Functional Analysis of Tumor-Infiltrating Myeloid Cells by Flow Cytometry and Adoptive Transfer

Summary

This protocol provides reliable methods of solid tumor dissociation and myeloid cell isolation in murine intradermal or subcutaneous tumor models. Flow cytometry allows for phenotypic characterization of heterogeneous myeloid populations within the tumor microenvironment and sorting will demonstrate their functionality in the context of adoptive transfer.

Abstract

The tumor-infiltrating myeloid cell compartment represents a heterogeneous population of broadly immunosuppressive cells that have been exploited by the tumor to support its growth. Their accumulation in tumor and secondary lymphoid tissue leads to the suppression of antitumor immune responses and is thus a target for therapeutic intervention. As it is known that the local cytokine milieu can dictate the functional programming of tumor-infiltrating myeloid cells, strategies have been devised to manipulate the tumor microenvironment (TME) to express a cytokine landscape more conducive to antitumor myeloid cell activity. To evaluate therapy-induced changes in tumor-infiltrating myeloid cells, this paper will outline the procedure to dissociate intradermal/subcutaneous tumor tissue from solid tumor-bearing mice in preparation for leukocyte recovery. Strategies for flow cytometric analysis will be provided to enable the identification of heterogeneous myeloid populations within isolated leukocytes and the characterization of unique myeloid phenotypes. Lastly, this paper will describe a means of purifying viable myeloid cells for functional assays and determining their therapeutic value in the context of adoptive transfer.

Introduction

The tumor microenvironment (TME) is comprised of rapidly proliferating neoplastic cells and a surrounding heterogeneous stromal cell compartment. As growing tumors are often poorly vascularized, the TME is a peripheral site uniquely characterized by hypoxia, nutrient deprivation, and acidosis¹. To survive in this landscape, tumor stress responses and metabolic reprogramming result in the secretion of soluble factors that promote tissue remodeling and angiogenesis as well as the selective recruitment of immune cells². As myeloid cells are one of the most abundant type of hematopoietic cells in the TME, there is increasing interest in examining the role of tumor-infiltrating myeloid cells in the TME.

Myeloid cells are a heterogenous and plastic group of innate immune cells including monocytes, macrophages, dendritic cells, and granulocytes. Although they have critical roles in tissue homeostasis and adaptive immune response regulation, their function can be polarizing depending on the composition of activation signals within the local microenvironment³. Tumors take advantage of myeloid cell characteristics through the secretion of soluble factors within the TME. These alternative signals can divert myelopoiesis towards immature differentiation and skew the function of existing tumor-infiltrating myeloid cells³. Indeed, myeloid cells within the TME often promote cancer progression and can suppress antitumor immune responses, leading to adverse effects on cancer therapy.

Although therapeutic strategies promoting the depletion of immunosuppressive myeloid cells have been shown to delay tumor growth⁴, the lack of target specificity risks the removal of immunostimulatory myeloid cells, which by contrast, aid in the resolution of cancer. These inflammatory myeloid cells can exert profound antitumor effects including direct tumor cell killing and activation of cytotoxic CD8⁺ T cells⁵. Alternatively, strategies normalizing the composition and function of myeloid cells in the TME have shown therapeutic success⁶; however, the biological mechanisms underlying their re-education towards an antitumor phenotype have still not been fully understood. Ultimately, a comprehensive characterization of tumor myeloid cells is necessary for further improvement of cancer therapy.

Unfortunately, reproducible disaggregation of tumors for myeloid cell isolation is challenging. Tumor-derived myeloid cells are sensitive to ex vivo manipulation compared to other leukocyte subsets, and the aggressiveness of tumor processing can lead to enzymatic epitope cleavage and reduced viability of recovered cells⁷. The purpose of this method is to provide a reliable means of tumor dissociation to preserve surface marker integrity for analysis and cellular vitality for functional study. In comparison to tumor-infiltrating leukocyte (TIL) isolation protocols that favor harsher enzymatic mixes to enhance the reproducible release of various cellular subsets, this method favors more conservative enzymatic digestion to maximize myeloid cell recovery. High-level multi-color flow gating strategies are also provided to identify murine tumor myeloid cell subsets for further characterization and/or sorting.

Protocol

NOTE: All animal studies complied with the Canadian Council on Animal Care guidelines and were approved by McMaster University's Animal Research Ethics Board. 1. Tumor harvest and dissociation Inoculate 6-8-week-old, female, C57BL/6 mice intradermally/subcutaneously with 2 × 105 B16 melanoma cells as described by Nguyen et al.8 Allow tumors to grow for 7 days before harvesting. Euthanize the mouse by cervical dislo…

Representative Results

The results demonstrate that this method produces a high yield of myeloid cells from solid murine tumors. The preservation of receptor integrity and cellular viability facilitates reliable functional analysis of the desired myeloid subsets. These improvements to myeloid cell isolation allowed the discernment of the changing function of intratumoral myeloid cells upon normalization of the TME with the class I histone deacetylase inhibitor (HDACi), MS-275, during adoptive T cell therapy. TIL isolation protocols typically d…

Discussion

Although tumor-infiltrating myeloid cells exist in varying activation and differentiation states within the tumor, several subsets have been identified including tumor-associated DCs (TADCs), tumor-associated neutrophils (TANs), myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAMs)¹². Unfortunately, the overlapping expression of cell-surface markers used to identify these myeloid cell subsets makes it currently challenging to phenotypically differentiate tumor myeloid c…

Materials

Alexa Fluor 700 Mouse Anti-Mouse CD45.2	BD Biosciences	560693	1:100
APC-Cy7 Mouse Anti-Mouse NK-1.1	BD Biosciences	560618	1:100
Biotin Mouse Anti-Mouse CD45.2	BD Biosciences	553771
BV421 Hamster Anti-Mouse CD11c	BD Biosciences	562782	1:100
BV650 Rat Anti-Mouse F4/80	BD Biosciences	743282	1:100
BV711 Rat Anti-Mouse CD8a	BD Biosciences	563046	1:100
Collagenase, Type IV, powder	Gibco	17104019
DNase I	Roche	10104159001
EasySep Mouse CD11b Positive Selection Kit II	Stemcell technologies	18970
EasySep Mouse CD11c Positive Selection Kit II	Stemcell technologies	18780
EasySep Release Mouse Biotin Positive Selection Kit	Stemcell technologies	17655
FITC Rat Anti-Mouse Ly-6C	BD Biosciences	553104	1:100
Fixable Viability Stain 510	BD Biosciences	564406	1:1000
Fixation/Permeabilization Solution Kit (BD Cytofix/Cytoperm)	BD Biosciences	554714
PE Rat Anti-CD11b	BD Biosciences	557397	1:100
PE-Cy7 Rat Anti-Mouse CD4	BD Biosciences	552775	1:100
PerCP-Cy5.5 Rat Anti-Mouse Ly-6G	BD Biosciences	560602	1:100
Perm/Wash (BD Perm/Wash)	BD Biosciences	554723
Purified Rat Anti-Mouse CD16/CD32 (Mouse BD Fc Block)	BD Biosciences	553141
iNOS Monoclonal Antibody (CXNFT), APC	Thermo Fisher	17-5920-82	1:100
Human/Mouse Arginase 1/ARG1 Fluorescein-conjugated Antibody	R&D Systems	IC5868F	1:100