Research ArticleExperimental Studies

Effect of Eribulin on Angiogenesis and the Expression of Endothelial Adhesion Molecules

ANDREA ABBONA, MATTEO PACCAGNELLA, SIMONETTA ASTIGIANO, STEFANIA MARTINI, NERINA DENARO, FIORELLA RUATTA, OTTAVIA BARBIERI, MARCO MERLANO and ORNELLA GARRONE
Anticancer Research June 2022, 42 (6) 2859-2867; DOI: https://doi.org/10.21873/anticanres.15767

Abstract

Background/Aim: Tumor vasculature is an important component of the tumor microenvironment and deeply affects anticancer immune response. Eribulin is a non-taxane inhibitor of the mitotic spindle. However, off-target effects interfering with the tumor vasculature have been reported. The mechanisms responsible of this effect are still unclear. Materials and Methods: We designed an in vitro study to investigate the effect of eribulin, with or without TGF-β, on neo-angiogenesis, and on the expression of the adhesion molecules ICAM-1 and VCAM-1. We also investigated the effects of paclitaxel and vinorelbine under the same experimental conditions. Results: Eribulin up-regulated the epithelial markers VE-cadherin and CD-31 in HUVEC and inhibited tube formation in HUVEC cells cultured in Matrigel. The drug effectively arrested tube formation even in the presence of TGF-β and counteracted the TGF-β-induced change in cell shape from the endothelial cobblestone-like morphology to an elongated spindle-shaped morphology. We also observed that eribulin was able to upregulate ICAM-1 and to counteract its down-regulation induced by TGF-β. Conclusion: Eribulin exerts different off-label effects: increases vascular remodeling, counteracts the endothelial-to-mesenchymal transition (EndMT) mediated by TGF-β and promotes tumor infiltration by immune cells via increasing the expression of ICAM-1 and transcription of CD31 and VE-cadherin. Moreover, eribulin was able to inhibit vasculature remodeling and the induction of EndMT mediated by TGF-β better than vinorelbine and paclitaxel. The effects observed in this study might have important therapeutic consequence if the drug is combined with immunotherapy.

Key Words:

Microtubules are important targets of cancer chemotherapy. Eribulin, as well as vinorelbine, paclitaxel, and other agents, directly target microtubules. Unlike these drugs, eribulin inhibits microtubule growth causing non-productive tubulin aggregates, without any effect on microtubule shortening. This inhibits the formation of the mitotic spindle leading to irreversible mitotic block at G2-M phase and apoptosis (1).

Eribulin is approved for the treatment of metastatic breast cancer by the European Medicines Agency (EMA) after at least one previous line of therapy, and by the Food and Drug Administration (FDA) for the treatment of metastatic breast cancer after at least two previous lines of therapy. However, the efficacy of eribulin as first-line treatment for primary breast osteosarcoma has been demonstrated in a patient derived orthotropic xenograft (PDOX) mouse model (2). Eribulin has also been used as first line chemotherapy in clinical trials for advanced or metastatic breast cancer, since it seems to suppress growth of new metastases (3, 4). Interestingly, the clinical response to eribulin treatment seemed to correlate with the persistence of high numbers of circulating lymphocytes (5, 6), suggesting the possibility that immune-mediated mechanism(s) might also play a role in the pharmacological effect of eribulin.

In addition to its main mechanism of action on microtubules, eribulin shows off-target effects, as it is able to interact with the tumor microenvironment (TME). Moreover, eribulin is able to interfere with neo-angiogenesis (7), and the expression of TGF-β and other cytokines both in experimental models and humans (8, 9).

TGF-β interferes with the tumor microenvironment leading to an immunosuppressive environment and strongly contributes to T-cell exclusion through many mechanisms such as the induction of a reactive stroma and neo-angiogenesis (10). Therefore, the modulation of TGF-β by eribulin might favor the homing of effector T cells in excluded tumors.

Many human breast cancers are immune excluded tumors, in particular triple negative breast cancer (11) and, as such, are characterized by TGF-β signature, neo-angiogenesis, and reactive stroma (10). These cancer characteristics may have contributed to the success of eribulin in this disease. The deep knowledge of the off-target effects of eribulin may support its use in combination with immune therapy.

We designed the following in vitro study to investigate the effect of eribulin on the human umbilical vein vascular endothelial cells (HUVEC) in terms of endothelial-to-mesenchymal transition (EndMT) and expression of endothelial adhesion molecules (ICAM-1 and VCAM-1).

Materials and Methods

Study design. Eribulin effect was evaluated on HUVEC with or without previous exposure to TGF-β. We also tested the effects of navelbine and paclitaxel under the same experimental conditions and compared the results with those achieved with eribulin and TGF-β alone.

Cell culture. HUVEC were purchased from Lonza (Walkersville, MD, USA; batch 0000440546 and 0000442486) and cultured in EBMTM-2 Basal Medium (Lonza) with EGM-2MV Single Quots (Lonza) in 25 cm2 gelatinized cell culture flasks at 37°C and 5% CO2. All the experiments were performed using cells at a passage number between 5 and 12.

Drugs. TGF-β, TNF-α, and IL-1β were purchased from Life Technologies Europe (Bleiswijk, the Netherlands), reconstituted, and stored according to manufacturer’s recommendations. Eribulin (Halaven®, EISAI, Tokyo, Japan), paclitaxel (Taxol® Bristol-Meyers Squibb, NY, USA) and vinorelbine (Navelbine 50® Pierre Fabre, Paris, France) were provided by the Santa Croce and Carle Hospital pharmacy and Ospedale Policlinico IRCCS S. Martino, Genova, and kept at 4°C for no longer than 7 days. Before treatments, the drugs were freshly diluted in cell medium at the chosen concentrations.

HUVEC morphology and tube formation assay on matrigel. HUVEC were seeded in 25 cm2 gelatinized cell culture flasks and treated for 24 h with 1 nM (2×IC50) (12) eribulin, or 10 ng/ml TGF-β (13), or the combination of the two. Images were acquired using inverted Leika DR IRB microscope associated to a Nikon camera. For tube formation on matrigel, 24-well-plates were coated with 200 μl of growth factor-depleted matrigel (BD, Franklin Lakes, NJ, USA) and incubated 30 min at 37°C. HUVEC were then seeded at 7×104 cells/well density in 500 μl of growth medium and monitored for network formation up to 24 h. Drugs were added during seeding, either alone or in combination, at the final concentration of 10 ng/ml TGF-β and 1 nM eribulin. Experiments were repeated four times. Images were acquired after 4 h using an Olympus stereo-microscope (Olympus Corp., Tokyo, Japan).

RNA isolation and quantitative real-time polymerase chain reaction (qRT-PCR). To measure the two epithelial markers (VE-cadherin and CD31) and the three mesenchymal markers (Vimentin, Snail and αSMA), in the first set of experiments HUVEC were seeded in 25 cm2 flasks and, at confluence, treated with either 1.0 nM eribulin, 0.8 nM paclitaxel, 10 nM vinorelbine or medium for 4 h. In the second set of experiments, cells were pre-treated with 10 ng/ml TGF-β for 4 h, subsequently cells were washed and treated with medium, eribulin, paclitaxel, or vinorelbine at the above-mentioned concentrations for 4 more h. Each experiment was performed in triplicate. Total RNA was extracted from each cell sample using RecoverAll™ Total Nucleic Acid Isolation Kit (Ambion Inc., Austin, TX, USA) according to the manufacturer’s instructions. Concentration and purity of the RNA samples were tested using NanoDrop ND 1000 (Thermo Fisher Scientific, Wilmington, DE, USA) at 260/280 nm. Total RNA sample was reverse transcribed to cDNA using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions, using Applied Biosystems Veriti 96 well Thermal Cycler 1000 (Thermo Fisher Scientific). The reaction conditions were: 10 min at 25°C, followed by 2 h at 37°C and 5 min at 85°C. The PCR assay was performed in duplicate in a total volume of 25 μl, using 12.5 μl SYBR™ green chemistry (SYBR™ green master mix, Applied Biosystems) 4 μl of c-DNA and 1.25 μl of 50 μM each primer for 40 cycles. Table I shows the genes analyzed and the primers used. mRNA fold change was calculated using the 2–ΔΔCt method (14) where GAPDH was used as the control gene.

View this table:
Table I.

Gene analyzed and primers used. Primer sequences and melting temperature (TM) of each primer used in the present study are listed.

Flow cytometry analysis. The effect of eribulin on the expression of the membrane proteins ICAM-1, and VCAM-1 was analyzed by flow cytometry. Endothelial cells were identified after staining with CD31 antibody. HUVEC were seeded overnight onto 6-well-plates coated with 0.1% gelatin, and then treated for 24 h with 1 or 10 ng/ml TGF-β, for VCAM-1 or ICAM-1 respectively, 10 ng/ml TNF-α (12), 5 nM eribulin (10×IC50), and combinations thereof. Cells were then detached by a brief incubation in 0.012% trypsin/0.1 mM EDTA and stained with the following monoclonal antibodies: PE mouse anti-human ICAM-1, PE mouse anti-human VCAM-1 or Alexa Fluor 647 mouse anti-human CD31 (all antibodies from BD). Samples were analyzed on Gallios Flow Cytometer (Beckman Coulter, Brea, CA, USA), and data were processed with FlowJo software (TreeStar, Ashland, OR, USA).

Statistical analysis. The statistical analyses were conducted using GraphPad Prism 5. Statistical significance for transcript analyses was determined by non-parametric Mann-Whitney U-test. Considering the number of comparisons planned, we adjusted for multiple testing using the Bonferroni correction. For flow cytometry analysis, statistical significance was determined by the paired Student t-test. Data are expressed as mean±SD.

To analyze the significance in molecular marker expression changes induced by the treatments, the data of the individual expression levels were collected in a ratio (E/M) calculated by the sum of the ratio of the 2 epithelial markers (VE-cadherin and CD31 in the numerator) and the sum of the ratio of the 3 mesenchymal markers (Vimentin, Snail and αSMA in the denominator). The calibrant used was the untreated HUVEC (CTR). If ΔΔCT is >0, ΔCT (CTR) > ΔCT (test) and the ratio (2–ΔΔCT) is <1, it indicates under-expression of the mRNA of interest in the treated sample, compared to CTR. On the contrary, if ΔΔCT <0, ΔCT (CTR) < ΔCT (test), and the (2–ΔΔCT) ratio is >1, it indicates increased expression of the mRNA in the treated sample compared to CTR.

Results

Eribulin affects the morphology and tube formation of HUVEC. To test whether eribulin influences EndMT and vascular remodeling, we first checked the morphological effect of the drug on HUVEC monolayers, either alone or in the presence of TGF-β, which is known to induce epithelial-mesenchymal transition (EMT) and EndMT (15).

Eribulin was administered for 24 h at the concentration of 1 nM. TGF-β was given at 10 ng/ml, according to the literature. TGF-β induced a change in cell shape from the endothelial cobblestone-like morphology to an elongated spindle-shaped morphology that is characteristic of EndMT. Eribulin did not induce HUVEC EndMT but effectively counteracted the effect of TGF-β (Figure 1A).

Figure 1.
Figure 1.

Morphogenesis of HUVEC incubated with eribulin and/or TGF-β. (A) Morphology was studied on HUVEC incubated in gelatinized flasks for 24 h with medium (a), 10 ng/ml TGF-β (b), 1 nM eribulin (c), or TGF-β eribulin (d). Images were obtained at ×500 magnification. (B) Tube formation was studied on HUVEC incubated on matrigel-coated plates with medium (a), 10 ng/ml TGF-β (b), 5 nM eribulin (c), TGF-β eribulin (d). Images were obtained at ×750 magnification, 4 h after seeding.

Eribulin has been reported to alter the tumor microenvironment also through vascular remodeling (7). Thus, we tested its influence on the ability of HUVEC to organize a reticulum after seeding on matrigel (16). In cells incubated with medium or TGF-β, a well-organized tubule network was detected 4 h after seeding (Figure 1B, a and b). On the contrary, in cells treated with eribulin, the reticulum was not completely organized, displaying shorter and disjoined ramifications (Figure 1B, a and c). The same phenotype was detected when eribulin was added together with TGF-β Figure 1B, c and d), indicating that also under these conditions, eribulin was able to overcome the TGF-β effect.

Eribulin modulates the expression of EndMT markers in HUVEC. To better elucidate the effect of eribulin on EndMT and vascular remodeling, we evaluated the variation of molecular markers involved in the EndMT, namely Vimentin, Snail, αSMA, CD31, and VE-cadherin (17) by qRT-PCR.

Moreover, to examine whether the effect of eribulin on EndMT was similar to that of other inhibitors of the mitotic spindle, we tested the effect of vinorelbine (10 nM) and paclitaxel (0.8 nM) on the same molecular markers (18, 19). Finally, TGF-β was also tested. Eribulin did not affect the transcription of the mesenchymal markers compared to CTR, while vinorelbine up-regulated Snail and paclitaxel down-regulated the transcription of both Vimentine and Snail. The transcription of CD31 and VE-cadherin was significantly enhanced by all the drugs. However, eribulin had the strongest effect on the increase of VE-cadherin mRNA, while vinorelbine effect was highest on the increase of CD31 (Figure 2A).

Figure 2.
Figure 2.

Analysis of the transcription of mesenchymal and epithelial molecular markers in HUVEC treated with drugs targeting the microtubule system. (A) Transcripts were measured by qRT-PCR analysis in HUVEC after 4 h of treatment with either 1.0 nM eribulin, 0.8 nM paclitaxel, 10 nM vinorelbine or medium (CTR). (B) qRT-PCR analysis was performed after 4 h of cell pre-incubation with 10 ng/ml TGF-β and 4 more h of drug treatment. Bars represent data obtained from the mean of four independent experiments (±1 SD); samples were normalized to CTR represented as the dashed line at 1. (**p<0.01, *p<0.05).

TGF-β has been shown to increase the transcription of the three mesenchymal markers and decrease the transcription of the two epithelial markers (20). We assessed how the three drugs affected TGF-β modulation of the EndMT markers by checking the effect on HUVEC pre-incubated with TGF-β. All treatments showed a significant down-regulation of Vimentin mRNA compared to CTR. The transcription of αSMA was significantly increased by vinorelbine, decreased by paclitaxel, and unaffected by eribulin. The expression of Snail was significantly down-regulated by eribulin (Figure 2B).

CD31 mRNA was significantly enhanced by all drugs, in particular by eribulin and vinorelbine, and to a lesser extent by paclitaxel. Eribulin and paclitaxel significantly increased the expression of VE-cadherin (Figure 2B).

To evaluate the overall effect of the drugs on both epithelial and mesenchymal markers, we calculated the ratio between the expression of epithelial and mesenchymal markers (E/M). Compared to CTR, a higher E/M ratio was found for each of the three drugs (Figure 3A). However, compared to paclitaxel and vinorelbine, eribulin counteracted better the TGF-β-induced EndMT, since the E/M ratio on TGF-β pre-treated HUVEC was significantly higher compared to the ratio of the other two drugs (Figure 3B).

Figure 3.
Figure 3.

Analysis of the ratio between epithelial and mesenchymal markers. The ratio was calculated between the sum of the ratio of the epithelial markers and the sum of the ratio of the mesenchymal markers (E/M). (A) Expression analysis after 4 h of drug treatment. (B) Expression analysis after 4 h of pre-treatment with TGF-β and 4 more h of drug treatment. On the y axis the ratio between the expression values of the endothelial genes and the expression values of the mesenchymal genes is indicated. (***p<0.001, **p<0.01, *p<0.05).

Eribulin increases ICAM-1 expression on HUVEC. Finally, we evaluated the effect of eribulin on the expression of ICAM-1 and VCAM-1 adhesion molecules, involved in the process of leukocyte adhesion, rolling, and extravasation (21). The inducibility of these markers was preliminarily checked by incubating the HUVEC with TNF-α, which greatly increased the expression of both ICAM-1 and VCAM-1 (Figure 4). In CTR, VCAM-1 was undetectable, while ICAM-1 was expressed at low levels (Figure 5). Adding eribulin to CTR significantly increased the expression of ICAM-1 without any effect on VCAM-1. TGF-β is known to down-regulate ICAM-1 and VCAM-1 (22, 23). We also analyzed the effect of eribulin on ICAM-1 and VCAM-1 expression modulated by TGF-β. Eribulin was able to counteract the down-regulation of ICAM-1 induced by TGF-β.

Figure 4.
Figure 4.

Flow cytometry evaluation of the inducibility of ICAM-1 and VCAM-1 expression in HUVEC. Cells were incubated for 24 h with medium (CTR) or 10 ng/ml TNF-β. Mean ±1 SD of three independent experiments.

Figure 5.
Figure 5.

Flow cytometry analysis of ICAM-1 and VCAM-1 expression in HUVEC treated with eribulin and/or TGF-β. Cells were incubated for 24 h with medium (CTR), 1 (VCAM-1) or 10 (ICAM-1) ng/ml TGF-β and/or 5 nM eribulin. Mean ±1 SD of three independent experiments. *p<0.05.

Discussion

In our study, eribulin exerted an anti-angiogenic activity on human endothelial cells. A similar effect was previously observed by Agoulnik et al. (12). In particular, we found that eribulin displays a dual antiangiogenic effect: i) it prevents the organization of the vascular reticulum during the first step of morphogenesis, and ii) it is also able to counteract the EndMT effect mediated by TGF-β.

Eribulin, exerts its therapeutic effect primarily by targeting the microtubule cytoskeleton; however, off-target effects have been reported and could have important implications in its therapeutic activity. We herein demonstrated that eribulin can affect the endothelial cell organization by interfering with the morphogenesis process. At the molecular level, the drug did not impact the transcription of any of the mesenchymal markers. However, it increased the transcription of the epithelial markers CD31 and VE-cadherin in both untreated and TGF-β-treated cells. Both CD31 and VE-cadherin are instrumental in endothelial cell organization and function by reshaping and strengthening the endothelial lining and by controlling vascular permeability (24). Our results provide the molecular basis to defining that eribulin treatment induces vascular remodeling associated with improved perfusion in breast cancer xenograft models (7).

CD31 and VE-cadherin are also instrumental in controlling leukocyte extravasation (24, 25). In particular, CD31 plays a fundamental role in the extravasation not only of myeloid and natural killer (NK) cells but of T-cells as well (26). Eribulin treatment also significantly increased ICAM-1 membrane expression in CTR and showed a clear trend toward the down-regulation of the molecules induced by TGF-β. ICAM-1 is down-regulated in tumor-derived endothelial cells (27), and since ICAM-1 is involved in leukocyte adhesion and endothelial cell extravasation (28), this phenomenon may also contribute to a reduction in the recruitment of T and NK cells into the tumors. The findings mentioned above supported the hypothesis that eribulin may in part exert its pharmacological effect by promoting tumor infiltration by immune effector cells through the induction of the expression of molecules involved in leukocyte extravasation. Indeed, in a xenograft tumor model, eribulin treatment was found to increase the number of tumor-infiltrating immune cells, and NK cell depletion reduced the antitumor effects of eribulin (29). These effects of eribulin on immune cell recruitment at the tumor site is important, since many of the human solid tumors, including breast cancer, show an excluded immunophenotype (9, 30).

We gathered evidence that eribulin may trigger another therapeutic mechanism. The induction of EMT by TGF-β is well known in literature, and several studies have indicated increased TGF-β signaling as a key effector of EMT in cancer progression and metastasis (31, 32). However, TGF-β has been found to play a central role also in inducing EndMT (33, 34), not surprisingly since EMT and EndMT are supported by almost identical mechanisms. TGF-β induces EndMT via Smad signaling, which results, among other effects, in increased transcription of Snail, αSMA, and Vimentin, and down-regulation of VE-cadherin and CD31 (35). Eribulin significantly inhibited the TGF-β mediated effect on the transcription of Snail, Vimentin, VE-cadherin, and CD31, suggesting that part of the pharmacological effect of the drug is the result of its ability to inhibit the induction of EndMT by TGF-β. Interestingly, we had previously observed that response to eribulin in breast cancer patients correlated to a reduction in circulating TGF-β (8).

On the basis of our results, we suggest that eribulin exerts its pharmacological effect also via three off target mechanisms: i) increases transcription of CD31 and VE-cadherin, which results in vascular remodeling, ii) promotes tumor infiltration by immune cells by increasing transcription of CD31 and VE-cadherin, and expression of ICAM-1, and iii) counteracts the EndMT mediated by TGF-β. These conclusions might indicate new therapeutic approaches. For instance, we could speculate that eribulin might achieve the best therapeutic effect in tumors displaying an immune excluded phenotype.

Vinorelbine and paclitaxel are known to exert antiangiogenic activity on HUVEC (18); however, we found that the transcription patterns are different between them and compared to eribulin. Vinorelbine up-regulated the transcription of Snail, CD31, and VE-cadherin in CTR, and of αSMA, Vimentin and CD31 in HUVEC pre-treated with TGF-β.

Paclitaxel reduced the transcription of Vimentin and Snail in CTR and of Vimentin and αSMA in TGF-β-treated cells but increased the transcription of the epithelial markers in both treated and untreated HUVEC. These findings suggest that each of the three drugs might affect endothelial cell function through distinct mechanisms. Indeed, treatment with eribulin showed a better effect than paclitaxel in a PDOX mouse model from a patient with triple-negative breast carcinoma (36). The individual gene expression values were separated in two groups and added; the first group was the sum of the expression values of the endothelial genes while the second was the sum of the expression values of the mesenchymal genes. A ratio between endothelial and mesenchymal characteristics (E/M) was calculated. Eribulin showed the same ratio as the other two drugs in the CTR samples, but a higher ratio in the samples treated with TGF.

This finding strengthens our hypothesis on the role of eribulin in EndMT, confirming that it counteracts the TGF-β-dependent polarization of the endothelial cells towards the mesenchymal phenotype. TGF-β is highly expressed in many solid tumors and in particular in excluded tumors, which are characterized by a TGF-β signature (10). Many human breast cancers show an excluded immunophenotype (11) and interestingly, we have observed that the response of breast cancer to eribulin correlates to a reduction in circulating TGF-β (8).

Therefore, considering the off-target effects of eribulin, this drug might favor the immune checkpoint inhibitor activity in excluded breast cancer, and possibly in other excluded solid tumors, by removing many mechanisms of T-cell exclusion.

Acknowledgements

This study was funded by the ARCO Foundation.

Footnotes

  • Authors’ Contributions

    A.A., M.P., S.A. and S.M. planned and carried out the experiments. M.P. performed also data analysis. N.D. and F.R. contributed to the drafting of the manuscript. O.B., M.C.M and O.G. planned and supervised the work and wrote the manuscript.

  • Conflicts of Interest

    The Authors declare no conflicts of interest in relation to this study.

  • Received March 18, 2022.
  • Revision received April 9, 2022.
  • Accepted April 11, 2022.

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Effect of Eribulin on Angiogenesis and the Expression of Endothelial Adhesion Molecules
ANDREA ABBONA, MATTEO PACCAGNELLA, SIMONETTA ASTIGIANO, STEFANIA MARTINI, NERINA DENARO, FIORELLA RUATTA, OTTAVIA BARBIERI, MARCO MERLANO, ORNELLA GARRONE
Anticancer Research Jun 2022, 42 (6) 2859-2867; DOI: 10.21873/anticanres.15767
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