Chemotherapy-induced peripheral neuropathy (CIPN) is a severe dose-limiting side effect of taxanes such as paclitaxel and docetaxel. Despite the high medical needs, insufficient understanding of the complex mechanism underlying CIPN pathogenesis precludes any endorsed causal therapy to prevent or relieve CIPN. In this study, we report that elevation of plasma galectin-3 level is a pathologic change common to both patients with taxane-treated breast cancer with CIPN and a mouse model of taxane-related CIPN. Following multiple intraperitoneal injections of paclitaxel in mice, galectin-3 levels were elevated in Schwann cells within the sciatic nerve but not in other peripheral organs or cells expressing galectin-3. Consistent with this, paclitaxel treatment of primary cultures of rat Schwann cells induced upregulation and secretion of galectin-3. In vitro migration assays revealed that recombinant galectin-3 induced a chemotactic response of the murine macrophage cell line RAW 264.7. In addition, perineural administration of galectin-3 to the sciatic nerve of naive mice mimicked paclitaxel-induced macrophage infiltration and mechanical hypersensitivity. By contrast, chemical depletion of macrophages by clodronate liposomes suppressed paclitaxel-induced mechanical hypersensitivity despite the higher level of plasma galectin-3. Deficiency (Galectin-3−/− mice) or pharmacologic inhibition of galectin-3 inhibited paclitaxel-induced macrophage infiltration and mechanical hypersensitivity. In conclusion, we propose that Schwann cell–derived galectin-3 plays a pronociceptive role via macrophage infiltration in the pathogenesis of taxane-induced peripheral neuropathy. Therapies targeting this phenomenon, which is common to patients with CIPN and mouse models, represent a novel approach to suppress taxane-related CIPN.
These findings demonstrate that the elevation of plasma galectin-3 is a CIPN-related pathologic change common to humans and mice, and that targeting galectin-3 is a therapeutic option to delay CIPN progression.
More than 50% of patients receiving taxanes, such as paclitaxel and docetaxel, develop a dose-limiting side effect, chemotherapy-induced peripheral neuropathy (CIPN; refs. 1, 2). CIPN is a serious impediment to effective cancer treatment because aggravation of its symptoms results in discontinuation of taxane treatment or dose reduction (3). However, the complex mechanisms underlying taxane-related CIPN pathogenesis are not fully understood. On the basis of studies in CIPN rodent models, several candidate factors have been proposed as key determinants of CIPN pathogenesis (4–6), but effective therapeutic targets that can also be applied to patients with CIPN have not yet been found. Identifying pathologic changes common to patients and animal disease models is an important step toward a better understanding of essential mechanisms and the development of effective therapeutic drugs.
Schwann cells are the myelinating glial cells in the peripheral nervous system (PNS; ref. 7), and participate in the maintenance of peripheral nerve functions via interactions with axons (8, 9). We previously demonstrated that paclitaxel reduces myelin-forming Schwann cells by promoting their dedifferentiation, a process characterized by elevated expression of low-affinity nerve growth factor receptor p75 (a marker of immature Schwann cells) and reduced expression of myelin-associated molecule (MBP, a marker of mature Schwann cells; ref. 10). Furthermore, we found that galectin-3 (a marker of dedifferentiated Schwann cells; ref. 11) is upregulated in primary cultures of rat Schwann cells following paclitaxel treatment and Schwann cells in the sciatic nerve of paclitaxel-related CIPN model mice (10).
The β-galactoside binding protein galectin-3 is localized in the cytoplasm, nucleus, and plasma membrane, and can be secreted into the extracellular compartment, where it serves as an immune cell chemoattractant (12). A number of earlier clinical studies showed that galectin-3 derived from cardiac fibroblasts are increased in plasma of heart failure patients with reduced ejection fraction (13–15). In addition, extracellular galectin-3 leads to microglial activation in the brain via Toll-like receptors or triggering receptor expressed on myeloid cells 2 (TREM2) signaling, thereby eliciting neuroinflammation associated with the progression of Alzheimer's disease (16). On the other hand, during Wallerian degeneration after peripheral nerve injury, dedifferentiated Schwann cells show high expression of galectin-3, which plays an important role in lectin-mediated phagocytosis of degraded material at the site of injury (11). Reports of pathogenic roles for extracellular galectin-3 in some diseases have raised the intriguing hypothesis that Schwann cell-derived galectin-3 is also released into the extracellular compartment (i.e., the blood circulation) and participates in the pathogenesis of taxane-related CIPN.
In this study, we found that the plasma galectin-3 level was elevated in both patients with taxane-treated breast cancer with CIPN and a mouse model of taxane-related CIPN. We then showed that the origin of plasma galectin-3 is Schwann cells within the sciatic nerves. Furthermore, we demonstrated for the first time that Schwann cell–derived galectin-3 plays a critical role via macrophage infiltration in the development of taxane-induced peripheral neuropathy.
Materials and Methods
Measurement of human plasma galectin-3
A self-controlled, prospective observational study was conducted. The clinical trial was approved by the Ethics Committee of Kyoto University Graduate School and Faculty of Medicine (G424) in accordance with Helsinki guidelines and registered with the University Hospital Medical Information Network. All subjects provided written informed consent. Plasma galectin-3 level was determined using a commercially available ELISA Kit (Human Galectin-3 Platinum ELISA, BMS279/4; Thermo Fisher Scientific).
All animal experiments were approved by the Kyoto University Animal Research Committee (permission number: Med Kyo 16067, 17103, 18103, 19103) and performed according to the guidelines of the animal ethics committee of Kyoto University. C57BL/6J JmsSlc (male, 5–7 weeks), C57BL/6-Tg(CAG-EGFP) C14-Y01-FM131Osb transgenic mice (male C57BL/6GFP transgenic mice), or C57BL/6.Cg-Lgals3tm1Poi/J transgenic (Galectin-3−/−) mice (male and female, 6–14 weeks) were used. Other information about experimental animals is described in the Supplementary Materials and Methods.
Reagents are described in the Supplementary Materials and Methods.
Mouse model of paclitaxel-induced CIPN
Mice were intraperitoneally injected with paclitaxel (20 mg/kg; Nippon Kayaku) or vehicle (16.7% ethanol) twice a week (on days 1 and 2) for a total of 1 to 8 weeks. Behavioral testing or sample collection for ELISA, qRT-PCR, and IHC analysis was conducted 4 or 5 days after the previous injection (shown in Fig. 1C).
von Frey filament test
Mechanical sensitivity was assessed by measuring the paw withdrawal score from a mechanical stimulus with a von Frey filament, as described previously (17) with minor modifications.
Primary culture of Schwann cells and treatment with anticancer agent
Primary Schwann cells were obtained from the sciatic nerves of neonatal Wistar/ST rats (P2–3) as described previously (10) with slight modification. After 4 to 7 days of purification, to induce differentiation of Schwann cells, the cells were cultured in differentiation medium. After 2 days of culture in differentiation medium, Schwann cells were treated with each drug.
Infection of MBP::Venus lentiviral vector into Schwann cells
At 1 day after seeding on plates, 60% confluent purified Schwann cells were exposed to MBP::Venus lentiviral vector (LVV) diluted 1:7,500 in maintenance medium for 24 hours. After incubation, medium containing LVV was washed out and replaced with fresh maintenance medium. After 2 days of culture, the medium was replaced with differentiation medium.
Measurement of galectin-3 level in mouse plasma or culture supernatant of Schwann cells
Galectin-3 concentration in mouse plasma or culture supernatant of rat primary Schwann cells was determined using the Mouse LGALS3/Galectin-3 ELISA Kit (#LS-F494; LifeSpan BioSciences) or Rat LGALS3/Galectin-3 ELISA Kit (#LS-F3877, LifeSpan BioSciences), respectively.
Under deep anesthesia with pentobarbital (50 mg/kg, i.p.; Nacalai Tesque), the mouse tissues and organs were harvested immediately after transcardial perfusion with PBS. Total RNA from the mouse tissue and rat primary cultured Schwann cells was isolated using the SV Total RNA Isolation system (Promega). Purified 0.2 to 1.0 μg of total RNA was used to prepare first-strand cDNA as described previously (10). Each gene was amplified in PCR solution with synthesized primers as described in Supplementary Materials and Methods and Supplementary Table S1.
Migration assay of RAW 264.7 cells
The murine macrophage cell line RAW 264.7 (TIB-71, RRID:CVCL_0493) was purchased from ATCC. Migration was monitored using the CytoSelect 96-Well Cell Migration Assay (8 μm, Fluorometric Format; Cell Biolabs) as described previously (18). RAW 264.7 was negative for Mycoplasma, which was confirmed using MycoAlert Mycoplasma Detection Kit (Lonza) most recently in October 2020. RAW 264.7 cells were passaged less than few times after thaw and used for each experiment.
Focal application of galectin-3 to the mouse sciatic nerve
Surgery was performed as previously reported (18) with minor modifications. Mice were anesthetized with pentobarbital (50 mg/kg, i.p.), and then the right sciatic nerve was exposed in the popliteal fossa without damaging the perineurium. The right sciatic nerve 2 to 3 mm proximal to the trifurcation was loosely enwrapped with gauze containing vehicle (PBS) or 10 nmol/L recombinant mouse galectin-3 (rGalectin-3; R&D Systems, #9039-GA) without inducing any nerve injury.
Immunocytochemistry, IHC, and Western blotting
For staining of Schwann cells, the fixed cells were incubated with the following primary antibodies: rat anti-MBP (Merck, #MAB386, RRID:AB_94975), mouse anti-p75 (Merck, #MAB365, RRID:AB_2152788), rabbit anti-p75 (Abcam, #ab38335, RRID:AB_2152644), or mouse anti–galectin-3 (Abcam, #ab2785, RRID:AB_303298). For IHC, sciatic nerves were collected from paclitaxel- or vehicle-injected mice according to a schedule shown in Figs. 1C, 5E, and 6D. The sciatic nerves of rGalectin-3–treated mice were obtained 3 or 7 days after the surgery. The section of each sciatic nerve was incubated with primary antibodies: mouse anti–galectin-3, rat anti-glial fibrillary acidic protein (GFAP; Merck, #345860, RRID:AB_211868), rabbit anti-ionized calcium binding adapter molecule 1 (Iba1; Wako Pure Chemical Industries, #019–19741, RRID:AB_839504), rat anti-CD31 (Becton, Dickinson and Company, #550274, RRID:AB_393571), or rat anti-inducible nitric oxide synthase (iNOS; Thermo Fisher Scientific, #14–5920–82, RRID:AB_2572890). For immunoblot detection, the polyvinylidene difluoride (PVDF) membrane was incubated with mouse anti–galectin-3 and mouse anti–β-actin (Sigma-Aldrich, #A1978, RRID:AB_476692).
Generation of GFP-bone marrow chimeric mice and flow cytometry assay
Bone marrow (BM) transplantation and flow cytometry to determine the purity of GFP-positive cells in peripheral blood after BM transplantation was conducted as reported previously (18, 19).
Data were analyzed using GraphPad Prism 8 (GraphPad Software; RRID:SCR_002798) and expressed as means ± SEM. Differences between two groups were compared with the unpaired t test or Mann–Whitney U test. Data from more than two groups were compared by one-way or two-way ANOVA followed by Bonferroni post hoc comparison test. In all cases, P < 0.05 was considered statistically significant. The additional methods are described in detail in the Supplementary Materials and Methods.
Plasma galectin-3 level is elevated in patients with taxane-treated breast cancer with CIPN
The clinical characteristics of 8 female patients with breast cancer [mean age (range): 51.1 (37–79)] and the regimens of taxane-based chemotherapy over 12 weeks are summarized in Supplementary Tables S2 and S3, respectively. Because the drugs used concomitantly with taxane-based chemotherapy (i.e., bevacizumab, trastuzumab, pertuzumab, cyclophosphamide, and doxorubicin) are not considered risk factors for CIPN (20–23), we considered it unlikely that they contributed to changes in plasma galectin-3 levels (Supplementary Table S3).
In plasma samples obtained from these 8 patients, we checked changes in galectin-3 concentration before chemotherapy (baseline), 6 weeks after the initiation of each regimen (6 weeks), and just before the final taxane administration (12 weeks; Fig. 1A). CIPN occurred in three of 4 patients who received 12 weeks of paclitaxel-based (patients nos. 2–4) or docetaxel-based chemotherapy (patients nos. 5, 6, and 8; indicated by filled circles in Fig. 1A and B). The average concentration of plasma galectin-3 among these 8 patients increased at 6 weeks, and then slightly decreased at 12 weeks after the beginning of chemotherapy (Supplementary Table S4). In patients who received therapy of paclitaxel plus bevacizumab (patient no. 3), paclitaxel/pertuzumab/trastuzumab (patient no. 4), docetaxel plus cyclophosphamide (TC; patient nos. 5 and 6), and docetaxel combined with cyclophosphamide/doxorubicin (patient no. 8), plasma galectin-3 levels in the presence of CIPN were higher than the corresponding baselines. During the study period, galectin-3 levels did not change in patients receiving weekly paclitaxel therapy (patient nos. 1 and 2). In a patient who received TC (patient no. 7), the plasma galectin-3 level increased after the initiation of chemotherapy, although CIPN did not occur.
To better understand the relationship between plasma galectin-3 levels and the development of CIPN, we next performed statistical analysis by classifying each galectin-3 concentration according to the presence or absence of CIPN. Plasma galectin-3 levels were significantly higher in the presence of CIPN (mean ± SD: 8.190 ± 3.398 ng/mL) than before development of CIPN (5.703 ± 2.435 ng/mL; Fig. 1B). Conversely, galectin-3 levels in the absence of CIPN were similar to those in subjects without cancer (5.353 ± 2.237 ng/mL, indicated by dashed line).
Plasma galectin-3 level is elevated in a mouse model of taxane-related CIPN
Unless otherwise noted, male mice were used for all experiments. Mice were intraperitoneally injected with vehicle or paclitaxel (20 mg/kg) twice a week for 8 weeks, and then subjected to analysis (Fig. 1C). Paclitaxel induced significant mechanical hypersensitivity in the hindpaw 2 weeks after the first injection relative to injection of vehicle alone (16.7% ethanol), and this effect persisted for 8 weeks (Fig. 1D). Plasma galectin-3 concentration increased in paclitaxel-treated mice compared with vehicle-administered mice from 1 to 4 weeks after the injection, reaching statistical significance 2 weeks after the first injection (Fig. 1E). Plasma galectin-3 recovered to the same level in the vehicle-treated group by 8 weeks after paclitaxel injection. Repeated injections of docetaxel (20 mg/kg, i.p.) also induced both significant mechanical hypersensitivity (Supplementary Fig. S1A and S1B) and increased plasma galectin-3 levels (Supplementary Fig. S1C) 2 weeks after the first injection. However, repeated injections of the platinum derivative oxaliplatin (4 mg/kg, i.p.) did not change the plasma galectin-3 concentration 2 weeks after the first injection (Supplementary Fig. S1D and S1F), despite the occurrence of significant mechanical hypersensitivity (Supplementary Fig. S1E).
Taxane treatment increases galectin-3 production and secretion from dedifferentiated Schwann cells
To verify that galectin-3 upregulation following taxane treatment was specific for p75-positive dedifferentiated Schwann cells, rather than p75-positive immature Schwann cells mixed in heterogeneous cell populations, we visualize mature Schwann cells by transducing cultured Schwann cells with an LVV encoding the MBP::Venus reporter (Fig. 2A). This culture system allows us to distinguish between dedifferentiated Schwann cells transitioned from Venus-positive mature Schwann cells upon paclitaxel treatment (i.e., Venus/p75 double-positive cells) and immature Schwann cells (i.e., Venus-negative/p75-positive cells) in mixed cell populations. Before cell differentiation, Schwann cells exhibited a considerable p75 immunoreactivity (IR), but only weak MBP IR and Venus fluorescence (Supplementary Fig. S2A). After 2 days of culture in differentiation medium, Schwann cells exhibited a differentiated phenotype, characterized by increased MBP IR and disappearance of p75 IR (Supplementary Fig. S2A). In these differentiated Schwann cells, Venus fluorescence was elevated and highly colocalized with MBP IR. However, exposure of differentiated Schwann cells to paclitaxel (10 nmol/L) for 48 hours decreased MBP IR (Fig. 2B) and increased both p75 and galectin-3 IR relative to Schwann cells treated with vehicle alone (0.1% DMSO; Fig. 2C). The increased p75 and galectin-3 IR colocalized with Venus fluorescence in paclitaxel-treated Schwann cells (Fig. 2C). In addition, treatment of mature Schwann cells with paclitaxel (10 nmol/L) for 48 hours increased Sox2 and Pax3 mRNA levels (Supplementary Fig. S2B), which are highly expressed by immature Schwann cells (24). These findings highlight the upregulation of galectin-3 in dedifferentiated Schwann cells derived from Venus-expressing mature Schwann cells, as illustrated in Fig. 2G. The marked morphologic changes, characterized by retraction of bipolar processes and a rounded shape, were also seen in Schwan cells 48 hours treatment with paclitaxel (10 nmol/L; Supplementary Fig. S3). These changes in paclitaxel-treated cells reverted 48 hours after washout of paclitaxel (Supplementary Fig. S3). The immunocytochemical data were supported by qRT-PCR and Western blot data showing that treatment with paclitaxel (10 nmol/L) or docetaxel (10 nmol/L) for 48 hours significantly increased Galectin-3 mRNA (Fig. 2D) and protein levels (Fig. 2E) in Schwann cells.
The 48-hour treatment with either paclitaxel (10 nmol/L) or docetaxel (10 nmol/L) significantly increased the galectin-3 concentration in the supernatant of Schwann cells (Fig. 2F). On the other hand, 48-hour exposure of oxaliplatin (3 μmol/L) decreased DAPI-positive Schwann cells (Supplementary Fig. S4A), but did not increase galectin-3 IR or the galectin-3 level in culture supernatant (Supplementary Fig. S4B and S4C), ruling out the possibility that secretion of galectin-3 induced by paclitaxel or docetaxel treatment reflected a general reduction in viability.
It is reported that galectin-3 expression is regulated by NF-κB (25). We found that paclitaxel (10 nmol/L) or docetaxel (10 nmol/L) treatment significantly increased level of phosphorylated NF-κB p65 in Schwann cells, suggesting the activation of NF-κB signaling pathway (Supplementary Fig. S5A). Cotreatment with a NF-κB inhibitor, SC75741 (1 μmol/L), which inhibits NF-κB binding to gene promoter regions (26), suppressed paclitaxel-induced increase in Galectin-3 mRNA expression (Supplementary Fig. S5B).
Paclitaxel increases galectin-3 expression specifically in the neuroaxis of mouse sciatic nerve, but not in other organs or cells
To determine whether Schwann cell–derived galectin-3 could be the origin of plasma galectin-3 following paclitaxel administration, we evaluated the changes in galectin-3 expression in Schwann cells of the mouse sciatic nerve over a time course. The experimental timeline and dose of paclitaxel were the same as described in Fig. 1C. Consistent with the elevation in the plasma galectin-3 level, the intensity of galectin-3 IR in the longitudinal section of the sciatic nerve from paclitaxel-injected mice increased from 2 to 4 weeks after injection, reaching significance at 2 weeks after the first administration of paclitaxel (Fig. 3A and C). The increase in galectin-3 IR was almost completely colocalized with IR of GFAP, which is expressed in Schwann cells (Fig. 3B). Likewise, expression of Galectin-3 mRNA was significantly increased in the neuroaxis of the sciatic nerve, which contains abundant Schwann cells, in paclitaxel-treated mice 2 and 4 weeks after the first injection (Fig. 3D). Repeated injections of docetaxel (20 mg/kg, i.p.) for 2 weeks also increased galectin-3 IR within sciatic nerve sections (Supplementary Fig. S6). Repeated injection of paclitaxel for 2 weeks did not increase the Galectin-3 mRNA level in the dorsal root ganglia (DRG), peripheral blood mononuclear cells (PBMC), liver, heart, lung, small intestine, colon, and kidney (Fig. 3E).
Paclitaxel induces infiltration of macrophages into the mouse sciatic nerve
Given that galectin-3 serves as a chemoattractant for macrophages/monocytes (27, 28), we hypothesized that Schwann cell–derived galectin-3 plays a critical role in macrophage infiltration leading to neuroinflammatory responses. As shown in Fig. 4A, repeated injection of paclitaxel significantly increased the number of Iba1-positive macrophages in the sciatic nerve 2 weeks after the first injection. The increased Iba1-positive cells decreased to the same level in the vehicle-treated group by 8 weeks after paclitaxel injection (Fig. 4B).
To further investigate whether macrophages infiltrated the sciatic nerve after repeated injection of paclitaxel, we used BM chimeric mice derived by crossing donor C57BL/6GFP transgenic and recipient C57BL/6J mice. Reconstitution of preirradiated recipient mice with donor BM cells from GFP transgenic mice resulted in replacement of 98.2% of peripheral blood cells in recipient mice (i.e., GFP-BM chimeric mice) by donor GFP-positive cells 6 weeks after transplantation (Fig. 4C). Repeated injection of paclitaxel increased GFP/Iba1 double-positive cells (i.e., GFP-BM–derived macrophages) in the sciatic nerves of GFP-BM chimeric mice (Fig. 4D). The number of GFP-positive cells in the sciatic nerve was significantly higher in paclitaxel-injected mice than in vehicle-injected mice (Fig. 4D). Furthermore, these increased GFP-positive macrophages in paclitaxel-treated GFP-BM chimeric mice were less colocalized with CD31-positive endothelial cells (Fig. 4E, white arrows), whereas GFP-positive cells in vehicle-treated mice were primarily adjacent to CD31-positive endothelial cells (Fig. 4E, white arrowheads), suggesting that BM-derived macrophages infiltrated the parenchyma of the sciatic nerve.
In addition, we investigated changes in the expression of pro- and anti-inflammatory macrophage-related genes such as Cd86 and iNos (a proinflammatory macrophage marker), Irf5 (a transcription factor involved in proinflammatory macrophage polarization), Il1β and Tnfα (proinflammatory cytokines), Cd206 (an anti-inflammatory macrophage marker), Irf4 (a transcription factor involved in anti-inflammatory macrophage polarization), and Il10 (anti-inflammatory cytokines) in the mouse sciatic nerve 2 weeks after repeated paclitaxel injection. iNos mRNA expression was significantly increased in the sciatic nerve of paclitaxel-treated mice at 2 weeks after the first injection (Fig. 4F). Consistently, iNOS IR was colocalized with Iba1 IR, and significantly increased in the mouse sciatic nerve (Fig. 4G). The repeated injection of paclitaxel tended to increase Irf5 and Il1β mRNA levels, but not Cd86, Tnfα, Cd206, Irf4, and Il10, in the mouse sciatic nerve (Fig. 4F).
Perineural recombinant galectin-3 application to the sciatic nerve induces the infiltration of macrophages and mechanical hypersensitivity in naive mice
In a chemotaxis assay, we observed significant migration of RAW 264.7 murine macrophage cells toward culture medium containing 2 pmol/L rGalectin-3 (Fig. 5A); this concentration was similar to that in culture supernatant of paclitaxel- or docetaxel-treated Schwann cells (see Fig. 2F; paclitaxel: 46.1 pg/mL = 1.77 pmol/L, docetaxel: 43.8 pg/mL = 1.69 pmol/L. Furthermore, RAW 264.7 cells exhibited chemotactic responses toward culture supernatant freshly collected from Schwann cells treated with 10 nmol/L paclitaxel for 2 days, but the effect was not significant (Supplementary Fig. S7). The increase in the chemotactic response was completely abolished by addition of a neutralizing antibody for galectin-3 (10 μg/mL) to culture supernatant from paclitaxel-treated Schwann cells (Supplementary Fig. S7). We did not detect significant changes in the expression of macrophage-related genes (proinflammatory: Cd86, iNos, Irf5, Il1β, and Tnfα; anti-inflammatory: Cd206, Irf4, and Il10) in RAW 264.7 cells 24 hours after treatment with rGalectin-3 (2 pmol/L; Supplementary Fig. S8). Perineural administration of 10 nmol/L rGalectin-3 to the sciatic nerve significantly increased the number of Iba1-positive macrophages in the sciatic nerve of naive mice 3 and 7 days after application (Fig. 5B and C), and induced significant mechanical hypersensitivity (Fig. 5D).
Depletion of macrophages with clodronate inhibits paclitaxel-induced mechanical hypersensitivity
As shown in Fig. 5E, mice were intraperitoneally injected three times with clodronate liposomes, a well-characterized macrophage-depleting compound (29), or control liposomes. Clodronate liposomes significantly decreased the number of Iba1-positive cells in the sciatic nerve of paclitaxel-injected mice relative to control liposomes (Fig. 5F and G). Consistent with this, clodronate liposomes significantly inhibited paclitaxel-induced mechanical hypersensitivity 2 weeks after injection (Fig. 5H). However, clodronate liposomes exacerbated the paclitaxel-induced increase in plasma galectin-3 level, which was significantly higher than that in mice treated with control liposomes (Fig. 5I).
Taxane-induced macrophage infiltration and mechanical hypersensitivity are suppressed in Galectin-3−/− mice
To investigate whether lack of galectin-3 could influence paclitaxel-induced macrophage infiltration and mechanical hypersensitivity, we repeatedly injected wild-type (WT) and Galectin-3−/− mice with vehicle (16.7% ethanol) or paclitaxel (20 mg/kg, i.p.) twice a week for 8 weeks. The mutation in Galectin-3−/− mice was confirmed by genomic PCR (Supplementary Fig. S9A). Flow cytometry analysis revealed no difference in the percentage of CD11b and F4/80 double-positive monocytes in the circulating blood cells between naive WT and Galectin-3−/− mice (Supplementary Fig. S9B and S9C). Nevertheless, the increase in Iba1-positive macrophages observed in the sciatic nerve of paclitaxel-treated WT mice was significantly reduced in paclitaxel-treated Galectin-3−/− mice 2 weeks after paclitaxel injection (Fig. 6A). Eight weeks after the injections, the levels of Iba1 IR in paclitaxel-injected WT and Galectin-3−/− mice were similar to those in the corresponding vehicle-injected mice (Fig. 6B). The paclitaxel-induced mechanical hypersensitivity observed in the WT mice was also abolished in Galectin-3−/− mice 1 and 2 weeks after injection (Fig. 6C). However, mechanical hypersensitivity gradually appeared in Galectin-3−/− mice from 4 to 8 weeks after multiple paclitaxel injections, and the extent of sensitivity was similar to that in paclitaxel-injected WT mice 6 to 8 weeks after injection.
The galectin-3 inhibitor TD-139 suppresses paclitaxel-induced macrophage infiltration and mechanical hypersensitivity
The galectin-3 inhibitor TD-139 (15 mg/kg) or vehicle (2.3% DMSO) was intraperitoneally injected into mice three times as indicated in Fig. 6D. TD-139 has high affinity for the carbohydrate recognition domain of galectin-3, and inhibits its binding to receptors and effector proteins (30). Treatment with TD-139 significantly suppressed macrophage infiltration into the mouse sciatic nerve 2 weeks after the first paclitaxel injection (Fig. 6E and F). In contrast, TD-139 did not affect the paclitaxel-induced increase in the plasma galectin-3 level (Fig. 6G). Under these conditions, paclitaxel-induced mechanical hypersensitivity was suppressed by TD-139 injection 2 weeks after paclitaxel injection (Fig. 6H).
Impact of galectin-3 on paclitaxel-related CIPN is ascertained in female mice
Finally, we examined sexual dimorphism in the paclitaxel-induced increase in galectin-3 level and galectin-3–dependent macrophage infiltration. Repeated injections of female mice with paclitaxel, plasma galectin-3 level, and the intensity of galectin-3 IR within the sciatic nerve 2 weeks after the first injection were elevated relative to the corresponding values in vehicle-injected mice (Supplementary Fig. S10A and S10B). Furthermore, paclitaxel treatment also induced a significant increase in the number of Iba1-positive macrophages in the sciatic nerve and mechanical hypersensitivity; these effects were almost abolished in Galectin-3−/− females (Supplementary Fig. S10C and S10D).
The first major finding of this study was that plasma galectin-3 level was increased in both patients with taxane-treated breast cancer with CIPN and a mouse model of CIPN (as illustrated in Fig. 7). In contrast, the plasma galectin-3 level in patients with taxane-treated breast cancer without CIPN was similar to that in noncancer subjects. These results suggest that breast carcinoma has only a little effect, if any, on plasma galectin-3 levels, and that the increase in plasma galectin-3 level is related to the taxane-induced onset of CIPN. However, unlike breast cancer, high expression of galectin-3 is reported in tissues such as pancreatic, liver, and kidney cancer, so the possibility that these types of cancer-derived galectin-3 affects blood galectin-3 levels in patients cannot be excluded (31). Plasma galectin-3 was also elevated in CIPN model mice 2 weeks after repeated injections of taxanes, but not oxaliplatin, and largely coincided with the appearance of mechanical hypersensitivity. Thereafter, the elevated plasma galectin-3 level in paclitaxel-treated mice gradually decreased by 8 weeks after administration. Similarly, the average plasma concentration of galectin-3 among 8 patients peaked 6 weeks after the beginning of chemotherapy. Taken together, these findings show that a transient increase in plasma galectin-3 level is a pathologic change related to CIPN that is common to humans and mice treated with taxanes.
On the basis of our previous report showing taxanes increase galectin-3 expression in p75-positive dedifferentiated cells (10), we hypothesized that dedifferentiated Schwann cells residing in peripheral sensory nerves were the origin of elevated levels of plasma galectin-3 following taxane injection. To test this idea, we first quantitatively confirmed that paclitaxel or docetaxel treatment of primary cultures of mature Schwann cells induced galectin-3 upregulation. However, these mature Schwann cells were composed of a heterogeneous population consisting of a large number of differentiated cells and a few immature cells. Using a Venus expression system dependent on Schwann cell maturation, we found that paclitaxel increased galectin-3 expression in p75-positive dedifferentiated Schwann cells derived from mature Schwann cells rather than p75-positive immature Schwann cells already present in the heterogeneous population. This finding is further supported by the qPCR data showing highly expression of stem cell marker (Sox2 and Pax3) in Schwann cells. Under these conditions, galectin-3 production and secretion from Schwann cells were elevated after treatment with taxanes, but not oxaliplatin, suggesting that Schwann cells have the ability to secrete galectin-3 following dedifferentiation by taxanes. The increased expression of galectin-3 may be mediated through the NF-κB signaling pathway, as reported previously in another type of cell (25). On the other hand, our results demonstrated that upregulation of galectin-3 in Schwann cells of the mouse sciatic nerve peaked 2 to 4 weeks after the first paclitaxel injection, consistent with the elevation of plasma galectin-3 levels. In contrast, galectin-3 expression was not altered in DRG or other peripheral organs/cells expressing galectin-3, including heart, lung, liver, small intestine, colon, kidney, and PBMCs (including monocytes; refs. 12, 32, 33) even after paclitaxel injection. Additional studies are needed to identify the molecular mechanisms underlying dedifferentiation of Schwann cells by taxanes. Nonetheless, our data emphasize that the transient elevation of plasma galectin-3 in taxane-related CIPN model mice is a consequence of promoting galectin-3 release from dedifferentiated Schwann cells. At present, we cannot convincingly explain why galectin-3 upregulation in the sciatic nerve peaked 2 weeks after taxane injection and decreases thereafter, even though mechanical hypersensitivity persisted. However, our data are essentially in agreement with a previous report showing that galectin-3 expression is rapidly induced in dedifferentiated Schwann cells in the PNS during Wallerian degeneration, but subsequently disappears within a week (34). Considering in conjunction with our results (Supplementary Fig. S3), the effect of taxanes on Schwann cells may be transient and reversible.
Consistent with previous reports (4, 35), we found that paclitaxel-induced macrophage infiltration into the parenchyma of the sciatic nerve. Earlier studies documented that extracellular galectin-3 modulates immune reactions via chemotaxis of macrophages/monocytes (12, 27). We demonstrated that the murine macrophage cell line RAW 264.7 exhibited a chemotactic response toward both recombinant galectin-3 and culture supernatant collected from paclitaxel-treated Schwann cells. Generally, macrophages infiltrate the sites of nerve injury and mature into the proinflammatory phenotype in response to microenvironment signals (36–38). The inflammatory factors produced by these macrophages in the PNS facilitate pain transmission from the periphery to the central nervous system (19, 37). Although in vitro treatment with recombinant galectin-3 did not induce macrophage polarization, paclitaxel increased proinflammatory macrophage-related genes (iNos, Irf5, and Il1β) in the mouse sciatic nerve. A repeated injection of paclitaxel also increased iNOS-positive macrophages in the sciatic nerve, suggesting that infiltrated macrophages were polarized toward proinflammatory phenotype and thereafter may release IL1β in the sciatic nerve. Furthermore, perineural administration of recombinant galectin-3 to the sciatic nerve of naive mice induced infiltration of macrophages and mechanical hypersensitivity. Overall, these results strongly suggest that Schwann cell–derived galectin-3 is an important determinant of macrophage infiltration into peripheral sensory nerves after paclitaxel administration.
We further demonstrated that Galectin-3−/− mice exhibited delayed onset of paclitaxel-induced mechanical hypersensitivity relative to WT mice, accompanied by a lack of macrophage expansion in the sciatic nerve. In addition, the galectin-3 inhibitor TD-139 almost completely abolished paclitaxel-induced macrophage infiltration and mechanical hypersensitivity. Thus, our data reveal a pronociceptive role of extracellular galectin-3 in the development of taxane-related CIPN. Importantly, chemical depletion of macrophages by clodronate liposomes suppressed paclitaxel-induced mechanical hypersensitivity, despite the higher level of plasma galectin-3. Our findings do not exclude the possibility that galectin-3 acts directly on peripheral nerves during CIPN pathogenesis, but they do emphasize the central role of macrophages in the effects of galectin-3. Specifically, galectin-3 released from Schwann cells induces macrophage infiltration into the sciatic nerve, which promotes taxane-induced mechanical hypersensitivity (Fig. 7).
Recent findings have provided new insights into the sex-specific pathogenic mechanisms of neuropathic pain, revealing that the role of macrophage- or glial cell–modulated immune response in neuropathic pain is sexually dimorphic (39, 40). However, our results suggest no sex differences in the role of Schwann cell–derived galectin-3 in taxane-related CIPN pathogenesis.
On the other hand, paclitaxel-induced mechanical hypersensitivity appeared gradually in Galectin-3−/− mice, 4 to 8 weeks after multiple paclitaxel injections. Taken together with the transient upregulation of galectin-3 in the sciatic nerve and plasma, this observation suggests that galectin-3–induced macrophage infiltration is involved in the early phases of paclitaxel-induced peripheral neuropathy. It is widely accepted that one of major cause of taxane-related CIPN is pathologic changes in sensory neurons due to neurotoxicity (41). On the basis of our findings, we speculate that direct impairment of sensory neurons by taxanes makes a major contribution specifically during later stages of taxane-induced peripheral neuropathy.
In conclusion, the data presented herein suggest that transient elevation of the plasma galectin-3 level is a CIPN-related pathologic change common to humans and mice following taxane treatment. Extracellular galectin-3 derived from dedifferentiated Schwann cells in the sciatic nerve may be involved in the early phase, rather than the late phase, of taxane-induced mechanical hypersensitivity mediated by macrophage infiltration. Thus, drugs that inhibit the galectin-3 signaling axis between Schwann cells and macrophages have the potential to delay CIPN progression and prolong the duration of treatment with taxane-based chemotherapy.
M. Koyanagi reports grants from Japan Society for the Promotion of Science during the conduct of the study. S. Imai reports grants from Japan Society for the Promotion of Science and Smoking Research Foundation during the conduct of the study. No disclosures were reported by the other authors.
M. Koyanagi: Conceptualization, data curation, formal analysis, funding acquisition, investigation, visualization, methodology, writing–original draft. S. Imai: Conceptualization, data curation, funding acquisition, validation, investigation, visualization, methodology, writing–original draft, project administration, writing–review and editing. M. Matsumoto: Investigation, methodology. Y. Iguma: Resources, writing–review and editing. N. Kawaguchi-Sakita: Resources, supervision, writing–review and editing. T. Kotake: Resources, writing–review and editing. Y. Iwamitsu: Investigation. M. Ntogwa: Investigation, methodology. R. Hiraiwa: Investigation. K. Nagayasu: Resources, investigation, methodology, writing–review and editing. M. Saigo: Investigation. T. Ogihara: Investigation, methodology. A. Yonezawa: Writing–review and editing. T. Omura: Writing–review and editing. S. Nakagawa: Writing–review and editing. T. Nakagawa: Conceptualization, funding acquisition, writing–review and editing. K. Matsubara: Conceptualization, supervision, writing–review and editing.
The authors thank BORN (Breast Oncology Research Network) BioBank, and all the participants in this study. This work was supported in part by Grants-in-Aid for Scientific Research (KAKENHI) from the Japan Society for the Promotion of Science [JSPS; Grants-in-Aid for Scientific Research (C) to S. Imai (17K08445), Scientific Research (B) to T. Nakagawa (17H04008, 20H03390), Challenging Exploratory Research to T. Nakagawa (17K19722), Scientific Research on Innovative Area “Thermal Biology” to T. Nakagawa (18H04696), “Integrated Bio-metal Science” to T. Nakagawa (20H05506) and Grant-in-Aid for JSPS Fellows to M. Koyanagi (18J21240)] and by grants from Smoking Research Foundation to S. Imai.