Oligo-Fucoidan supplementation enhances the effect of Olaparib on preventing metastasis and recurrence of triple-negative breast cancer in mice

Abstract

Background: Seaweed polysaccharides have been recommended as anticancer supplements and for boosting human health; however, their benefits in the treatment of triple-negative breast cancers (TNBCs) and improving immune surveillance remain unclear. Olaparib is a first-in-class poly (ADP-ribose) polymerase inhibitor. Oligo-Fucoidan, a low-molecular-weight sulfated polysaccharide purified from brown seaweed (Laminaria japonica), exhibits significant bioactivities that may aid in disease management.

Methods: Macrophage polarity, clonogenic assays, cancer stemness properties, cancer cell trajectory, glucose metabolism, the TNBC 4T1 cells and a 4T1 syngeneic mouse model were used to inspect the therapeutic effects of olaparib and Oligo-Fucoidan supplementation on TNBC aggressiveness and microenvironment.

Results: Olaparib treatment increased sub-G1 cell death and G2/M arrest in TNBC cells, and these effects were enhanced when Oligo-Fucoidan was added to treat the TNBC cells. The levels of Rad51 and programmed death-ligand 1 (PD-L1) and the activation of epidermal growth factor receptor (EGFR) and adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) facilitate drug resistance and TNBC metastasis. However, the combination of olaparib and Oligo-Fucoidan synergistically reduced Rad51 and PD-L1 levels, as well as the activity of EGFR and AMPK; consistently, TNBC cytotoxicity and stemness were inhibited. Oligo-Fucoidan plus olaparib better inhibited the formation of TNBC stem cell mammospheroids with decreased subpopulations of CD44^high/CD24^low and EpCAM^high cells than monotherapy. Importantly, Oligo-Fucoidan plus olaparib repressed the oncogenic interleukin-6 (IL-6)/p-EGFR/PD-L1 pathway, glucose uptake and lactate production. Oligo-Fucoidan induced immunoactive and antitumoral M1 macrophages and attenuated the side effects of olaparib, such as the promotion on immunosuppressive and protumoral M2 macrophages. Furthermore, olaparib plus Oligo-Fucoidan dramatically suppressed M2 macrophage invasiveness and repolarized M2 to the M0-like (F4/80^high) and M1-like (CD80^high and CD86^high) phenotypes. In addition, olaparib- and Oligo-Fucoidan-pretreated TNBC cells resulted in the polarization of M0 macrophages into CD80(+) M1 but not CD163(+) M2 macrophages. Importantly, olaparib supplemented with oral administration of Oligo-Fucoidan in mice inhibited postsurgical TNBC recurrence and metastasis with increased cytotoxic T cells in the lymphatic system and decreased regulatory T cells and M2 macrophages in tumors.

Conclusion: Olaparib supplemented with natural compound Oligo-Fucoidan is a novel therapeutic strategy for reprogramming cancer stemness, metabolism and the microenvironment to prevent local postsurgical recurrence and distant metastasis. The combination therapy may advance therapeutic efficacy that prevent metastasis, chemoresistance and mortality in TNBC patients.

Keywords: Cancer stem cells; Glucose uptake; IL-6/EGFR/PD-L1 signaling pathway; Lactate production; M1/M2 macrophage polarization; Olaparib; Oligo-Fucoidan; Triple-negative breast cancer.

Fig. 1

Olaparib and Oligo-Fucoidan cooperatively promote TNBC cell death. A Cell viability was examined following olaparib (0–50 μM) treatment for 24 h and was examined by WST-1 assays. B The levels of PARP, cleaved PARP (Asp214), p21, γ-H2AX, p-AMPK (Thr172), AMPK and Rad51 were analyzed after olaparib (0–50 μM) treatment for 48 h. C Cell viability was analyzed by WST-1 assays after olaparib (50 μM) and/or Oligo-Fucoidan (400 μg/ml) treatment for 24 h. D Cell cycle profiling was performed after olaparib (50 μM) and/or Oligo-Fucoidan (400 μg/ml) administration for 48 h. E Histograms reveal the cell cycle patterns and comparisons between experimental settings. One-way ANOVA with Duncan’s test was used to calculate statistical significance (A, C, E)

Fig. 2

Olaparib combined with Oligo-Fucoidan further inhibits TNBC cell growth and stemness properties. A, B Clonogenic formation of MDA-MB-231 cells was examined after treatment with different doses of olaparib (A) or Oligo-Fucoidan (B) for 2 weeks (n = 6). C The colony formation capacities of 4T1, HCC1395 and MDA-MB-231 cells were examined after olaparib (50 μM) and/or Oligo-Fucoidan (400 μg/ml) treatment for 2 weeks (n = 6). D–E MDA-MB-231 mammosphere formation (≥ 50 µm in diameter) and the expression levels of cancer stemness markers (EpCAM, Nanog and Sox2) were measured by qRT–PCR on Day 10 in response to olaparib (0–50 μM) treatment (n = 5). F–G MDA-MB-231 mammosphere formation (≥ 50 µm in diameter) and the expression levels of cancer stemness markers (CD24, CD44 and Snail) were measured by qRT–PCR on Day 10 in response to Oligo-Fucoidan (0–1000 μg/ml) treatment for 2 weeks (n = 5). The data represent the mean ± SD. One-way ANOVA with Duncan’s test was used to define statistical significance

Fig. 3

Olaparib and Oligo-Fucoidan synergistically inhibit TNBC cell stemness. A, B Mammosphere formation abilities of MDA-MB-231 cells (A) (n = 6–10) and 4T1 cells (B) (n = 7–10) were analyzed in response to olaparib (50 μM) and/or Oligo-Fucoidan (500 μg/ml) treatment for 16 and 5 days, respectively. C The mRNA expression levels of EpCAM, Nanog and ALDH1A1 in MDA-MB-231 mammospheres (Day 16) were quantified by qRT–PCR (n = 5). D EpCAM(+) subpopulations in MDA-MB-231 mammospheres (Day 14) were examined by flow cytometry after the indicated treatments (n = 4–5). E, F CD44(+)/CD24(−) subpopulations were analyzed in MDA-MB-231 mammospheres (Day 14) (E) and 4T1 mammospheres (Day 5) (F) after the indicated treatments. The data are expressed as the mean ± SD. One-way ANOVA with Duncan’s test was used to determine statistical significance

Fig. 4

Olaparib plus Oligo-Fucoidan is superior to monotherapy in inhibiting M2 macrophage plasticity and invasiveness. A, B THP-1 monocytes were treated with olaparib (50 μM) and/or Oligo-Fucoidan (400 μg/ml) for 48 h. The populations of CD80(+) M1 (A) and CD163(+) M2 (B) macrophages were evaluated by flow cytometry (n = 5). C Intracellular markers of M1 (iNOS and p-p38) and M2 (Arginase 1 and IL-10) macrophages were compared before and after olaparib (50 μM) treatment of THP-1 monocytes for 24 h. D, E The expression levels of M0 (F4/80), M1 (CD80, CD86) (n = 5) (D) and M2 (CD163, CD206 and TGF-β) (n = 5) (E) markers in response to the indicated treatments were evaluated by qRT–PCR. F–I THP-1 monocytes were pretreated with PMA (100 ng/ml) for 24 h and IL-4 (50 ng/ml) for another 24 h to activate differentiation of M2 macrophages. M2 invasion abilities (n = 3) (F) and the expression levels of M0 (F4/80) (n = 5) (G), M1 (CD80 and CD86) (n = 5) (H) and M2 (CD163 and CD206) (n = 5) (I) markers were analyzed after the indicated treatments for 48 h. The data are expressed as the mean ± SD. One-way ANOVA with Duncan’s test was used to estimate statistical significance

Fig. 5

Olaparib- and Oligo-Fucoidan-treated TNBC cells promote M1 macrophage plasticity and inhibit the oncogenic and glycolytic pathways. A The F4/80(+) M0 macrophage population was examined by flow cytometry (n = 6) after THP-1 monocytes were treated with PMA (100 ng/ml) for 72 h. B, C MDA-MB-231 cells were pretreated with olaparib (50 μM) and/or Oligo-Fucoidan (400 μg/ml) for 48 h and cultured in serum-free medium for 48 h, after which the conditioned medium (CM) was collected and incubated with M0 macrophages for 48 h and 72 h to examine the polarity of CD80(+) M1 (B) and CD163(+) M2 macrophages (C) by flow cytometry (n = 6). D IL-6, EGFR/p-EGFR (Tyr1068), AMPK/p-AMPK (Thr172), PD-L1 and glucose metabolism factors (Glut1, PFKL, RPIA, PKM2/p-PKM2 (Tyr105), HK2, MCT4) were examined in MAD-MB-231 and 4T1 cells after olaparib (50 μM) and/or Oligo-Fucoidan (400 μg/ml) treatment for 24 h. E, F Glycolytic pathways, such as glucose uptake (E) and lactate production (F), were analyzed in TNBC cell lines after the indicated treatments for 24 h (n = 4). The data represent the mean ± SD. Student’s t test (A) and one-way ANOVA with Duncan’s test (B, C, E, F) were used to calculate statistical significance

Fig. 6

Oligo-Fucoidan combined with olaparib therapy better prevent TNBC aggressiveness than olaparib alone. A The trajectories of 4T1 cell migration were analyzed and compared after olaparib (50 μM) and/or Oligo-Fucoidan (400 μg/ml) treatment for 48 h (n = 20). B–E 4T1 cells (1 × 10⁴) were orthotopically inoculated into BALB/c mice, and the primary breast tumors were surgically removed at week 2 followed by olaparib (50 mg/kg) treatment via i.p. injection twice per week for 2 weeks or combined with Fucoidan (150 mg/kg) by oral feeding twice per week for 5 weeks (n = 6). PBS was used as the control treatment (n = 5). The volumes of primary mammary tumors and the recurred breast tumors in response to the indicated treatments were examined (B). At the endpoints of the indicated treatments (week 7), the recurrent tumor loads (C) and plasma IL-6 levels (D) were measured. The incidences of postsurgical mammary tumor relapse and distant metastasis indicated the therapeutic efficacies (E). The data are presented as the means ± S.E.M. One-way ANOVA and Newman–Keuls test were used to analyze the data

Fig. 7

Olaparib plus Oligo-Fucoidan improves anti-TNBC immunity. Splenic immune cells, tumor-associated macrophages and T cells were analyzed in postsurgical mice bearing TNBC and treated with olaparib (50 mg/kg) and/or Oligo-Fucoidan (150 mg/kg). A Immunohistochemical analysis of CD163(+) M2 macrophages in recurrent mammary tumors. B, C Flow cytometry analysis of CD8( +) cytotoxic T cells in spleen (B) and CD4(+)/CD25(+)/CD127(−) Tregs in recurrent breast tumor after the indicated treatments (C). One-way ANOVA with Duncan’s test was used to calculate statistical significance. D Summary diagram of the effects of olaparib plus Oligo-Fucoidan treatment on suppressing the oncogenic IL-6/p-EGFR/PD-L1 pathway and AMPK activation, which impact TNBC cell growth, stemness and metabolism. The combined treatment prevents TNBC relapse and metastasis, as well as immune evasion, by decreasing immunosuppressive M2 macrophages and Tregs and increasing cytotoxic T cells