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. 2024 Nov 5;51:102168. doi: 10.1016/j.tranon.2024.102168

SPL-108 mitigates metastasis and chemoresistance in tubo-ovarian carcinoma

Olivia D Lara a, Elke Van Oudenhove b, Luiza Pereira b, Selim Misirlioglu b, Douglas A Levine c, Kari E Hacker b,⁎
PMCID: PMC11577183  PMID: 39504709

Highlights

  • •

    Unveiling new treatment strategies in tubo-ovarian carcinoma (TOC) remains a high unmet need.

  • •

    The small molecular inhibitor, SPL-108, has anti-metastatic properties and may play a role in reversing chemoresistance in preclinical models of TOCs through MDR1 inhibition.

  • •

    SPL-108 warrants further clinical development in combination with cytotoxic agents that are MDR1 substrates.

Keywords: Ovarian cancer, Chemoresistance, Metastasis, Therapeutic approaches, Targeted therapies

Abstract

Background

Overcoming the heterogeneous mechanisms of metastasis and chemoresistance will improve outcomes for women with tubo-ovarian carcinomas (TOCs). CD44 expression has been shown to be associated with poor prognosis and advanced disease in TOCs. In addition, studies have shown a link between chemoresistance and CD44 pathways. Given the therapeutic implications of targeting CD44, this manuscript examines the biologic effects of a novel CD44 modulator, SPL-108, in TOCs.

Materials and Methods

We assessed the effects of SPL-108 on chemosensitivity and migration in a panel of ovarian cancer cell lines with varied CD44 and MDR1 expression. In vitro experiments (cell viability assay, Western blot analysis, Calcein AM fluorescence assay, and migration assay) were carried out to determine the functional effects of SPL-108 in TOCs.

Findings

Ovarian cancer cell lines OVCAR5 and OVCAR8 expressed higher protein levels of CD44 as demonstrated through Western Blot analysis. SPL-108 treatment significantly decreased the number of migrating cells in OVCAR8, OVCAR5 and OVCAR3 cell lines and migratory response was independent of CD44 expression. Treatment with SPL-108 led to significant accumulation of the MDR1 substrate Calcein in OVCAR5, OVCAR8 and OVCAR3 cells lines compared to verapamil treated positive control cells. Retention of Calcein after SPL-108 treatment was seen in cell lines with high MDR1 protein expression and no Calcein retention was seen in cells lacking MDR1 expression, suggesting SPL-108 inhibits MDR1.

Conclusions

SPL-108 treatment has anti-metastatic properties and may play a role in chemoresistance in preclinical models of TOCs independent of CD44 expression. Ongoing in vitro and in vivo studies will help guide further clinical development of SPL-108.

Introduction

Despite practice-changing advances in gynecologic cancer treatment, the number of deaths from tubo-ovarian carcinomas (TOCs) remains considerable [1]. While the combination of cytoreductive surgery and platinum-doublet chemotherapy in the treatment of ovarian cancer (OC) results in remission for ∼80% of patients, the majority of patients will experience recurrence and eventually die of disease [2]. Breakthroughs in the understanding of ovarian cancer molecular biology and tumorigenesis have led to the development of targeted agents including monoclonal antibodies, small molecule inhibitors, and peptides [[3], [4], [5]]. Despite these new treatments, there remains an unmet need for novel and effective treatment options for patients with recurrent and advanced-stage TOC.

CD44, a cell surface adhesion receptor, is expressed in most tubo-ovarian carcinomas [6,7]. Several publications demonstrate the association of CD44 with poor prognosis and advanced disease [[8], [9], [10]]. In addition, preclinical studies have demonstrated a link between chemoresistance and CD44 pathways, including P-gp mediated efflux of chemotherapeutic agents [11,12]. CD44 has been shown to inhibit P-gp degradation, thus increasing drug resistance in vitro [13,14].

Members of the ABC family, such as P-gp (ABCB1 or MDR), can induce drug resistance to multiple chemotherapeutic drugs including paclitaxel, vinblastine and doxorubicin [15,16]. Increased P-gp expression is observed in 30% of ovarian tumors and is associated with poor outcomes in ovarian, breast and prostate cancers [17,18]. Promoter fusions involving ABCB1 and SLC25A40 drive drug resistance through increased P-gp expression [19]. Additionally, ABCB1 fusion events correlate with increased numbers of prior therapeutic lines and type of chemotherapy.

Improved understanding of tumor adaptation and chemotherapy resistance mechanisms is critical. Clearly defining resistance pathways will allow for the development of more effective treatment strategies. In the present study, we tested the anticancer effects of Splash-108 (SPL-108), an 8- amino acid peptide [acetyl-KPSSPPEE-amino] with sequence homology within the hyaluronic acid binding domain of CD44, in preclinical models of ovarian carcinoma [20,21]. This region of CD44 is critical to conformational integrity; thus binding of SPL-108 to CD44 impacts a structurally and functionally important region [20]. We hypothesize that SPL-108 prevents binding of CD44 to hyaluronic acid and impairs downstream cancer signaling pathways. SLP-108 has demonstrated a very favorable side effect profile in early phase clinical trials and has been tested in persistent or recurrent epithelial ovarian, fallopian tube or primary peritoneal carcinoma. [22]

Methods

Cell line maintenance

Patient derived cell line AOCS 18.5 (containing a SLC25A40-ABCB1 fusion resulting in overexpression of P-gp; gifted from Dr. David Bowtell, Australian Ovarian Cancer Study) and ovarian cancer cell lines OVCAR5, OVCAR8, CAOV3, OVCAR4, and OVCAR3 were maintained in RPMI 1640 medium (GE Life Sciences) supplemented with 10% FBS (#35–010-CV, Corning Inc), penicillin and streptomycin solution (#30,001,137, Corning Inc) [19]. All cells were cultured at 37 °C with 5% CO2. Mycoplasma testing of all cell lines was performed using an ATCC Universal Mycoplasma Detection Kit. All in vitro experiments were conducted with 80% confluent cultures and low passage of cells.

Cell viability assays

Cell viability assays were performed by testing cells’ ability to reduce a resazurin-based solution (PrestoBlue Cell Viability Reagent; ThermoFisher, A13261). Ovarian cancer cell lines were treated for 24 h with 10 µm SPL-108 (Splash Pharmaceuticals, San Diego California; Lot #AN60402). These cells were then harvested and seeded in a 96-well plate. Twenty-four hours later, SPL-108 pre-treated cells were exposed to varying concentrations of paclitaxel for 4 h. Media was then exchanged to drug-free media and cells were incubated for 72 h. All cells were treated with 10% PrestoBlue for 2 h at 37 °C. The excitation/emission at 560/590 was determined and percent viability was calculated relative to a 0.01% DMSO treated control.

Immunoblotting

Cells were harvested and lysed with RIPA buffer (1% Triton X-100, 25 mM Tris, 150 mM NaCl, 0.1% SDS, 0.5% sodium deoxycholate) supplemented with fresh protease and phosphatase inhibitors (5872S, Cell Signaling Technology). Protein quantification was performed using a BCA Protein Assay Kit (#23,225; Thermo Fisher Scientific) following the manufacturer's protocol. Thirty micrograms of cell lysate protein was loaded onto SDS-PAGE gels. After separation, proteins were transferred to nitrocellulose membranes and blocked with 5% nonfat dry milk (#1,706,404, Bio-Rad Laboratories) in TBS-T (0.1% Tween-20) for 1 hour at room temperature. After blocking, indicated antibodies were diluted in 5% milk in TBS-T and placed on membranes overnight at 4 °C. The membranes were then washed three times with TBS-T for 10 min with light agitation. Afterward, a species-specific IRDye conjugated secondary antibody (Licor) was placed on membranes for 2 h at room temperature. The membranes were washed three times in TBS-T and finally developed on the Odyssey Clx Imaging system. The primary antibody dilutions were as follows: anti-MDR1, 1:1000 (#12,683; Cell Signaling Technology); anti-CD44, 1:100 (#37,259; Cell Signaling Technology), anti-β-actin, 1:10,000 (sc-69,879; Santa Cruz Biotechnology).

Migration assay

Migration assays were performed using a Transwell system (8-μm pore size; Corning Inc.). Cells were treated with 10uM SPL-108 or DMSO for 24 h and 3 × 105 cells were seeded onto the apical side of a Transwell chamber (six-well insert) in serum-deprived culture media. The basal compartment was supplemented with 10% FBS to serve as a chemoattractant. For the SPL-108 treated groups, 10μM of the drug was added to both top and bottom chambers. The cells were allowed to migrate overnight for 24 h. Cells were fixed with ice-cold methanol for 10 min at 4 °C and stained with 0.5% crystal violet for 30 min at room temperature. The cells that remained on the apical side of the chamber were gently scraped off with cotton swabs. The migrating cells (cells adherent to bottom chamber) were quantified in 4 microscopic fields at a magnification of 20X.

Calcein AM MDR assay

The Vybrant™ multi-drug resistance assay (Invitrogen) utilizes Calcein AM as a substrate for P-gp activity by measuring intracellular Calcein fluorescence. Ovarian cancer cell lines were treated with SPL-108 or verapamil (positive control for MDR inhibition) and cultured in 96-well plates for 24 h. Cells were incubated in Calcein AM for 30 min, washed and centrifuged twice with cold PBS. Cell fluorescence was measured at a wavelength of 490 nm on a microplate spectrofluorometer. All experiments were performed in triplicate.

Immunofluorescence

Cells were plated on a coverslip and allowed to adhere overnight. They were fixed with 10% formalin acetate for 15 min at room temperature and permeabilized with 0.3% Triton-X in 1% bovine serum albumin (BSA) for 1 hour at room temperature. Following cell fixation, cells were incubated overnight at 4 °C with anti-CD44 (#3570S; Cell signaling), washed with PBS, and stained with a donkey anti-mouse IgG conjugated to Alexa Fluor 488 (A-21,202; ThermoFisher). Nuclei were counterstained using mounting media containing DAPI (AB104139; Abcam). Antibodies dilutions were 1:400 for the primary and 1:2000 for the secondary, both in 0.3% Triton-X in 1% BSA. Images were acquired in 20X magnification using an EVOS FL Cell Imaging System microscope (ThermoFisher).

Statistical analysis

Student t-test (for comparison of two groups) and ANOVA (for comparison of all groups) were used to calculate P values for normally distributed data. All statistical data were analyzed using the Prism software program (GraphPad Software). Two-tailed P values less than 0.05 were considered significant. All statistical tests were two-sided.

Results

Characteristics of ovarian cancer cell lines

We measured CD44 expression in a panel of six ovarian cancer cell lines and compared it with that in CD44 expressing HeLa cells (positive control) [23]. OVCAR5 and OVCAR8 ovarian cancer cell lines expressed high levels of CD44 whereas expression was undetectable in OVCAR4, OVCAR3, CAOV3 and AOCS18.5 cells (Fig. 1A). Western blot findings were confirmed with immunofluorescence (IF) staining for CD44 (Fig. 1B). Expression of MDR1 was evaluated in the same ovarian cancer cell lines and compared to expression in the HepG2 positive control. Varied levels of MDR1 expression were seen among ovarian cancer cells with higher expression noted in OVCAR5 and OVCAR8 (Fig. 1C). MDR1 expression trends did not correlate with levels of CD44 expression in this panel of OC cell lines.

Fig. 1.

Fig 1:

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Characteristics of ovarian cancer cell lines (A) Western blot analysis of CD44 expression in a panel of ovarian cancer cell lines compared with HeLa cells (positive control). The adjoining graph quantifies their expression normalized to HeLa. (B) Representative immunofluorescence (IF) staining for CD44 (green) and DAPI (blue). (C) Western blot analysis of MDR1 expression in a panel of ovarian cancer cell lines compared with HepG2 (positive control). The adjoining graph shows their expression normalized to HepG2.

Effects of SPL-108 on ovarian cancer MDR1 activity and migration

We analyzed the effect of SPL-108 treatment on MDR1 (P-gp) protein pump activity via a Calcein-AM assay. Calcein-AM is a substrate of MDR1. Decreased MDR1 activity leads to intracellular accumulation of fluorescent Calcein-AM and increased intracellular fluorescence. Effects of SPL-108 were compared to Verapamil, a specific first-generation MDR1 inhibitor [24]. First, we performed a Calcein-AM assay with the highest MDR1 expressing cell line OVCAR5 at increasing doses of SPL-108 or Verapamil at varied time points (Supplemental Figure 1). We found intracellular accumulation of Calcein-AM at low 1 µM dose of both agents and peak accumulation of Calcein-AM at 10 µM dosing after 24 h of treatment (Fig. 2A). Therefore, we treated all cell lines with 10 µM of SPL-108 and evaluated Calcein-AM retention 24 h after treatment. Intracellular accumulation of Calcein-AM was highest in OVCAR5, OVCAR8 and OVCAR3 (SPL-108 1.9, 1.7 and 1.2-fold change respectively compared to control (DMSO); P < 0.01). No differences in retention were seen in the low-MDR1 expressing cell lines OVCAR4, CAOV3 and AOCS18.5 (Fig. 2B). We then performed western blot analysis to determine the impact of 10 µM SPL-108 treatment on MDR1 expression. We saw no significant effects on overall expression of MDR1 at varied time intervals (Supplemental Figure 2). These results suggest that SPL-108 is able to inhibit MDR activity in P-gp expressing OC cell lines but does not significantly affect P-gp expression levels.

Fig. 2.

Fig 2:

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SPL-108 leads to calcein-AM accumulation in MDR ovarian cancer cell lines (A) Representative fluorescence microscopy images of calcein-AM fluorescence in OVCAR5 cell line after 24 h treatment with SPL-108. Adjoining graph shows quantification of intracellular calcein-AM at increasing doses of SPL-108. (B). Calcein-AM fluorescence intensity determined 24 h after treatment with DMSO (untreated), 10 µM SPL-108 or verapamil. Data represent mean fold change of triplicate experiments. Error bars, SEM. All statistical tests were two-sided. NS = not significant.

To assess SPL-108′s antimigratory effects, we performed a migration assay in high CD44 protein expressing (OVCAR5, OVCAR8) and low CD44 protein expressing (OVCAR3) cell lines. Following 24 h of treatment with 10 µm SPL-108, all cancer cell lines demonstrated decreased number of migratory cells compared to DMSO treated control cells. The greatest effect was seen in the non-CD44 expressing cell line OVCAR3 (mean, 50 migratory cells vs 111 migratory cells, P < 0.01). High CD44 expressing cell lines OVCAR 5 (mean, 134 migratory cells vs, 177 migratory cells, P < 0.05) and OVCAR 8 (mean, 88 migratory cells vs 149 migratory cells, P < 0.05) were also noted to have a significant decrease in the number of migratory cells (Fig. 3A).

Fig. 3.

Fig 3:

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Effect of SPL-108 on migration in ovarian cancer cell lines. (A) Results of migration assay performed at 24 h after treatment with 10 µM SPL-108 compared to DMSO treated (control) cells. The adjoining graph shows corresponding mean number of migratory cells 24 h after treatment. Error bars, SEM. All statistical tests were two-sided.

Effect of SPL-108 on chemotherapy sensitivity in ovarian cancer cell lines

A number of solid tumors have been treated with SPL-108 with no cytotoxicity noted [21,25,26]. Paclitaxel is cytotoxic chemotherapy used in the primary treatment of TOC and is an MDR1 substrate. After determining that SPL-108 treatment decreases MDR1 substrate export in cells expressing higher levels of P-gp, we hypothesized that SPL-108 may increase sensitivity to MDR1 substrates. Therefore, we performed cell viability assays to assess the effect of SPL-108 on ovarian cancer cell lines alone and in combination with Paclitaxel. Patient derived cell line AOCS 18.5, which contains the ABCB1-SLC25A40 fusion, was the most resistant line to single agent paclitaxel therapy compared to OVCAR5, OVCAR8, OVCAR3, OVCAR4 and CAOV3 (Fig. 4A). No cell viability effects were noted following treatment with single agent SPL-108, consistent with prior publications (Fig. 4B) [21,25]. All ovarian cancer cell lines were pre-treated for 24 h with 10 µM of SPL-108. Cells were then treated with increasing doses of Paclitaxel for four hours. After 4 h, media was exchanged and cell viability was analyzed 72 h after paclitaxel treatment to allow for maximum effect. The addition of SPL-108 led to a non-significant trend towards sensitizing the fusion positive cell line AOCS 18.5 to paclitaxel, but had no effect on the other ovarian cancer cell lines (Fig. 4C).

Fig. 4.

Fig 4

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Effect of SPL-108 on cell viability in ovarian cancer cell lines. (A) Cell viability 72 h after treatment with paclitaxel or (B) 24 h after treatment with 10 µM SPL-108. (C) Ovarian cancer cell viability after treatment with 10 µM SPL-108 for 24 h followed by paclitaxel treatment for 72 h. (D) Associated IC50 (nM). Error bars, SEM. All statistical tests were two-sided.

Discussion

Our study sought to investigate the effects of SPL-108 in ovarian cancer cell lines and the ABCB1-SLC25A40 fusion positive patient-derived cell line AOCS 18.5. We found that SPL-108 treatment of ovarian cancer cell lines results in decreased migration and impacted the functionality of P-gp drug efflux. We noted antimigratory effects in CD44 expressing and non-expressing cell lines. We also demonstrated that the effect of SPL-108 on efflux of the Calcein substrate is correlated with the expression of MDR1 and CD44. SPL-108 may also increase sensitivity to Paclitaxel, an MDR1 substrate, in specific TOC ABCB1-SLC25A40 fusion positive patient-derived cell lines.

Our findings that SPL-108 effects on migration is independent of CD44 expression contradicts prior research which highlighted the antimigratory activity of SPL-108 (A6 peptide) in a CD44-dependent manner in a subset of ovarian, breast, lung and prostate cancer cell lines [21,[25], [26], [27]]. There are two potential explanations for these discrepant results. We used anti-CD44 clone E2K2Y to detect CD44 expression in ovarian cancer lines. Prior studies have that demonstrated CD44 expression varies based on the anti-CD44 antibody epitope used. Specifically, the binding of anti-CD44 antibodies DF1485, B-F24, F-4 and IM7 showed varied expression of CD44 among OVCAR3 [21]. Additionally, the significance of CD44 expression in epithelial ovarian carcinoma has remained a controversial topic [6]. Some studies have shown patients with CD44 positive tumors have significantly shorter progression free survival than CD44 negative tumors [8]. Alternatively other studies have found no association between CD44 with ovarian cancer metastasis or survival [28,29]. The authors have hypothesized that different results between studies may be attributed to technical factors including the use of differing antibodies or variations in technique [6,29]. Thus, it's possible that detection of CD44 in this study is limited by these technical factors and “low expressing” cell lines express different isoforms of CD44 that were unable to be evaluated by the anti-CD44 antibody used in this study. The second explanation for our conflicting results involves the duration of SPL-108 exposure. Our series of experiments were carried out after 24 h of SPL-108 treatment, where greatest anti-migratory effect was seen, compared to 30 min of treatment in prior studies [21]. Given this discrepant data, it is possible that prolonged treatment with SPL-108 effects cell adhesion via a CD44 independent mechanism.

The high prevalence of eventual chemoresistance in ovarian cancers represents an important therapeutic opportunity using SPL-108. Increased expression of MDR1 is a well described resistance mechanism to ovarian cancer chemotherapeutics including doxorubicin, paclitaxel and PARP (poly (ADP-ribose) polymerase) inhibitors (PARPis) [30,31]. Preclinical models of brain and breast carcinoma have suggested increased expression of MDR transporters in PARP resistant cell lines [32,33]. The use of SPL-108 in combination with PARPi requires further exploration. In addition, a growing body of evidence suggests that ovarian cancer stem cells survive standard chemotherapy regimens which give rise to chemoresistant recurrent tumors [[34], [35], [36]]. In addition to mediating tumor growth and migration, CD44 has been used as a marker of ovarian cancer stem cells [37,38]. Further investigation of the impact of SPL-108 on ovarian cancer stemness may yield innovative therapeutic strategies.

The safety and efficacy of SPL-108 has been demonstrated in a number of phase I clinical trials [39,40]. A phase II trial conducted in patients with persistent or recurrent ovarian, fallopian tube or primary peritoneal carcinoma to evaluate the efficacy of A6 peptide confirmed minimal toxicity however revealed minimal clinical efficacy as monotherapy [22]. More recently the effect of SPL-108 with weekly paclitaxel in patients with platinum resistant CD44 positive ovarian, primary peritoneal or fallopian tube cancer was examined [41]. Fourteen patients with strong or moderate CD44 expression based on immunohistochemistry were treated with this combination and there was an overall response rate of 36%. Seventy-two percent of patients demonstrated a clinical benefit [41]. Patients who had progression of disease had tumors with p53 loss of function mutations. The authors hypothesize that a lack of p53 may lead to CD44 overexpression offsetting the effect of SPL-108 [41]. Given these findings and our own work, further investigation is needed to evaluate the efficacy of SPL-108 in p53 wild-type CD44 positive and negative tumors.

Limitations to our study include restricting our investigations to ovarian cancer cell lines and only exploring SPL-108 efficacy in one patient derived cell line. Our findings suggest that SPL-108 is efficacious regardless of CD44 expression. Expanding to additional patient derived cell lines would allow for further exploration to assess biomarkers of response. Current studies are underway to assess SPL-108 treatment in vivo models as well.

In this manuscript, we report that SPL-108 has promising anti-migratory and MDR1 inhibition effects in preclinical models of ovarian cancer. While the anti-migratory effects of SPL-108 were independent of CD44 expression, the accumulation of the MDR1 substrate Calcein in ovarian cancer cell lines was correlated with high MDR1 and high CD44 expression. In vitro and in vivo studies are needed to further evaluate the mechanisms through which SPL-108 mediates its effects on ovarian cancer and to develop rational drug combinations in this patient population with a high unmet need.

Financial support

This work is supported in part by US Department of Defense Award: W81XWH-15–1–0429, NIH:P30 CA016087, The V Foundation for Cancer Research, The Honorable Tina Brozman Foundation for Ovarian Cancer Research, and Arnold Chavkin and Laura Chang.

CRediT authorship contribution statement

Olivia D. Lara: Writing – original draft, Methodology, Investigation, Data curation, Conceptualization. Elke Van Oudenhove: Data curation, Conceptualization. Luiza Pereira: Data curation. Selim Misirlioglu: Data curation. Douglas A. Levine: Writing – review & editing, Project administration, Methodology, Data curation, Conceptualization. Kari E. Hacker: Writing – review & editing, Formal analysis, Data curation, Conceptualization.

Declaration of competing interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: D.A.L had consulting/advisory role for Tesaro/GSK, Merck, received research funding to institution from Merck, Tesaro, Clovis Oncology, Regeneron, Agenus, Takeda, Immunogen, VBL Therapeutics, Genentech, Celsion, Ambry, Splash Pharmaceuticals. He also is a founder of Nirova BioSense, Inc. and Resident Diagnostics, Inc. D.A.L. is currently a full-time employee of Merck & Co., Inc. K.E.H reports that her spouse receives a salary from Strata Oncology.

Acknowledgments

Cell viability assays were performed on Multi-Mode Microplate reader through the Small Instrument Fleet instrumentation core facility at NYU Langone Hospital. We thank Splash Pharmaceuticals, Inc for providing SPL-108 and David Bowtell (Peter MacCallum Cancer Center, Victoria Australia) for the gift of ABCB1-SLC25A40 cells.

Footnotes

Supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.tranon.2024.102168.

Appendix. Supplementary materials

mmc1.docx (505.3KB, docx)

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