Fighting breast cancer stem cells through the immune-targeting of the xCT cystine-glutamate antiporter

Abstract

Tumor relapse and metastatic spreading act as major hindrances to achieve complete cure of breast cancer. Evidence suggests that cancer stem cells (CSC) would function as a reservoir for the local and distant recurrence of the disease, due to their resistance to radio- and chemotherapy and their ability to regenerate the tumor. Therefore, the identification of appropriate molecular targets expressed by CSC may be critical in the development of more effective therapies. Our studies focused on the identification of mammary CSC antigens and on the development of CSC-targeting vaccines. We compared the transcriptional profile of CSC-enriched tumorspheres from an Her2⁺ breast cancer cell line with that of the more differentiated parental cells. Among the molecules strongly upregulated in tumorspheres we selected the transmembrane amino-acid antiporter xCT. In this review, we summarize the results we obtained with different xCT-targeting vaccines. We show that, despite xCT being a self-antigen, vaccination was able to induce a humoral immune response that delayed primary tumor growth and strongly impaired pulmonary metastasis formation in mice challenged with tumorsphere-derived cells. Moreover, immunotargeting of xCT was able to increase CSC chemosensitivity to doxorubicin, suggesting that it may act as an adjuvant to chemotherapy. In conclusion, our approach based on the comparison of the transcriptome of tumorspheres and parental cells allowed us to identify a novel CSC-related target and to develop preclinical therapeutic approaches able to impact on CSC biology, and therefore, hampering tumor growth and dissemination.

Keywords: Breast cancer; Cancer stem cell; NIBIT 2017; Tumorsphere; Vaccine; xCT.

Fig. 1

Topological model of the x_c-transporter. xCT is composed by 12 transmembrane domains spanning the cellular membrane (red cylinders), 7 intracellular and 6 extracellular domains (ECD1-6, all represented as red lines) and is associated to CD98, the heavy chain of the x_c-transporter spanning the cellular membrane (blue cylinder). A scheme of the x_c-transporter function is also reported, with extracellular cystine (CySS) being imported in exchange for intracellular glutamate (Glu) and reduced to cysteine, the rate-limiting precursor for the synthesis of GSH, which then scavenges intracellular ROS

Fig. 2

Schematic representation of the different vaccination protocols. a Therapeutic anti-tumor setting. Female BALB/c mice are challenged with a subcutaneous injection of TUBO tumorsphere-derived cells. When tumors become palpable, mice are vaccinated twice at 2-week interval and tumor growth is monitored for the following days. b Prophylactic anti-metastasic setting. Female BALB/c mice are vaccinated twice at 2-week interval. 1 week after the second vaccination, mice are challenged with an intravenous injection of TUBO tumorsphere-derived cells. Few weeks after the challenge, mice are euthanized and lungs analyzed for the presence of metastasis. c Therapeutic anti-tumor and anti-metastasis setting. Female BALB/c mice are challenged with a subcutaneous injection of 4T1 tumorsphere-derived cells. When tumors become palpable, mice are vaccinated twice at 2-week interval and the tumor growth is monitored for the following days. Few weeks after challenge, mice are euthanized and lungs analyzed for the presence of metastasis

Fig. 3

Anti-xCT vaccination induces a specific humoral response. Sera were collected from BALB/c mice vaccinated twice at 2-week interval with either BoHV-4-mxCT, pVAX1-mxCT, AX09-0M6 or AX09-0M3 or control vectors (pVAX1, BoHV-4-A29 or MS2) 2 weeks after the second vaccination, and tested by ELISA on wells coated with: a full-length mouse xCT protein, b full-length human xCT protein, or peptides corresponding to c mouse/human xCT ECD6, d mouse xCT ECD3 and e human xCT ECD3. ELISA was performed from pools (each composed by 5 mice) of sera from 6 independent experiments. Graphs show mean ± SEM of the OD₄₅₀ of each pool after the subtraction of the OD₄₅₀ of the corresponding pool from untreated mice. Sera from mice vaccinated with the empty vectors did not bind to either full length xCT or its ECD, while those from BoHV-4-mxCT, pVAX–mxCT, AX09-0M6 and AX09-0M3 vaccinated mice recognized both recombinant xCT proteins and their ECD, although with variable efficiency