Tumor Infiltrating Lymphocytes:

Isolation and Phenotyping of Infiltrating Cells in Advanced Cell Models

Tumor Infiltrating Lymphocytes: Why study them?

Immunotherapies are utilizing the power of the host immune system to target malignant cells. Sufficient numbers of Tumor Infiltrating Lymphocytes (TILs) must penetrate the tumor, survive, proliferate and kill the cancerous cells within the microenvironment for optimal pathological response to treatment. In vitro modelling of T-cell infiltration into solid tumors may allow identification of methods to increase the efficiency of cytotoxic TILs, leading to increased potency of immunotherapies and better prognosis for patients.

Comprehensive in vitro analysis of tumor infiltrating lymphocytes (TILs) using advanced high-throughput flow cytometry

  • A turnkey solution for the in vitro quantification and phenotyping of TILs and non-infiltrating lymphocytes in advanced cell models.
  • Ease of use workflow; utilizing a 96-well plate format from start to finish.
  • Increase confidence by minimizing variability; include more replicates without increasing acquisition time.

Key Advantages

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Gentle dissociation of 3D tumor models for the quantification of T-cell infiltration.

Easy to follow protocol for the dissociation of 3D cell models to allow quantification of tumor infiltrating lymphocytes.

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Identify immune cell phenotypes in physiologically relevant advanced cell models.

Immuno-phenotype infiltrating and non-infiltrating cells to assess activation profiles and cell subsets.

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Real-time data acquisition and analysis with the integrated ForeCyt software.

Single software solution for acquisition and analysis, delivering actionable results faster.

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Unlock your productivity

Rapid sampling speeds enables increased replicates for highly variable biology.

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Gentle dissociation of 3D tumor models for the quantification of T-cell infiltration.

Figure 1. Schematic of the protocol enabling in vitro analysis of tumor infiltrating lymphocytes using the iQue3.

Identify immune cell phenotypes in physiologically relevant advanced cell models.

  • Quantify immune cell infiltration in response to stimuli.
  • Reveal phenotypic differences of activation profiles between infiltrated and non-infiltrated cells to identify effects of the tumor microenvironment.

 
 

 
Figure 2. (A) Concentration-dependent increases of CD3+ PBMC infiltration into breast cancer spheroids. BT474 (4,000 cells per well) spheroids were formed for 72 hours before PBMCs (20,000 cells per well) and CD3/CD28 Dynabeads were added. Activation of PBMCs with CD3/CD28 Dynabeads causes CD3 T-cells to infiltrate into breast cancer spheroids in a concentration dependent manner. At the highest density of beads (20K) PBMCs began to kill the tumor spheroid, making infiltration reduce as the spheroid was attacked. Each data point represents mean ±SEM, n=8.

(B) and (C) Infiltrating cells exhibit higher expression of activation markers compared to non-infiltrating cells. Non-infiltrated cells were collected and labeled using the T-Cell Activation Cell and Cytokine profiling kit (TCA). Spheroids were then dissociated and infiltrated cells labeled. Infiltrated cells express much higher levels of CD69 (early), CD25 (mid) and HLA-DR (late) activation markers. Each data point represents mean ±SEM, 8 replicates.

Identify phenotypes of infiltrating cells in physiologically relevant advanced cell models.

Figure 3. T-cell infiltration into tumor spheroids is cell type specific and modulated by stromal cells. (A) A549, SKOV-3 or BT474 (5,000 cells per well) spheroids were formed for 72 h, then pre-activated (CD3/CD28 Dynabeads®) PBMCs (25,000 cells per well) were added. Data represents 3 donors per cancer type. CD3 infiltration is dependent on cancer type. (B) CD8:CD4 ratio also differs dependent on cancer type, an indicator of treatment outcomes and likelihood of pathological complete response (pCR). (C) BT474 alone or mixed 1:1 with CCD1068SK fibroblasts formed spheroids for 72 h and were then seeded with pre-activated PBMCs. Inclusion of fibroblasts substantially reduces (>60%) the infiltration of CD3+ cells into the spheroid.

Real-time data acquisition and analysis with the integrated ForeCyt software.

  • Acquire and analyze data from multiple plates in parallel to get answers faster.

Figure 4. The integrated ForeCyt software enables fast analysis, reducing data handling time. The TCA kit has a predefined gating template allowing a “set up and walk away” experience. The gating can be adjusted by defining positive (green) and negative (red) populations, which automatically updates the plate view readout. The plate view can then be translated into a concentration response curve via a simple to use wizard.

Unlock your productivity.

  • Rapid sampling speeds allows increased replicates to alleviate some of the issues associated with highly variable biology.

Figure 5. CD3 cell infiltration was quantified using the iQue3 after spheroid dissociation to assess replicate number affects. Utilizing the high-throughput capacity of the iQue3 enables increased replicate numbers, without extending acquisition time, thus facilitating the study of variable biological effects.

96-well plate:
– 4 replicates (washing every 4, shaking every 4, 5 second sip) 21mins 32sec
– 8 replicates (washing every 8, shaking every 4, 5 second sip) 19mins 08sec

Validation

 

 

Figure 6: IncuCyte images verify the success of washing steps and evidence of infiltration. (A) Spheroids were washed as per the protocol and imaged after each wash to identify remaining PBMCs (labelled with CytoLight Rapid Green). (B) Green integrated intensity (GCU x µm2) metric from the IncuCyte quantifies infiltration, highlighting the activation-dependence of PBMC entry into the tumor.

Ordering Information

T Cell Activation Cell and Cytokine Profiling Kit 
PlatformCompatible with iQue3 VBR configuration
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Available SizesCatalog Numbers
1 x 96 well
90560
T Cell Activation Cell and Cytokine Profiling Kit
5 x 96 wells
90561
1 x 384 wells
90562
5 x 384 wells
90563
IncuCyte® Reagent 
PlatformCompatible with iQue3 VBR configurationOrder Now                                                  
CytoLight Rapid Green Reagent                   Catalog Numbers                 
1 vial for labelling 10-100 million cells4705