Investigations on Zinc Isotope Fractionation in Breast Cancer Tissue Using in vitro Cell Culture Uptake-Efflux Experiments

Zinc (Zn) accumulates in breast cancer tumors compared to adjacent healthy tissue. Clinical samples of breast cancer tissue show light Zn isotopic compositions (δ66Zn) relative to healthy tissue. The underlying mechanisms causing such effects are unknown. To investigate if the isotopic discrimination observed for in vivo breast cancer tissue samples can be reproduced in vitro, we report isotopic data for Zn uptake-efflux experiments using a human breast cancer cell line. MDA-MB-231 cell line was used as a model for triple receptor negative breast cancer. We determined Zn isotope fractionation for Zn cell uptake (Δ66Znuptake) and cell efflux (Δ66Znefflux) using a drip-flow reactor to enable comparison with the in vivo environment. The MDA-MB-231 cell line analyses show Zn isotopic fractionations in an opposite direction to those observed for in vivo breast cancer tissue. Uptake of isotopically heavy Zn (Δ66Znuptake = +0.23 ± 0.05‰) is consistent with transport via Zn transporters (ZIPs), which have histidine-rich binding sites. Zinc excreted during efflux is isotopically lighter than Zn taken up by the cells (Δ66Znefflux = −0.35 ± 0.06‰). The difference in Zn isotope fractionation observed between in vitro MDA-MB-231 cell line experiments and in vivo breast tissues might be due to differences in Zn transporter levels or intercellular Zn storage (endoplasmic reticulum and/or Zn specific vesicles); stromal cells, such as fibroblasts and immune cells. Although, additional experiments using other human breast cancer cell lines (e.g., MCF-7, BT-20) with varying Zn protein characteristics are required, the results highlight differences between in vitro and in vivo Zn isotope fractionation.


INTRODUCTION
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)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(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).
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)(31)(32)(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)(31)(32)(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 neuronlike 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).

Cell Proliferation
The cell proliferation experiments were conducted to investigate if isotopic mass controls cell growth. Isotopically enriched metal powders of 64 Zn (>99.00 %) and 68 Zn (>99.00 %) (Isoflex, San Francisco, CA, US) were dissolved separately in 6 M HCl. Chloride solutions were diluted with 18 MΩ cm purified H 2 O (MilliQ) to make 10 mM ZnCl2 solutions which were then added to the exposure medium. The exposure medium consisting of Dulbecco's modified Eagle's medium (DMEM) + 10% fetal calf serum (FBS, Gibco Life, heat inactivated) + 1% penicillin/streptomycin (Sigma-Aldrich, UK) + 10 mM ZnCl 2 solution was combined, adjusted to pH 7.4 if necessary and then equilibrated overnight. This exposure medium was added over two passages of MDA-MB-231 cells to enable full isotopic equilibration. At a cell density of 1 × 10 5 , cells were seeded in six well-plates (n = 4) for negative control (0 µM Zn, -ve), positive control (20 µM Zn, +ve), isotopically light (20 µM, 64 Zn) and isotopically heavy (20 µM, 68 Zn) exposures and grown under normal oxygen conditions. The cells were counted at 48, 72, and 96 h using a Cellometer Auto T4 (Nexcelom Bioscience, MA, US) to monitor the growth rate. To ensure constant cell proliferation the exposure media were refreshed after 48 h for the cells growing for 72 or 96 h.

Cell Culturing
In our uptake-efflux experiment, MDA-MB-231 (ATCC HTB-26) cells were first cultured under sterile conditions inside a class II biological safety cabinet at 37 • C. Cells were grown in Dulbecco's modified Eagle's medium (DMEM) + 10% fetal calf serum (FBS, Gibco Life, heat inactivated) + 1% penicillin/streptomycin (Sigma-Aldrich, UK) without Zn addition. After 48 h, the cells were transferred to petri dishes containing medium without additional Zn. At this step cells were grown on microscopic slides until they reached 10 5 cells/cm 2 . Microscopic slides with MDA-MB-231 were transferred to media spiked with 15 µM isotopically natural Zn solution. A concentration of 15 µM ZnCl 2 was selected as it is within the reference range of intracellular and plasma homeostatic Zn (11-24 µM) (41). As "free" Zn is toxic to cells, even at nanomolar levels (42), we added Zn complexed with histidine (Zn-His), a stable octahedral compound (43). MDA-MB-231 cells on microscopic slides were incubated for 24 h at 37 • C before being transferred to the drip flow biofilm reactor for the efflux experiment.  Figure 2 shows the experimental setup for the efflux experiment using a drip flow-through biofilm reactor (BioSurface Technologies, Corp., USA). The experiment was conducted in a class II biological safety cabinet. Prior conducting the efflux experiment with a cell line, the setup was tested for Zn blanks using a 20 mM metal-free HEPES input solution. The polysulfone drip flow-through biofilm reactor has a 10 • inclination and consists of four chambers. The 20 mM metal-free HEPES input fluid was supplied through four channels imposed by a peristaltic pump and ran through the system for 1 and 2.5 h, respectively, and the effluent was collected separately for each channel.

Efflux Experiment
For the cell line efflux experiment, microscopic slides with cells cultured for 24 h in Zn-His spiked medium were washed three times with 20 mM metal-free HEPES solution before placed in the drip flow-through biofilm reactor to remove detached cells. This step ensured that the collected effluent solution of the efflux experiment only contained Zn excreted by the cells. The initial cell density and initial Zn isotope composition of these cells prior to the efflux experiments were determined by harvesting the cells from a microscopic slide with trypsin (Lonza). The harvested cells were counted using the Cellometer Auto T4 cell counter (Nexcelom, Bioscience). The 20 mM metal-free HEPES solution was directly dripped onto the cells with a rate of 1 ml min −1 . Due to inclination of the drip flow-through reactor the residence time of the fluid passing the chambers was extremely short (<1 min) resulting in short contact time of the fluid and the cells. The effluent was collected in 30 min intervals in acid-washed metalfree centrifuge tubes (VWR). After the experiment, the cells on the microscopic slides were harvested using trypsin. Harvested cells, effluent and aliquots of the medium, and starting input HEPES solution were stored at −20 • C, until required.
Prior to isotope analysis, harvested cells were lysed using a MARS 5 Digestion Microwave System (CEM Corp., UK). The harvested cell suspension was transferred in acid cleaned XP-1500 Plus (PTFE) vessels and 3 ml of quartz sub-boiled distilled nitric acid (15.4 N) and 2 ml hydrogen peroxide (Romil Ltd) were added. A blank sample consisting of only 3 ml of nitric acid and 2 ml hydrogen peroxide was processed for quality control. The samples were pre-digested at room temperature overnight. Microwave-assisted acid digestion was performed by ramping up the temperature stepwise to 210 • C and 250 psi over 60 min, and held there for 30 min to ensure complete digestion.
For Zn isotope analysis, all samples (e.g., acid-digested cells, efflux solution) were double-spiked consisting of mixture of 64 Zn and 67 Zn. The double-spike mixture had a 64 Zn/ 67 Zn = 4.2336 and was added to each sample in an ideal sample/spike mixture of 1. To ensure the ideal sample/spike mixture, a small sample aliquot (20 µl) was spiked with an appropriate volume of 64 Zn + 67 Zn spike and the sample Zn concentration was determined by isotope dilution. Once sample-Zn was known, a larger aliquot of the sample was prepared for Zn isotopic analysis. Zinc was separated and purified using AG-MP1 resin (BioRad, 100-200 mesh) following the method described by (1,44). The Zn columns with 250 µl AG-MP1 resin were rinsed with 10 mL of each 0.1N HNO 3 and double-deionized water, conditioned with 6N HCl, and equilibrated with 4 x 0.5 ml 1N HCl. The spiked sample re-dissolved in 1 ml 1N HCl was loaded on the column and subsequently rinsed with 8 mL of 1N HCl. In the The extent of isotope fractionation was determined and described as: based on the difference between Zn isotopic composition of cells after incubation for 24 h (δ 66 Zn cells ) and the Zn isotopic composition of the effluent (δ 66 Zn effluent ) collected for 0.5 h. The in-house London Zn was used as standard for samplestandard bracketing and the precision of the unprocessed London Zn solution was ± 0.04‰ (n = 26) over two analytical days. The precision of δ 66 Zn for processed pure Zn IRMM-3702 reference material was −0.13 ± 0.08‰ (2SD, n = 6). If not stated otherwise, the precision of 2SD for δ 66 Zn (‰) refers to repeated analysis of London Zn bracketing standards. Figure 3 shows the average cell growth of MDA-MB-231 in a 64 Zn and 68 Zn isotope enriched medium as well as the control without Zn (-ve) and natural Zn (+ve). In all experiments, regardless of the isotopic composition of the Zn in solution, the cell density increased by 5-fold within 96 h reaching an average density of 5.3 × 10 5 ± 0.5 × 10 5 cells. If cell growth is driven by the mass of an isotope, we would expect to see faster growth for the lighter isotopes compared t the heavier isotopes of Zn (Figure 3 and Table 1).

Cell Efflux
Control of blank levels. We assessed blank contributions because Zn usually has a high background level, which can significantly alter the measured isotopic composition. The microwave control blank was 0.9 ng Zn ml −1 and procedural blanks for the anion exchange chromatography were 0.24 ± 0.20 ng Zn ml −1 (n = 4). The Zn blank for the experimental setup ranged between 0.5 and 2.7 ng Zn ml −1 after 1 h effluent collection and decreased to 0.6-1.4 ng Zn ml −1 after 2.5 h ( Table 2). This corresponds to 0.7-4.8% of Zn (mean: 1.9 ± 2.0% Zn) collected in the effluent of the cell line efflux experiment. The sum of all blanks (microwave, procedural and efflux) corresponds to 0.008 and 0.81% of the initial Zn in medium and harvested cells before the efflux experiment. This observation demonstrates that precise and reliable Zn isotope data can be achieved for cell line experiments using the drip flow-through biofilm reactor.
Mass balance. Our current experimental design does not allow for a complete isotopic mass balance. Cellular concentrations and isotopic changes of Zn can only be monitored assuming a similar distribution and behavior of cells on each microscopic slide. Based on the Zn concentration of harvested cells from two microscopic slides, we calculated that about 0.15% of total 15 µM Zn from the medium was incorporated into MDA-MB-231 cells.

DISCUSSION
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 ( 64 Zn) or heavy ( 68 Zn) 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 δ 66 Zn 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 ( 66 Zn 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 66 Zn by MDA-MB-231 may reflect previously reported upregulation of histidine-rich ZIPs in breast cancer cells (25)(26)(27)(28)(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 × 10 9 Zn atoms/cells by MDA-MB-231 in our experiments compared to Zn required by mammalian cells (10 8 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 64 Zn 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 66 Zn 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 66 Zn efflux suggests diffusion via non-selective channels. During diffusion light 64 Zn diffuses faster out of the cell than 66 Zn. The efflux experiment shows that MDA-MB-231 cells preferentially 'excrete' light 64 Zn with 66 Zn 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  66 Zn 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 66 Zn 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 Zn 2+ across cell membranes (54) and with that most likely isotopically heavy Zn 2+ 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 64 Zn due to faster diffusion of light 64 Zn. Obviously conducting an uptakeefflux experiment with free Zn 2+ could be a better approach, but even if Zn is added to the medium as free Zn 2+ , culture medium contains millimolar levels of ligands (e.g., histidine, cysteine and phosphate) which rapidly complex free Zn 2+ (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)(56)(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.

Future Directions
Our results support the relevance of in vitro cell culture models to identify and understand factors driving changes in Zn isotopic composition caused by cancer development and progression. However, with respect to in vivo relevance, the results should be regarded as preliminary, and additional experiments are required using other human breast cancer cell lines, e.g., MCF-7, BT-20, with different expressions of ZnT and ZIP ( Table 1). This is important, because, for instance, upregulation of ZIP6 in estrogen treated MCF-7 (61) ( Table 1) might lead to more pronounced uptake of heavy 66 Zn. As almost every cancer is unique, it may be advantageous to determine average Zn isotope fractionation for cancer cells based on Zn biology (e.g., MT expression, ZnT and ZIP regulation, cell growth rate) moreover the use of 3D cell cultures as spheroids may be a representative approach and enable other stromal cell types to be incorporated into the workstream.

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

AUTHOR CONTRIBUTIONS
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.