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, driving metastasis development^7,8. In this context, cancer neuroscience has emerged as a prominent research field, offering new perspectives in oncology by elucidating the interactions between tumor cells and the fundamental components of the peripheral nervous system (PNS) that may contribute to these processes^8,9.

The PNS consists of both peripheral nerves and ganglia, which are heterogeneous structures composed of diverse cell types, including neurons of varying sizes and functions, Schwann and other glial cells, fibroblasts, macrophages, mast cells, melanocytes, and vascular endothelial cells that form the local vasculature¹⁰. The PNS is a critical component of the tumor microenvironment (TME), and reciprocal communication between cancer cells and nerves, either directly through physical contact or indirectly through the release of neurotrophic factors, neurotransmitters, growth factors, and other signals, can facilitate neural invasion and promote more aggressive phenotypes⁸.

In vitro models can be used to characterize the processes that contribute to nerve maintenance and repair and are increasingly being used to examine cellular interactions between nerves and cancer cells^11,12. A frequently used in vitro model involves extracted mouse dorsal root ganglia (DRG) co-cultured with tumor cells¹¹. These models often use two-dimensional (2D) monolayer culture of dissociated DRG cells to study neuronal biology in vitro and have been used to model cellular interactions in the PNS^13,14. However, 2D cultures lack the complexity of the nerve microenvironment and poorly represent its in vivo architecture. Three-dimensional (3D) whole mounts of peripheral neurons or ganglia offer an alternative that more effectively recapitulates peripheral nerve physiology to represent nerve-environment interactions more holistically^15,16,17,18. However, the heterogeneity of this model limits the ability to visualize or quantify cell-cell interactions, and these models are more effective for observing overall peripheral nerve responses than for exploring specific cellular interactions and mechanistic responses to stimuli. Thus, it is clear that dissociated and whole mount neuronal cultures are complementary and that evaluation of 2D and 3D models in parallel is optimal to assess both overall effects and individual cell responses to stimuli.

This protocol describes methods for generating complementary dissociated (2D) and whole-mount (3D) nerve cultures that can be used to evaluate nerve and cancer cell interactions at both cellular and tissue levels, providing a more comprehensive view of these interactions than methods using only one type of culture^1,13. These models can be used to visualize and assess cellular processes, including nerve cell morphology and neurite outgrowth, as well as tumor cell responses to nerve-secreted chemotactic agents, such as neurotrophic factors, and crosstalk between sensory neurons and tumor cells. It also allows exploration of potential interactions between tumor cells and other components of the neural microenvironment, such as immune cells, fibroblasts, and glia. We highlight the application of these models to study nerve-cancer cell interactions over time, visualized by live cell staining or immunofluorescence, as examples of approaches to assess or quantify cell interactions and responses. Together, the results generated from the use of these methods can further our understanding of how the nervous system promotes the growth and spread of cancer, helping us to develop strategies to target these mechanisms.

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 be performed in a biological safety cabinet. For better understanding, schematic representations of the steps mentioned were created (Figure 1).

1. Preparation of stock solutions

1. Prepare Papain solution and Collagenase IV/Dispase II solution. Dissolve Papain in sterile PBS (Phosphate-buffered Saline) at a concentration of 30 U/mL; store in 1 mL aliquots at -20 °C. Dissolve Collagenase Type IV at 1.25 mg/mL and Dispase II at 2.5 mg/mL in PBS. Store in 1 mL aliquots at -20 °C.
2. Prepare Neurobasal culture media (F12) supplemented with 2% B27 Supplement (50x stock solution), 10% fetal bovine serum (FBS), and 1% Penicillin-Streptomycin (Pen-Strep) (100x stock solution).
3. Prepare Hank's Balanced Salt Solution (HBSS) with 1% Penicillin-Streptomycin (100x stock solution).
   NOTE: To support initial cell survival and attachment, F12 media supplemented with 10% FBS is used to improve the establishment of the cultures. However, FBS also promotes the proliferation of non-neuronal cells, including fibroblasts. Reduced or serum-free media conditions (e.g., F12 supplemented with B27 only) may be used to minimize non-neuronal cell growth in the DRG culture and cancer co-culture but may affect survival, particularly of dissociated neuron cultures. Serum-free media should also be considered when collecting DRG conditioned media to assess its effects on cancer cell behavior.

2. Preparing reagents and materials for the dissection of DRGs (Figure 1A)

1. Autoclave surgical instruments. Surface-sterilize the dissecting microscope with 70% ethanol and set up on a clean bench.
2. Add 6-8 mL of HBSS-Pen-Strep (step 1.3) to two 10 cm culture plates and place on ice.
   NOTE: These plates will be used to collect spinal columns after dissection. Depending on the number of spinal cords being dissected, additional plates might be needed.
3. Add 8-10 mL of HBSS-Pen-Strep to a 15 mL conical tube, and place on ice.
4. Add 3 mL of HBSS-Pen-Strep to a 3 cm culture plate, and place on ice.
   NOTE: The 15 mL tube prepared in step 2.3 will be used to collect DRGs for dissociation, while the 3-cm culture plate (step 2.4) will be used to collect DRGs for the whole-mount model.

3. Preparing reagents and materials for whole-mount DRG culture

NOTE: Some steps in this section might require overnight preparation. Although any type of tissue culture plate can be used for DRG cultures, the volumes and instructions in this protocol were optimized for 8-well glass bottom slides, which provided optimal imaging quality for our analyses. Keep 8-well glass bottom slide, pipette tips, and Matrigel on ice at all times.

1. Thaw Matrigel solution at 4 °C overnight (or at least 3 h) before use. Make sure to keep Matrigel covered with ice at all times.
2. Chill pipette tips and 8-well glass bottom slide at -20 °C overnight to minimize Matrigel sticking to the tips and prevent premature Matrigel polymerization in the plate.
3. Warm supplemented media (step 1.2) to 37 °C in a water bath.

4. Preparing reagents and materials for dissociated DRG culture

1. Thaw Papain solution and Collagenase IV/Dispase II solution (step 1.1) in a water bath at 37 °C until ready for use.
   NOTE: For each mouse dissected, use 1 aliquot (1 mL) each of Papain and Collagenase IV/Dispase II solution.
2. Warm supplemented culture media (step 1.2).

5. Harvesting the spinal column (Figure 1B) and dissecting the DRGs (Figure 1C)

1. Euthanize 4-8-week-old mice by isoflurane overdose followed by cervical dislocation. Lay the euthanized animal on its abdomen and shave its fur, focusing on the area over the spinal column (Figure 1B, steps 1,2).
2. Spray the back of the animal with 70% ethanol and remove any loose fur, wiping it with paper towels from tail to skull to prevent fur from contaminating the dissection.
3. Using sterilized scissors, make a straight incision along the shaved dorsal surface, from the tail to the skull. Retract the skin on both sides of the incision using blunt forceps to expose the spinal column (Figure 1B, step 3).
4. Using a fresh pair of forceps and scissors to prevent contamination with fur, make a horizontal incision at the base (tail end) of the spinal column (Figure 1B, step 4).
5. Make two parallel longitudinal incisions on each side of the spinal column to release it. Use forceps to gently lift the spinal column to remove the surrounding connective tissue (Figure 1B, step 5).
6. To fully release the spinal column, make an incision at the top of the spinal column (proximal to the skull). Transfer the spinal column to the prechilled 10 cm plates containing HBSS-Pen-Strep on ice (step 2.2) (Figure 1C, step 6).
   NOTE: It is convenient to collect all of the spinal columns in the same 10 cm plate and move individual columns one at a time to a separate plate for further dissection steps.

6. Dissecting the Dorsal Root Ganglia (DRGs)

1. Using spring scissors, remove any residual connective tissue around the spinal column, exposing the vertebrae (Figure 1C, step 7). This allows clearer visualization of the vertebra and facilitates cutting along the midline. Collect trimmed columns in a fresh, prechilled 10 cm plate (step 2.2).
2. One at a time, transfer the trimmed spinal cord to the microscope stage for dissection. Carefully split the spinal column in the sagittal plane using spring scissors (Figure 1D, step 8).
3. Using fine-tipped forceps, remove the spinal cord from the spinal column, exposing the DRGs bilaterally along the roots of the spinal nerves (Figure 1D, step 9).
   NOTE: Extra caution should be taken in this step as DRGs can be removed with the spinal cord if it is handled too roughly.
4. Cut the spinal nerves with spring scissors and remove any connective tissue around the DRGs (Figure 1D, step 10).
   NOTE: Removing connective tissue in this step minimizes debris in the DRG cultures.
5. Using fine-tipped forceps, gently pull the DRG to extract it from the dorsal root. For whole mount culture, place the DRGs in the 3 cm plate (prepared in step 2.4) (Figure 1D, step 11). Pool the remaining DRGs in a 15 mL conical tube (prepared in step 2.3) to be used for dissociation.
   NOTE: Keep plates and tubes on ice throughout the dissection process.
6. Repeat steps 6.4 to 6.5 until all the DRGs are extracted. Repeat all the steps above for any additional spinal columns.
   NOTE: Mice have 30-31 pairs of DRG depending on the strain, which will allow for the collection of up to 60 DRGs from each animal. If collecting DRGs only from the lumbar portion of the spinal cord, 10 DRGs can be extracted. Using DRGs from the same animal to establish both culture models reduces intra-experimental variability and ensures consistent and comparable results between complementary experiments.
   NOTE: All the steps following the extraction of the DRGs must be performed in a biological safety cabinet.

7. Whole-mount DRG culture (Figure 2)

1. Using prechilled tips (step 3.2), pipette 2 µL of chilled Matrigel to form a droplet in each well of an 8-well glass bottom slide placed on ice (Figure 2A, step 1). To prevent Matrigel from polymerizing prematurely before adding the DRG, pipette only a single droplet at a time. To prevent scratching of the slide surface, place the slide in a 10 cm plate before placing on ice.
2. Using fine-tipped forceps, transfer one DRG to a dry plate and gently move it around to remove any excess HBSS, preventing further dilution of the Matrigel when adding the DRG to the Matrigel droplet. Transfer the DRG to the Matrigel drop (Figure 2A, step 2).
   NOTE: Use fine-tipped forceps to gently push the DRG into the Matrigel droplet, ensuring that the DRG is fully immersed and centrally positioned within the droplet.
3. Repeat steps 7.1 to 7.2 for each DRG added to a Matrigel droplet.
4. Incubate the 8-well glass bottom slide at 37 °C for 5 min in a humidified incubator to allow the Matrigel to polymerize (Figure 2A, step 3).
5. Carefully add 200 µL of warm supplemented culture media to each well and incubate at 37 °C with 5% CO₂ (Figure 2B, step 4). Replace the media with fresh supplemented culture media every 72 h.
6. Monitor the DRG using brightfield microscopy to assess its viability and neuronal outgrowth every 24 h (Figure 2C and Figure 3).
   NOTE: Viability can be indicated by an increase in length and density of neurites. Images can be captured over time to demonstrate the development of the DRG cell extensions (Figure 3).

8. Dissociated DRG culture (Figure 4)

NOTE: Any multiwell plate can be used to seed the dissociated DRG neurons. However, in this protocol, 8-well glass-bottom slides were used because they provided better cell confluency and imaging quality for immunofluorescence analyses.

1. Coating culture plates with collagen
   1. Prepare a 50 µg/mL working solution of acidified collagen I in sterile 0.01 M HCl in a biological safety cabinet.
   2. Pipette 100 µL of collagen I solution into each well of the 8-well glass bottom slide. Tilt and gently rotate the slide to ensure the collagen solution covers the entire bottom surface of each well evenly. Incubate the slide at 37 °C in a humidified incubator for 1 h to ensure collagen I polymerization.
      NOTE: During incubation time, proceed with steps 8.2 and 8.3.
   3. After incubation, gently pipette off any residual unpolymerized collagen I solution from each well. Rinse each well with 100 µL of PBS. Pipette off excess PBS, leaving a thin residual layer to prevent the collagen from drying out.
      NOTE: The 8-well glass bottom slide can remain in the biological safety cabinet until the dissociated DRGs are ready for the seeding step.
2. Enzymatic digestion
   1. Gently centrifuge the 15 mL tube containing extracted DRGs (step 6.5) for 5 min at 200 × g at room temperature to pellet the DRGs. Gently remove the HBSS using a pipette.
   2. Rinse DRGs in prewarmed supplemented media, gently tapping the tube to ensure all the contents are being washed and centrifuge for 5 min at 200 × g to pellet the DRGs.
   3. Pipette off the media and add 1 mL of Papain solution (Figure 4A, step 1). Mix gently and incubate for 20 min at 37 °C in a tissue culture incubator. Gently shake the tube to agitate it every 5 min.
   4. After 20 min, add 4.5 mL of supplemented media and mix gently to neutralize Papain. Centrifuge tube for 5 min at 400 × g to pellet DRGs. Carefully remove the supernatant using a pipette.
   5. Add 1 mL of Collagenase IV/Dispase II solution and incubate for 20 min at 37 °C in the incubator. Gently shake the tube every 5 min (Figure 4A, step 1).
   6. After 20 min, add 4.5 mL of supplemented media and gently mix to neutralize the Collagenase IV/Dispase II solution. Centrifuge tube for 5 min at 400 × g to pellet DRGs. Carefully remove the supernatant using a pipette.
   7. Add 2 mL of supplemented media and add DNase I to a final concentration of 0.2 mg/mL. At this point, the DRGs are thoroughly digested and may appear as a loose clump (Figure 4A, step 2).
3. Mechanical dissociation
   1. Gently triturate the DRG, pipetting up and down 4-5x using a 1000 µL filter pipette tip (Figure 4A, step 2).
   2. Perform a second trituration step using a 200 µL pipette tip 4-5x. After this step, the sample appears cloudy with some visible clumps of tissue remaining (Figure 4A, step 2).
   3. Filter the cell suspension through a 70 µm cell strainer into a 50 mL conical tube. Gradually rinse the strainer with 10 mL of media to ensure complete filtration of the cells through the strainer (Figure 4A, step 3).
   4. Centrifuge tube for 5 min at 1,000 × g to pellet cells. Remove media from pellet and resuspend in 500 µL of supplemented media (Figure 4A, step 4).
   5. Remove any residual PBS from wells of the 8-well glass bottom slide. Distribute the dissociated DRG suspension between the collagen-coated wells. Incubate the slides at 37 °C in a humidified incubator with 5% CO₂. Add more media as needed and replace media with fresh supplemented culture media every 48 h (Figure 4B, step 5).
      NOTE: DRG-dissociated cells can be counted using a hemocytometer or cell counting chamber and trypan blue staining to ensure a consistent number of cells per well. For this protocol, approximately 1 × 10⁵ cells were seeded in each well.
   6. Monitor DRG-derived cells using brightfield microscopy to assess viability and neuronal outgrowth (Figure 4C and Figure 5).
      NOTE: Neurons appear as rounded cell bodies with defined borders and may take 24 h or more to adhere, while non-neuronal cells typically settle before the neurons. Some neurite outgrowth might be observed after 24 h and increases over time up to 7 days in culture. Dissociated DRG-derived cells and whole-mount DRG are preincubated for 72 h before adding cancer cells and establishing co-cultures.

9. Preparing cancer cell suspensions and establishing co-cultures

NOTE: Adherent cancer cells of many kinds can be used. Growth media and cell numbers for co-cultures will be cell line-dependent. We recommend preliminary testing before the experiment to determine timing for cell collection and optimal cell seeding numbers. The protocol described here was based on the use of human pancreatic cancer cell lines PanC1, and MiaPaCa2, and prostate cancer cell line DU-145.

1. Wash cells with serum-free media and trypsinize them for approximately 3 min with 2 mL of trypsin/10 cm plate.
2. Neutralize the trypsin by adding 6 mL of supplemented media and gently pipette up and down to ensure a single-cell suspension. Count the cells using a hemocytometer and trypan blue stain.
3. Suspend the cancer cells at a concentration of 2 × 10⁵ cells/mL in supplemented media (step 1.2).
   NOTE: Optimal cell density may vary depending on cell size, doubling time, and the duration of co-culture and can be adjusted as needed.
4. Whole-mount DRG and cancer cell co-culture (Figure 6B)
   1. Aspirate the media off the whole mount DRG cultures in the 8-well glass bottom slide.
   2. Gently pipette 200 µL of the cancer cell suspension (step 9.3) on top of the Matrigel droplet containing the whole mount DRG.
      NOTE: This ensures a uniform distribution of cancer cells around the droplet. Be careful not to disturb the Matrigel droplet with the pipette tip.
   3. Incubate the co-culture slide in a humidified incubator at 37 °C with 5% CO₂. Refresh media daily by washing with 200 µL of warmed supplemented media once, then adding 300 µL of supplemented media to each well.
      NOTE: Gently aspirate the media off using a pipette to avoid disturbing DRG-cancer cells co-culture. Over time, as the co-cultures become more complex and organized, they also become more susceptible to disturbances that may impact cellular interactions and co-culture integrity.
   4. Monitor the co-culture using brightfield or fluorescence microscope at 24 h intervals to assess cancer cell behavior and neuronal interactions (Figure 6B).
      NOTE: Observation frequency can be adjusted based on the experimental timeline and cell interactions of interest.
5. Dissociated DRG and cancer cell co-culture (Figure 6C)
   1. Aspirate the media off the dissociated cultures in the 8-well glass-bottom slide.
   2. Gently distribute 200 µL of the cancer cell suspension (step 9.3) into each well. Follow the same incubation, media refreshment, and monitoring steps as outlined for whole-mount co-cultures (steps 9.4.3 and 9.4.4).
      NOTE: For better visualization of cellular processes and cancer cell-neuron interactions, fluorescent cell trackers can be added to the culture, following the manufacturer's protocol.

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 generated from each model to be integrated more easily, providing a more comprehensive view of cell dynamics and cell-cell interactions. These culture models can be adapted to many applications; however, here we have focused on their use in the context of cancer-nerve interactions.

This protocol offers an efficient workflow for DRG dissection, extraction, and co-culture of viable DRGs with cancer cells while preserving DRG structure and maximizing viability of DRG-derived cells (Figure 1). However, since DRG cultures can easily become contaminated due to carryover of fur during the initial dissection steps, which prevents successful long-term culture, strategies to prevent contamination are essential. Contamination risk can be minimized by switching to fresh sterile scissors and forceps after the initial incisions are made, and before direct contact with the DRGs occurs. Adding antibiotics to the HBSS used when collecting the spinal columns and DRGs also minimizes contamination risk (Figure 1, steps 6, 7, and 11). It is critical to handle DRGs carefully throughout all steps to minimize damage and disruptions of cellular architecture and ensure their integrity and viability.

Investigators should consider the effects of culture media conditions in establishing DRG culture models. In this protocol, Neurobasal media supplemented with B27 and FBS was used for optimal attachment and survival of DRG cells and stimulation of robust neurite outgrowth suitable for co-cultures. Establishment of DRG cultures was less efficient in the absence of supplements and neurite extension was markedly reduced (Figure 7). Depending on application, DRG can be cultured in reduced serum or treated with purified neurotrophic factors to stimulate neurites and minimize overgrowth by other non-neural cell types.

An important consideration in establishing DRG cultures is the age of the animals. Although we see some variability in the extent of neurite formation between DRGs, whole-mount DRGs isolated from younger animals (<4 weeks old) generally produced extensive glial proliferation, which makes visualization of individual neurites challenging. In contrast, whole-mount DRGs from older animals (4-8 weeks) produced less profuse and more distinguishable neurite extension and minimal glial cells, facilitating visualization of nerve-cancer cell interactions (Figure 8). Another critical consideration when setting up this model is the variability in DRG size along the spinal column-cervical DRGs are smaller, with the size gradually increasing as they get closer to the dorsal portion of the spinal column. For optimal consistency, co-cultures of DRGs from the same region of the spinal column should be compared when possible.

The dissociation of DRG neurons (Figure 4) was optimized for maximal neuron-recovery efficiency through two steps of enzymatic dissociation (Papain and Collagenase IV/Dispase II treatments) followed by a step of mechanical dissociation with DNase I. Lower neuron recovery was achieved if only a single enzymatic digestion step (Collagenase IV/Dispase II) was performed. Dissociated neurons need to be homogeneously dispersed in wells in preparation for co-culture with cancer cells, but excessive pipetting should be avoided, as it can damage the cells. We recommend gentle pipetting up and down up to 5x before seeding and monitoring integrity and distribution of dissociated neurons using brightfield or fluorescence microscopy (Figure 4C).

DRG cultures are heterogeneous, including neurons and non-neuronal cells. The presence of non-neuronal cells, such as Schwann cells, macrophages, and fibroblasts, within the DRG culture becomes evident within 24 h of culture, as their morphology becomes distinct from the neurons, which maintain a well-delimited rounded cell body (Figure 4C and Figure 5). Neurite sprouting from the DRG-derived cells was observed within 24 h of culture and further increased after 72 h (Figure 5). However, care must be taken as other cell types within the culture, such as Schwann cells, can also form cell extensions similar to neurites in appearance. Additional strategies can be used to differentiate specific cell types within this heterogeneous population. For example, immunostaining can be used to distinguish neuronal nuclei (NeuN) and axons (β-tubulin III) (Figure 9), and the presence of non-neuronal cells (β-tubulin III-negative, NeuN-negative) and other cell types can be recognized by differences in morphology and actin organization (phalloidin).

As an example of application, dissociated DRG neurons can be used in transwell assays to assess cellular responses to DRG-secreted factors. Cancer cells seeded in Matrigel-coated transwell inserts invade towards DRG dissociated neurons but not towards negative controls (HEK293 cells) after 24 h incubation (Figure 10). Our preliminary findings show that significantly more cancer cells undergo directional invasion towards the DRG neurons in response to secreted attractant molecules.

Whole-mount DRG cultures were optimized to investigate direct neuron-cancer cell interactions while maintaining complex tissue integrity more similar to the in vivo nerve structure (Figure 2). Dissected DRGs were immersed in a Matrigel droplet and incubated to allow neurite extension outward through the matrix towards the periphery. Careful positioning of the DRG centrally in the droplet provided the maximal neurite extension without overgrowing the borders of the Matrigel droplet (Figure 2 and Figure 3), allowing evaluation of chemotactic interactions between tumor and nerve cells. If neurites extend beyond the droplet, they can come in direct contact with cancer cells, which will no longer respond to a chemotactic gradient.

While Matrigel is broadly used as an extracellular matrix in co-culture and preclinical in vivo models^16,19,20, alternative natural or synthetic matrices could also be considered depending on the experimental goals. Natural alternatives include Type I collagen²¹ or decellularized extracellular matrix²², which retains many tissue-specific bioactive components that mimic native neural environments, but like Matrigel, these also have inter-batch variability²³. PEG-based hydrogels²⁴, as semi-synthetic matrices, allow for precise control over stiffness, adhesiveness and biochemical composition, but the incorporation of necessary peptide cues can be costly. These alternatives offer more defined and tunable systems, which may be particularly useful in studies requiring precise control over the extracellular environment.

To further investigate the cancer-neuron interactions, we established direct co-culture models using whole-mount (Figure 11A) and dissociated DRG (Figure 11B). As cancer cells generally proliferate rapidly, DRG cultures were allowed to establish and extend neurites before adding the cancer cells to optimize viewing of cell-cell interactions (Figure 6). Neurites often appear 24 h after plating the DRG, with continued outgrowth observed after 48 h and 72 h. Before adding the cancer cells, DRG cultures can be labeled by the addition of fluorescent dyes such as CellTrace Calcein Red-Orange (red), which facilitates imaging of neurites (Figure 11A,B).

After 12 h of co-culture, cancer cells settled around the border of the Matrigel droplet containing the DRG (Figure 12A). Imaging these co-cultures over time allowed us to observe the movement of cancer cells into the matrix droplet. After 72 h of co-culture, cancer cells had invaded the Matrigel and moved toward the DRG and direct interaction between DRG cell extensions and cancer cells could be observed (Figure 7, Figure 11A, and Figure 12B). Invasion can be quantified by comparison of the area of invasion after 12 h and 72 h (Figure 12). In an example of this application, co-culture with whole mount DRG significantly increased invasion of cancer cells into a Matrigel droplet compared to no DRG conditions (Figure 12).

The final time point for co-cultures will depend on the invasive potential of the cancer cell type chosen, and longer times of co-culture may be needed for slower growing cell types. The 72 h timepoint was found to be optimum for a 2 µL Matrigel droplet and the number of cancer cells seeded, as it allowed the maximum outgrowth of DRG extensions without exceeding the limits of the Matrigel droplet (Figure 3). If longer periods of culture are necessary, adjusting the Matrigel droplet size should be considered to ensure clear visualization of neurites and/or cells invading the droplet. Whole-mount DRG co-culture with cancer cells allowed us to recapitulate part of the complexity present in vivo; however, because of the cellular density of the DRG, the specific cell-cell interactions were not easily visualized in this 3D setting (Figure 11A and Figure 12B). For this reason, co-culture of dissociated DRG-derived cells with cancer cells provides a complementary model that allows detailed visualization of individual cell interactions.

As with the DRG whole-mount, cancer cells were added to the dissociated DRG cultures after 72 h of incubation and direct cell-cell interactions observed using brightfield or confocal microscopy (Figure 6C). To facilitate imaging and differentiate between nerve-derived and cancer cell populations within the co-cultures, cancer cells expressing fluorescent markers, such as EGFP-tagged cells (green), can be used. Alternatively, cells can be labeled by treatment with fluorescent dyes such as CellTrace Calcein Red-Orange (red) that can be added to the DRG cultures before the addition of cancer cells and allow differential tracking of the cell populations (Figure 11B). Live cell imaging of co-cultures over time can be used to quantify the progressive outgrowth of neurites from DRG cells and assess changes in contact between cancer cells and neurons (Figure 6C).

Finally, immunofluorescence staining of these co-cultures at end points can provide high-resolution visualization of the extent and complexity of cancer-nerve interactions. For example, the degree and pattern of pancreatic ductal adenocarcinoma cell invasion through Matrigel matrix and along neuronal extensions can be identified by immunofluorescence in whole-mount DRG (Figure 13). In contrast, direct interactions and specific cell contact points between cancer cells and neuronal extensions can be visualized in dissociated DRG co-cultures (Figure 14).

Figure 1: Dissection and extraction of mouse dorsal root ganglia. Overview of the main steps for dissecting and extracting DRG from mice. (A) The bench area is prepared, including sterilized instruments and dissecting microscope. Prepare and chill HBSS + 1% Penicillin-Streptomycin (Pen-Strep) on ice. (B) Preparing the mouse and removing the spinal column. (1) Mouse is positioned on its abdomen and (2) fur is shaved along the dorsal midline. (3) Skin is removed to expose the spinal column, and (4,5) incisions are made to release the spinal column. (C) Spinal column trimming. (6) Place isolated spinal columns in collection media on ice. (7) Trim to remove excess connective tissue. (D) Isolation of DRGs. (8,9) Cut along the midline to expose the spinal cord and remove the spinal cord to expose the DRGs. (10) Extract DRG by cutting the spinal nerves and gently pulling the ganglia free. (11) Collect DRGs in cold HBSS-Pen-Strep for subsequent steps. Abbreviations: DRG = dorsal root ganglion; HBSS = Hank's balanced salt solution. Please click here to view a larger version of this figure.

Figure 2: Workflow for embedding DRG in Matrigel for whole-mount culture. (A) DRG embedding in Matrigel droplet.(1) A 2 µL droplet of chilled Matrigel is pipetted into the well using a chilled pipette tip; (2) harvested DRGs are placed in the center of the Matrigel droplet. (3) Matrigel is allowed to polymerize for ~3 min. (B) Neurite outgrowth, (4) supplemented media is added to the well, and DRG cultures are monitored every 24 h for neurite outgrowth. (C) Imaging whole-mount DRG cultures. Brightfield or fluorescence microscopy imaging of whole-mount DRGs with CellTrace Calcein Red-Orange reveals extensions (arrows) after 72 h of culture. Scale bars = 200 µm. Abbreviation: DRG = dorsal root ganglion. Please click here to view a larger version of this figure.

Figure 3: Time-lapse visualization of whole-mount DRG cultures showing progressive neurite outgrowth. Schematic diagrams (upper panels) and representative brightfield images (lower panels) showing DRG cultures at indicated times after embedding in Matrigel: (A) whole DRG and (B) Zoom view of region in box, at initial time of embedding in Matrigel (0 h); neurite extension after (C) 24 h; (D) 48 h; (E) 72 h. White arrows indicate neurite projections. Images captured under brightfield microscopy. Scale bars = 500 µm (A); 200 µm (B-E). Abbreviation: DRG = dorsal root ganglion. Please click here to view a larger version of this figure.

Figure 4: Workflow for preparation of dissociated DRG cultures. (A) DRG dissociation (1) Collected DRGs are treated with two successive steps of enzymatic digestion. (2) Mechanical trituration is performed using a p1000 pipette tip, followed by trituration with p200 pipette tip. (3) Suspension is filtered to remove debris. (4) Pellet containing dissociated DRG are collected after centrifugation. (B) Neurite outgrowth: (5) DRGs were dissociated into individual cells and cultured in supplemented media. (C) Imaging dissociated DRG culture. Representative brightfield and immunofluorescence images of dissociated DRG cultures after 72 h. Neurons (arrows) and Schwann cells (arrowheads) in culture are indicated. Scale bars = 200 µm. Abbreviation: DRG = dorsal root ganglion. Please click here to view a larger version of this figure.

Figure 5: Visualization of neurite outgrowth of dissociated DRG neurons in culture. Dissociated DRG neurons isolated from 8-week-old C57BL/6 mice and cultured in Neurobasal medium (F12) supplemented with B27 and 10% FBS. Representative images show the extent of neurite outgrowth (white arrows) observed at initial plating (0 h) and after 24 h and 72 h of culture. Images were captured using a brightfield microscope. Scale bars = 50 µm. Abbreviation: DRG = dorsal root ganglion. Please click here to view a larger version of this figure.

Figure 6: Timeline for preparing whole mount and dissociated DRG cultures and co-cultures with cancer cells. (A) Illustration of timeline for DRG culture (top, purple) and subsequent co-culture with PanC1 pancreatic adenocarcinoma cells (bottom, blue). (B,C) Brightfield images of (B) DRG whole mount in the centre of a Matrigel droplet (dashed circles) and (C) Dissociated DRG cultures, at the corresponding timepoints indicated in A. (B,C) Left Panels show DRG cultures at initial isolation and after 72 h in culture. Arrows indicate neurite extensions and arrowheads indicate cancer cells. Right panels show DRG co-cultured with PanC1 cells (arrowheads) at 0 h and after 72 h of co-culture. Scale bars = 500 µm for whole-mount DRG; 100 µm for dissociated DRGs at 0 h; 50 µm for dissociated DRGs at 72 h. Abbreviation: DRG = dorsal root ganglion. Please click here to view a larger version of this figure.

Figure 7: Effects of Fetal Bovine Serum or Glial Cell line-derived Neurotrophic Factor (GDNF) on neurite outgrowth in whole-mount DRG cultures. Representative fluorescence images of whole-mount DRG cultured for 72 h under the indicated conditions. DRG were stained with CellTrace Calcein Red-Orange for better visualization of the extensions. (A) Minimal extension of neurites is observed in the absence of FBS. (B) Moderate growth of neurites is observed in DRGs culture with 5% FBS. (C) Extensive growth of neurites is observed in DRGs cultured in the presence of 10% FBS. (D) Media supplemented with GDNF in the absence of FBS promotes neurite outgrowth in DRGs. Scale bars = 100 µm. Abbreviations: DRG = dorsal root ganglion; FBS = fetal bovine serum. Please click here to view a larger version of this figure.

Figure 8: Effects of mouse age on DRG cultures. Representative brightfield images of whole-mount DRG from C57BL/6 mice cultured in Neurobasal medium (F12) supplemented with B27 and 10% FBS for 72 h. Left, mouse-derived DRGs from a young (3-week-old) animal. Right, DRGs from an older (8-week-old) animal. Arrows indicate neurite extensions. Images demonstrate how animal age influences Schwann cell density, extensions, and organization, affecting neurite visualization. Scale bars = 200 µm. Abbreviations: DRG = dorsal root ganglion; FBS = fetal bovine serum. Please click here to view a larger version of this figure.

Figure 9: Immunofluorescence staining of DRG cultures to identify neurons. Fluorescence images of dissociated DRG neurons isolated from adult C57BL/6 mice and cultured for 72 h. Cells were fixed and stained for β-tubulin III (green, neuronal cytoskeleton), NeuN (red, neuronal nuclear marker), and Hoechst (blue, nuclei) and images captured by confocal microscopy. Merged image shows co-localization of NeuN and Hoechst stain in neuron nuclei (white arrows). Scale bar = 50 µm. Abbreviation: DRG = dorsal root ganglion. Please click here to view a larger version of this figure.

Figure 10: DRG neurons promote cancer cell invasion in an indirect co-culture model. (A) Schematic illustration of the transwell assay shown in B and C. Representative images of Transwell invasion assay showing cancer cells invasion toward (B) HEK293 and (C) DRG dissociated neurons. (D) Quantification graph shows significantly enhanced invasion of cancer cells in the presence of DRG compared to HEK293 cells after 24 h of co-culture (n = 4; *p < 0.05). Scale bars = 100 µm. Abbreviations: DRG = dorsal root ganglion. Please click here to view a larger version of this figure.

Figure 11: Co-culture of cancer cells and DRG neurons. DRG neurons isolated from adult C57BL/6 mice were cultured for 72 h and stained with CellTrace Calcein Red-Orange. EGFP-tagged PanC1 pancreatic adenocarcinoma cells were added and incubated for an additional 72 h. (A) Representative image of a whole-mount DRG (red) co-cultured with PanC1 cells (green), showing cancer cells (arrows) invading the Matrigel droplet (dashed line) towards the DRG. Scale bars = 200 µm.(B) Representative fluorescence image of dissociated DRG with PanC1 cells showing extension of neurites (arrowheads) in the presence of cancer cells (arrows). Scale bars = 50 µm. Abbreviations: DRG = dorsal root ganglion; EGFP = enhanced green fluorescent protein. Please click here to view a larger version of this figure.

Figure 12: DRG neuron co-culture significantly increases cancer cell invasion. Representative brightfield images of cancer cells co-cultured with DRG for 72 h. (A,B) In the presence of DRG, cancer cells exhibit directional migration and interaction with neurites extending from the core of the DRG. (C,D) Invasion over time is significantly enhanced compared with cancer cells in the absence of DRG. (E) Quantification of cancer cell invasion in the presence and absence of DRG after 72 h. White arrows indicate invasive cancer cells. Images were captured using brightfield microscopy at 10x magnification. Scale bars = 200 µm. Abbreviations: DRG = dorsal root ganglion. Please click here to view a larger version of this figure.

Figure 13: Visualization of DRG and cancer cell association in co-culture. Whole mount DRG neurons isolated from adult C57BL/6 mice were cultured for 72 h before EGFP-tagged PanC1 pancreatic adenocarcinoma cells were added and incubated for a further 72 h of co-culture. Nuclei are stained with Hoechst (blue), actin filaments with phalloidin (green), and neuronal structures with β-Tubulin III (red). Confocal image shows PanC1 cells (arrows, blue nucleus, green cytoskeleton) invading through the Matrigel droplet (dashed line) along neurites (red) towards the DRG. Scale bars = 100 µm. Abbreviations: DRG = dorsal root ganglion; EGFP = enhanced green fluorescent protein. Please click here to view a larger version of this figure.

Figure 14: Visualization of dissociated DRG and cancer cell association in co-culture. Dissociated DRG neurons isolated from adult C57BL/6 mice were cultured for 72 h before EGFP-tagged PanC1 pancreatic adenocarcinoma cells were added and incubated for a further 72 h of co-culture. Nuclei are stained with Hoechst (blue), actin filaments with phalloidin (green), and neuronal structures with β-Tubulin III (red). The image shows a PanC1 cell in direct contact with a DRG-derived cell (red cytoskeleton and blue-stained nuclei). Neuronal processes (arrows) can also be observed around the cancer cell. Scale bars = 10 µm. Abbreviations: DRG = dorsal root ganglion; EGFP = enhanced green fluorescent protein. Please click here to view a larger version of this figure.

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 cellular behaviour and complex tissue responses. Our protocol further optimizes nerve dissection and dissociation conditions to maximize neurite outgrowth and maturation of the nerve environment before addition of tumor cells. We also provide additional methodological details, to address common technical challenges, reduce contamination and increase efficiency of establishment of cultures to ensure reproducibility of conditions and expand the applicability of this protocol across different experimental models. In this regard, we have used this protocol successfully to establish nerve cultures from mice of several genetic backgrounds and have had similar success with wildtype inbred or outbred strains (e.g., C57BL/6, CD-1) and immune-compromised animals (e.g., Rag2IL2Rγc KO), confirming broad applicability of the method.

While whole-mounted DRGs embedded into a Matrigel droplet offer the ability to investigate biological mechanisms in a complex three-dimensional environment, the dissociated DRG culture allows us to observe individual cell behavior and structure, as well as allowing high-resolution imaging of these interactions²⁵. In each case, these models maintain DRG cellular heterogeneity, including both neural and non-neural cell types (Figure 8 and Figure 10). Schwann cells, the primary glial cell of the peripheral nerve, with key roles in matrix remodeling and neurotrophic support for neurite outgrowth and cell migration in the nerve environment, are maintained in these 2D and 3D nerve cultures. Growth factors and neurotrophic factors secreted by these cells, as well as neural fibroblasts, are essential for accurate recapitulation of the in vivo nerve environment and cell interactions^26,27 and also contribute to cancer cell proliferation and invasion in human cancers²⁸. Further, these DRG culture models are also broadly applicable to other investigations. This method can be adapted for assessment of drug sensitivity or efficacy¹⁸, for neuroprotection or nerve regeneration¹⁶. Nerve cultures may have particular value in accelerating drug discovery or validation for nervous system conditions, such as Parkinson's disease or neuropathic pain²⁹. This protocol may also provide valuable tools for exploring gene-of-origin in knockout animal models related to the development of nervous system disorders.

The whole-mount DRGs and the dissociated DRG-derived cell models presented here are ideal for evaluating cancer cell interactions with peripheral nerves under different conditions and over time (Figure 9, Figure 10, and Figure 11). Whole-mount cultures allow assessment of the invasive capacity of cancer cells towards and into the ganglia in response to peripheral nerve-derived signals, while direct interactions between nerve and cancer cells can be visualized and quantified in dissociated DRG cultures. Because cancers differ in growth rate and invasiveness, this protocol may require optimization for use with cell lines of different cancer types. For rapidly proliferating cancer cells, reducing the number of cells seeded or allowing additional time for neuron culture before adding cancer cells may be required. For slower growing cells, increasing cancer cell numbers seeded and extending coculture conditions may be more successful. Because of these cell-specific variations in growth, we recommend checking cultures daily so that timing or endpoint of the experiment can be adjusted as needed. We have found that nerve cultures can remain viable up to 10 days after plating and cocultures can be analyzed more than 5 days after initiation.

When establishing a primary culture of differentiated cells such as DRG-derived cells in vitro, the age of the animal from which these DRGs are extracted is an important variable¹⁸. We observed that DRGs from younger mice form extensive glial proliferation, while DRGs from older mice display minimal glial cell extension, facilitating visualization of nerve cell extensions (Figure 7). The increased number of glial cells around DRGs extracted from a younger mouse can not only impair the visualization of neuronal extension but can affect the microenvironment by increasing the nature or levels of molecules normally secreted by those cells. Previous studies have shown a reduction in the number of glial cells with advancing age, as well as a decrease in mitochondrial volume in these cells, suggesting loss of functionality^30,31. Reduced glial cell density and functionally can also affect the microenvironment secretome composition, decreasing release of neurotrophic factors that interact with sensory neurons^32,33 and altering nerve-cancer cell interactions, and impacting experimental reproducibility. The mouse strain used for DRG isolation should also be considered since varied responses in neurite outgrowth have been observed among genetically different mouse strains³⁴. Further, DRGs also vary within the same animal, with cervical DRGs being significantly smaller than DRGs from the lumbar portion of the spinal column and differing in the proportions of different neuron subtypes present³⁵. When coculture models are used to evaluate the effects of different treatments or cell conditions (e.g., gene knockout or knockdown), we recommend using matched DRGs from similar positions along the spinal column to ensure similar size and cellular composition of the ganglia and provide more comparable conditions.

Primary tissue explants and cocultures provide cost-effective and versatile models to assess the nerve microenvironment and cancer cell nerve invasion. Isolated DRGs allow multiple experiments to be performed with relatively few animals, optimizing preliminary validation steps, allowing fine-tuning of assays before transition to more focused preclinical animal models. The DRG co-culture system enables detailed observation of cancer-nerve interactions, including neurite remodeling, cancer-induced neuritogenesis, and the directional migration of tumor cells along neurites^11,36. However, the in vivo nerve microenvironment is highly complex, rich in stromal and immune cells, vascular components, and biochemical gradients, all these structures influence tumor progression and perineural invasion^37,38. Such complexity is not fully recapitulated in vitro; their systemic factors like immune surveillance and mechanical forces are not represented. Nevertheless, in vitro systems such as DRG co-culture, remain a powerful and ethically accessible tool for exploring tumor-nerve crosstalk and testing targeted therapeutic strategies under controlled conditions.

Overall, these observations highlight the versatility of the models presented here in assessing the invasiveness of cancer cells mediated by DRG neurons from different perspectives. The application of these two models offers a complementary approach, from assessment of direct cell-cell interactions with dissociated DRGs to a more integral view of cancer-nerve interactions and their impacts on the interplay and spatial dynamics of these cells in a three-dimensional context.

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