Isolation and analysis of tumor-infiltrating leukocytes (TILs)

  • Tumor dissociation with preserved cell surface epitopes
  • TILs pre-enrichment for improved downstream analysis
  • From manual single-sample separation up to fully automated high-throughput separation

Application data for TILs by workflow step

Storage of tumor tissue

 

Freshly dissected tumor tissue can be stored for up to 48 h in MACS® Tissue Storage Solution without compromising cell viability or immune cell composition.

The MACS Tissue Storage Solution preserves TIL composition 
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The MACS Tissue Storage Solution preserves TIL composition 
 

Flow cytometry analysis of TIL populations after storage in MACS Tissue Storage Solution show no changes in TIL composition.

Tumor dissociation

 

Enzymatic tumor dissociation for efficient recovery of TILs from B16-F10 tumors. 
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Enzymatic tumor dissociation for efficient recovery of TILs

Standardized processing of tumor samples using the Tumor Dissociation Kit together with the gentleMACS™ Dissociator enhances reproducibility and was essential for recovery of key populations of TILs. 
 

IFN- γ release of TILs prepared with the gentleMACS Dissociator compared to overnight digestion. 
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Preserved anti-tumor functionality of TILs

Secretion of IFN-γ is one of the hallmark functions of antigen-specific T cells encountering the respective tumor antigen. Combined mechanical and enzymatic processing of TILs with the gentleMACS Dissociator is gentle to cells and results in profound secretion of IFN-γ upon tumor cell co-culture.

Magnetic isolation of human tumor-infiltrating leukocytes

 

The amount and composition of TILs is highly variable, complicating the analysis of individual subpopulations. When working with large cohort sizes, even immunophenotyping of TILs by flow cytometry is time consuming and data processing highly work intensive. Therefore, pre-enrichment of TILs is desirable to increase the sensitivity of analysis and save time and effort during downstream analysis such as sequencing and flow cytometry. To this end, Miltenyi Biotec offers optimized MicroBeads for tumor tissue samples.
 

Automated isolation of human CD45+ TILs from xenograft tumors 
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Automated isolation of human CD45+ TILs from xenograft tumors with MACS Technology

 

Automated isolation of CD45+ TILs using CD45 (TIL) MicroBeads and the autoMACS® Pro Separator results in an increase of CD45+ TIL frequencies from 29.6% and 29.3% (Tumor 1 and 2) to 88.6% and 91.9%, respectively.

Isolation of MicroBead- and label-free CD45+ TILs from a dissociated ovarian tumor  
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REAlease Technology delivers beads- and label-free CD45+ human TILs 

Isolation of human CD45+ TILs using the REAlease® CD45 (TIL) MicroBead Kit results in high cell purities of 89%. Directly after isolation, cells show staining of biotin (MicroBead-free CD45+ cells), whereas the label-free CD45+ cells after the REAlease Biotin Complex release are negative for biotin similar to the non-labeled cells before separation, indicating efficient MicroBead and label release when applying REAlease Technology. 


Isolation of CD4+/CD8+ TIL subpopulation with REAlease Technology followed by dead cell removal improves cell vitality  

Using the REAlease CD4/CD8 (TIL) MicroBead Kit, human directly on dissociated tumor tissue provides highly pure CD4+/CD8+ human TILs and delivers MicroBead- and label-free cells. Isolated cells are suitable for further processing, e.g., with the MicroBead-based Dead Cell Removal Kit, human, thereby improving the vitality of CD4+/CD8+ TILs from 80% to nearly 90%. 
 

Magnetic isolation of mouse tumor-infiltrating leukocytes

 

Automated isolation of human CD45+ TILs from xenograft tumors 
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Isolation of CD4+, CD8+, and pan T cells from syngeneic mouse tumors

Using CD4+, CD8+, and pan T cell specific MACS MicroBeads for magnetic cell isolation (MACS Technology) directly from dissociated tumor tissue delivers high purities of isolated TILs above 80%.  

This pre-enrichment of TILs greatly reduces time and costs of downstream analysis while the quality of data analysis significantly increased.

Isolation and analysis of CD45+ T cells
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Isolation of CD45+ T cells from syngeneic mouse tumor

Isolation of TILs directly from dissociated tumor tissue can be improved by using the CD45 (TIL) MicroBeads for magnetic cell sorting. 

Pre-enrichment of TILs significantly improves subset analysis and does not affect the composition of infiltrating immune cell populations.

Serial sorting of tumor-infiltrating leukocyte subpopulations
 

One major goal in immunotherapies is to understand how the composition of tumor-infiltrating myeloid and lymphoid cells contributes to patient stratification. However, TIL numbers can be very low and small subpopulations might be lost in background noise, particularly in single-cell analyses. Therefore, serial sorting of TIL subpopulations is highly advantageous in order to investigate the immune cell composition.
 

Sequential sorting of tumor-infiltrating NK and B cells 
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Gentle serial sorting of B and NK cells from syngeneic mouse tumors after a CD45+ TIL pre-enrichment

CD45+ TILs can be sorted into single TIL subpopulations using the MACSQuant® Tyto® Cell Sorter. Thanks to releasable antibodies and to the MACSQuant Tyto Cartridge, a serial sorting of tumor-infiltrating NK and B cells is possible in two steps. Flow cytometry analysis using the MACSQuant Analyzer demonstrates a selective enrichment of target cell populations with purity of >94%, without affecting cell viability.

Flow analysis of tumor-infiltrating leukocytes

 

Background-free flow analysis of TILs 

Using hybridoma-derived antibodies to identify TILs leads to a gross overestimation of the frequency of immune cell subpopulations present in the tumor. This experimental artifact is most likely caused by unspecific binding of hybridoma-derived antibodies to FcγRs on immune cells, as it is significantly reduced when using an FcR blocking reagent.  
In contrast, assessment of TIL frequency using REAfinity™ Recombinant Antibodies is unchanged in the presence or absence of FcR blocking, providing a more exact analysis of respective cell populations, even in the absence of FcR blocking. 
REAfinity Antibodies are highly specific recombinant antibodies that provide superior lot-to-lot consistency and purity compared to mouse or rat hybridoma-derived monoclonal antibodies.  
 

Optimized multicolor flow cytometry panels

Phenotypic characterization of TILs by flow cytometry can be performed using optimized panels of fluorochromes-conjugated REAfinity Recombinant Antibodies. Our Miltenyi Biotec tested panels (MBTPs) for flow cytometry applications are developed and validated by Miltenyi Biotec’s R&D experts, as well as by customers who use our antibodies.  

Relevant flow panels for TILs:

Panel 1
 
CloneFluorochromePanel 2
 
CloneFluorochrome
CD8βREA793VioBlue®CD8βREA793
 
VioBlue
Viobility™ 405/520Viobility 405/520
Lag-3REA776Vio® Bright 515
 
CD103REA789Vio Bright 515  
 
TIM-3REA602PECXCR3REA724PE
CD4REA604PE-Vio 615CD4REA604PE-Vio 615
PD-1REA802APCCD39REA870APC
CD44REA664APC-Vio 770CD44REA664APC-Vio 770

Omics analysis of tumor-infiltrating leukocytes
 

TIL numbers can be very low and small subpopulations might escape analysis when they are lost in background noise. This can be especially challenging when performing single-cell analysis where a debris-free single-cell suspension with a viability above 80% is required. We offer innovative tools to eliminate debris and enrich viable cells that will consequently improve your single-cell sequencing results.
 

Magnetic isolation of TILs increases the sensitivity of single-cell receptor sequencing
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Magnetic isolation of TILs increases the sensitivity of single-cell receptor sequencing

Single-cell TCRs (A) and BCRs (B) were sequenced prior to and after the isolation of T or B cells from a tumor sample. The graphs show the top 50 TCR (A) and top 25 BCR (B) CD3 clonotypes ranked by their abundancy in the isolated T or B cell fraction (dark purple). The light purple bars show the number of cells containing the same CDR3 clonotype of the respective receptor in the bulk sample.

Characterization of the tumor microenvironment by ultra high content imaging
 

Understanding the complex interactions between TILs, stroma fibroblasts, resident immune cells, endothelial cells, and extracellular matrix (ECM) proteins in the tumor microenvironment (TME) still remain a challenge in cancer research.

Miltenyi Biotec’s MICS (MACSimaTM Imaging Cyclic Staining) technology impressively overcomes these limits as it allows the fluorescence microscopic analysis of hundreds of markers on a single sample, without any harm, in a fully automated manner.
 

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