Modeling Tumor-Associated Macrophages In Vitro: The Role of Secreted Factors in Tumor Microenvironment Dynamics

Summary

This manuscript presents an in vitro protocol for generating tumor-associated macrophage-like cells by exposing human monocytes to conditioned media from tumor cell lines.

Abstract

Macrophages exhibit remarkable plasticity, enabling them to acquire distinct functional states in response to a variety of environmental cues. In solid tumors, they frequently constitute the most abundant immune cell population within the tumor microenvironment, where they are referred to as tumor-associated macrophages (TAMs). TAMs closely interact with malignant cells, actively promoting tumor progression, angiogenesis, and immune evasion. Tumor cells can reprogram macrophages through direct cell-cell contact and a wide array of secreted factors, shaping their polarization toward pro-tumoral phenotypes. To study these TAM polarization mechanisms in vitro, we propose a method that exposes human monocytes to soluble factors present in tumor cell line-conditioned media. This protocol enables the generation of TAM-like macrophages under controlled conditions, facilitating the analysis of phenotypic and functional changes induced by tumor-derived signals. Our approach offers a reproducible and physiologically relevant model to investigate tumor-macrophage interactions, deepens our understanding of macrophage reprogramming, and supports research into novel therapeutic strategies in cancer immunology and immunotherapy.

Introduction

Macrophages are multifunctional innate immune cells whose remarkable plasticity enables them to adopt diverse phenotypes in response to environmental cues¹. Within solid tumors, macrophages often become the most abundant immune cell population and differentiate into tumor-associated macrophages (TAMs)². These cells are actively reprogrammed by the tumor microenvironment to support tumor growth, promote angiogenesis, facilitate metastasis, and suppress effective antitumor immune responses. Tumor cells drive this polarization through both direct cell-cell interactions and a complex repertoire of secreted factors, including....

Protocol

Peripheral blood samples were collected from healthy donors after informed consent in accordance with the Declaration of Helsinki. This protocol was approved by the Institutional Ethics Committee of the University of Buenos Aires. See the Table of Materials for details about all materials and reagents used in this protocol.

NOTE: Maintain sterile conditions throughout the whole protocol, working in a Biosafety level 2 (BSL-2) cabinet, and ensure cell viability >90%. For disposal, all biological waste must first be decontaminated by soaking in a 10% sodium hypochlorite solution for at least 30 min, and then placed in the....

Results

We generated triple-negative breast cancer tumor-associated macrophages (TAM)-like cells by treating monocyte-derived macrophages with 50% conditioned media (CM) from the MDA-MB-231 cell line. Monocytes were sorted from peripheral blood mononuclear cells (PBMC) of six independent healthy donors and differentiated following our protocol. The CM preparation utilized MDA-MB-231 cells at 90 % confluency (Figure 1). CM was added to the macrophage .......

Discussion

The protocol described here provides a reproducible and practical approach to study tumor-associated macrophage-like polarization by exposing human monocyte-derived macrophages to tumor cell line-conditioned media. This system captures the influence of soluble factors secreted by tumor cells, which are known to play a pivotal role in macrophage reprogramming within the tumor microenvironment^{7,9,10,15.......}

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Alexa Fluor 488 mouse anti-human  CD206 Antibody clone 15-2	BioLegend, San Diego, CA, USA.	Cat# 321113; RRID:AB_571874
Alexa Fluor 647 mouse anti-human CD64 Antibody clone 10.1	BioLegend, San Diego, CA, USA.	Cat# 305012; RRID:AB_528867
BD Cytofix/Cytoperm	BD Biosciences, San Jose, CA, USA	Cat# 554714	fixative/permeabilization kit
Dulbecco's Modified Eagle Medium (DMEM), high glucose	Gibco Thermo Fisher Scientific, Waltham, MA USA	Cat# 11965092
EasySep Human CD14 Positive Selection Kit; EasySep Positive Selection Cocktail; EasySep magnetic Nanoparticles; Easy Sep Magnet	STEMCELL Technologies, Vancouver, Canada	Cat# 18058
FACS Canto cytometer	Becton Dickinson, Franklin Lakes, New Jersey, USA.
Fetal Bovine Serum, qualified, New Zealand	Gibco™ Thermo Fisher Scientific, Waltham, MA USA	Cat# 10091148
Ficoll-Paque PLUS Cytiva	GE Healthcare - Life Sciences, USA	Cat# 17144003
FlowJo	FlowJo LLC.	RRID:SCR_008520
GraphPad Prism	GraphPad Software Inc.	RRID:SCR_002798
Partec CyFlow space cytometer	Sysmex Partec, Germany
PE anti-human HLA-DR Antibody clone L243	BioLegend, San Diego, CA, USA.	Cat# 307605; RRID:AB_314683
PE/Cy7 rat anti-mouse/human CD11b Antibody clone M1/70	BioLegend, San Diego, CA, USA.	Cat# 101216; RRID:AB_312799
PerCP/Cy5.5 mouse anti-human CD163 Antibody clone GHI/61	BioLegend, San Diego, CA, USA.	Cat# 333607; RRID:AB_1134006
RPMI 1640 Medium	Gibco Thermo Fisher Scientific, Waltham, MA USA	Cat# 11875119
Sodium pyruvate solution	Sigma-Aldrich Merck, San Luis, MO, USA.	Cat#  S8636
Trypsin-EDTA (0.25%), phenol red	Gibco Thermo Fisher Scientific, Waltham, MA USA	Cat# 25200056
Zombie Violet Fixable Viability Kit	BioLegend, San Diego, CA, USA.	Cat# 423113