Usefulness of the measurement of neurite outgrowth of primary sensory neurons to study cancer-related painful complications

Abstract

Abnormal outgrowth of sensory nerves is one of the important contributors to pain associated with cancer and its treatments. Primary neuronal cultures derived from dorsal root ganglia (DRG) have been widely used to study pain-associated signal transduction and electrical activity of sensory nerves. However, there are only a few studies using primary DRG neuronal culture to investigate neurite outgrowth alterations due to the underlying cancer-related factors and chemotherapeutic agents.

In this study, primary DRG sensory neurons derived from mouse, non-human primate, and human were established in serum and growth factor-free condition. A bovine serum albumin gradient centrifugation method improved the separation of sensory neurons from satellite cells. The purified DRG neurons were able to maintain their heterogeneous subpopulations, and displayed an increase in neurite growth when exposed to cancer-derived conditioned medium, while they showed a reduction in neurite length when treated with a neurotoxic chemotherapeutic agent. Additionally, a semi-automated quantification method was developed to measure neurite length in an accurate and time-efficient manner. Finally, these exogenous factors altered the gene expression patterns of murine primary sensory neurons, which are related to nerve growth, and neuro-inflammatory pain and nociceptor development.

Together, the primary DRG neuronal culture in combination with a semi-automated quantification method can be a useful tool for further understanding the impact of exogenous factors on the growth of sensory nerve fibers and gene expression changes in sensory neurons.

Graphical Abstract

1. INTRODUCTION

Recent evidence suggests that crosstalk between the peripheral nervous system and cancer plays an important role in both cancer development and cancer-associated pain [1–3]. In head and neck cancer, tumors innervated by peripheral nerves are known to be more aggressive than those with less innervation [4]. Recent studies using mouse models have revealed that the sympathetic nervous system regulates prostate cancer tumorigenesis and bone metastasis [5], and that denervation can even attenuate tumorigenesis and metastasis in several types of cancer [5–8]. Moreover, it has been indicated that cancer-associated pain may be a negative indicator of survival in cancer patients [9], suggesting that sensory nerves influence disease progression in patients with poor prognoses. This accumulating evidence indicates that the peripheral nervous system within the tumor microenvironment may enhance disease progression.

Conversely, cancer cells are also known to affect neuronal activities. Cancer often causes pain by interacting with sensory nerves. Especially, when cancer cells metastasize to the bone, patients experience bone pain, referred to as cancer-induced bone pain (CIBP). Bone is richly innervated by sensory nerves [10], and it has been suggested that the acidic environment surrounding cancer, or factors, such as growth factors, cytokines, or chemokines, secreted by cancer cells can all stimulate receptors on sensory nerves to induce CIBP [11–14]. Additionally, recent studies have also demonstrated that sensory nerve outgrowth (or sprouting) is one of the potential mechanisms of CIBP [15–19]. A neurotrophic factor nerve growth factor (NGF) is known to be involved in the regulation of survival and growth of neurons [20]. NGF derived from osteosarcoma cells stimulated neuroma-like formation in the periosteum of cancer-inoculated mouse bones, resulting in CIBP [19]. When an NGF antagonist was administered to cancer-bearing mice, CIBPs as well as the accompanying nerve sprouting were attenuated [19]. Consistently, abnormal sensory nerve sprouting also causes pain behaviors in other chronic pain conditions [10, 21–23].

Not only nerve growth, but also nerve damage is associated with the induction of chronic pain conditions. Chemotherapy-induced peripheral neuropathy (CIPN) is a consequence of the neurotoxic effects of chemotherapy agents [24–26]. Indeed, paclitaxel reduced the density of intraepidermal nerve fibers in hind paw skin and produced pain-related behaviors in rodents [26, 27].

These findings suggest (i) that nerves and cancer interact very closely with one another and (ii) abnormal plasticity or remodeling of sensory neurons may contribute to cancer-induced pain. Therefore, it is crucial to understand how nerve growth is regulated in the context of cancer and its treatments. In pursuit of this goal, animal studies are often used to investigate the systemic effects of disease states and therapies on peripheral nerves, but they are not always suitable to study local interactions. For that reason, various in vitro neuronal culture systems have been developed to understand the mechanisms of neurite outgrowth. The mouse pheochromocytoma cell line PC12, which is known to form neurite-like structures in response to NGF [28], has been previously used to assess the effects of cancer-derived factors on neurite outgrowth. Conditioned medium (CM) derived from pancreatic cancer cells [29] or head and neck cancer-derived exosomes [30] were shown to enhance neurite sprouting of PC12 cells. Cell lines are easy to maintain and handle but may not fully recapitulate the phenotype of sensory neurons. Moreover, only few neuronal cell lines which extend neurites are currently available. The use of primary culture of dorsal root ganglia (DRG) neurons may overcome this hurdle. To date primary DRG culture has mainly been used to investigate physiological alterations in neuronal activity [31] and to a lesser degree neurite outgrowth.

In the current study, we established methods for reliable in vitro primary cultures of sensory neurons derived from mouse, non-human primate, and human DRGs. Additionally, we found that these primary DRG neuronal cultures were suitable to study the neurite length alterations and changes in gene expression associated with nerve growth, and neuro-inflammatory pain and nociceptor development, mediated by CM obtained from cancer cells or paclitaxel-induced neurotoxicity. We also developed a semi-automated method for the measurement of neurite outgrowth, resulting in the reduction of overall analysis time.

2. MATERIALS AND METHODS

All methods were carried out in accordance with relevant guidelines and regulations. All human studies and all animal studies followed the Declaration of Helsinki and the Institutional Animal Care and Use Committee Guidelines, respectively.

2.1. Human subjects research certification

All human studies are approved by the Institutional Review Board (IRB #00056846) at Wake Forest University Health Sciences. Informed consent was obtained from all subjects involved in the current study.

2.2. Animal care and use certification

All animal studies are approved by the Institutional Animal Care and Use Committee (Protocol A18–026 for mouse and Protocol A18–161 for non-human primate) at Wake Forest University Health Sciences.

2.3. Murine DRG isolation and primary neuronal culture establishment

Primary DRG neuronal culture was prepared, according to previously published protocols [32, 33] with modifications. Nine-twelve lumbar DRGs (L2-L4) of male C57BL/6 mice (8–12 weeks of age, Jackson Laboratory, Bar Harbor, ME) were dissected and directly placed into a 15 mL conical tube, containing 14 mL of ice-cold 1 × Hanks’ balanced salt solution (HBSS) without Mg⁺⁺/Ca⁺⁺ (Thermo Fisher Scientific, Gibco, Waltham, MA). Isolated DRGs were enzymatically digested in 3 mL of papain solution [30 U/mL papain (Worthington Biochemical Corp., Lakewood, NJ), 0.1% saturated NaHCO3 solution (Sigma-Aldrich, St. Louis, MO), 0.3 mg/mL L-Cys (Sigma-Aldrich) in HBSS without Mg⁺⁺/Ca⁺⁺)] for 30 min at 37°C with 5% CO₂, and then incubated in 3 mL of collagenase type II (CLS2)/dispase type II (Dispase II) solution [4 mg/mL CLS2 (Worthington Biochemical Corp.) and 4.7 mg/mL dispase (Sigma-Aldrich) in HBSS without Mg⁺⁺/Ca⁺⁺] for 30 min at 37°C with 5% CO₂. The DRGs were mixed gently every 10 mins. The resulting DRGs were centrifuged at 200 × g for 2 min and washed with HBSS without Mg⁺⁺/Ca⁺⁺. The pellet was transferred to a 15 mL conical tube, containing 500 μL of neuronal growth (NG) medium [Neurobasal-A (Thermo Fisher Scientific, Gibco), 1% N2 (Thermo Fisher Scientific), 2% B-27 (Thermo Fisher Scientific), 2 mM L-glutamine (Thermo Fisher Scientific), 1% penicillin-streptomycin (Thermo Fisher Scientific), and 0.4% glucose (Sigma-Aldrich)]. The DRGs were triturated 15–20 times using p1000 and then p200 pipette tips and filtered through a stainless mesh sieve (40 μm, Thermo Fisher Scientific) to obtain single-cell suspensions and remove undigested tissue debris. After bovine serum albumin (BSA) purification [3.5% (W/V) BSA solution] (see below), 500–1,000 cells of DRGs in 30 μL of warm NG medium were seeded onto the center of 12 mm round coverslips (MatTek Corp., Ashland, MA), pre-coated with Poly-D-lysine (50 ug/mL, overnight at 4°C, Thermo Fisher Scientific, Corning) and laminin (20 μg/mL, 1 h at 37°C, Thermo Fisher Scientific, Corning), in 24-well plate. After 1–2 h, 1 mL of warm NG medium was gently added to the sides of wells and the cells were maintained at 37°C with 5% CO₂.

2.4. BSA purification

After trituration and filtration, single-cell suspensions from DRGs were centrifuged through 3.5% (W/V) BSA solution (Thermo Fisher Scientific) (5 mL of BSA solution:1ml of HBSS without Mg⁺⁺/Ca⁺⁺; 1 mL of cell suspension in a 15 mL conical tube) at 14 × g for 20 min at room temperature (RT) to separate sensory neurons (in pellet) from satellite cells and debris (in BSA layer).

2.5. Non-human primate DRG isolation and primary neuronal culture establishment

Non-human primate primary neuronal culture was prepared similarly as described in the murine primary neuronal culture with minor modifications. DRGs (T12 and L1) of a healthy 8-year old female Rhesus macaque (Worldwide Primates, Inc., Miami, FL) were dissected and directly placed in a 15 mL conical tube containing 14 mL of ice-cold HBSS without Mg⁺⁺/Ca⁺⁺ and transferred to the laboratory for the further procedures. Isolated DRGs were cleaned up by removing non-nervous tissues and nerve root under the laminar flow hood using sterile blade, scissors, and forceps, and cut into small pieces achieved at approximately 1–3 mm side length of a cubical volume in ice-cold HBSS without Mg⁺⁺/Ca⁺⁺ and transferred to a 15 mL conical tube containing papain solution. DRGs were enzymatically digested in 6 mL of papain solution for 1 h at 37°C with 5% CO₂ and then incubated in 6 mL of CLS2/Dispase II solution for 1 h at 37°C with 5% CO₂. During the digestion, DRGs were triturated 10–15 times using p1000 tips every 20 min and filtered through a stainless mesh sieve (100 μm, Thermo Fisher Scientific) to obtain single-cell suspensions and remove undigested tissue debris. After BSA purification [3.5% (W/V) BSA solution], 600 cells of DRGs in 30 μL of warm NG medium were seeded onto the center of 12 mm round coverslips, pre-coated with Poly-D-lysine and laminin, in 24-well plate. After 1–2 h, 1 mL of warm NG medium was gently added to the sides of wells and the cells were maintained at 37°C with 5% CO₂.

2.6. Human DRG isolation and primary neuronal culture establishment

Human primary neuronal culture was prepared similarly as the non-human primate neuronal culture with minor modifications, according to previously published studies [34–38]. DRG (T10) of a male patient (41 years old age) were dissected during a T10 corpectomy for correction of kyphotic deformity secondary to discitis/osteomyelitis and directly placed in a 15 mL conical tube containing 14 mL of ice-cold HBSS without Mg⁺⁺/Ca⁺⁺ and transferred to the laboratory for the further procedures. Isolated DRGs were cleaned up by removing non-nervous tissues and nerve root under the laminar flow hood using sterile blade, scissors, and forceps, and at approximately 1–3 mm side length of a cubical volume in ice-cold HBSS without Mg⁺⁺/Ca⁺⁺ and transferred to a 15 mL conical tube containing papain solution. DRGs were enzymatically digested in 9 mL of papain solution for 1 h at 37°C with 5% CO₂ and then incubated in 9 mL of CLS2/Dispase II solution for 1.5 h at 37°C with 5% CO₂. During the digestion, DRGs were triturated 10–15 times using p1000 tips every 20 min and filtered through a stainless mesh sieve (100 μm) to obtain single-cell suspensions and remove undigested tissue debris. After BSA purification [3.5% (W/V) BSA solution, DRGs in 30 μL of warm NG medium were seeded onto the center of 12 mm round coverslips, pre-coated with Poly-D-lysine and laminin, in 24-well plate. After 1–2 h, 1 mL of warm NG medium was gently added to the sides of wells and the cells were maintained at 37°C with 5% CO₂.

2.7. Cancer cell or normal prostate epithelial cell derived conditioned medium generation

To collect the conditioned medium (CM) from cancer cells, 5 × 10⁵ cells of a human breast cancer cell line established from a pleural effusion, MDA-MB-231 [American Type Culture Collection (ATCC), Manassas, VA], a human prostate cancer cell line established from bone metastasis, PC-3 (ATCC), a human lung adenocarcinoma cell line established from lung carcinomatous tissue, A549 (ATCC), or a murine lung carcinoma cell line established from a primary tumor nodule from the Lewis lung carcinoma model, LL/2 (ATCC) were seeded onto a 10 cm dish with 10 mL of growth medium. These cancer cell lines were chosen since they have been shown to grow in the bone and/or can induce CIBP [39–42]. The passage number for all cell lines used was less than 25 passages. MDA-MB-231 and LL/2 cells were cultured in Dulbecco’s modified eagle medium (DMEM) (Thermo Fisher Scientific, Gibco) containing 10% fetal bovine serum (FBS, Thermo Fisher Scientific, Gibco), 1% penicillin-streptomycin and glutamine (PSG) (Thermo Fisher Scientific, Gibco), while PC-3 and A549 cells were culture in Roswell park memorial institute (RPMI) 1640 medium (Thermo Fisher Scientific, Gibco) containing 10% FBS and 1% PSG. At 24 h, the growth medium was removed and replaced with 10 mL of serum free DMEM or RPMI 1640 medium (Thermo Fisher Scientific, Gibco) containing 1% PSG. For the control medium, 10 mL of serum free DMEM or RPMI medium were added to a 10cm dish without adding any cancer cells. To collect the CM from non-cancer cells, 5 × 10⁵ cells of a human normal epithelial prostate cell line, PWR-1E (ATCC) were seeded onto a 10 cm dish with 10 mL of Keratinocyte-serum free medium (K-SFM) containing human recombinant EGF (5 ng/mL) and bovine pituitary extract (0.05 mg/mL) provided with the K-SFM kit (Thermo Fisher Scientific, Gibco). The passage number for all cell lines used was less than 25 passages. At 24 h, the growth medium was removed and replaced with 10 mL of K-SFM. For the control medium, 10 mL of K-SFM were added to a 10cm dish without adding any cells. After 24 h of incubation at 37°C with 5% CO₂, the CM were collected and filtered through a 0.2 μm syringe filter (Thermo Fisher Scientific) to remove any cell debris. Additionally, Cancer-derived CM were concentrated to 4 × using an Amicon Ultra-15 centrifugal filter tube (MilliporeSigma, Burlington, MA, NMWL10K) performed at 4,000 × g for 20 min at RT. The CM was stored at 4°C until use.

2.8. Neurite outgrowth assay

After 48 h (murine and non-human primate) or 72 h (human) of primary DRG neuronal culture establishment (the time required to achieve 20–30% nerve fiber occupancy on the slide based on Fig. 3A), half (500 μL) of medium were replaced with 500 μL of either control, normal epithelial cell-derived CM, or cancer-derived CM, and then the cells were incubated another 48–72 h (the time required to achieve 70–80% nerve fiber occupancy on the slide based on Fig. 3A). In some case, the cells were treated with 500 μL of NG medium containing either dimethyl sulfoxide (DMSO) control or paclitaxel (Sigma-Aldrich) for 24 h. At the termination of the experiments, the cells were fixed in 500 μL of 4% paraformaldehyde (PFA) for 10 min at RT, and immediately subjected to immunofluorescence or stored in 1 × D-PBS at 4°C until use.

Fig. 3. Characterization of murine primary DRG sensory neurons.

(A) Representative bright field images of murine primary DRG culture (9–12x lumbar DRGs (L2-L4) from 8–12-week-old male C57BL/6 mice) after a BSA gradient centrifugation [3.5% (W/V) BSA solution] seeded onto Poly-D-lysine/laminin coated coverslips at 24, 48, 72, and 96 h. Magnification 4x. (B) Quantification analysis of the cell size distribution of murine primary DRG sensory neurons (n = 467). Areas of soma (μm²) of cells from (A) were measured using image J software. (C) Representative immunofluorescence images of murine primary DRG sensory neurons after a BSA gradient centrifugation. Sensory neurons were stained with antibodies against 200kD neurofilament (NF200) (Green) (all myelinated A-fibers), calcitonin gene-related peptide (CGRP) (red) (peptidergic myelinated thin Aδ-fibers and unmyelinated C-fibers), and isolectin B4 (IB4) (light blue) (non-peptidergic unmyelinated C-fibers). Magnification 10 x. Bar = 100 μm.

2.9. Immunofluorescence assay

Fixed cells were blocked with immunofluorescence (IF) buffer [1x D-PBS supplemented with 5% Normal Donkey serum (Jackson ImmunoResearch, West Grove, PA) and 0.03% Triton X-100 (Sigma-Aldrich) for 1h at RT and incubated with primary antibodies overnight at 4°C in IF buffer. Primary antibodies used: mouse anti-β III tubulin antibody (1:1,000, Biolegend, San Diego, CA, cat #: 801201); chicken anti-200kD neurofilament (NF200) antibody (1:3,000, Neuromics, Cambridge, MA, cat #: ab134459); rabbit anti-protein gene product 9.5 (PGP9.5) antibody (1:1,000; Cederlane, Rosemont, IL, cat #: 14730–1-AP); rabbit anti-calcitonin gene-related peptide (CGRP) antibody (1: 5,000, Sigma-Aldrich, cat #: C8198); biotinylated isolectin B4 (IB4) antibody (1: 2,500, Sigma-Aldrich, cat #: L2140); rabbit anti-S100 antibody (1: 1,000, Abcam, Cambridge, UK, cat #: ab868); or goat anti-glial fibrillary acidic protein (GFAP) antibody (1: 200, Santa Cruz, Dallas, TX, cat #: sc-6170). Thereafter, the cells were incubated with secondary antibodies for 2h at RT in IF buffer. The specific choices of secondary antibodies were made based on primary antibodies used. Secondary antibodies used: anti-rabbit cyanine 3 (CY3) (1:700, Jackson ImmunoResearch, cat #: 711–165-152); anti-chicken CY2 (1:600, Jackson ImmunoResearch, cat #: 703–225-155); anti-streptavidin CY5 (1:500, Jackson ImmunoResearch, cat #: 016–170-084); anti-goat CY5 (1:500, Jackson ImmunoResearch, cat #: 705–175-147); or anti-mouse CY2 (1:600, Jackson ImmunoResearch, cat #: 715–225-150). After washing 5 times with 1x D-PBS, the cells were mounted with ProLong Gold antifade mountant with DAPI (Thermo Fisher Scientific).

2.10. Imaging and quantification of immunofluorescence

For each group, 2–3 coverslips were quantified, and 6–10 images were taken from each coverslip using a Nikon Eclipse Ni fluorescent microscope system (Nikon, Tokyo, Japan). Images were saved in nd2 or tiff files for further analysis using Visiopharm (Hørsholm, Denmark) or Image J (NIH, Bethesda, Maryland) software, respectively.

2.11. Image J analysis

Neuron J, a plugin for Image J software was used to manually analyze nerve density, as previously described [43, 44]. Briefly, images were opened in Image J (https://imagej.net/Fiji/Downloads) and converted to 8-bit, which were then individually opened in Neuron J plugin. Thereafter, total neurite lengths of neuronal markers NF200 or β III tubulin positive neurites per each image were traced and normalized with the numbers of neuron, which were counted using a multi-point tool of Image J.

2.12. Visiopharm image analysis

An algorithm (called an “APP”) was created in the Visiopharm software, which uses digital masks and associated labels to automatically detect and count the somas of NF200 positive neurons, as well as detect and measure total NF200 positive neurite length. In some cases, PGP9.5, β III tubulin, or activating transcription factor 3 (ATF3) were used as markers instead of NF200 for neurite length or soma quantification. The APP was designed to exploit the observations that somas are usually brighter in fluorescence intensity than neurites, and neurites are long, thin structures whereas somas are round and larger in diameter. This APP uses thresholds, which were set manually according to the set of images to be analyzed, as sets of images can vary in fluorescence intensity between experiments due to staining and microscope setting differences. The APP includes post-processing for cleaning up the classification (i.e. small object removal, skeletonization, erosion, dilation, etc.) and the calculation of the variable included in our measurements.

2.13. Real time qPCR

After murine primary DRG neuron cultures were treated with (i) either control or cancer-derived CM (48 h), or (ii) either DMSO or paclitaxel (10 μM) (24 h), cells were lysed in 350 μL RLT-β-ME buffer. RNA was extracted using the RNeasy plus micro kit (Qiagen, Germantown, MD) and cDNA was generated using Invitrogen SuperScript II Reverse Transcriptase (Invitrogen, Carlsbad, CA). Real time PCR (qPCR) was performed using Taqman gene expression master mix (Applied Biosystems, Foster City, CA) and Taqman gene expression assays on the Bio-Rad CFX Connect instrument (Hercules, CA). Taqman gene expression assays used: growth associated protein 43 (GAP43) (Mm00500404_m1), NF200 (Mm01191456_m1), CGRP (Mm00801463_g1), Substance P (Mm01166996_m1), Bradykinin (Mm04207315_s1), tumor necrosis factor-α (TNF-α) (Mm00443258_m1), suppressor of cytokine signaling 3 (SOCS3) (Mm00545913_s1), immunoglobulin superfamily containing leucine rich repeat 2 (ISLR2) (Mm00623260_s1), and GAPDH (Mm99999915_g1). For interleukin-6 (IL-6), cDNAs were amplified by qPCR using SsoAdvanced Universal SYBR green supermix (Bio-rad laboratories, Hercules, CA). Primers used: mouse IL-6 Forward: 5’-TTCCTACCCCAATTTCCAAT-3’ and Reverse: 5’-CCTTCTGTGACTCCAGCTTATC-3’ (Integrated DNA Technologies, Newark, NJ). Data is presented using the delta-delta Ct method, with GAPDH used as the reference gene.

2.14. Statistical analysis

Numerical data are expressed as mean ± the standard error of the mean (SEM). Statistical analysis was performed by unpaired two-tailed Student’s t test or one-way ANOVA with Tukey’s multiple comparisons, using the GraphPad Prism statistical program (GraphPad Software, San Diego, CA) with significance at p ≤ 0.05. The chi-square goodness-of-fit test was used to test whether the distribution of the three soma size groups (0–599 μm², 600–1,199 μm², and 1,200–1,300 μm²) of one species was the same as that of the other species.

3. RESULTS

3.1. Murine DRGs can survive, and sensory neurons can grow neurites even in serum-free condition

To establish a primary 2D DRG neuron culture, L2–4 DRGs were collected from C57BL/6 mice and dissociated into single cell suspension by enzymatic digestion. The resulting DRGs were plated onto Poly-D-lysine/laminin-coated coverslips and cultured in neuronal growth medium. Unlike other studies [45–48], DRGs were maintained in the condition that serums and nerve-related growth factors (e.g. NGFs, GDNFs) were withdrawn to avoid their effects on the survival of neurons and neurite outgrowth. As shown in Fig. 1A, cells obtained from DRGs, including sensory neurons and satellite cells, could maintain viability for at least 15 days in serum and growth factor-free condition. Although approximately 90% of sensory neurons extended neurites, it was very difficult to observe under the light microscope since two cell types overlapped with each other. To visualize sensory neurons, DRGs were stained with DAPI and three different neuronal markers: 200kD neurofilament (NF200) (all myelinated A-fibers); calcitonin gene-related peptide (CGRP) (peptidergic myelinated thin Aδ-fibers and unmyelinated C-fibers) and substance P (peptidergic unmyelinated C-fibers). Sensory neurons expressed these markers, whereas satellite cells failed to do so (Fig. 1 B).

Fig. 1. Establishment of a murine primary 2D DRG neuron culture.

(A) Representative bright field images of murine primary DRG culture (9–12x lumbar DRGs (L2-L4) from 8–12-week-old male C57BL/6 mice) seeded onto Poly-D-lysine/laminin coated coverslips at Day 3, 9, and 15. Magnification 4 x. (B) Representative immunofluorescence images of (A) at Day 15. Sensory neurons were stained with antibodies against 200kD neurofilament (NF200) (green) (all myelinated A-fibers), calcitonin gene-related peptide (CGRP) (red) (peptidergic myelinated thin Aδ-fibers and unmyelinated C-fibers), and substance P (blue) (peptidergic unmyelinated C-fibers). DAPI (gray) is used for nuclear staining. Magnification 10 x. Bar = 100 μm.

3.2. Murine DRG sensory neurons were purified using a BSA gradient centrifugation

It has been demonstrated that, in the peripheral nervous system, satellite cells are involved in the regulation of survival and axonal growth of neurons [49, 50]. Since one of our main purposes of establishment of primary DRG neuronal culture is to investigate the direct effects of exogenous factors on the neurite outgrowth of sensory neurons or neurite toxicity, our next attempt was to separate sensory neurons from satellite cells. To do so, density gradient centrifugation was performed using 3.5% BSA solution. After purification, sensory neurons were enriched in pellet fraction, while satellite cells were concentrated in BSA layer. When cells in pellet fraction were plated, the numbers of satellite cells (S100 positive) were reduced, compared to those before purification (Fig. 2A). To further confirm the quality of purification, both cells in pellet fraction and BSA layer were plated. As expected, cells in pellet fraction could extend neurites, while cells in BSA layer failed to do so (Fig. 2B).

Fig.2. Purification of murine primary DRG sensory neurons using a BSA gradient centrifugation.

(A) DRG cells were seeded onto Poly-D-lysine/laminin coated coverslips before or after a BSA gradient centrifugation [3.5% (W/V) BSA solution]. Representative bright field images of murine DRGs before and after a BSA gradient centrifugation (top panels). Magnification 4 x. Representative immunofluorescence images murine DRGs before and after BSA centrifugation (bottom panels). Sensory neurons were stained with antibodies against 200kD neurofilament (NF200) (green), and satellite cells were stained with antibodies against S100b (red), DAPI (blue) is used for nuclear staining. Magnification 20 x. Bar = 100 μm. (B) After a BSA gradient centrifugation, cells obtained from the pellet fraction (pellet) and BSA layer were seeded onto Poly-D-lysine/laminin coated coverslips. Representative bright field images of cells from the pellet fraction and BSA layer at Day 1 (top panels) and 3 (bottom panels). Magnification 4 x.

Afterwards, the neurite outgrowth of neuronal cells in the pellet fraction were followed over time. The nerve fiber occupancy on the slides increased in a time dependent manner (10% nerve fiber occupancy at 24 h, 20–30% nerve fiber occupancy at 48 h, 50–60% nerve fiber occupancy at 72 h, and 70–80% nerve fiber occupancy at 96 h) (Fig. 3A). Neuronal cells in the pellet fraction were also segregated into three groups based on soma size (Fig. 3B). The size of soma was divided into small (≤ 599 μm²), medium (600–1,199 μm²) and large (1,200–1,300 μm²), which has been used for in vivo characterization of rodent DRGs [51–55]. The majority of murine sensory neuron somas were smaller than 599 μm² (Fig. 3B). Additionally, cells were stained with NF200, CGRP, and isolectin B4 (IB4) (non-peptidergic unmyelinated C-fibers) to further characterize the subpopulation of the DRG neurons. Interestingly, NF200 positive neurons always co-localized with CGRP positive neurons, but not IB4 positive neurons (Fig. 3C). Additionally, some CGRP positive neurons overlapped with IB4 positive neurons (Fig. 3C). Moreover, small size neurons consisted of NF200 negative/CGRP negative/IB4 positive neurons; medium size neurons consisted of NF200 negative/CGRP positive/IB4 positive, NF200 negative/CGRP positive/IB4 negative, or NF200 positive/CGRP positive/IB4 negative neurons; and large size neurons consisted of NF200 positive/CGRP positive/IB4 negative neurons (Fig. 3C).

3.3. Cancer-derived factors stimulated the neurite outgrowth of murine sensory neurons, whereas a chemotherapeutic agent reduced.

Next, to test whether the growth of sensory neurons can be manipulated in vitro by exogenous factors, which again is our main purpose of the establishment of primary sensory neuron cultures, murine primary sensory neurons were treated with either cancer-derived CM or paclitaxel. We chose cancer-derived CM treatments since it is known to (i) enhance neurite sprouting [29, 30] and (ii) induce pain behaviors in rodents when delivered with an intraplantar injection [56]. When sensory neurons were exposed to CM derived from human breast cancer cell line MDA-MB-231 cells, human prostate cancer cell line PC3 cells, human lung cancer cell line A549 cells, or murine lung cancer cell line LL/2 cells, the sprouting of sensory neurons significantly increased, compared to those exposed to control CM (Fig. 4A&B). However, CM derived from normal prostate epithelial cells did not affect the growth of sensory nerves (Fig. 4C). Interestingly, we observed more nerve sprouting in sensory neurons treated with CM derived from lung cancer cell lines (A549 and LL/2). This finding might be consistent with orthopedic surgeons’ anecdotal evidence that bone metastasis from lung cancer are more painful than those from other cancer types (e.g. prostate cancer and breast cancer) (personal communications), although further studies to confirm this observation are clearly warranted. On the other hand, when sensory neurons were treated with paclitaxel, the growth of sensory nerve fibers was reduced in a dose dependent manner (Fig. 4D&E). Mouse sensory neurons were treated with 10 μM paclitaxel, since this dose is close to a physiological-dose [57].

Fig. 4. Manipulation of the sprouting of murine primary DRG sensory neurons by exogenous factors.

(A) Murine primary DRG sensory neurons were treated with either control medium (Control CM) or conditioned medium (CM, 0% serum) derived from human breast cancer cell line MDA-MB-231 cells (MDA-MB-231 CM), human prostate cancer cell line PC3 cells (PC3 CM), human lung cancer cell line A549 cells (A549 CM), or murine lung cancer cell line LL/2 cells (LL/2 CM) for 48 h. Sensory neurons were stained with antibodies against 200kD neurofilament (NF200). Quantification of the average of the total neurite length. Mean ± SEM. Student’s t-test, significance at p ≤ 0.05. The figure is a representative of two independent experiments, and 6 random images from each coverslip (n = 2) were analyzed in a blind manner. (B) Representative immunofluorescence images of murine primary DRG sensory neurons treated with either control or human lung cancer cell line A549 CM. Magnification 10 x. Bar = 100 μm. (C) Murine primary DRG sensory neurons were treated with either control medium (Control CM) or CM derived from human normal prostate epithelial cell line PWR-1E cells (PWR-1E CM) for 48 h. Sensory neurons were stained with antibodies against NF200. Quantification of the average of the total neurite length. Mean ± SEM. Student’s t-test. The figure is a representative of one independent experiment, and 7–8 random images from each coverslip (n = 3) were analyzed in a blind manner. (D) Murine primary DRG sensory neurons treated with either DMSO or paclitaxel (100 nM, 10 μM) for 24 h. Sensory neurons were stained with antibodies against PGP9.5. Quantification of the average of the total neurite length. Mean ± SEM. Student’s t-test, significance at p ≤ 0.05. Experiment was performed once, and 8–10 random images from each coverslip (n = 3) were analyzed in a blind manner. (E) Representative immunofluorescence images of murine primary DRG sensory neurons treated with either DMSO or paclitaxel (10 μM) for 24 h. Magnification 10 x. Bar = 100 μm.

For quantification of neurite outgrowth, we used a commercially available image analysis software, Visiopharm to automatically measure a total neurite length and Image J to count the numbers of the soma in each coverslip. Then, a total neurite length was normalized with soma count. To validate whether an automated method can accurately be used to measure neurite outgrowth, we first created a novel algorithm (called an “APP”) using Visiopharm that enable us to detect the somas and measure the length of their neurites based on structural differences and fluorescence intensity, respectively (Fig. 5A). Then, values obtained automatically (Visiopharm) were compared to those obtained manually (Image J). As shown in Fig. 5B, total neurite lengths obtained using these two different methods were highly correlated (r²=0.8975). However, soma counts were not as highly correlated as total neurite lengths (r²=0.1561) (Fig. 5C). This might in part be due to the difficulty to segregate the cluster of the somas into a single soma. These studies demonstrated the following: (i) the total neurite length obtained using an automated method were similar to that obtained manually; (ii) a manual counting method provided more accurate information regarding the numbers of soma than an automated method; and (iii) by manually, counting somas was not as time consuming as measuring neurite length. Therefore, we chose the quantification strategy described above.

Fig. 5. Quantification of total neurite length by automated imaging analysis.

(A) Representative images of the image-processing steps performed in the Visiopharm APP used for automated neurite length quantification and soma count, starting from the original fluorescent image of 200kD neurofilament (NF200) stained mouse DRG neurons (top left) to the final quantified image (bottom right) with relevant masks for neurite length (red) and soma count (green). Black arrow: direction of processing steps. White arrow: two neurons inaccurately counted as one. (B) 316 images of murine primary DRG sensory neurons stained with antibodies against NF200 were analyzed using the Visiopharm APP and by manual tracing using ImageJ software and values for total neurite length were plotted (μm): Linear regression analyses, significance at p ≤ 0.05. (C) 164 images of murine primary DRG sensory neurons stained with antibodies against NF200 were analyzed using the Visiopharm APP and by manual counting using ImageJ software and values for total soma numbers were plotted (counts): Linear regression analyses, significance at p ≤ 0.05.

3.4. The impact of cancer-derived factors and a chemotherapeutic agent on the expression of genes, associated with nerve growth and neuro-inflammatory pain, in murine sensory neurons.

Next, to determine whether the alterations of sensory neuron length mediated by exogenous factors are associated with pain, gene expression analyses were performed by qPCR on murine primary sensory neurons treated with either cancer-derived CM or paclitaxel. In this case, we chose CM derived from A549 cells (A549 CM), since the greatest enhancement of murine primary sensory neuron sprouting was observed when treated with A549 CM (Fig. 4A). To determine whether our observations in the neurite length changes mediated by exogenous factors correlate to gene expression patterns, the levels of growth associated protein 43 (GAP43), a nerve growth marker [58], and NF200 gene expression in A549 CM or paclitaxel-treated murine primary sensory neurons were measured. Consistent with our findings in the neurite length changes, A549 CM significantly increased GAP43 expression, while paclitaxel exerted opposite effects (Fig. 6A). A similar trend was observed in changes in NF200 gene expression patterns as those of GAP43, although it did not reach statistical significance (Fig. 6B). Then, gene expression levels of pain-related molecules, including neuropeptides (CGRP, Substance P, Bradykinin) and cytokines involved in pathological pain conditions [tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6)] [59, 60] were measured to determine the association between nerve growth and pain. Interestingly, patterns of gene expression changes were different depending on the treatments used. Neither A549 CM nor paclitaxel affected CGRP expression levels (Fig. 6C), while A549 CM significantly decreased Substance P expression levels (Fig. 6D), and both A549 CM and paclitaxel significantly increased Bradykinin expression levels in murine primary sensory neuron (Fig. 6E). Paclitaxel significantly increased TNF-α expression levels (Fig. 6D), whereas A549 CM significantly decreased IL-6 expression levels (Fig. 6G). Additionally, the gene expression levels of suppressor of cytokine signaling 3 (SOCS3) and immunoglobulin superfamily containing leucine rich repeat 2 (ISLR2), which were significantly increased in DRGs derived from patients with neuropathic pain, compared to those derived from patients without pain [36], were measured. SOCS3 and ISLR2 are known to be involved in development of cancer-related pain [61] and nociceptive sensory neuron development [62], respectively. In these cases, A549 CM significantly impacted on the gene expression of SOCS3 (increased, Fig. 6H) and ISLR2 (decreased, Fig. 6I), while paclitaxel failed to do so.

Fig. 6. Changes in expression of genes associated with nerve growth and pain in murine primary DRG sensory neurons by exogenous factors.

Murine primary DRG sensory neuron cells were treated with control medium (Control CM) or human lung cancer cell line A549 cell-derived conditioned medium (A549 CM) for 48 h, and DMSO or paclitaxel (10 μM) for 24 h. Expression of (A) GAP43 and (B) NF200, which are genes associated with nerve growth. Expression of (C) CGRP, (D) Substance P, (E) Bradykinin, (F) TNF-α, (G) IL-6, (H) SOCS3, and (I) ISLR2, which are genes associated with neuro-inflammatory pain and nociceptor development. GAPDH was used reference gene. Delta-delta Ct method. Results were displayed as Mean ± SEM. Student’s t-test. ^*p ≤ 0.05, ^**p ≤ 0.01, ^***p ≤ 0.001, ^****p ≤ 0.0001.

GAP43: growth associated protein 43; NF200: 200kD neurofilament; CGRP: calcitonin gene-related peptide; TNF-α: tumor necrosis factor-α; IL-6: interleukin-6; SOCS3: suppressor of cytokine signaling 3; ISLR2: immunoglobulin superfamily containing leucine rich repeat 2.

3.5. Sensory neurons obtained from non-human primate and human DRGs act similar to those of mice.

To enhance translational potential, non-human primate primary DRG sensory neuron culture was established using similar methods to establish murine primary sensory neuron culture. Consistent with murine culture, the BSA purification method could largely reduce satellite cell population, and about 90% of the viable neurons grew neurite in serum and growth factor-free condition (Fig. 7A). Additionally, the majority of non-human primate sensory neuron somas were between 600 μm² to 1,199 μm² (Fig. 7B). Furthermore, non-human primate sensory neurons responded to cancer-derived CM (Fig. 7C&D) and paclitaxel treatment (Fig. 7E&F), similar to murine sensory neurons. Non-human primate and human sensory neurons were treated with 1 μM paclitaxel, since a concentration of 10 μM used for mouse sensory neurons was too toxic to them (Data not shown).

Fig. 7. Establishment of a non-human primate primary DRG sensory neuron culture.

(A) Representative immunofluorescence images non-human primate DRGs (T12 and L1 of a healthy 8-year old female Rhesus macaque) before and after BSA centrifugation [3.5% (W/V) BSA solution]. Sensory neurons were stained with antibodies against β-III tubulin (green), and satellite cells were stained with antibodies against S100b (red), DAPI (gray) is used for nuclear staining. Magnification 10 x. Bar = 100 μm. (B) Quantification analysis of the cell size distribution of non-human primate primary DRG sensory neurons (n = 309). Areas of soma (μm²) of cells from (A) were measured using image J software. (C) Representative immunofluorescence images of non-human primate primary DRG sensory neurons treated with either control medium (Control CM) or human lung cancer cell line A549 cell-derived conditioned medium (A549 CM) (0% serum) for 72 h. Sensory neurons were stained with antibodies against β-III tubulin. Magnification 10 x. Bar = 100 μm. (D) Quantification of the average of the total neurite length per image of (C): Mean ± SEM. Student’s t-test, significance at p ≤ 0.05. The figure is a representative of two independent experiments, and 8–12 random images from each coverslip (n = 3) were analyzed in a blind manner. (E) Representative immunofluorescence images of non-human primate primary DRG sensory neurons treated with either DMSO or paclitaxel (1 μM) for 24 h. Sensory neurons were stained with antibodies against PGP9.5. Magnification 10x. Bar = 100 μm. (F) Quantification of the average of the total neurite length per image of (E): Mean ± SEM. Student’s t-test, significance at p ≤ 0.05. Experiment was performed once, and 8–12 random images from each coverslip (n = 3) were analyzed in a blind manner.

Primary sensory neuron culture of human DRGs was also established. Consistent with murine and non-human primate culture, the BSA purification method could reduce satellite cell population, and about 93% of the viable neurons grew neurite in serum and growth factor-free condition (Fig. 8A), and the majority of the soma size was smaller than 599 μm² (Fig. 8B). Since sensory neurons from three different species showed different size distributions of cell population, we sought to determine whether there are any similarities in the soma size distribution among murine, non-human primate, and human sensory neurons. Interestingly, the chi-square goodness-of-fit test showed that the distributions of the soma size were different among the three species (all p-values from pairwise comparisons < 0.0001). Additionally, when neurons were treated with cancer-derived CM, the lengths of neurite were significantly longer than those treated with control CM (Fig. 8C&D). On the other hand, when neurons were treated with paclitaxel, the growth of neurite was significantly reduced compare to the neurons treated with vehicle (Fig. 8E&F).

Fig. 8. Establishment of a human primary DRG sensory neuron culture.

(A) Representative immunofluorescence images human primary DRGs (T10 of a 41-year-oldmale patient with discitis/osteomyelitis) before and after BSA centrifugation [3.5% (W/V) BSA solution]. Sensory neurons were stained with antibodies against β-III tubulin (green), and satellite cells were stained with antibodies against glial fibrillary acidic protein (GFAP) (blue), DAPI (gray) is used for nuclear staining. Magnification 10x. Bar = 100 μm. (B) Quantification analysis of the cell size distribution of human primary DRG sensory neurons (n = 72). Areas of soma (μm²) of cells from (A) were measured using image J software. (C) Representative immunofluorescence images of human primary DRG sensory neurons treated with either control medium (Control CM) or human lung cancer cell line A549 cell-derived conditioned medium (A549 CM) (0% serum) for 72 h. Sensory neurons were stained with antibodies against β-III tubulin. Magnification 10x. Bar = 100 μm. (D) Quantification of the average of the total neurite length per image of (C): Mean ± SEM. Student’s t-test, significance at p ≤ 0.05. The figure is a representative of two independent experiments, and 8–13 random images from each coverslip (n = 2) were analyzed in a blind manner. (E) Representative immunofluorescence images of human primary DRG sensory neurons treated with either DMSO or paclitaxel (1 μM) for 24 h. Sensory neurons were stained with antibodies against PGP9.5. Magnification 10x. Bar = 100 μm. (F) Quantification of the average of the total neurite length per image of (E): Mean ± SEM. Student’s t-test, significance at p ≤ 0.05. Experiment was performed once, and 8–10 random images from each coverslip (n = 2) were analyzed in a blind manner.

4. DISCUSSION

In this study, we demonstrated four important findings: (1) primary DRG sensory neuron cultures can be derived from mouse, non-human primate, and human and cultured in serum and growth factor-free condition, (2) a BSA gradient centrifugation method is useful to separate sensory neurons form satellite cells, (3) DRG sensory neurons maintain their heterogeneous subpopulation, and (4) the length of nerve fibers and the gene expression patterns, associated with nerve growth, and neuro-inflammatory pain and nociceptor development, of sensory neurons were altered by responding to exogenous factors, such as cancer-derived CM and reduced by responding to a chemotherapeutic agent. We developed a semi-automated quantification method which allowed us to analyze the neurite growth in an accurate and time-efficient manner. Together, these findings suggest that a semi-automated measurement of the neurite growth, along with gene expression analyses, of primary DRG sensory neurons can be a useful in vitro tool to study the impact of exogenous factors on the growth of sensory neurons. The schematic workflow of the primary DRG sensory neuron culture preparation and analyses, is described in Fig. 9.

Fig. 9. The schematic workflow of the primary DRG sensory neuron culture preparation and analyses.

Dorsal root ganglia of mice, non-human primate, or human, were dissociated by enzymatic digestion and mechanical dissociation to obtain single cell suspensions. Then, the resulting cells were segregated into sensory neurons and satellite cells using a BSA gradient centrifugation. Thereafter, sensory neurons were plated onto Poly-D-lysine and laminin-coated coverslips to establish monolayer culture. In some cases, sensory neurons were exposed to exogenous factors, such as cancer-derived conditioned medium (CM) or chemotherapeutic agents, and then changes in nerve fiber length and gene expression were analyzed by immunohistochemical analyses and qPCR, respectively.

Animal models have been widely used to study pathological mechanisms underlying the human diseases and to test therapeutic strategies. Although in vivo studies are useful for addressing large-scale biological questions, in vitro methods are better suited for exploring micro-level mechanisms. In this study, using primary neuronal cultures, we demonstrated that (i) cancer-derived CM induced the sprouting of nerve fibers and (ii) the chemotherapeutic agent, paclitaxel inhibited the neurite outgrowth. One of the most important aspects of our studies is that the structural changes observed in our in vitro setting recapitulate pathological conditions observed in in vivo rodent models. For example, the inoculation of cancer cells into the bone of rodents resulted in increased neurite density around the bone, which is associated with CIBP behaviors [17, 18]. Similarly, the systemic administration of paclitaxel to rodents led to a reduction of intraepidermal neuronal fiber density in the skin, which is a hall mark of CIPN [26, 27]. Our in vitro primary DRG sensory culture manipulations were in line with these in vivo structural changes in peripheral sensory neurons. Thus, in vitro primary DRG sensory neuron culture system established in this study is an alternative tool to study the impact of soluble factors and/or direct cell-cell interactions on the architectural remodeling of sensory neurons.

Even though the use of rodent in vivo and in vitro models is essential for the advancement of science, the results of pre-clinical studies do not always translate to the human condition. In fact, a recent meta-analysis of 2,000 scientific studies using animal models revealed that more than half of these studies (~60%) either could not be replicated or failed to move forward to clinical trials [63]. To overcome this hurdle, studies using human tissues are receiving growing attention. Therefore, an additional salient finding of our studies is the reproducibility of pathologic neurite outgrowth following cancer-derived CM and paclitaxel-induced neurotoxicity in non-human primate and human primary neuronal cultures, similar to murine neuronal cultures. Our findings also indicate that our approach with rodent sensory neurons provides an alternative to the use of non-human primate or human primary sensory neuron samples so that these precious resources can be used specifically for translational assessments after more thorough characterization with mouse neuron cells. We also recognized that the use of non-human primate or human primary sensory neurons is not trivial and requires a great deal of resources to obtain fresh DRGs. In fact, one of the major challenges that we faced in the course of our studies was the coordination of multiple departments and laboratories. However, these constraints become minor when there is solid coordination and commitment among different departments and laboratories, and a frequent communication and engagement between physicians and basic scientists.

Our study also uncovered that our approach has some limitations. For example, although successful, the numbers of sensory neurons recovered from human DRG were relatively lower than those from mice and non-human primate, which limited the number of conditions performed in each experiment. This may be in part due to the difficulty in the digestion step of human DRGs. Human DRGs contains more connective tissues, including fibroblasts, blood vessels, and base membrane, than murine and non-human primate DRGs [64]. Consistent with this notion, during preparation of culture, the large number of fibroblasts were observed in human DRG, while few or no fibroblasts were seen in murine and non-human primate DRGs. Since thick connective tissues surrounding neurons may interfere enzymatic reaction, it is crucial to cut the human DRGs into small pieces prior to the digestion step. Another potential limitation is the fact that the most abundant population of neurons in human DRG was small neurons in contrast to murine and non-human primate DRGs. Although the result is in line with previous findings [65, 66], this suggests that our approach might not be ideal for the study targeting medium or large size of human sensory neurons. However, it is important to note that the cell size characterization method used in this study might not translate into non-human primate and/or human sensory neurons. Not only cell size differences, but also there may be differences in responses to exogenous factors among DRGs derived from these species at a molecular and functional levels, although the effects of cancer-derived CM and paclitaxel treatments on nerve growth were consistent in all species. Indeed, nicotine evoked currents as well as nicotinic receptor expression patterns are significantly different between human and rodent DRGs [34]. Therefore, further subpopulation, molecular, and functional characterizations of murine, non-human primate, and human DRGs are clearly warranted.

Once logistic and technical optimization issues were overcome, we found that the image analysis of neurite length represents a challenge due to the lack of reliable methods to accurately measure the length of nerve fiber, and perhaps this is the reason why very few studies include this outcome. We found that manually measurement is accurate when performed by the same experimenter using a blinded method, but at the same time it is extremely time consuming and experimenter biases might reduce its reproducibility. These challenges prompted us to develop a method of image analysis aimed to reduce potential experimenter biases and increase efficiency in analysis time. In this study, we successfully developed a semi-automated method using the Visiopharm platform for the measurement of neurite length. The highly correlated values between this method and the manual approach using Image J demonstrate that our unbiased approach using the Visiopharm interface is accurate enough to consistently detect neurite length. One of the limitations of our approach however is the lack of tight correlation between automatically and manually generated soma counts. This might be attributed to the use of only one marker for two different cellular structures; i.e. NF200 staining was used to detect both soma count and neurite length. We uncovered that this approach does not allow the algorithm (APP) to differentiate a cluster of multiple neurons from individual neurons, something that the human eye could discern or infer. This limitation could be overcome by using different markers for neuronal soma (or nucleus) from neurites. Thus, the APP in this study was designed to maximize the accuracy of its neurite length measurements at the expense of accurate soma counts, since manually counting somas is significantly easier than manually tracing neurites. We are currently working to develop soma quantification APPs using nuclear stains for future studies capable of generating counts that are highly correlated with manual methods.

Although the importance of nerves for cancer progression has been appreciated [1], there are no treatments yet available which target the nerve-cancer interaction (e.g. cancer-induced nerve sprouting). In addition, chemotherapy is still the gold standard for cancer treatments and results in CIPN in many patients due to drug neurotoxicity. It is, therefore, critical to understand the interactions between cancer and/or chemotherapeutic agents, and sensory nerves, as well as how these interactions affect the progression of these painful complications, so that safer and more effective treatments can be developed. Although further electrophysiological analyses, phenotypical analyses, and chemical neural stimulation (e.g. capsaicin or KCL) on neuronal cell bodies might strengthen our findings, the measurement of neurite length and gene expression changes mediated by exogenous factors (e.g. cancer-derived factors, chemotherapeutic agents) can be a useful tool to elucidate the causes and mechanisms of these painful symptoms. For example, it has been suggested that nerve-related growth factors [29] or exosomes [30, 56] derived from cancer cells induce cancer-related pain by stimulating neurite outgrowth [15–19], and that axonal damages mediated by chemotherapeutic agents through mitochondrial dysfunction in nerve cells and oxidative stress on nerve cells are responsible for CIPN symptoms [67]. We believe that our developed approaches could be used to reveal these underlying mechanisms. Moreover, the use of higher species’ neuronal tissues in tandem with the development of an accurate and time-efficient methodology to measure neurite length under different pathological conditions will enhance clinical translational of our discovery. For success of this approach, a well-coordinated multidisciplinary team between physicians and basic scientists, which we began to establish, is clearly essential.