Isolation and separation of murine tumor-associated macrophages (TAMs) subpopulations from orthotopic 4T1 breast tumors

Abstract

Tumor-associated macrophages (TAMs) are highly heterogenous regarding their intratumoral localization, surface marker expression, and molecular properties. This protocol describes the complete procedure for isolation and digestion of murine breast cancer samples and fluorescence-activated cell sorting (FACS) of TAMs from murine orthotopic 4T1 breast tumors. This includes steps to separate PoEMs (podoplanin-expressing macrophages) and non-PoEMs (podoplanin-negative macrophages). Our FACS separation approach could also be used for other tumor types with TAM infiltration. For complete details on the use and execution of this protocol, please refer to Bieniasz-Krzywiec et al. (2019).

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

Graphical abstract

Figure 1

Exemplary tumor growth curve of 4T1 breast cancer cells implanted orthotopically in WT→WT or Pdpn KO→WT bone marrow chimeras Figure reprinted with permission from (Bieniasz-Krzywiec et al., 2019).

Figure 2

A representative photo of a 4T1 tumor pellet following digestion and straining through a 70 μm strainer, where the volume of the red blood cell lysis buffer can be appreciated (marked with a black arrow).

Figure3

Gating strategy for the isolation of various populations of 4T1 tumor-infiltrating TAMs, including total TAMs (single, viable, CD11b⁺, F4/80⁺), PoEMs (single, viable, CD11b⁺, F4/80⁺, PDPN⁺) and non-PoEMs (single, viable, CD11b⁺, F4/80⁺, PDPN^-).