Optimized protocol for immunophenotyping of melanoma and tumor-bearing skin from mouse

Abstract

While isolating immune cells from spleens and lungs is routinely achieved using flow cytometry, it is challenging to isolate viable immune cells from skin. Here, we describe a step-by-step protocol for skin digestion using a murine melanoma model, which is amenable for detection of low abundant immune cell populations including group 2 innate lymphoid cells.

Keywords: Cancer; Cell isolation; Flow Cytometry/Mass Cytometry; Immunology; Model Organisms.

Graphical abstract

Figure 1

Application of 4-Hydroxytamoxifen (4-HT) for the induction of Melanoma formation (A) Mice are sedated with 3% Isoflurane. (B) Mice are shaved at the lower back near the base of tail. (C) Hair depilatory cream is applied for 2–3 min to remove residual fur. (D) Wet swab used to remove the cream. (E) Dry and inspect skin for any residual fur. (F) Place mice back in the cage with a heating lamp to allow recovery. (G) Using a pipette tip, add 2μl of 5mM 4-HT to the shaved, exposed skin. (H) Use the side of a pipette tip to spread 4-HT on the skin and allow it to dry before placing the mouse back into the cage

Figure 2

Melanoma progression following 4-HT treatment (A) Day 7 following 4-HT treatment. During this timepoint, no pigmented lesions yet visible. (B) Day 12 – 14 following 4-HT treatment. During this timepoint, pigmented lesions will start to form. (C) Day 24 – 28 following 4-HT treatment. During this timepoint, measurable tumors will start to form. (D) Any time after Day 40 following 4-HT treatment, mice are monitored closely for human endpoints.

Figure 4

Skin and melanoma harvest (A) Euthanize mice according to institutional guidelines, typically by sedation with 5% Isoflurane, followed by asphyxiation and physical confirmation of death by cervical dislocation. (B) Shave fur from tumor sites and peripheral area. (C) Harvest tumors using scissors and forceps. (D) Place tumors in cold PBS. (E) Place tumors in 10cm dishes on ice. (F) Cut tumors into strips. (G) Cut strips into 1–2 mm² squares. (H) Place tumors squares into a 6-well plate and add 5 mL digestion media. (I) Place plate containing tumor squares on a rocker in a cold room and incubate for 16–20 h. (J) Place tumors and digestion media through a 70μM cell strainer. (K) A 3 mL or 5 mL syringe plunger is used to mash the tumor on the strainer and wash the strainer twice with DMEM containing 5% FBS. (L) Filter the tumor and media through a 50μM cell strainer, wash, and count cells with Trypan Blue and a hemocytometer.

Figure 3

Single cell suspensions of melanomas were prepared using distinct enzymatic treatments and cell viabilities were determined by flow cytometric analysis. (A) Collagenase D, (B) Collagenase P, (C) Collagenase IV at 30^°C for 1 h, (D) Liberase TM

Figure 5

ILC2 gating strategy (A) Forward and sideward scatter gating to exclude debris. (B) Singlet cell gating. (C) Live cell gating. (D) CD45⁺ cell gating. (E) Lineage-negative cell population gating. (F) GATA3⁺ cell population gating. (G) ILC2 were characterized as GATA3⁺ST2⁺ cell population.

Figure 6

Regulatory T-cells (Tregs) gating strategy (A) Forward and sideward scatter gating to exclude debris. (B) Singlet cell gating. (C) Live cell gating. (D) CD45⁺ cell gating. (E) CD11b and F4/80 double-negative gating. (F) CD11c negative gating. (G) CD3ε⁺ T cell gating. (H) CD4⁺ T cell and CD8⁺ T cell gating. (I) CD4⁺FOXP3⁺ gating to identify Tregs

Figure 7

Non-lymphoid tissue dendritic cells (NLT DCs) gating strategy (A) Forward and sideward scatter gating to exclude debris. (B) Singlet cell gating. (C) Live cell gating. (D) CD45⁺ cell gating. (E) CD11b negative gating. (F) CD3ε and CD8ε double negative gating. (G) F4/80^Low, CD11c^Mid gating to identify NLT DCs.

Figure 8

Myeloid-derived suppressor cells (MDSC) gating strategy (A) Forward and sideward scatter gating to exclude debris. (B) Singlet cell gating. (C) Live cell gating. (D) CD45⁺ cell gating. (E) CD11b positive gating. (F) Ly-6G^Low and Ly-6C^High gating for M-MDSCs and Ly-6G^High and Ly-6C^Mid for G-MDSCs. (G) F4/80^High gating to identify M-MDSCs. (H) F4/80^Low gating to identify G-MDSCs

Figure 9

Monocytic tissue dendritic cells (mDCs) gating strategy (A) Forward and sideward scatter gating to exclude debris. (B) Singlet cell gating. (C) Live cell gating. (D) CD45⁺ cell gating. (E) CD8α^All and Ly-6C^Low gating. (F) F4/80^-CD3ε^- for mDCs. (G) CD8α^Low and CD11c^{Mid to High} to identify mDCs

Figure 10

Macrophage gating strategy (A) Forward and sideward scatter gating to exclude debris. (B) Singlet cell gating. (C) Live cell gating. (D) CD45⁺ cell gating. (E) F4/80^High population gating. (F) CD11c^High and CD206^Low gating for M1 Macrophages, and CD11c^Low and CD206^{Mid to High} gating for M2 Macrophages. (G) M2 Macrophages express high levels of Arginase-1.

Figure 11

Detection of ILC2s at 3 different timepoints during melanoma progression (mouse model shown in Figure 2) ILC2s reduce in number in melanomas characterized on Day 40, compared to Days 7 and 14. One-way ANOVA. All values are represented as mean ± SD. ∗∗P ≤ 0.01.