Flow cytometry analysis of endothelial cells and subsets of exhausted CD8+ T cells in murine tumor models

Abstract

Here, we present a protocol for flow cytometry analysis of endothelial cells (ECs) and CD8+ T cells in murine tumor models, at baseline and after cancer immunotherapy with anti-PD-1/anti-CTLA-4 antibodies. We provide gating strategies for identification of specific cell subsets including ECs from tumor-associated high endothelial venules (TA-HEVs), stem-like, and terminally exhausted CD8+ T cells. This protocol represents a valuable tool for the analysis of rare subsets of tumor ECs and CD8+ T cells with critical roles in antitumor immunity. For complete details on the use and execution of this protocol, please refer to Asrir et al. (2022).

Keywords: Cancer; Cell Biology; Cell isolation; Flow Cytometry/Mass Cytometry; Health Sciences; Immunology; Model Organisms; Single Cell.

Graphical abstract

Figure 1

Tumor resection (A) Subcutaneous tumor 10 days after inoculation of tumor cells (MCAprog). (B) Visualization of the tumor and the draining lymph node (dLN) following initial dissection.

Figure 2

Tumor digestion (A) Resected tumor (MCAprog) in the collection tube. (B) Minced tumor. (C) Tumor fragments in the digestion tube before enzymatic digestion. (D) Digestion tubes placed on a shaker for enzymatic digestion in a warm room. (E) Tumor cell suspension and remaining fragments after enzymatic digestion. (F) Filtration of the tumor cell suspension and mechanic dissociation of remaining fragments with a syringe plunger in the 50 mL tube overlaid with a cell strainer. (G) Flushing of the cell strainer and syringe plunger. (H) Visualization of the 25 mL of tumor single-cell suspension after entire processing. (I) Tumor-derived cells in the 96-well plate prepared for flow cytometry analysis.

Figure 3

Gating strategy for analysis of tumor endothelial cells (A) Forward and sideward scatter gating to exclude debris. (B) Single cell gating. (C) Single cell gating. (D) CD45-CD31high cell gating to identify total tumor-derived endothelial cells. (E) Identification of MECA-79- tumor-associated endothelial cells (TA-ECs) and MECA-79+ tumor-associated HEV endothelial cells (TA-HECs). (F) FMO MECA-79 control.

Figure 4

Analysis of CD62P and CD62E expression in MECA-79+ TA-HECs (A) Histogram showing CD62P expression in MECA-79+ TA-HECs. (B) Histogram showing CD62E expression in MECA-79+ TA-HECs.

Figure 5

Gating strategy for analysis of tumor-infiltrating CD8+ T cells (A) Single cell gating. (B) Forward and sideward scatter gating to identify lymphoid cells. (C) Live CD45+ cell gating. (D) CD8+ cell gating. (E) CD44+ cell gating. (F) PD-1+ cell gating. (G) Identification of SLAMF6+TIM3- stem-like and SLAMF6-TIM3+ terminally exhausted CD8+ T cells. (H) FMO SLAMF6 control. (I) FMO TIM3 control.

Figure 6

Analysis of TCF-1 and TOX expression in tumor-infiltrating exhausted CD8+ T cells (A) Histograms showing TCF-1 expression in SLAMF6+TIM3- stem-like (SLAMF6+) and SLAMF6-TIM3+ terminally exhausted (TIM3+) CD8+ T cells. (B) Histograms showing TOX expression in SLAMF6+TIM3- stem-like (SLAMF6+) and SLAMF6-TIM3+ terminally exhausted (TIM3+) CD8+ T cells.

Figure 7

Impact of immune checkpoint blockade on tumor-associated endothelial cells (A–D) Representative images of flow cytometry analysis of total tumor-derived endothelial cells (A and C) and MECA-79+ TA-HECs (B and D) in the MCAprog tumor model following treatment with Isotype control (Iso) or combined anti-PD-1 and anti-CTLA-4 antibodies (ICB).

Figure 8

Impact of immune checkpoint blockade on tumor-infiltrating CD8+ T cells (A–H) Representative images of flow cytometry analysis of tumor-infiltrating CD8+ T cells (A and E), PD-1+ CD8+ T cells (B and F), SLAMF6+TIM3- stem-like and SLAMF6-TIM3+ terminally exhausted CD8+ T cells (C and G) and Ki67high SLAMF6+TIM3- stem-like CD8+ T cells (D and H) in the MCAprog tumor model following treatment with Isotype control (Iso) or combined anti-PD-1 and anti-CTLA-4 antibodies (ICB).