Treatment of ovarian cancer by targeting the tumor stem cell-associated carbohydrate antigen, Sialyl-Thomsen-nouveau

Abstract

Recurrent ovarian cancer (OvCa) is thought to result in part from the inability to eliminate rare quiescent cancer stem cells (CSCs) that survive cytotoxic chemotherapy and drive tumor resurgence. The Sialyl-Thomsen-nouveau antigen (STn) is a carbohydrate moiety present on protein markers of CSCs in pancreatic, colon, and gastric malignancies. We have demonstrated that human OvCa cell lines contain varying levels of cells that independently express either STn or the ovarian CSC marker CD133. Here we determine co-expression of STn and CD133 in a subset of human OvCa cell lines. Analyses of colony and sphere forming capacity and of response to standard-of-care cytotoxic therapy suggest a subset of OvCa STn⁺ cells display some CSC features. The effect of the anti-STn antibody-drug conjugates (ADCs) S3F-CL-MMAE and 2G12-2B2-CL-MMAE on OvCa cell viability in vitro and in vivo was also assessed. Treatment with S3F-CL-MMAE reduced the viability of two of three OvCa cell lines in vitro and exposure to either S3F-CL-MMAE or 2G12-2B2-CL-MMAE reduced OVCAR3-derived xenograft volume in vivo, depleting STn⁺ tumor cells. In summary, STn⁺ cells demonstrate some stem-like properties and specific therapeutic targeting of STn in ovarian tumors may be an effective clinical strategy to eliminate both STn⁺ CSC and STn⁺ non-CSC populations.

Keywords: antibody-drug conjugate; cancer stem cell; ovarian cancer; sialyl-Tn; tumor-associated carbohydrate antigen.

Figure 1. STn⁺ and CD133⁺ are co-expressed in human ovarian cancer cell lines

A representative example of flow cytometric scatter plots for STn⁺ and CD133⁺ levels in OV90, OVCAR3 and OVCAR4 cells is shown along with quantification of the four populations analyzed (STn^-/CD133^-, STn^-/CD133⁺, STn⁺/CD133^- and STn⁺/CD133⁺). Error bars represent the mean ± SEM of three independent experiments.

Figure 2. STn⁺ and CD133⁺ cells have enhanced colony formation capacity and are enriched in spheres

(A) The indicated sub-populations of OV90, OVCAR3 or OVCAR4 cells were isolated by FACS and seeded in soft agar. Unsorted OV90, OVCAR3 or OVCAR4 cells were seeded in parallel as a control. After 21 days, colonies ≥20 cells were counted and the percent colony formation efficiency of each population was calculated as described in Material and Methods and compared to that of unsorted cells. (B) OV90, OVCAR3 and OVCAR4 cells were cultured under either monolayer or sphere conditions for 10-12 days. At the end of this incubation period, the relative frequency of each sub-population was determined by flow cytometry. Error bars represent the mean ± SEM of a minimum of three independent experiments ^*p < 0.05.

Figure 3. STn⁺ and CD133⁺ cells display chemoresistance

(A) OV90, OVCAR3 and OVCAR4 cells were cultured for 72 hours in the presence of either vehicle control or carboplatin (10 μM). Following treatment, adherent cells were trypsinized and analyzed by flow cytometry to determine the relative frequency of STn^-/CD133^-, STn⁺/CD133^-, STn^-/CD133⁺ and STn⁺/CD133⁺ sub-populations. (B) In a parallel analysis, the total number of live, apoptotic and necrotic cells remaining after 72 hours of exposure to vehicle control or carboplatin (10 μM) was determined by annexin assay. Error bars represent the mean ± SEM. ^*p < 0.05. All experiments were repeated a minimum of three times.

Figure 4. Anti-STn ADC decreases cell viability in vitro

The indicated cell lines were treated in quadruplicate with increasing concentrations of S3F-CL-MMAE for either 72 (A) or 144 hours (B). The effect of S3F-CL-MMAE on metabolic activity was determined by MTT assay and assessed relative to the untreated control. Error bars represent the SEM. ^*p <0.05. All experiments were repeated four times.

Figure 5. S3F-CL-MMAE decreases CSC frequency in vitro

The extreme limiting dilution assay was used as surrogate to determine the effect of S3F-CL-MMAE on CSC frequency. Seventy two (A) and 144 hours (B) following treatment with vehicle or 10 nM S3F-CL-MMAE, OV90, OVCAR3 and OVCAR4 cells (1-10-20 or 40/well) were plated under tumorsphere culture conditions and the number of spheres that formed after 7-8 days in culture was determined. Stem cell frequencies were calculated as described in Materials and Methods. Error bars represent the SEM. ^*p < 0.05. All experiments were repeated four times.

Figure 6. Treatment with the anti-STn, S3F-CL-MMAE, on CD133 and STn populations in vitro

OV90, OVCAR3 and OVCAR4 cell lines were harvested 72 (A) or 144 (B) hours post treatment with vehicle or S3F-CL-MMAE (10 nM), and the relative frequency of STn^-/CD133^-, STn⁺/CD133^-, STn^-/CD133⁺ and STn⁺/CD133⁺ fractions was determined by flow cytometry. Error bars represent the mean and SEM. ^*p < 0.05. All experiments were repeated four times.

Figure 7. Anti-STn ADCs impede OvCa xenograft growth in vivo and target STn⁺ cells

(A) Mice bearing tumors derived from OVCAR3 were treated with vehicle, unconjugated S3F, S3F-CL-MMAE, unconjugated 2G12-2B2, or 2G12-2B2-CL-MMAE. Tumors were measured twice weekly, and the percentage change in tumor volume compared to the baseline volume (100%) is shown. Error bars represent the mean ± SEM ^*p < 0.05. (B) Xenografts collected at the end of the in vivo treatment were examined to assess the relative frequency of STn^-/CD133^-, STn⁺/CD133^-, STn^-/CD133⁺ and STn⁺/CD133⁺ sub-populations.