Establishing In Vitro Models of Dorsal Root Ganglia Culture: Complementary Approaches for Investigating Cancer-Nerve Crosstalk

Summary

This protocol describes methods for establishing whole mount and dissociated cultures of mouse Dorsal Root Ganglia (DRG) and their use to assess tumor-nerve interactions.

Abstract

The contribution of the nervous system to the tumor microenvironment and the importance of neural invasion as a route for cancer dissemination are being increasingly recognized. Interactions of cancer cells with neurons can promote their invasion around and into nerves, a feature of many cancers with poor clinical outcomes. In vitro models to study reciprocal interactions between neurons and cancer cells provide valuable tools for understanding cancer spread and identifying approaches to mitigate it.

Here, we describe a protocol for murine dorsal root ganglia (DRG) isolation and the establishment of both whole mount and dissociated monolayer cultures that can be used to visualize neuron morphology and neurite outgrowth over time. Whole DRGs mounted in Matrigel preserve nerve architecture and responses to stimuli in a heterogeneous environment more similar to the in vivo nerve, while dissociated nerve cultures allow assessment of direct cell-cell interactions more closely. Once DRG cultures are established, cancer cells can be added to generate co-cultures that can be used to visualize changes in neurite outgrowth and nerve morphology in response to cancer cells. Growth or motility of cancer cells in response to nerve-derived signals over time or under conditions of growth stimulation or inhibition can be assessed, as well as visualizing the effects of direct contact between cancer cells and nerve extensions.

As both co-culture models can be generated simultaneously, this protocol provides a more comprehensive view of the impact of cancer-neuron interactions and facilitates comparisons of treatment conditions and integration of information from the cellular level and whole ganglia. This protocol will facilitate the study of nerve-tumor interactions and can be used for a wide range of applications, including studies of cell signaling, drug screening, or study of the heterogeneity of the tumor-nerve environment and the mechanisms of tumor dissemination along nerves.

Introduction

Tumor dissemination, through local invasion and regional or distant metastasis, is the primary cause of cancer-related death. Although research has focused on vascular or lymphatic routes of spread, neural invasion of tumor cells is frequently observed in many solid tumors, including pancreatic^1,2, prostate³, head and neck⁴, breast⁵, and colorectal⁶ cancers. In recent years, numerous studies have highlighted the emerging role of nerves in promoting tumor growth and progression, and as routes of dissemination, drivin....

Protocol

All mouse work was reviewed and approved by the Office of the University Animal Care Committee at Queen's University (Protocol number 2524). Animals were maintained and euthanized for tissue collection in compliance with Canadian Council on Animal Care, and National Centre for the Replacement, Refinement & Reduction on Animals in Research (UK) ethical guidelines. In this protocol, we used tissue isolated from C57BL/6 animals but also successfully used other genotypes, including Rag2IL2Rγc KO animals. Sections 1-6 can be performed in a clean environment on a surface-sterilized lab bench (see Figure 1), while sections 7-9 must ....

Results

Our protocol describes the isolation of primary mouse DRG-derived cells and the establishment of two complementary culture models: dissociated DRG and whole-mounted DRG cultures, which allow us to investigate nerve-cancer cell interactions at different levels of complexity. A key advantage of this protocol is that both types of culture can be generated using DRGs isolated from the same animal, which minimizes inter-animal and inter-experimental variability and allows the information gener.......

Discussion

The protocol presented here describes improved methods to establish 2D and 3D DRG primary nerve cultures and their application in co-culture with cancer cells. This protocol advances on previous methods by establishing the two models in parallel. As 2D and 3D models are established at the same time, using primary nerves isolated from the same animals, interanimal and interexperimental variation is minimized, and data from both models can be more easily compared or integrated facilitating complementary observations of cel.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Tools
70 µm Cell Strainer	Progene	71-229483-ULT	Used for filtering cell suspension
8-well glass bottom slides	IBidi, Munich, Germany	80807
Dumont #5 - Fine Forceps	Fine Science Tools, Foster City, USA	11254-20
Dumont #5 Forceps	Fine Science Tools, Foster City, USA	11252-20
Fine Scissors	Fine Science Tools, Foster City, USA	91460-11
Pattern Forceps	Fine Science Tools, Foster City, USA	91121-12
Spring Scissors	Fine Science Tools, Foster City, USA	91500-09
Reagents
1x Phosphate buffered saline	Sigma, St. Louis, USA	D8662-500ML	Used for dissolving enzymes
B-27 Supplement (50x)	Thermo Fisher Scientific, Massachusetts, USA	17504044	Used at 2% concentration in F12 media
Bovine Type I Collagen	Corning Incorporated, New York, USA	354231	Used for coating plates, Working solution 50 µg/mL in 0.01 M HCl
Collagenase Type IV	Sigma-Aldrich, St. Louis, USA	C4-BIOC	Dissolve in PBS at 1.25 mg/mL, aliquot, store at -20 °C
Dispase II	Roche, Basel, Switzerland	4942078001	Dissolve in PBS at 2.5 mg/mL, aliquot, store at -20 °C
DNase I	Roche, Basel, Switzerland.	11284932001	Added at final concentration of  0.2 mg/mL during dissociation
Fetal Bovine Serum (FBS)	Sigma-Aldrich, St. Louis, USA	F1051	Used at 10% concentration in F12 media
HBSS (Hank's Balanced Salt Solution)	Sigma, St. Louis, USA	H6648-500ML	Prepared with 1% Penicillin-Streptomycin
Matrigel Growth Factor Reduced	Corning Incorporated, New York, USA	356231	Thaw at 4 °C overnight, keep on ice
Nutrient Mixture F-12 Ham	Sigma-Aldrich, St. Louis, USA	N6658-500ML	Basal medium for preparation of supplemented culture media
Papain	Roche, Basel, Switzerland.	10108014001	Dissolve in PBS at 30 U/mL, aliquot, store at -20 °C
Penicillin-Streptomycin (100x)	Sigma-Aldrich, St. Louis, USA	P4333-100ML	Used at 1% concentration in media
Trypan Blue Solution	Sigma-Aldrich, St. Louis, USA	T8154-100ML	To assess cell viability
TrypLE Express	Thermo Fisher Scientific, Massachusetts, USA	12605-028	For harvesting cancer cells
Antibodies
β-Tubulin III antibody	Abcam (Cambridge, UK)	AB15568	Dilution 1:200
Alexa Fluor 488	Thermo Fisher Scientific, Massachusetts, USA	A-11008	Dilution 1:2000
Alexa Fluor 594	Thermo Fisher Scientific, Massachusetts, USA	A-21442	Dilution 1:2000
Alexa Fluor 647	Thermo Fisher Scientific, Massachusetts, USA	A-21245	Dilution 1:2000
NeuN antibody	Sigma-Aldrich, St. Louis, USA	MAB377	Dilution 1:100
Fluorescent Dyes & Probes
ActinGreen 488 ReadyProbes Reagent (Phalloidin)	Thermo Fisher Scientific, Massachusetts, USA	R37110	Dilution 1:40
CellTrace Calcein Red-Orange	Thermo Fisher Scientific, Massachusetts, USA	C34851	Cell tracker used to visualize neurons under fluorescence microscope
Hoechst 33342	Thermo Fisher Scientific, Massachusetts, USA	H3570	Dilution 1:5000