Over the last decade analysis of natural metal isotopes has emerged as an interdisciplinary field of substantial biomedical potential, including diagnosis and defining disease mechanisms (1–14). Compared to metabolic studies where radioactive and single isotope tracers are used to monitor concentration changes, high precision measurements of natural isotopic fractionations are especially useful because they can inform on mechanisms, e.g., why changes in concentration occur due to altered uptake, secretion or excretion. Two recently published breast cancer studies found that Zn dyshomeostasis linked to carcinogenesis is reflected in enrichment of light Zn isotopes in malignant breast tissue compared to adjacent histologically healthy tissue (3, 5).

Zinc homeostasis in humans is maintained by multiple proteins which tightly regulate intracellular Zn concentrations. Among these proteins are influx-controlling Zn importers (SLC39A; ZIP1-ZIP14), efflux-controlling Zn transporters (SLC30A; ZnT1-ZnT10), and Zn-sequestering proteins (e.g., metallothionein) [e.g., (15–19)]. Histidine-, glutamate-, aspartate- and cysteine-residues serve as binding ligands for Zn in these proteins. In general, histidine-rich loops form the primary Zn-binding sites of ZIPs and ZnTs (20), while Zn-binding on metallothionein occurs on cysteine-rich ligands (21, 22). Cancer-induced Zn dyshomeostasis has been related to up- or down-regulation of Zn proteins (23) and changes in their coordination and ligand chemistry (24). For breast cancer, upregulation of ZIP6, ZIP7, ZIP10, ZnT2 and overexpression of metallothionein have been proposed to implicate higher Zn levels in malignant relative to healthy tissue (25–29).

Isotopic fractionation of Zn isotopes in cells is linked to coordination chemistry. Lower atomic weight is associated with lower bond energy, so heavier isotopes are enriched in the strongest ligand bonds, assuming equilibrium isotope fractionation (30–33). Thus, cysteine (S-ligands) preferentially binds isotopically light Zn, while normally tighter binding histidine (N-ligands) and aspartate (O-ligands) preferentially complex isotopically heavy Zn (30–33). If the normal Zn-ligand binding environments are disturbed by Zn dyshomeostasis, isotopic compositions in various body reservoirs (e.g., blood, urine, tissue) can be perturbed. Thus, Zn isotope analysis of clinical samples could be a method to probe the role of Zn uptake and secretion in cancer development and progression (1–3, 5, 11, 34).

In vitro cell lines are powerful simplified models to study processes at the molecular and cellular levels including the study of cancer-induced isotopic fractionation. Various human cell lines have been used to better understand uptake and transport mechanisms causing isotope fractionation of copper (35, 36), iron (37), uranium (38, 39) and Zn (38). Differentiated neuron-like cells (SH-SY5Y) preferentially incorporate and accumulate light isotopes of copper, uranium, and Zn (36, 38). Similar observations have been made for differentiated intestinal (Caco-2) cells showing the preferential uptake of isotopically light iron (37). Under oxidative stress, however, cancerous liver cells incorporate copper that is isotopically heavy (35, 40). These observations contradict the expected correlation between in vitro and in vivo studies on metal ion isotope fractionation.

There is little information about the processes at the molecular and cellular level causing systematic enrichment of light Zn isotopes in malignant breast tissue (3, 5). Our work aimed to improve the mechanistic understanding of the isotopic behavior of Zn in breast cancer, with the long-term objective of biomarker development. We conducted experiments using in vitro breast cancer cell lines (MDA-MB-231) spiked with isotopically-natural Zn and determined the isotopic fractionation induced due to active uptake and efflux. The results highlight differences between in vitro and in vivo Zn isotope processing and will inform future studies on using Zn isotope fractionation as a biomarker (Figure 1).

The results show nearly identical proliferation of MDA-MB-231 under single isotope and natural Zn exposure conditions; these observations demonstrate that isotopic mass does not control cell growth. The addition of 20 μM Zn did not enhance or suppress the proliferation, as the cell density for light (⁶⁴Zn) or heavy (⁶⁸Zn) isotopically spiked Zn conditions are similar to those for the no-Zn control and Zn with natural isotope composition (Figure 3).

Metabolism in cancerous breast tissue is generally thought to be enhanced in order to provide sufficient energy and anabolic substrate for proliferation (45, 46). Adaptation in Zn-protein expression in neoplasms is not driven by preferential uptake of any Zn isotopologue. This means that the isotopic shift reported in breast tumors (3, 5) likely reflects metabolic changes for Zn in cancerous breast cells, and not an energetic advantage related to the isotopic composition possessed by the neoplasm. As such, observed isotopic shifts in breast cancer tissue can be used purely as a monitor of neoplastic driven metabolic changes. Furthermore, single stable isotope tracer studies are established as providing better insight into the processes under investigation without inducing or exacerbating pathological processes (47).

Notably, our MDA-MB-231 cell line analyses show isotopic fractionations in the opposite direction to those determined for in vivo breast cancer tissue samples. Malignant breast tumors have significantly lower δ⁶⁶Zn than healthy tissues which has been interpreted as an indicator for increased metallothionein expression (3, 5). The difference in Zn isotope fractionation observed between in vitro MDA-MB-231 cell line experiments and in vivo breast tissue might be due to differences in Zn transporter levels or intercellular Zn storage mechanisms (e.g., metallothionein expression).

A positive direction of Zn isotope fractionation for the cell uptake (Δ⁶⁶Zn_uptake = +0.23 ± 0.1‰, 2SD, n = 2) can be mechanistically explained by Zn adsorption, diffusion through non-selective channels into the cell or an active cell uptake via ZIP proteins. Adsorption studies on bacterial cells and biofilms show preferential binding of heavy Zn isotopes with equilibrium isotopic fractionation between +0.50 and +1.30‰ (48, 49). However, adsorption may be less likely in our studies as the 2:1 Zn (His)₂ complexes are uncharged (43) and cells were rinsed with metal-free HEPES to remove any loosely adsorbed Zn. Instead, the preferential uptake of ⁶⁶Zn by MDA-MB-231 may reflect previously reported upregulation of histidine-rich ZIPs in breast cancer cells (25–29). ZIP proteins (ZIP1-ZIP14) control intracellular Zn levels by transporting Zn into the cells across the cell membrane. Upregulation of ZIPs has been observed in most cancers to meet the demand of increased rate of proliferation and metabolism (46). An indicator for ZIP upregulation is one-order of magnitude higher Zn uptake of 5.4 × 10⁹ Zn atoms/cells by MDA-MB-231 in our experiments compared to Zn required by mammalian cells (10⁸ Zn atoms/cell) (50).

Zinc isotope fractionation for Zn uptake by cells can be associated with Zn speciation, Zn-binding domains of proteins or exchange reactions. A study with neuron-like cells showed a preferential uptake of light ⁶⁴Zn with an isotopic fractionation of 1.14‰ (39). However, uptake and efflux of Zn-His should not fractionate Zn isotopes due to a lack in coordination change for complexed Zn species (51). Caldelas and Weiss (51), however, argued that non-quantitative uptake of complexed Zn can result in an enrichment of ⁶⁶Zn in plant cells. We can assume a similar isotopic effects of non-quantitative uptake for Zn-His because only 0.15% of Zn from the medium has been taken up by MDA-MB-231 cells. However, our data cannot directly resolve whether diffusion through non-selective channels or uptake via ZIPs, or other proteins, is the predominant pathway for Zn uptake by MDA-MB-231 cells.

Negative Δ⁶⁶Zn_efflux suggests diffusion via non-selective channels. During diffusion light ⁶⁴Zn diffuses faster out of the cell than ⁶⁶Zn. The efflux experiment shows that MDA-MB-231 cells preferentially 'excrete' light ⁶⁴Zn with Δ⁶⁶Zn_efflux of −0.42 ± 0.12‰ (Figure 4). An existing concentration gradient with high Zn in cells and no Zn in the efflux solution may allow diffusion of Zn-His via non-selective channels. Coutaud et al. (49) reported preferential excretion of lighter Zn isotopes with Δ⁶⁶Zn of −0.50 ± 0.20‰ due to desorption from the biofilm. Also, lighter Zn isotopes can diffuse faster through biological membranes or due to a gradient within the boundary layer (52). This leaves the cell culture enriched in heavy ⁶⁶Zn over time resulting in opposite isotopic signal of what has been observed for in vivo malignant breast tissue. These in vivo studies found that Zn isotope compositions are about 0.17‰ lower in malignant breast than healthy tissues (3, 5).

The ZnTs family (ZnT1-ZnT10) regulates intracellular Zn levels by transporting Zn from the cytoplasm into the extracellular space. To decrease the toxic effects of high Zn in breast cancer cells, ZnT1 is overexpressed to prevent apoptotic cell death and increase Zn secretion. Wang et al. (53) observed that the mRNA expression level of ZnT-1 increases significantly in MDA-MB-231 cells when they are exposed to Zn sulfate. In vivo, ZnTs transport only free Zn²⁺ across cell membranes (54) and with that most likely isotopically heavy Zn²⁺ out of the cell due to their histidine-rich structure. In our experiments, however, Zn-His might be able to pass cell membranes via different channels and the diffusion out of the cell is similar to Zn-His uptake. This explains the preferential efflux of light ⁶⁴Zn due to faster diffusion of light ⁶⁴Zn. Obviously conducting an uptake-efflux experiment with free Zn²⁺ could be a better approach, but even if Zn is added to the medium as free Zn²⁺, culture medium contains millimolar levels of ligands (e.g., histidine, cysteine and phosphate) which rapidly complex free Zn²⁺ (42). Moreover, conducting an experiment with “free” Zn can result in apoptosis because “free” Zn is a cell toxin at even nanomolar levels (42).

In vivo, tumors are heterogenous, have a three-dimensional shape and are often mixed with connective tissue, immune cells, and stromal tissues (55–57). More specifically, breast cancer varies in its origin and genetic lesions, leading to distinct phenotypes (58, 59) which is also reflected in different ZIP and ZnT expression between various breast cancer cell lines. Therefore, we tested our findings from MDA-MB-231 in a large panel of breast cancer cell lines (60) to further our understanding in cell lines representing patient subtypes (59) (Table 3). Using a well-defined list of human ZnT and ZIP proteins (23), we identified ten Zn proteins which are differentially expressed (FDR-adjusted P < 0.1) in MDA-MB-231 compared to other basal-like breast cancer cell lines (e.g., Her2, Luminal A and Luminal B) (Table 3). It shows that more ZIPs and ZnTs are up- than downregulated which might explain the high uptake and efflux. Other studies on the hormone-dependent breast cancer cell lines T-47D and MCF-7 showed that ZnT2 and metallothionein are overexpressed, providing protection from Zn hyperaccumulation and preventing apoptosis by either removing Zn from the cell or redistributing it among cellular compartments (27, 29). Consequently, whether breast cancer cell lines and the experimental approach with initially ligand complexed Zn (Zn-His) reflect that of in vivo carcinoma remains an important issue to resolve before drawing any reliable conclusion on Zn isotope fractionation in clinical samples.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

KS and FL designed the experiments. KS performed the data analysis, interpretation of the data, and drafting of the manuscript. ALH provided lab space at MRC Weatherall Institute of Molecular Medicine to conduct the uptake-efflux experiment. The isotopic analyses were conducted in the Oxford Earth Sciences isotope facility of ANH. SH provided the bioinformatic data and wrote the bioinformatic parts of the manuscript. All authors contributed to the article and approved the submitted version.

This work was funded by Cancer Research UK (No. C5255/A18085), through the CRUK Oxford Centre and John Fell Oxford University Press Research Fund (No. 153/035).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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