating cells (BTICs), the rare cells within a heterogeneous tumor possessing stem cell properties, provides new insights into human brain tumor pathogenesis. We have refined specific culture conditions to enrich for BTICs, and we routinely use flow cytometry to further enrich these populations. Self-renewal assays and transcript analysis by single cell RT-PCR can subsequently be performed on these isolated cells.

Brain tumors are typically comprised of morphologically diverse cells that express a variety of neural lineage markers. Only a relatively small fraction ...

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

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

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

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

Cancer Research

Isolation and Flow Cytometric Analysis of Immune Cells from the Ischemic Mouse Brain

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident cells. A key mechanism of this inflammatory response is the migration of circulating immune cells to the ischemic brain facilitated by chemokine release and increased endothelial adhesion molecule expression. Brain-invading leukocytes are well-known contributing to early-stage secondary ischemic injury, but their significance for the termination of inflammation and later brain repair has only recently been noticed.
Here, a simple protocol for the efficient isolation of immune cells from the ischemic mouse brain is provided. After transcardial perfusion, brain hemispheres are dissected and mechanically dissociated. Enzymatic digestion with Liberase&#160;is followed by density gradient (such as Percoll) centrifugation to remove myelin and cell debris. One major advantage of this protocol is the single-layer density gradient procedure which does not require time-consuming preparation of gradients and can be reliably performed. The approach yields highly reproducible cell counts per brain hemisphere and allows for measuring several flow cytometry panels in one biological replicate. Phenotypic characterization and quantification of brain-invading leukocytes after experimental stroke may contribute to a better understanding of their multifaceted roles in ischemic injury and repair.

Inflammation plays a central role in the pathogenesis of ischemic stroke. Increasing evidence suggests that it acts as a double-edged sword which exacerbates early brain injury, but also contributes to later repair. This protocol describes the isolation of immune cells from the ischemic brain and their subsequent flow cytometric phenotyping.

Ischemic stroke initiates a robust inflammatory response that starts in the intravascular compartment and involves rapid activation of brain resident ...

Analysis of Microglia and Monocyte-derived Macrophages from the Central Nervous System by Flow Cytometry

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main macrophage populations in the CNS: (i) the microglia, which are the resident macrophages of the CNS and are derived from yolk sac progenitors during embryogenesis, and (ii) the monocyte-derived macrophages (MDM), which can infiltrate the CNS during disease and are derived from bone marrow progenitors. The roles of each macrophage subpopulation differ depending on the pathology being studied. Furthermore, there is no consensus on the histological markers or the distinguishing criteria used for these macrophage subpopulations. However, the analysis of the expression profiles of the CD11b and CD45 markers by flow cytometry allows us to distinguish the microglia (CD11b+CD45med) from the MDM (CD11b+CD45high). In this protocol, we show that the density gradient centrifugation and the flow cytometry analysis can be used to characterize these CNS macrophage subpopulations, and to study several markers of interest expressed by these cells as we recently published. Thus, this technique can further our understanding of the role of macrophages in mouse models of neurological diseases and can also be used to evaluate drug effects on these cells.

This protocol provides an analysis of the macrophage subpopulations in the adult mouse central nervous system by flow cytometry and is helpful for the study of multiple markers expressed by these cells.

Numerous studies have demonstrated the role of immune cells, in particular macrophages, in central nervous system (CNS) pathologies. There are two main ...

Quantitative Immunohistochemistry of the Cellular Microenvironment in Patient Glioblastoma Resections

With the growing interest in the tumor microenvironment, we set out to develop a method to specifically determine the microenvironment components within patient samples of glioblastoma, the deadliest and most invasive brain cancer. Not only are quantitative methods beneficial for accurately describing diseased tissues, they can also potentially contribute to more accurate prognosis, diagnosis, and the development of tissue-engineered systems and replacements. In glioblastoma, glial cells, such as microglia and astrocytes, have been independently correlated with poor prognosis based on pathologist grading. However, the state of these cells and other glial cell components has not been well-described quantitatively. This can be difficult due to the large processes that mark these glial cells. Furthermore, most histological analyses focus on the overall tissue sample or only within the bulk of the tumor, as opposed to delineating quantifications based on regions within the highly heterogeneous tissue. Here, we describe a method for identifying and quantitatively analyzing the populations of glial cells within the tumor bulk and adjacent regions of tumor resections from glioblastoma patients. We used chromogenic immunohistochemistry to identify the glial cell populations in patient tumor resections and ImageJ to analyze percent coverage of staining for each glial population. With these techniques we are able to better describe the glial cells throughout regions of the glioma tumor microenvironment.

This protocol was developed to quantitatively identify tumor microenvironment components in glioblastoma patient resections using chromogenic immunohistochemistry and ImageJ.

With the growing interest in the tumor microenvironment, we set out to develop a method to specifically determine the microenvironment components within ...

Flow Cytometric Analysis of Lymphocyte Infiltration in Central Nervous System during Experimental Autoimmune Encephalomyelitis

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible genetic background. Experimental autoimmune encephalomyelitis (EAE) is a typical disease model of MS widely used for investigating the pathogenesis in which T lymphocytes specific for myelin antigens initiate an inflammatory reaction in CNS. It is very important to assess how lymphocytes in the CNS regulate the development of disease. However, the approach for measuring the quantity and quality of infiltrated lymphocytes in the CNS is very limited due to the difficulties in isolating and detecting infiltrated lymphocytes from the brain. This manuscript presents a protocol for that is useful for the isolation, identification, and characterization of infiltrated lymphocytes from the CNS and will be helpful for our understanding of how lymphocytes are involved in the development of the CNS autoimmune disease.

This manuscript presents a protocol to induce active experimental autoimmune encephalomyelitis (EAE) in mice. A method for the isolation and characterization of the infiltrated lymphocytes in the central nervous system (CNS) is also presented to show how lymphocytes are involved in the development of CNS autoimmune disease.

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) caused by the combination of environmental factors and susceptible ...

Replicating Glioblastoma Invasion with Hyaluronan-Collagen I Hydrogel System

A Photopolymerizable Hyaluronic Acid-Collagen Model of the Invasive Glioma Microenvironment with Interstitial Flow

Glioblastoma recurrence is a major hindrance to treatment success and is driven by the invasion of glioma stem cells (GSCs) into healthy tissue that are inaccessible to surgical resection and are resistant to existing chemotherapies. Tissue-level fluid movement, or interstitial fluid flow (IFF), regulates GSC invasion in a manner dependent on the tumor microenvironment (TME), highlighting the need for model systems that incorporate both IFF and the TME. We present an accessible method for replicating the invasive TME in glioblastoma: a hyaluronan-collagen I hydrogel composed of human GSCs, astrocytes, and microglia seeded in a tissue culture insert. Elevated IFF can be represented by applying a fluid pressure head to the hydrogel. Additionally, this model can be tuned to replicate inter- or intra-patient differences in cellular ratios, flow rates, or matrix stiffnesses. Invasion can be quantified, while gels can be harvested for a variety of outcomes, including GSC invasion, flow cytometry, protein or RNA extraction, or imaging.

Author Spotlight: Patient-Informed 3D Model for Studying Glioblastoma Invasion via Interstitial Fluid Flow

We present a method for replicating the glioma tumor microenvironment at the invasive front that incorporates interstitial fluid flow. This model is a hyaluronan-collagen I hydrogel in a tissue culture insert where a fluid pressure head can be applied. Invasion can be quantified, and cells can be isolated or lysed.

Glioblastoma recurrence is a major hindrance to treatment success and is driven by the invasion of glioma stem cells (GSCs) into healthy tissue that are ...

Bioengineering

Isolating Tumor-Infiltrating Immune Cells from a Murine Brain

Source: Baker, G. J., et al., Lowenstein, P. R. <a href="https://app.jove.com/v/53676">Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells.</a> J. Vis. Exp. (2015)
The video demonstrates the isolation of tumor-infiltrating peripheral blood mononuclear cells from a mouse brain using mechanical dissociation and enzymatic digestion. This is followed by density gradient centrifugation to collect the immune cells for further analysis.

All procedures involving sample collection have been performed in accordance with the institute&#39;s IRB guidelines.
Prepare density centrifugation media ...

Isolation and Flow Cytometric Analysis of Glioma-infiltrating Peripheral Blood Mononuclear Cells

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