Abstract
Background
Cisplatin (CP) is a potent chemotherapy drug used for the management of several cancers. However, the use of CP is associated with painful peripheral neuropathy. Effective therapies are currently unavailable for CP-induced peripheral neuropathy (CisIPN). Given the known association between pro-inflammatory cytokines and CisIPN, we explored the role of the pro-inflammatory cytokine, the macrophage migration inhibitory factor (MIF), in CisIPN.
Methods
CisIPN was induced in normal and breast cancer mouse models. Circulating MIF levels in CisIPN mice were examined using ELISA. The effect of MIF inhibition on CisIPN severity was evaluated using small molecule inhibitors. Mechanical and cold sensitivities of CisIPN mice were examined using Von Frey filament and cold acetone assays. Macrophage infiltration and expression of MIF in dorsal root ganglia (DRG) and sciatic nerves of CisIPN mice were examined using immunostaining. Molecular changes downstream of MIF were examined in Schwann Cells (SCs) using western blot.
Results
Circulating MIF levels were increased in CisIPN mice, indicating a potential role for MIF in CisIPN. Pharmacological inhibition of MIF suppressed the mechanical hypersensitivity component associated with CisIPN, substantiating a facilitatory role for MIF in CisIPN. There was no significant change observed in macrophage abundance in DRGs and sciatic nerves of CisIPN mice. ELISA, immunostaining, and western blot assays indicated that sensory neurons and SCs are potential sources for increased MIF in CisIPN. We also found fluctuations in ERK1/2 signaling downstream of MIF in SCs, suggesting it as a potential mechanism underlying MIF-dependent facilitation of CisIPN.
Conclusions
Overall, our results indicate that MIF facilitates CisIPN, suggesting its potential role as a therapeutic target for CisIPN.
Keywords: breast cancer, CIPN, cisplatin, mechanical hypersensitivity, MIF, peripheral neuropathy
Key Points.
Cisplatin-induced peripheral neuropathy (CisIPN) accompanies upregulation of circulating MIF driven by sensory neurons and Schwann Cells.
Pharmacological inhibition of MIF suppresses CisIPN.
MIF modulates ERK1/2 signaling in Schwann Cells (SCs).
Importance of the Study
Despite being a painful complication, effective therapies are unavailable for chemotherapy-induced peripheral neuropathy, especially cisplatin-induced peripheral neuropathy (CisIPN). Although several mechanisms have been postulated to underlie CisIPN, none of them have translated into therapeutic intervention points. Here, we demonstrate for the first time that MIF facilitates CisIPN, supporting a potential role for MIF as a therapeutic target for CisIPN. The study was conducted in regular and breast cancer mouse models to demonstrate the reproducibility of the findings. This novel finding deserves attention and warrants further investigation in humans.
Current advances in targeted therapies have transformed the entire landscape of cancer treatment. However, conventional cytotoxic drugs continue to be a major part of cancer chemotherapy regimens due to their high efficacy in rapidly eliminating proliferating cells. Unfortunately, their use is often associated with unbearable side effects. Along these lines, chemotherapy-induced peripheral neuropathy (CIPN) is a painful complication associated with cytotoxic drugs.1,2 CIPN predominantly affects sensory nerves, leading to abnormal and unpleasant sensations, including hyperalgesia and allodynia.2 The incidence and severity of CIPN vary depending upon the specific chemotherapy agent used, with platinum compounds, taxanes, vinca alkaloids, and bortezomib having been reported to induce higher incidences of CIPN.3 The unpleasant pain and nerve dysfunctions associated with CIPN often lead to premature discontinuation or dose reduction of the causative drug, leaving patients with suboptimal treatment options.
Cisplatin (CP) shows the highest incidence of CIPN among platinum compounds used in cancer therapy.4 At the same time, CP remains a key therapeutic choice for various solid cancers, including the most aggressive BRCA1 mutant triple-negative breast cancer (TNBC).5-7 The underlying molecular mechanism of CP-induced peripheral neuropathy (CisIPN) is largely unknown, posing a significant challenge to the development of effective management strategies. Several previous studies have suggested a role for neuroinflammation in promoting CisIPN.8 However, therapies targeting the neuroinflammatory arm have not been successfully developed for the management of CisIPN, indicating that further exploration of this arm with specific attention to the molecular mediators is critical to develop effective therapies. In this study, we explored the role of neuroinflammation in CisIPN using 3 different animal models, including a breast cancer model. Interestingly, our experiments revealed a critical role for macrophage migration inhibitory factor (MIF), a cytokine responsible for neuroinflammation, in CisIPN.
Methods
Breast Cancer Animal Model
Adult female athymic nude mice (Crl: NU(NCr)-Foxn1nu) purchased from Charles River Laboratories were used for breast cancer induction. Briefly, 2x105 MDA-MB-231 cells suspended in 1:1 DMEM/F12 medium: Cultrex matrix (Biotechne, Cat No. 3433-010-01) were injected into either the 4th or 2nd mammary fat pad of animals for separate experiments. After 4 weeks of cell injections, the animals were subjected to CisIPN experiments, as described below.
Experimental Model of CisIPN
Two to five months old CDI mouse, athymic nude mouse (Crl: NU(NCr)-Foxn1nu), and an inhouse bred Cx3crCreERT2 -Rosa26R-EYFP mouse (C57BL/6 strain) models were used for the experiments. The parent mice for the Cx3crCreERT2 -Rosa26R-EYFP crossbred strain, the B6.129P2(C)-Cx3cr1tm2.1(cre/ERT2) Jung/J and B6.129X1-Gt(ROSA)26Sortm1(EYFP)Cos/J mice, were purchased from Jackson laboratories. CD1 mice were purchased from Charles River Laboratories. All animal experiments were approved by the Animal Research Ethics Board (AREB) at the University of Saskatchewan. For the induction of CisIPN, the mice were given intraperitoneal injections of 2.3 mg/kg CP (Sigma-Aldrich, Cat No. 232120) for days 1-5. This was followed by a 5-day treatment interval (days 6-10) and a second cycle of CP dosing (days 11-15). The induction of neuropathy was confirmed by performing a mechanical sensitivity assay.
Measurements of MIF Using ELISA
Blood samples were collected from the submandibular vein of experimental animals. Prior to blood collection, the animals were anesthetized using isoflurane. The blood samples were then centrifuged at 3,000 rpm for 10 minutes at 4°C to collect plasma. The plasma concentrations of MIF were measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Antibodies Online, Cat No. ABIN6574190). A standard curve was generated using recombinant MIF available in the kit, and the absorbance of both standard and test samples was read at 450 nm using a spectrophotometer. MIF concentrations in the test samples were calculated from the standard curve. MIF levels in sensory neuron culture supernatants were also measured by ELISA.
Pharmacological Treatment
For Dexamethasone (Dex) treatment, the animals were administered 2mg/kg of Dex (Millipore Sigma, Cat No. D1756) intraperitoneally 30 minutes before CP dosing. For in vivo MIF inhibition, the MIF inhibitors CPSI-1306 (Cayman Chemical, Cat No. 29905) and ISO-1 (Selleckchem, Cat No. S7732) were used. The animals were intraperitoneally injected with either CPSI-1306 (20 mg/kg ip) or ISO-1 (10 mg/kg ip) 15 minutes prior to CP dosing. The control animals received saline.
Mechanical Sensitivity Measurements
Mechanical sensitivity of animals was assessed using Von Frey Filament (VFF) assay. Mice were individually habituated in the testing apparatus by placing them in cubicles for 5 minutes before conducting the tests. Briefly, VFFs of varying size/number were applied to the left hind paw of each animal, and the threshold stimulus required for the paw withdrawal response was recorded. A response was considered a “positive response” if paw withdrawal was observed 3 or more times out of the 5 trials at a specific filament size. If less than 3 positive responses occurred, the next filament size was tested. A minimum of 3 baseline measurements were taken before starting CP dosing. The average baseline threshold was then calculated for each animal. The mechanical sensitivity recordings were continued at frequent intervals after CP dosing began. The percentage change in mechanical sensitivity was calculated for each animal relative to their baseline sensitivity threshold.
Cold Sensitivity Measurements
Cold sensitivity was evaluated in mice after spraying acetone on their hind paw. The animals’ response to the cold stimulus was recorded as: 0—no response, 1—paw withdrawal, 2—paw withdrawal + paw licking, 3—paw withdrawal + paw licking + scratching, and 4—jumping or peeing. Three trials were conducted for each mouse, and the average of the three trials was taken as the final reading. Multiple baseline measurements were also done for each animal before CP dosing, and the average was calculated to determine the mean baseline value for each animal. The cold sensitivity measurements were continued at frequent intervals after starting CP dosing. Finally, the percentage change in cold sensitivity was calculated for each animal relative to their baseline sensitivity threshold.
Schwann Cell (SC) Culture
The SC line S16 was purchased from ATCC, USA. The cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) containing 10% FBS (ThermoFisher Scientific, Cat No. 12483020) and an antibiotic-antimycotic solution (Fisher Scientific, Cat No. SV3007901). The cultures were maintained in a CO2 incubator at 37°C.
Immunohistochemistry
Dorsal root ganglia (DRG) and sciatic nerves were isolated from experimental animals at the end of the experiment. The isolated tissues were fixed in Zamboni’s buffer (2% paraformaldehyde [Fisher Scientific, Cat No. F79-500] and 0.5% picric acid [Ricca Chemical, Cat No.5860-16] in PBS) for 24 h at 4°C. The tissues were then washed in PBS for 3 times before incubating them in 20% sucrose (Fisher Scientific, Cat No. BP220-1) for another 24 h at 4°C. The tissues were mounted in optimal cutting temperature compound (OCT) and stored at −80°C for a minimum 24 h before making sections. 10 m thick sections of DRG and sciatic nerve samples were made using a cryostat and the sections were collected onto slides. The slides containing the sections were stored at −80°C for a minimum 24 h before immunostaining. For immunostaining, slides were washed in PBS for 5 minutes and incubated in immunoblocker (5% donkey serum [Fisher Scientific, Cat No. 56-646-05ML] and 0.3% Triton-X [Millipore Sigma, Cat No. T8787] in PBS) for 30 minutes. Slides were then washed in PBS for 5 minutes and incubated with primary antibodies for 1 h at room temperature (RT). The primary antibodies used were Iba-1 (FUJIFILM Wako, Cat No. 019-19741), CD68 (Abcam, Cat No. ab53444; Abcam, Cat No. ab31630), NF200 (Millipore Sigma, Cat No. N0142), βIII tubulin (Merck, Cat No. MAB1637), GFAP (ThermoFisher Scientific, Cat No.PA1-10004), and MIF (Abcam, Cat No. ab187064). After 1h incubation, slides were washed three times in PBS for 5 minutes and then incubated with secondary antibody for another 1 h at RT. The secondary antibodies used were anti-rabbit-Alexa fluor 488 (ThermoFisher Scientific, Cat No. A-11034), anti-mouse-Alexa fluor 488 (ThermoFisher Scientific, Cat No. A-11001), anti-mouse- Alexa fluor 647 (ThermoFisher Scientific, Cat No. A-21235), anti-rabbit- Alexa fluor 647 (ThermoFisher Scientific, Cat No. A-21245), and anti-rat-Alexa fluor 647 (ThermoFisher Scientific, Cat No. A-21247). After 1 h incubation, slides were washed in PBS for 5 minutes three times and then mounted using Diamond Antifade Mountant with DAPI (ThermoFisher Scientific, Cat No. S36973). The images were taken using an Axio Observer (Zeiss) fluorescence microscope.
Western Blot
Protein from sciatic nerves were isolated using RIPA buffer (Fisher Scientific, Cat No. PI8990). An equal amount of protein was loaded for gel electrophoresis. The resolved proteins were transferred onto a polyvinylidene flouride (PVDF) membrane (Bio-Rad, Cat No. 1620177). The blocking of the membrane was done using 5% skim milk powder containing 0.1% Tween-20 (Fisher Scientific, Cat No. BP337-500) in TBS for 1 h. Following blocking, the membrane was washed twice and incubated with a primary antibody for overnight at 4°C. The primary antibodies used were MIF (Abcam, Cat No. 187064), ERK1/2 (Cell Signaling Technology, Cat no. 4696S), p-ERK1/2 (Cell Signaling Technology, Cat no. 9101S), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Millipore Sigma, Cat No. SAB3500247), and β-actin (Fisher Scientific, Cat No. AM4302). After washing, the membrane was incubated with a secondary antibody for 1h at RT. The secondary antibodies used were goat anti-rabbit HRP (Bio-Rad, Cat No.1706515), goat anti-mouse HRP (Bio-Rad, Cat No. 1706516), and goat anti-chicken HRP (ThermoFisher Scientific, Cat No. A16054). The blots were developed using an ECL substrate (Bio-Rad, Cat No. 1705060) and imaged using a GelDoc (Bio-Rad).
Quantification of Macrophage Density
Macrophages were quantified in DRG and sciatic nerve sections. Macrophages in randomly selected regions (multiple regions) of the sciatic nerve or DRG sections or entire DRGs were counted using ImageJ software. The number of macrophages was then normalized to the area considered for the quantification to calculate macrophage density.
Statistical Analysis
Statistical analyses were performed using the GraphPad Prism software. All data are presented as mean ± SE. Statistical tests performed included standard “t” test, One-way ANOVA and two-way ANOVA with Tukey’s post hoc analysis, as appropriate. The specific details of the statistical tests performed is provided in the corresponding figure legend. P-values of <0.05 were considered statistically significant.
Results
Dexamethasone Protects Animals from CisIPN
To examine whether suppressing neuroinflammation protects animals from CisIPN, we treated adult male CD1 mice with a potent anti-inflammatory drug Dex, 30 minutes before CP injection, for 5 days. The basal sensitivity of animals to mechanical stimuli was recorded using VFF assay for two days before starting CP dosing, and the assay was repeated at frequent intervals for 8 days after CP dosing (Figure 1A). The CP-treated animals showed increased sensitivity to stimuli by thinner filaments compared to control animals, confirming induction of CisIPN (Figure 1A and B). Interestingly, Dex pretreatment showed a clear trend to suppress CP-induced mechanical hypersensitivity in mice, suggesting that suppressing neuroinflammation may alleviate CisIPN symptoms.
Figure 1.
Dexamethasone suppresses cisplatin-induced peripheral neuropathy. (A) Line graph generated from Von Frey Filament (VFF) assay shows mechanical sensitivity of animals (male CD1 mice) in the indicated groups (mean ± SEM; n = 5; Two-way ANOVA, Tukey’s multiple comparisons test; **P < .01, ***P < .001 compared to CP group). (B) Percentage change in mechanical sensitivity of animals (male CD1 mice) on day 8 compared to the basal mechanical sensitivity (mean ± SEM; n = 5; One-way ANOVA, Tukey’s multiple comparisons test; **P < .01). (C) Line graph generated from VFF assay shows mechanical sensitivity of animals before and after breast cancer induction (Pre-T: Pre-Tumor, Post-T: Post-Tumor [n = 10]). (D) Bar graph generated from VFF assay shows mechanical hypersensitivity of animals before and after (average of days 26 and 28) breast cancer induction (mean ± SEM; n = 10; paired t-test, *P < .05). (E) Line graph generated from VFF assay shows suppression of CP-induced mechanical hypersensitivity in the Dex pre-treatment group (mean ± SEM; n = 5; Two-way ANOVA, Sidak’s multiple comparisons test; **P < .01, ***P < .001, ****P < .0001). (F) Bar graph generated from VFF assay shows suppression of CP-induced mechanical hypersensitivity on Day 15 in the Dex pre-treatment group (mean ± SEM; n = 5, Standard ‘t’ test; P-value is shown). (G) Representative images of tumors in CP and CP+Dex treated animals (breast cancer model). (H) Tumor volumes in CP and CP + Dex groups (mean ± SEM; n = 5; Standard ‘t’ test).
Dex Protects Breast Cancer Animals from CisIPN
Systemic inflammation induced by cancer can promote neuroinflammation initiated by chemotherapy agents,9,10 and thus cancer can influence the severity of CIPN. In line with this, experimental therapies for CIPN should be evaluated in cancer models too.11 Therefore, we evaluated the effect of Dex in CisIPN using a breast cancer animal model.
Breast cancer was induced by injecting MDA-MB-231 cells (TNBC cells) into the 4th mammary fat pad of adult female athymic nude mice. At first, we examined whether breast cancer has an independent role in inducing peripheral neuropathy by measuring the mechanical sensitivity of animals before and after cancer induction (Figure 1C). We found that animals respond to lower-size filaments after breast cancer induction, indicating that breast cancer can independently induce a neuropathy response (Figure 1C and D). Next, we evaluated whether Dex could suppress CisIPN in breast cancer animals. The VFF assay showed that pretreatment with Dex effectively suppressed CP-induced mechanical hypersensitivity in breast cancer animals (Figure 1E and F). Overall, our results showed that Dex could suppress CP-induced hypersensitivity in breast cancer animals. There was no worsening of tumor growth in the CP+Dex group compared to the CP group, indicating that Dex did not compromise the tumor-suppressive effect of CP (Figure 1G and H).
Circulating MIF Level Is Increased in CP-Treated Animals
Next, to explore whether Dex-mediated suppression of CisIPN involves neuroinflammatory molecules, we curated literature for neuroinflammatory molecules that have a common connection with peripheral neuropathies and Dex. We found that the cytokine MIF promotes neuropathies associated with chronic diabetes, GBS, and spinal cord injuries.12-14 Interestingly, Dex and MIF were shown to have an inverse relationship, with studies demonstrating that Dex reduces plasma levels of MIF in animal models of experimental sepsis.15 Similarly, MIF has been shown to antagonize the growth regulatory effects of Dex in glioma cells.16 Inclined to these reports, we found a trend for the downregulation of MIF in sciatic nerves of breast cancer animals that received a combination treatment of Dex and CP compared to individual CP treatment (Supplementary Figure S1A and B).
No previous studies had explored a role for MIF in CIPN, especially in CisIPN. We found that plasma level of MIF was increased in adult female Cx3cr1CreERT2-Rosa26R-EYFP mouse after CP treatment, suggesting a potential role for MIF in CisIPN (Figure 2A). We also noted that, compared to normal mouse, the basal level of MIF was higher in mouse induced with breast cancer. For example, the basal plasma levels of MIF in normal mice were in the range of 3 to 5000 pg/mL, while MIF levels in breast cancer mice were in the range of 5400-14,500 pg/mL (Figure 2A and B [Day 0]). We found that untreated breast cancer mice had a steady decline in plasma MIF levels over time (Figure 2B; day 0 to day 15). However, such a decline was not observed in CP-treated breast cancer mice, indicating that CP induces MIF or at least prevents the downregulation of MIF in the breast cancer model, further substantiating the effect of CP in modulating MIF levels (Figure 2C).
Figure 2.
CP induces MIF levels and inhibition of MIF suppresses CP-induced mechanical hypersensitivity. (A) ELISA assay shows significant induction of plasma levels of MIF after CP treatment in female Cx3cr1CreERT2-Rosa26R-EYFP mice (mean ± SEM; n = 5; paired t-test; *P < .05). (B) ELISA assay shows plasma levels of MIF in breast cancer animals before (Day 0) and after (Day 15) vehicle treatment (mean ± SEM; n = 5; paired t-test; *P < .05]. (C) ELISA assay shows plasma levels of MIF in breast cancer animals before (Day 0) and after (Day 15) CP treatment (mean ± SEM; n = 3; paired t-test). (D) Line graph generated from VFF assay shows mechanical sensitivity of animals (female Cx3cr1CreERT2-Rosa26R-EYFP mice) in the indicated groups (mean ± SEM; n = 5; Two-way ANOVA, Tukey’s multiple comparisons test; ****P < .0001 [significance shown is compared to CP group]). (E) Bar graph generated from VFF assay shows that pretreatment with the MIF inhibitor CPSI-1306 significantly suppresses CP-induced mechanical hypersensitivity in CisIPN animals (female Cx3cr1CreERT2-Rosa26R-EYFP mice) on Day-17 (mean ± SEM; n = 5; One-way ANOVA with Tukey’s multiple comparisons test; ****P < .0001). (F) Line graph generated from VFF assay shows mechanical sensitivity of animals (female CD1 mice) in the indicated groups (mean ± SEM; n = 5 [n = 4 for CPS1-1306 group]); Two-way ANOVA, Tukey’s multiple comparisons test; *P < .05, **P < .01, ***P < .001, ****P < .0001 (significance shown is compared to CP group). (G) Bar graph generated from VFF assay shows that pretreatment with MIF inhibitors, CPSI-1306 and ISO-1, significantly suppresses CP-induced mechanical hypersensitivity in CisIPN animals (female CD1 mice) on Day-15 (mean ± SEM; n = 5 [n = 4 for CPS1-1306 group]); One-way ANOVA with Tukey’s multiple comparisons test; **P < .01, ***P < .001.
Inhibition of MIF Protects Animals from CP-Induced Mechanical Hypersensitivity
A rise in plasma MIF levels after CP treatment suggests that MIF may contribute to CisIPN. Therefore, we examined whether inhibition of MIF could protect animals from CisIPN. Mechanical sensitivity experiments using Cx3cr1CreERT2-Rosa26R-EYFP mice showed that the MIF inhibitor CPSI-1306 significantly suppresses CP-induced mechanical hypersensitivity, indicating that MIF inhibition protects animals from CisIPN (Figure 2D and E). To check the reproducibility of the result, we repeated the experiments using CisIPN models generated from another mouse strain (CDI mouse strain). Two MIF inhibitors, CPSI-1306 and ISO-1, were used for this experiment. Consistent with our previous observation, CP-treated animals showed a paw withdrawal response at lower mechanical thresholds, indicating hypersensitivity, while animals that received saline, MIF inhibitor (CPSI-1306 or ISO-1), or the combination treatments (CP+CPS1-1306 and CP+ISO-1) maintained their basal sensitivities or responded to comparatively higher mechanical thresholds, showing that MIF inhibition could protect animals from CP-induced mechanical hypersensitivity (Figure 2F and G).
Effect of MIF Inhibition on Cold Sensitivity
We next examined whether CP increases cold sensitivity and whether MIF inhibition suppresses the same. The paws of adult female CD1 mice were stimulated using acetone, and behavioral responses were ranked on a scale of 0 to 4. Although there was a mild increase in cold sensitivity in CP-treated animals compared to control, it was not statistically significant. Pretreatment with ISO-1 appeared to prevent the CP-driven mild increase in cold sensitivity. However, it was also not statistically significant (Supplementary Figure S2A and B).
Inhibition of MIF Protects Breast Cancer Mice from CP-Induced Mechanical Hypersensitivity
We next evaluated the effect of MIF inhibition on CisIPN in a breast cancer model. Breast cancer was induced in adult female athymic nude mice by injecting MDA-MB-231 cells into the second mammary fat pad. As expected, mice responded by withdrawing paws to lower-size filaments after breast cancer induction, substantiating that breast cancer has an independent role in inducing neuropathy (Figure 3A and B). CP treatment further raised the mechanical hypersensitivity in these animals on day 15. Pretreatment with MIF inhibitors (CPSI-1306 or ISO-1) suppressed CP-induced mechanical hypersensitivity, however, the effect was significant only after CPSI-1306 pretreatment (Figure 3C and D). Representative images of breast tumor in each experimental group are shown in Supplementary Figure S2C.
Figure 3.
Inhibition of MIF suppresses CP-induced mechanical hypersensitivity in breast cancer animals. (A) Line graph generated from VFF assay shows mechanical sensitivity of animals before and after breast cancer induction (Pre-T: Pre-Tumor, Post-T: Post-Tumor) (mean ± SEM; n = 25). (B) Bar graph generated from VFF assay shows mechanical hypersensitivity of animals before and after (day 35) breast cancer induction (mean ± SEM; n = 25; paired t-test, ***P < .001). (C) Line graph generated from VFF assay shows mechanical sensitivity of breast cancer animals in the indicated groups (mean ± SEM; n = 5 [control and ISO-1 groups], n = 4 [CPSI and CP+ISO-1 groups], n = 3 [CP and CP+CPSI groups]; Two-way ANOVA, Tukey’s multiple comparisons test). (D) Bar graph generated from VFF assay shows that pretreatment with CPSI-1306 significantly suppresses CP-induced mechanical hypersensitivity in breast cancer animals on Day-15 (mean ± SEM; n = 5 [control and ISO-1 groups], n = 4 [CPSI and CP+ISO-1 groups], n = 3 [CP and CP+CPSI groups]; One-way ANOVA with Tukey’s multiple comparisons test; *P < .05).
CP Has No Major Influence on the Overall Abundance of Macrophage in Nerves
Increased infiltration of immune cells, including macrophages, into nerves is a salient feature of neuroinflammation. Therefore, we examined the overall abundance of macrophages in DRGs and sciatic nerves of CisIPN mice to examine if macrophage infiltration occurs after CP treatment, and if so, whether MIF inhibitors suppress the same. We used two well-known macrophage markers, CD68 and Iba-1, to stain macrophages.17,18 The total number of macrophages was normalized to the area of tissue analyzed. Three to five biological replicas were considered per experimental group. The data are presented as relative fold change in macrophage density compared to control. Strikingly, we found no significant difference in the overall abundance of macrophages in DRGs and sciatic nerves of CisIPN mice compared to control mice, suggesting that infiltrating macrophages may not play a major role in CisIPN, at least at the time points we considered (Supplementary Figures S3 and S4).
Sensory Neurons and SCs Are Potential Source for MIF
Since we found no major change in macrophage influx after CP treatment, we explored the potential cellular source for MIF in peripheral nerves. Immunostaining showed that MIF is preferentially expressed in sensory neurons, especially in NF200poor small-caliber sensory neurons, in the DRGs of control breast cancer animals (Figure 4A). We also noted gain of MIF in NF200high large caliber sensory neurons after CP treatment (Figure 4A). We next quantified the total population of sensory neurons expressing MIF in control and CP-treated CD1 mice. For this, DRG sensory neurons were co-stained with MIF and the pan-neuronal marker βIII tubulin (Figure 4B). Although not statistically significant, our results showed a clear trend for more sensory neurons gaining MIF expression after CP treatment (Figure 4C). Further, we quantified the MIF released from primary sensory neurons in culture. For this, primary sensory neurons were treated with either saline (control) or CP (500 ng/mL) for 8h and the secreted MIF in the supernatant was measured using ELISA. We found increased release of MIF from sensory neurons after CP treatment, substantiating that sensory neurons are potential source for increased MIF in CisIPN.
Figure 4.
Sensory neurons are potential source for MIF in DRGs. (A) Representative images of NF200 (yellow arrows) and MIF (white arrows) in the DRGs of control (saline) and CP treated animals (breast cancer model). Co-localization of NF200 and MIF is shown by red arrows in the merged CP group panel (scale bar, 50µm). (B) Representative images of βIII tubulin and MIF in the DRGs of control (saline) and CP treated mice (female CD1 mice). βIII tubulin positive neurons expressing MIF are shown by yellow arrows (scale bar, 100µm). (C) Quantification of βIII tubulin positive neurons expressing MIF in the DRGs of control (saline) and CP treated mice (female CD1 mice). (D) Quantification of MIF released from primary sensory neurons after treatment with saline (control) or CP (500 ng/mL) for 8h (mean ± SEM; n = 3; ‘t’ test; *P < .05).
Next, we examined the expression of MIF in sciatic nerves of CD1 mice. Immunostaining showed no co-localization of MIF and NF200 in control and CP-treated mice, indicating that axons may not be the primary source for MIF in nerves (Figure 5A). Interestingly, MIF was co-expressed with GFAP in nerves, suggesting that SCs are potential source for MIF in nerves (Figure 5B). To explore this further, we treated S16 cells with CP and evaluated MIF expression using western blot. Interestingly, MIF expression in S16 cells was found to fluctuate significantly after CP treatment. For example, 1 µg/mL CP consistently downregulated MIF in S16 cells at 24 h and 48 h, while a comparatively lower dose of 0.5 µg/mL CP showed a trend to upregulate MIF at 24h followed by downregulating its expression at 48 h (Figure 5C). To query this further, we examined MIF expression in S16 cells at a shorter timepoint of 8 h after CP treatment. Although a dose-dependent fluctuation in MIF levels was still apparent, 0.5 µg/mL CP consistently induced MIF expression even at this timepoint (Figure 5D). Altogether, this observation indicates that SCs respond to CP treatment by modulating MIF levels, especially inducing its upregulation in a transient manner, suggesting that SCs are an additional source of MIF at least during the initial treatment periods.
Figure 5.
SCs are potential source for MIF in peripheral nerves. (A) Representative images of NF200 (yellow arrows) and MIF (white arrows) in the sciatic nerves of control (saline) and CP treated mice (female CD1 mice) show no co-localization of MIF in NF200+ axons. (B) Representative images of GFAP (yellow arrows) and MIF (white arrows) in the sciatic nerves of control (saline) and CP treated mice (female CD1 mice). Co-localization of MIF in GFAP+ SCs is shown by red arrows in the merged panel (scale bar, 50µm). (C, D) Western blot shows the expression of MIF in SCs after CP treatment for 24h, 48h and 8h. The blot images are shown on the left and corresponding quantification is shown on the right. GAPDH is used as the loading control (mean ± SEM; n = 3; One-way ANOVA with Tukey’s multiple comparisons test; *P < .05, **P < .01, ***P < .001).
MIF Modulates ERK1/2 Signaling in SCs
We next addressed the implications of the transient increase of MIF in SCs and sensory neurons after CP treatment. Since nerve-resident SCs are drivers of neuropathies,19 we postulated that MIF derived from sensory neurons and SCs may modify neuropathy-related signaling in SCs. ERK signaling plays a major role in neuropathic pain,20 and thus we initiated the investigation by exploring ERK signaling downstream of MIF in S16 cells. For this, we treated S16 cells with 25 ng/ml MIF and examined the changes in ERK1/2 expression and activation. Dose-dependent experiments showed significant upregulation and activation of ERK1/2, evident by increased expression of parent and phosphorylated ERK1/2 in S16 cells at 15 minutes, followed by reversal of the same at longer timepoints of 4h and 8h (Figure 6A-D). A further reactivation of ERK1/2 was also noted at 24h of MIF treatment in S16 cells, strongly indicating that MIF modulates ERK1/2 signaling in SCs. Given the known role of ERK1/2 signaling in neuropathic pain,20 the modulation of this signaling by MIF in SCs may potentially underlie the mechanism of how MIF facilitates CP-induced neuropathy.
Figure 6.
MIF modulates MAPK signaling in SCs. (A-F) Western blot shows time dependent changes in the expression of p-ERK1/2 and ERK1/2 in SCs after MIF treatment. The blot images are shown on the left and the corresponding quantification is shown on the right. GAPDH is used as the loading control (mean ± SEM; n = 3; One-way ANOVA with Tukey’s multiple comparisons test; *P < .05, **P < .01, ****P < .0001).
Discussion
Despite being a debilitating side effect of cancer chemotherapy, no effective treatment is currently available, other than discontinuing or reducing the dose of the chemotherapy agent, to completely reverse CIPN.21 Several causative mechanisms have been suggested for CIPN in the past, however, none of them have proven to be effective intervention points for developing CIPN therapies. Moreover, unique CIPN mechanisms associated with individual classes of chemotherapeutic agents further complicate the identification of effective therapies. This work is specifically focused on CisIPN. Previous reports suggest that neuroinflammation can potentially influence CisIPN progression.10 Surprisingly, we did not observe significant changes in macrophage infiltration, a hallmark of inflammation, in DRGs and nerves after CP treatment, even though we found that the anti-inflammatory drug Dex could suppress CisIPN. Thus, it is likely that CisIPN may involve neuroinflammation driven by resident macrophages or neuroinflammatory molecules released by other cell types, such as neurons and glial cells.
We found that circulating MIF levels are elevated in mice after CP treatment. Individuals with spinal cord injuries were shown to have increased levels of circulating MIF.12 Patients with diabetic neuropathy also showed increased expression of MIF in their footpad lesions.14 Additionally, MIF levels were shown to elevate in the cerebrospinal fluid of multiple sclerosis patients.22 All these pathological conditions are associated with neuropathy, substantiating the involvement of MIF in neuropathies. Here, for the first time, we demonstrated the involvement of MIF in CisIPN.
We considered regular (Cx3cr1CreERT2-Rosa26R-EYFP and CD1 mice) and tumor (TNBC) bearing mouse models for this study. The regular animal models are rapid screening models, while tumor models provide insights into the role of MIF in CisIPN under the influence of a solid cancer. CP is a well-established therapy for TNBC, supporting the relevance of the cancer model we used in this study.7,23 Importantly, cancer itself can induce neuropathy.11 Especially, the emerging evidence of a crosstalk between solid cancers and peripheral nerves supports the independent role of cancers in inducing neuropathy.24-27 Our observation of elevated levels of MIF in tumor-bearing mice, as compared to healthy mice, suggests a potential role for MIF in mediating cancer-induced neuropathy too. TNBC patients were shown to express higher levels of MIF in tumors compared to adjacent normal tissues, and this observation further supports a role for MIF in cancer-driven neuropathy and warrants additional investigation.28
Mechanical and cold hypersensitivities are two well-known measurable indices of CIPN. Previous studies showed that direct injection of recombinant MIF produces a dose-dependent increase in mechanical sensitivity in mouse models, indicating that MIF can independently promote mechanical hypersensitivity.29 Interestingly, our results consistently demonstrated that pharmacological inhibition of MIF using CPSI-1306 and ISO-1 effectively suppressed CP-induced mechanical hypersensitivity, providing strong evidence that MIF facilitates neuropathic pain in CisIPN. Previous studies showed that MIF inhibitors suppress neuropathic pain in other neurological conditions. For example, pharmacological inhibition of MIF was shown to suppress pain-like behaviors in animal models of nerve injury.30 Our experiments did not show significant induction of cold hypersensitivity following CP treatment. A previous study also showed that CP is weak compared to other platins in inducing cold hypersensitivity, and this aligns with our observations.31
Although unexpected, we did not find significant changes in macrophage infiltration in nerves and DRGs after CP treatment, suggesting that recruited macrophages may not be the primary source for MIF in CisIPN. A recent study also showed that CP has no major influence on macrophage infiltration into nerves, matching the observations we made.32 We evaluated macrophage infiltration after 10 doses of CP, and hence, we do not know if macrophage infiltration occurs during early dosing periods. We also did not perform M1/M2 subpopulation-specific macrophage analysis, and this is also a limitation in our study. Regardless, the lack of macrophage infiltration in nerves and DRGs, at least at the timepoints we considered, posed questions on the source of elevated circulating MIF in CisIPN mice. We observed that MIF is expressed by sensory neurons in the DRGs, with a sub-population of large-caliber sensory neurons gaining MIF expression after CP treatment. Additionally, we demonstrated increased MIF secretion by CP-treated sensory neurons. Similarly, we showed that SCs treated with CP respond by modulating MIF levels. Thus, it is clear from our study that sensory neurons and SCs are sources of MIF in CisIPN. However, additional investigations are warranted to assess whether macrophages, especially self-renewing nerve-resident macrophages, also induce MIF in CisIPN.33
We found fluctuations in ERK1/2 signaling in SCs following MIF treatment. Combining the observations we made in this study, it is likely that CP-driven MIF in sensory neurons and SCs may modulate ERK1/2 signaling in SCs. Given the known role of ERK1/2 signaling in peripheral neuropathies,20 the fluctuating ERK1/2 expression and activation in SCs downstream of MIF may have implications in CisIPN. Additional investigation in this direction is, however, warranted.
Overall, this study, for the first time, has identified that MIF facilitates CisIPN. Our findings suggest that targeting MIF may represent a promising therapeutic strategy, especially for alleviating the mechanical hypersensitivity component of CisIPN. These encouraging results warrant further investigation in male models of CisIPN and across other forms of CIPN.
Supplementary Material
Contributor Information
Hya El-Baroudy, Department of Anatomy, Physiology, and Pharmacology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada; Cameco MS Neuroscience Research Centre, Saskatoon City Hospital, Saskatoon, Saskatchewan, Canada.
Bhadrapriya Sivakumar, Department of Anatomy, Physiology, and Pharmacology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada; Cameco MS Neuroscience Research Centre, Saskatoon City Hospital, Saskatoon, Saskatchewan, Canada.
Ximena Mejia Delgadillo, Department of Anatomy, Physiology, and Pharmacology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada; Cameco MS Neuroscience Research Centre, Saskatoon City Hospital, Saskatoon, Saskatchewan, Canada.
Nishi Bamania, Department of Anatomy, Physiology, and Pharmacology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada; Cameco MS Neuroscience Research Centre, Saskatoon City Hospital, Saskatoon, Saskatchewan, Canada.
Isabelle Monsman, Department of Anatomy, Physiology, and Pharmacology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada; Cameco MS Neuroscience Research Centre, Saskatoon City Hospital, Saskatoon, Saskatchewan, Canada.
Shubham Dwivedi, Department of Anatomy, Physiology, and Pharmacology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada; Cameco MS Neuroscience Research Centre, Saskatoon City Hospital, Saskatoon, Saskatchewan, Canada.
Bari Chowdhury, Department of Anatomy, Physiology, and Pharmacology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada; Cameco MS Neuroscience Research Centre, Saskatoon City Hospital, Saskatoon, Saskatchewan, Canada.
Jay Patel, Department of Anatomy, Physiology, and Pharmacology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada; Cameco MS Neuroscience Research Centre, Saskatoon City Hospital, Saskatoon, Saskatchewan, Canada.
Nickson Joseph, Department of Anatomy, Physiology, and Pharmacology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada; Cameco MS Neuroscience Research Centre, Saskatoon City Hospital, Saskatoon, Saskatchewan, Canada.
Snigdha Pathak, Department of Anatomy, Physiology, and Pharmacology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada; Cameco MS Neuroscience Research Centre, Saskatoon City Hospital, Saskatoon, Saskatchewan, Canada.
Sandra Mitzkus, Department of Anatomy, Physiology, and Pharmacology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada; Cameco MS Neuroscience Research Centre, Saskatoon City Hospital, Saskatoon, Saskatchewan, Canada.
Hanrong Li, Department of Anatomy, Physiology, and Pharmacology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada; Cameco MS Neuroscience Research Centre, Saskatoon City Hospital, Saskatoon, Saskatchewan, Canada.
Shahid Ahmed, Medical Oncology, Saskatchewan Cancer Agency, Saskatoon, Saskatchewan, Canada (S.A).
Anand Krishnan, Department of Anatomy, Physiology, and Pharmacology, College of Medicine, University of Saskatchewan, Saskatoon, Saskatchewan, Canada; Cameco MS Neuroscience Research Centre, Saskatoon City Hospital, Saskatoon, Saskatchewan, Canada.
Supplementary Material
Supplementary material is available online at Neuro-Oncology Advances (https://academic.oup.com/noa).
Author Contributions
HEB and BS performed experiments, analyzed data, and contributed to preparing the initial version of the manuscript and edited the final version of the manuscript. XMD, NB, IM, SD, BC, JP, NJ, SP, SM, and HL contributed to data generation, analysis, and edited the final version of the manuscript. SA contributed to data analysis and edited the manuscript. AK conceived the concept, provided the resources for the work, analyzed the data, prepared the initial and revised versions of the manuscript, and finalized the manuscript.
Conflict of Interest Statement
The authors declare no competing financial interests.
Funding
This work was supported by the operating grants from the Cancer Research Society-CIHR Institute of Cancer Research (#941001; #CR3-184663) and the Breast Cancer Canada (#2022-006) to A.K. This work was also supported by the Establishment Grant (#6106) from the Saskatchewan Health Research Foundation (SHRF) and CoMRAD grant from the College of Medicine (University of Saskatchewan) to A.K.
Ethics Approval Statement
The animal experiments described in this work have been approved by the Animal Research Ethics Board (AREB) at the University of Saskatchewan.
Data Availability
All data associated with this article are available from the corresponding author upon reasonable request.
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Associated Data
This section collects any data citations, data availability statements, or supplementary materials included in this article.
Supplementary Materials
Data Availability Statement
All data associated with this article are available from the corresponding author upon reasonable request.