Human malignant mammary epithelial cell lines MDA-MB-231 and MDA-MB-468 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) validated by STR profiling. All cell lines were routinely screened for mycoplasma contamination employing established qPCR protocol and specific primers [25]. These cell lines were selected for this research as NF-κB activation has been well-characterised [26]. These studies used staurosporine (Sigma-Aldrich; Saint Louis, MO, USA; Cat#S5921) and lipopolysaccharide (LPS) isolated from Escherichia Coli O111:B4 (Sigma-Aldrich; Cat# L2630).

Breast cancer cell lines were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) with high glucose (Thermo Fisher Scientific, Waltham, MA, USA; Cat#D5796), supplemented with 10% Fetal Bovine Serum (FBS; Thermo Fisher Scientific; Cat#A5670701), 1% penicillin/streptomycin (Life Technologies, Carlsbad, CA, USA; Cat#15240062). Cells were then maintained at 37 °C in a humidified incubator with 5% CO₂.

At 80–90% confluency, cells were seeded into sterile, flat-bottom 24-well plates (Greiner Bio-One, Kremsmünster, Austria; Cat#662160). MDA-MB-231 cells were seeded at 0.1 $\times$ 10⁶ cells per well, MDA-MB-468 cells at 0.5 $\times$ 10⁵ per well, in 500 µL of complete medium. After 24 h to allow cell attachment, media were replaced with fresh DMEM containing LPS at final concentrations of 0, 5, 10, 25, or 50 µg/mL. Untreated wells served as negative controls, while wells treated with 1µM staurosporine served as positive controls. Staurosporine induces cellular stress and programmed cell death by blocking the activity of protein kinases [27], with calcitonin receptor expression previously demonstrated as an early biomarker of this process [16]. For dose-response experiments, cells were harvested 19 h after treatment according to previous studies using CalRexin^TM antibody [16]. For time-course experiments, cells were treated with 10 µg/mL LPS and collected after 6, 24, or 48 h. For NF-κB nuclear translocation experiments, cells were treated with10 µg/mL LPS and collected after 4 h, according to previous studies on NF-κB activation [28]. Cells were washed twice with Dulbecco’s Phosphate-Buffered Saline (DPBS; Gibco, Thermo Fisher Scientific; Cat#14190144) and detached using 0.25% trypsin-EDTA (Thermo Fisher Scientific; Cat#25200056) prior to flow cytometry procedure. All groups were treated identically, and trypsin exposure was minimised.

At each time point and LPS concentration, 0.3 $\times$ 10⁶ cells were harvested and washed once with ice-cold DPBS, and once with freshly prepared FACS buffer (DPBS containing 1% FBS and 0.05% sodium azide; Sigma-Aldrich; Cat#S8032). Cells were incubated with Human BD Fc Block (1:50 dilution; BD Pharmingen, San Jose, CA, USA; Cat# 564220) for 15 min at room temperature, washed with FACS buffer, and centrifuged at 300 $\times$g for 5 min at 4 °C.

Viability staining was performed using propidium iodide (PI; 1.3 µg/mL; BioLegend, San Diego, CA, USA; Cat# 421301) for 5 min at room temperature, followed by washing with FACS buffer. Calcitonin receptor was detected using CalRexin^TM:647 (Apop Biosciences, Melbourne, Victoria, Australia, 1 mg/mL) at a 1:50 dilution, with incubation for 30 min at 37 °C, according to the manufacturer’s instructions. As CalRexin^TM binds an extracellular epitope of calcitonin receptor and is accumulated into live cells, CalRexin^TM positivity may be a combination of cell surface expression and intracellular accumulation. Cells were then washed in FACS buffer and stained with APC/Fire^TM 750 Annexin V (BioLegend; Cat# 640953) at a 1:100 dilution in Annexin V binding buffer (BioLegend; Cat# 422201) for 15 min at room temperature. Cells were resuspended in 200 µL Annexin V binding buffer and transferred to FACS tubes.

Flow cytometry was performed using a Cytek Aurora spectral flow cytometer (Cytek Biosciences, Fremont, CA, USA), with acquisition of a minimum of 30,000 events per sample. Unstained cells were used to assess autofluorescence. Heat-killed breast cancer cells (60 °C for 5 min) served positive control for PI staining. Single-stained controls were generated using cells treated with 1 µM staurosporine and stained individually with each fluorophore to enable spectral unmixing and background correction. Data were analysed using FlowJo v10.10.0 (FlowJo LLC, Ashland, OR, USA). Marker expression was quantified as geometric mean fluorescence intensity (GMFI). Debris was excluded using forward scatter (FSC-A), and doublets were excluded using FSC-H versus FSC-A gating.

Breast cancer cells were seeded at 0.5 $\times$ 10⁵ cells per chamber onto 8-chamber slides (Sarstedt, Nümbrecht, Germany; Cat# 6170.802) and incubated overnight at 37 °C in a humidified incubator with 5% CO₂. Cells were treated with 10 µg/mL LPS or 1 µM staurosporine for 19 h, then were fixed with fresh 4% paraformaldehyde for 20 min at room temperature. Cells were permeabilised with 0.1% Triton X-100 for 10 min at room temperature. Each step is followed by gentle washed twice with PBS.

Blocking was performed using 10% goat serum (Sigma-Aldrich; Cat# G9023) in PBS for 1 h at room temperature. Slides were gently washed, then incubated overnight at 4 °C with a primary polyclonal antibody against NF-κB p65 (Abcam, Cambridge, UK; Cat# ab16502; 1:300 dilution) in PBS containing 1% bovine serum albumin (BSA; Sigma-Aldrich, Cat# A5611). Following washing, slides were incubated with Alexa Fluor 568-conjugated goat anti-rabbit IgG (H+L) secondary antibody (Invitrogen, Carlsbad, CA, USA; Cat# A-11036; 1:500 dilution) for 1 h at room temperature. Slides were then incubated overnight at 4 °C with CalRexin^TM:488 (Apop Biosciences, Australia, 1 mg/mL; diluted 1:200). All slide incubations were performed in a humidified chamber unless otherwise stated.

After thorough washing, slides were counterstained with DAPI (Sigma-Aldrich; Cat# MBD0015) for 5 min, followed by coverslipping with fluorescence mounting medium (Agilent, Santa Clara, CA, USA; Cat# S3023). Images were acquired using a confocal laser scanning microscope (Carl Zeiss LSM 900, Oberkochen, Germany) operated with Zen Blue software 3.3 (Carl Zeiss, Oberkochen, Germany).

Confocal images acquired using a 20$\times$ objective were imported into Fiji, and channels were split prior to analysis. Identical threshold settings were applied across all control, treated, and positive samples within each experiment. Fields for analysis were chosen randomly from multiple regions of each slide, including corners and central areas.

CalRexin^TM:488 intensity was quantified by measuring the percentage of pixel intensity above threshold in the green channel. NF-κB nuclear translocation was assessed by colocalisation analysis using JACoP (Just Another Colocalization Plugin) in Fiji. Colocalisation was evaluated between the DAPI (nuclear) and Alexa Fluor-568 (NF-κB p65) channels. Pearson’s correlation coefficient was used to quantify spatial colocalisation [29].

Data were obtained from two independent experiments, with eight randomly selected fields per experiment, mean values were calculated for each condition. All results were exported for subsequent statistical analysis and data visualisation.

All statistical analyses were performed using Prism v10.10 (GraphPad Software, San Diego, CA, USA). Differences between the control and treated groups were assessed using ordinary one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test. A p-value 0.05, which is indicated by an asterisk, was considered statistically significant. All statistical tests were two-tailed. Data are presented as mean $\pm$ SEM.

To determine whether LPS alters calcitonin receptor positivity (detected using CalRexin^TM antibody) in breast cancer cells, MDA-MB-231 and MDA-MB-468 cells were treated with increasing concentrations of LPS for 19 h and analysed by flow cytometry.

In MDA-MB-231 cells, calcitonin receptor positivity, quantified as CalRexin^TM GMFI, was increased following treatment with 10 µg/mL and 25 µg/mL compared to untreated controls (Fig. 1A,C; p 0.0001). No significant change was observed at 5 µg/mL and 50 µg/mL LPS. This is consistent with previous studies showing low, intermediate and high concentrations of LPS can have different biological effects. For example, low concentrations of LPS fail to activate NF-κB, high concentrations may lead to a tolerance response, and intermediate concentrations are required to maximally activate NF-κB [30, 31]. In contrast, Annexin V GMFI remained unchanged across all LPS concentrations tested, indicating that LPS did not induce apoptotic signalling in these conditions (Fig. 1B,D). Treatment with the cytotoxin staurosporine resulted in marked increases in fluorescence intensity of both CalRexin^TM and Annexin V, consistent with previous findings in other cell lines [16].

Fig. 1.

Dose-dependent effect of LPS on calcitonin receptor fluorescence in MDA-MB-231 cells. (A) Calcitonin receptor and (B) Annexin V GMFI quantifying on cells treated with 5, 10, 25, 50 µg/mL LPS and 1 µM staurosporine (STA) after 19 h, or untreated Controls. Representative flow cytometry-based plots showing the fluorescence cell count following treatment with 10 µg/mL LPS and 1 µM staurosporine (STA) for (C) CalRexin^TM:647 and (D) APC-Fire-750 Annexin V, when compared to untreated control cells. Data are from two independent experiments conducted in triplicate, mean $\pm$ SEM, *p 0.05 compared to untreated Control.

In MDA-MB-468 cells, treatment with 10, 25, and 50 µg/mL LPS (p = 0.002, 0.007, and 0.001 respectively) resulted in an increase in fluorescence intensity of CalRexin^TM relative to untreated controls (Fig. 2A,C). As observed in MDA-MB-231 cells, Annexin V GMFI did not differ from control levels at any LPS concentration (Fig. 2B,D). Staurosporine treatment resulted in increased fluorescence intensity of both CalRexin^TM and Annexin V. Together, these findings indicate that LPS increases calcitonin receptor availability in a concentration-dependent manner without triggering apoptosis in either breast cancer cell line.

Fig. 2.

Dose-dependent effect of LPS on calcitonin receptor fluorescence in the MDA-MB-468 cell. (A) Calcitonin receptor and (B) Annexin V GMFI quantifying on cells treated with 5, 10, 25, 50 µg/mL LPS and 1 µM staurosporine (STA) after 19 h, or untreated Controls. Representative flow cytometry-based plots showing the fluorescent cell count following treatment with 10 µg/mL LPS and 1 µM staurosporine (STA) for (C) CalRexin^TM:647 and (D) APC-Fire-750 Annexin V, when compared to untreated control cells. Data are from two independent experiments conducted in triplicate, mean $\pm$ SEM, *p 0.05 compared to untreated Control.

To visualise calcitonin receptor subcellular distribution, immunofluorescence staining was performed following LPS or staurosporine treatment. Minimal CalRexin^TM staining was detected in untreated control cells (Fig. 3A–C). Staurosporine treatment for 19 h resulted in markedly increased calcitonin receptor staining in MDA-MB-231 cells (Fig. 3G–I), consistent with previous findings in HeLa human cervical adenocarcinoma cells [32] and MG63 osteosarcoma cells [16]. CalRexin^TM fluorescence signal was predominantly localised to cytoplasmic and perinuclear regions, with punctate staining patterns evident following staurosporine treatment. In contrast, LPS-treated cells displayed moderate CalRexin^TM fluorescence signal with fewer puncta (Fig. 3D–F).

Fig. 3.

CalRexin^𝐓𝐌 immunofluorescence staining of breast cancer cells following LPS or staurosporine (STA) treatment. (A–C) Representative immunofluorescence images of MDA-MB-231 cells untreated, or (D–F) treated with 10 µg/mL LPS or (G–I) 1 µM staurosporine for 19 h. Cells were stained with DAPI (A,D,G; blue) to visualise nuclei and CalRexin^TM antibody (B,E,H; green) to detect calcitonin receptor. Merged images are shown (C,F,I). All images were acquired using identical confocal settings. Scale bar = 20 µm.

To examine the temporal dynamics of calcitonin receptor positivity, MDA-MB-231 and MDA-MB-468 cells were treated with 10 µg/mL LPS and analysed after 6, 24, and 48 h.

In MDA-MB-231 cells, a modest increase in CalRexin^TM GMFI was observed after 6 h of LPS treatment (p = 0.03), with no changes detected at later time points (Fig. 4A). The majority of cells remained negative for both CalRexin^TM and Annexin V (Fig. 4B). In comparison, GMFI for CalRexin^TM was increased when cells were treated with staurosporine for 48 h (Fig. 4C; p 0.0001). The Annexin V single positive population was a small percentage of the total cells, while CalRexin^TM single positive cells were high at 6 h, with double positive cells increasing at 24 and 48 h (Fig. 4D,E).

Fig. 4.

Time-dependent effects of LPS and staurosporine (STA) on CalRexin^𝐓𝐌 and Annexin V staining in MDA-MB-231 cells. (A) CalRexin^TM GMFI and (B) CalRexin^TM/Annexin V positivity in cells treated with 10 µg/mL LPS at the 6, 24 and 48-h time points. (C) CalRexin^TM GMFI and (D) CalRexin^TM/Annexin V positivity in cells treated with 1 µM staurosporine at the 6, 24 and 48-h time points. Stacked bar charts represent the percentage of total live cell expressing CalRexin^TM+, CalRexin^TM+ Annexin V⁺, Annexin V⁺, and CalRexin^TM– Annexin V^–. (E) Representative flow cytometry plot of the cells treated with 1 µM staurosporine at the 6, 24 and 48-h time points; Q1 quadrant; Annexin V⁺, Q2; CalRexin^TM– Annexin V^–, Q3; CalRexin^TM+ Annexin V⁺, and Q4 CalRexin^TM+ populations. Cells were stained with CalRexin^TM –AF647 for CalRexin^TM and APC–Fire750 antibodies for Annexin V detection. Data are from two independent experiments conducted in triplicate, mean $\pm$ SEM, *p 0.05 compared to 0 h untreated Control.

In MDA-MB-468 cells, CalRexin^TM fluorescent signal was not significantly altered at early time points; however, an increase was observed at 48 h of LPS treatment (Fig. 5A; p = 0.01). Similar to MDA-MB-231, most of the cells treated with LPS do not express CalRexin^TM nor Annexin V (Fig. 5B). Staurosporine treatment resulted in increased CalRexin^TM GMFI at 24 and 48 h (Fig. 5C; p 0.0001). CalRexin^TM single-positive cells began to appear after 24 h of treatment while the Annexin V single-positive population appeared after 6 h (Fig. 5D,E). Cells positive for both markers accounted for the majority of the population after 48 h.

Fig. 5.

Time-dependent effects of LPS and staurosporine (STA) on CalRexin^𝐓𝐌 and Annexin V staining in MDA-MB-468 cells. (A) CalRexin^TM GMFI and (B) CalRexin^TM/Annexin V positivity in cells treated with 10 µg/mL LPS at the 6, 24 and 48-h time points. (C) CalRexin^TM GMFI and (D) CalRexin^TM/Annexin V positivity in cells treated with 1 µM staurosporine at the 6, 24 and 48-h time points. Stacked bar charts represent the percentage of total live cell expressing CalRexin^TM+, CalRexin^TM+ Annexin V⁺, Annexin V⁺, and CalRexin^TM– Annexin V^–. (E) Representative flow cytometry plot of the cells treated with 1 µM staurosporine at the 6, 24 and 48-h time points; Q1 quadrant; Annexin V⁺, Q2; CalRexin^TM– Annexin V^–, Q3; CalRexin^TM+ Annexin V⁺, and Q4 CalRexin^TM+ populations. Cells were stained with CalRexin^TM: AF647 for CalRexin^TM and APC–Fire750 antibodies for Annexin V detection. Data are from two independent experiments conducted in triplicate, mean $\pm$ SEM, *p 0.05 compared to 0 h untreated Control.

To assess cell viability in response to treatment with 10 µg/mL LPS or 1 µM staurosporine, MDA-MB-231 and MDA-MB-468 cells were stained with propidium iodide (PI), analysed by flow cytometry, and the percentage of non-viable cells was quantified by gating the PI-positive population. For this study, both live and heat-killed cells were used to define the boundary of the PI-positive population which represented dead cells. LPS treatment did not increase the proportion of PI-positive MDA-MB-231 (Fig. 6A,C) or MDA-MB-468 (Fig. 6B,D) cells, indicating that LPS did not adversely affect cell viability under these conditions. In contrast, staurosporine treatment reduced viability to below 90% in MDA-MB-231 and approximately 70% in MDA-MB-468 cells, consistent with induction of cell death.

Fig. 6.

Viability study of breast cancer cells using PI staining with flow cytometry over time. (A) Percentage of live MDA-MB-231 and (B) MDA-MB-468 cells treated with 10 µg/mL LPS and 1 µM staurosporine (STA) at the indicated time points (6, 24, and 48 h). (C) Representative flow cytometry PI scatter plots displaying the distribution of the MDA-MB-231 and (D) MDA-MB-468 cells population after 48 h of treatment with 10 µg/mL LPS and 1 µM staurosporine. Data are from two independent experiments conducted in triplicate, mean $\pm$ SEM.

To investigate whether LPS-induced calcitonin receptor positivity was associated with NF-κB activation, immunofluorescent co-staining for CalRexin^TM and NF-κB was performed in MDA-MB-231 cells. Minimal CalRexin^TM staining was observed in untreated controls, with NF-κB staining localised to the cytoplasm (Fig. 7A–D). Following 4 h of LPS treatment, CalRexin^TM positive cells were increased and NF-κB was detected (Fig. 7E–H). In staurosporine-treated cells, CalRexin^TM staining was evident and NF-κB staining remained predominantly cytoplasmic (Fig. 7I–L). Quantitative analysis demonstrated that CalRexin^TM fluorescence signal was increased by LPS and staurosporine treatment compared with controls (Fig. 7M; LPS p = 0.003, STA p = 0.001), and there was increased colocalisation between NF-κB and nuclei DAPI staining when cells were treated with LPS (Fig. 7N; p = 0.001). These findings indicate that NF-κB activation occurs alongside increased CalRexin^TM fluorescence signal in LPS-treated breast cancer cells but not in staurosporine-treated cells.

Fig. 7.

CalRexin^𝐓𝐌 and NF-κB immunofluorescence staining of breast cancer cells following LPS or staurosporine (STA) treatment. (A–D) MDA-MB-231 cells were untreated controls, (E–H) treated with 10 µg/mL LPS , or (I–L) treated with 1 µM staurosporine (STA) for 4 h. Representative images of DAPI in nuclei (A,E,I; blue), CalRexin^TM (B,F,J; green) and NF-κB-p65 (C,G,K; red), and the merged image of all three stains (D,H,L). (M) Percentage of CalRexin^TM positive cells and (N) colocalisation of DAPI (nuclei) and NF-κB-p65. Images were acquired on an LSM 900 confocal microscope using a 20$\times$ objective lens, all images (A–L) are the same scale with 20 µm scale bar shown in A, E, and I. Data are from two independent experiments conducted in triplicate, *p 0.05 compared to untreated Control.