Cystine/glutamate antiporter xCT deficiency reduces metastasis without impairing immune system function in breast cancer mouse models

Abstract

Background: The upregulation of antioxidant mechanisms is a common occurrence in cancer cells, as they strive to maintain balanced redox state and prevent oxidative damage. This includes the upregulation of the cystine/glutamate antiporter xCT, which plays a crucial role in protecting cancer cells from oxidative stress. Consequently, targeting xCT has become an attractive strategy for cancer treatment. However, xCT is also expressed by several types of immune cells where it has a role in proliferation and effector functions. In light of these observations, a comprehensive understanding of the specific role of xCT in the initiation and progression of cancer, as well as its potential impact on the immune system within the tumor microenvironment and the anti-tumor response, require further investigation.

Methods: We generated xCT^null BALB/c mice to investigate the role of xCT in the immune system and xCT^null/Erbb2-transgenic BALB-neuT mice to study the role of xCT in a mammary cancer-prone model. We also used mammary cancer cells derived from BALB-neuT/xCT^null mice and xCT^KO 4T1 cells to test the contribution of xCT to malignant properties in vitro and in vivo.

Results: xCT depletion in BALB-neuT/xCT^null mice does not alter autochthonous tumor initiation, but tumor cells isolated from these mice display proliferation and redox balance defects in vitro. Although xCT disruption sensitizes 4T1 cells to oxidative stress, it does not prevent transplantable tumor growth, but reduces cell migration in vitro and lung metastasis in vivo. This is accompanied by an altered immune cell recruitment in the pre-metastatic niche. Finally, systemic depletion of xCT in host mice does not affect transplantable tumor growth and metastasis nor impair the proper mounting of both humoral and cellular immune responses in vivo.

Conclusions: xCT is dispensable for proper immune system function, thus supporting the safety of xCT targeting in oncology. Nevertheless, xCT is involved in several processes required for the metastatic seeding of mammary cancer cells, thus broadening the scope of xCT-targeting approaches.

Keywords: Breast cancer; Immune system; Metastasis; Metastatic niche; SLC7A11; xCT.

Fig. 1

Initiation of autochthonous mammary tumors in cancer-prone mice is not dependent on xCT. A Percentage of disease free-survival and B tumor multiplicity from disease onset in BALB-neuT/xCT^wt (WT) and BALB-neuT/xCT^null (null) mice. C Percentage of mice affected by lung metastasis at sacrifice. Numbers of mice are reported in the panel legends. Statistical analysis: Log-rank (Mantel-Cox) test (panel A), unpaired t test (panel B), or Fisher’s exact test (panel C). Where not indicated, p value is not significant. In some instances, p values are represented in numbers when not significant. In panel B, mean values ± SD are depicted

Fig. 2

Immune response is preserved in xCT^null mice. A Immunization schedule of xCT^wt (WT) and xCT^null (null) BALB/c mice with the empty pVAX1 or the RHuT plasmids. B Percentage of immune cell populations in the blood of WT and null healthy mice. C  Percent of lysis of splenocytes stained with CFSE, pulsed with the target antigen, and injected in mice immunized with pVAX1 or RHuT, as assessed by flow cytometry. D ELISA assay of pre-vax (Pre) and post-vax (Post) sera from vaccinated mice, recognizing Human or Rat ERBB2 extracellular (EC) portion. E Experimental scheme of BM-derived cells culturing and conditioning. F Brightfield microscopy images (10X) of WT or null BM-derived cells cultured for 7 days with CM of 4T1 cells, with or without the addition of 100 µM β-ME. Percentage of G B lymphocytes, H polarized macrophages, I monocytes/mMDSC, J neutrophils/PMN-MDSC and K mature dendritic cells on their respective parent populations indicated in the y-axis legend. L Histogram representation of spleen weight at sacrifice of mice challenged subcutaneously with 10.000 4T1 cells. M Percentage of CD45+ tumor infiltrating cells on total single cells of mice described in panel L. N Growth curves of tumors deriving from 10.000 4T1 cells injected subcutaneously in WT or null mice. O Number of superficial metastases in lungs from mice described in panel N. Number of replicates: Each dot represents a mouse. Statistical analysis: unpaired t test. In panels G-K, t test was performed between xCT^null(null) cells grown in CM without β-ME versus all the other groups. Unpaired t test performed between pre-vax and post-vax values, or between pVAX and RHuT values. * p<0.05; ** p<0.01. Where not indicated, p value is not significant. In some instances, p values are represented in number when not significant. Histograms represent mean values. Error bars (SD) are shown only when n ≥ 5

Fig. 3

Generation and characterization of cell lines from BALB-neuT/xCT^wt and BALB-neuT/xCT^null tumors. A Fold change of the mean fluorescent intensity (MFI) of DCF signal or Oxydized Bodipy C11 signal 72 h after removal or not of β-ME from the growth medium, assessed through FACS analysis. B Percentage of dead (DAPI+) cells 72 h after removal of β-ME from the growth medium. C Proliferation curves, assessed by MTT assay, of SUT32-2H9 cells or WT27 cells in the presence or absence of β-ME. D Growth curves of 1 x 10⁶ SUT32-2H9 or WT27 cells injected subcutaneously in BALB/c mice. Number of replicates: each dot depicts a mouse (n = 5 per group in panel D) or an independent biological replicate. Statistical analysis: ratio paired t test (panel A) or unpaired t test (panels B and C). * p<0.05; ** p<0.01; *** p<0.001; Where not indicated, p value is not significant. Histograms (panels A, B) or lines (panel C) represent mean values. Error bars are not shown as n < 5

Fig. 4

Generation and characterization of 4T1 xCT^KO cell clones. A Western blot analysis of xCT expression, using vinculin as loading control. B Selenocystine uptake assay (used as a surrogate of cystine uptake), expressed as % fluorescence emitted as compared to parental 4T1 cells. Cells incubated with SAS or Erastin are used as control. C Cell proliferation curves of parental 4T1 cells, WT and KO clones, assessed by MTT assay. D Left: Colony-forming efficiency assay. Right: Percentage of plate surface occupied by colonies. E Left: FACS analysis of ROS content following 4 h incubation with 100 µM of tBHP, indicated by 2’,7’-Dichlorofluorescin diacetate (DCF-DA) fluorescent signal. Right: DCF fluorescent signal represented as fold change of DCF MFI, normalized on cells incubated in growth medium alone. F Right: Representative images of migrating cells in a wound-healing assay at 0 and 48 h post scratching. Left: Percentage of wound closure (of initial wound area) at 48 h. G Left: Percentage of transwell area covered by migrated cells. Right: Representative images of migrating cells in a transwell migration assay. Number of replicates: each dot represents an independent biological replicate, which is the result of at least two technical replicates, except for experiments of flow cytometry, where only a technical replicate for biological sample was performed. Statistical analysis: unpaired t test (panels C-F, G) or ratio paired t test (panels B, E). *p<0.05; ** p<0.01; *** p<0.001; **** p<0.0001. Where not indicated, p value is not significant, except in panel B, where dots and lines depicting comparisons with negative controls are omitted for a better visualization. Lines (panel C only) and histograms represent mean values. Error bars are shown only when n > 5, and represent SD

Fig. 5

xCT depletion in cancer cells impairs metastasis formation and alters the metastatic niche. A Western blot analysis of xCT expression in WT 4T1 cells and the pool of 10 xCT^KO clones (xCT-KO Pool). Vinculin is used as loading control. B Growth curves of tumors deriving from 10.000 4T1 (WT or KO pool) injected subcutaneously in BALB/c mice. C Tumor weight at sacrifice. D Left: representative slices of FFPE lungs from mice described in panel B, stained with H&E. Right: percentage of slice area occupied by metastases. E Left: representative slices of FFPE lungs (following i.v. injection of 10.000 4T1, either WT or KO pool) stained with H&E. Right: percentage of slice area occupied by metastases. Percentage of selected immune cell populations over total leukocytes (CD45+) F infiltrating the lungs or G isolated from peripheral blood of tumor-bearing mice described in panel B. H Left: experimental protocol used to assess alterations in the immune pre-metastatic niche. Right: Mean tumor growth curves of mice are shown for a better visualization. Percentage of I PMN-MDSC or J NK over total leukocytes (CD45+) infiltrating the lungs of tumor-bearing mice (described in panel H) at different stages of tumor growth and of healthy, unchallenged mice. Number of replicates: number of mice is reported in panels B and H; each dot represents a mouse. For flow cytometry data, each dot depicting a mouse is the result of a single technical replicate. Statistical analysis: unpaired t test. * p<0.05; ** p<0.01; *** p<0.001. Where not indicated, p value is not significant. Lines (panel H only) and histograms represent mean values. Error bars are shown only when n > 5, and represent SD