In vitro detection of breast and cervical cancer cells using a novel fluorescent choline derivative

In vitro detection of breast and cervical cancer cells using a novel fluorescent choline derivative | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article In vitro detection of breast and cervical cancer cells using a novel fluorescent choline derivative Anna Eleonora Caprifico, Luca Vaghi, Peter Spearman, Gianpiero Calabrese, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4643928/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 20 Nov, 2024 Read the published version in BMC Medical Imaging → Version 1 posted 9 You are reading this latest preprint version Abstract Introduction Cervical and breast cancers can be easily preventable but they still represent the main causes of cancer-related deaths of women worldwide. Detecting cancer at its early stages is key since treatment of pre-invasive lesions is more efficient than treating an invasive disease. However, there no screening method that is highly sensitive and specific, as well as affordable and practical is currently available. Therefore, the identification of markers complementing traditional cyto/histopathological assessments is needed. Alterations in choline metabolism represents a hallmark of many malignancies, including cervical and breast cancers. Choline radiotracers are widely used for several imaging purposes for the detection of tumours, even though there are many risks associated with the use of radioactivity. Therefore, the aim of this work was to synthesize and characterize a choline tracer based on fluorinated acridine scaffold (CFA) for the in vitro detection of cervical and breast cancer cells. Methods CFA was synthesized, fully characterized and tested for cytotoxicity on breast (MCF-7) and cervical (HeLa) cancer cell lines. CFA’s uptake by cancer cells was investigated by confocal microscopy and its intracellular intensity was studied by fluorescence means; a comparative uptake between living normal and cancer cells was also conducted. Results An enhanced intensity of CFA was recorded in breast cancer cells compared to cervical cancer cells in both confocal and fluorescence microscope analysis ( p ≤ 0.001). Weak signal intensity of CFA was recorded in normal cells ( p ≤ 0.0001). CFA was toxic at much higher concentrations (HeLa IC 50 = 200 ±18 µM and MCF-7 IC 50 =105 ±3 µM) than the one needed for its detection in cancer cells (5 µM). Conclusions Results showed that CFA preferentially accumulated in cancer cells rather than in normal ones. This suggests that CFA may be a potential diagnostic probe in discriminating healthy tissues from malignant ones, due to its specific and highly sensitive features; CFA may also represent a useful tool for in vitro investigations of choline metabolism in cervical and breast cancers. Choline fluorescent dye biomarker tracer cellular uptake cervical cancer breast cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Cervical and breast cancers represent the leading causes of cancer-related deaths of women worldwide even though they are easily preventable [ 1 ]; cervical and breast screening programs are offered to all women from the age of 25 and 50, respectively [ 2 ]. Cervical cancer starts from the uterine cervix, mostly caused by an enduring infection with the human papillomavirus (HPVs), activating oncogenes and leading to the development of a pre-invasive cervical intra-epithelial neoplasia and invasive cervical carcinoma [ 3 ]. Detection of infection is considered a useful marker for its diagnosis [ 3 , 4 ]. Instead, breast cancer may have multitude causes: the presence of steroid hormone receptors (HRs) along with tumour-infiltrating lymphocytes (TILs) represent the main prognostic factors [ 5 ]. Breast cancer is currently detected in clinics using several techniques such as gene expression assays, Breast Cancer Index (BCI) [ 6 ], and Genomic Grade Index (GGI) [ 7 ]. In addition, mammography using low-dose x-rays is employed as a preventative measure; indeed, this technique can provide high quality images, detecting breast cancer before it is palpable and has spread to the axillary nodes [ 8 ]. The early detection of cancer is crucial because the treatment of pre-invasive lesions is significantly more effective than treating an invasive disease. Yet, there is no screening method available that is both highly sensitive and specific, as well as affordable and practical. Consequently, there is a pressing need to identify markers that can complement traditional cyto/histopathological assessments and detect abnormal cells within healthy tissues [ 9 ]. Abnormal and uncontrolled cell proliferation are key features of cancer, mainly because of genetic and epigenetic changes regulating cell growth, differentiation and cell death [ 9 ]. A commonly used biomarker for the detection of cancer is choline, due to its high metabolism in cancer cells. Choline is an essential nutrient, involved in several cellular functions besides being involved in lipid metabolism and in the composition of cellular membranes [ 10 ]. Morozov et al .[ 11 ] provided a review on the role of choline in cell life and its potential as a biomarker of different pathologies. Indeed, in the last 20 years, much research has focused on targeting cancer cells by means of choline phospholipid metabolism [ 12 – 14 ]. However, the main drawback of research involving choline metabolism is the predominant use of radiotracers ( 11 C-choline or 18 F-fluoromethylcholine). Radiotracers are highly specific and produce real time and defined images, but these radiotracers have a short half-life and need to be stored and handled safely [ 15 ]. To overcome the problems associated with radioactivity, our group previously generated a non-radioactive choline tracer, using its self-fluorescence for detection in living breast cancer cells, and demonstrating a preferential accumulation in breast cancer cells compared to normal ones [ 16 ]. In the present study, we have synthesized a novel probe in which choline is linked to a fluorinated acridine (choline fluorinated acridine – CFA ( 1 ), Fig. 1 ) which acts both as fluorescent dye and DNA intercalator, enabling its intracellular visualization by fluorescence means while also being a powerful and selective cytotoxic agent [ 17 – 19 ]. We selected fluorinated acridine scaffold to increase bioavailability by enhancing the hydrophobic interactions with choline transporters as well as to exploit peculiar optical properties of fluorinated acridine systems [ 19 – 21 ]. The choline derivative tracer was tested on both cervical and breast tumour cells in terms of cellular uptake and cytotoxicity. CFA is a water-soluble molecule and results showed its cytotoxicity in a dose-dependent manner. Its emission lies in the visible region, therefore easily visualized in tumour living cells by fluorescence means. These results suggest a potential employment of CFA ( 1 ) as cervical and breast cancer cells in vitro tracer. Materials and methods Materials All reagents and solvents needed for the synthesis of CFA ( 1 ) were purchased from commercial sources (Fluorochem Co.; Tokyo Chemical Industry Co. BLD Co and Aldrich Chemical Co.) and used as received. Chromatographic purifications were performed using Merck 9385 silica gel, pore size 60 Å (230–400 mesh). Dulbecco’s Minimum Essential Medium (DMEM), penicillin/streptomycin, phosphate buffer saline (PBS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), foetal bovine serum (FBS), were purchased from Merck Millipore (UK). Fibroblast Growth Medium (FGM) was purchased from Promocell (Heidelberg, Germany). Chemical Characterization Melting points were measured with a Stanford Research Systems Optimelt apparatus. Infra-red (IR) spectra were recorded with a Perkin Elmer Spectrum 100 FT-IR spectrometer equipped with universal ATR sampling accessory. 1 H, 13 C and 19 F NMR were recorded with a Bruker AVANCE III HD 400 MHz spectrometer ( 1 H: 400 MHz, 13 C: 101 MHz, 19 F: 376 MHz). Chemical shifts (δ) are expressed in parts per million (ppm), and coupling constants are given in Hz. Splitting patterns are indicated as follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad. Elemental analyses were obtained with an Elementar vario MICRO cube instrument. Synthesis 2-[ethyl(5,6,7,8-tetrafluoroacridin-2-yl)amino]ethanol ( 3 ). A solution of pentafluorobenzaldehyde ( 2 ) (0.7 g, 3.6 mmol), DABCO (1,6 g, 14.4 mmol) and 2-[(4-aminophenyl)(ethyl)amino]ethanol sulfate (2.0 g, 7.2 mmol) in decahydronaphthalene (40 mL) was heated at 175°C for 72 h under nitrogen atmosphere. After cooling to rt, the crude mixture was purified by column chromatography (SiO 2 , CH 2 Cl 2 /EtOH 98:2) to afford 3 (650 mg, 53%). Orange solid; mp 184°C; 1 H NMR (400 MHz, CDCl 3 ) δ 8.59 (s, 1H), 8.14 (d, J = 9.7 Hz, 1H), 7.64 (dd, J = 9.7, 2.9 Hz, 1H), 6.90 (d, J = 2.8 Hz, 1H), 3.94 (t, J = 5.8 Hz, 2H), 3.67 (t, J = 5.8 Hz, 2H), 3.61 (q, J = 7.1 Hz, 2H), 1.84 (br, 1H), 1.29 (t, J = 7.1 Hz, 3H); 13 C NMR (101 MHz, CDCl 3 ) δ 146.4 (s), 144.9 (s), 143.6–142.9 (m), 141.7–140.3 (m), 139.1–137.7 (m), 130.5 (s), 128.5 (s), 124.7 (s), 124.0 (s), 122.9–122.3 (m), 115.4 (d, J = 15.9 Hz), 113.3 (s), 101.8 (s), 60.3 (s), 52.4 (s), 45.7 (s), 12.1 (s); 19 F NMR (376 MHz, CDCl 3 ) δ -152.17 (t, J = 16.4 Hz), -153.00 (t, J = 15.8 Hz), -157.28 (t, J = 16.2 Hz), -159.35 (t, J = 17.0 Hz); IR (ATR) 3315, 2979, 2955, 2893, 1679, 1615, 1593, 1501, 1491, 1463, 1426, 1367, 1339, 1286, 1250, 1238, 1163, 1127, 1082, 1058, 1025, 998, 950, 901, 822, 806, 774, 665, 603, 575 cm − 1 ; Anal. Calcd. For C 17 H 14 F 4 N 2 O: C, 60.36; H, 4.17; N, 8.28. Found: C, 60.58; H, 4.12; N, 8.19. (Additional file 1) N -(2-bromoethyl)- N -ethyl-5,6,7,8-tetrafluoroacridin-2-amine ( 4 ).2-[ethyl(5,6,7,8-tetrafluoroacridin-2-yl)amino]ethanol ( 3 ) (650 mg, 1.92 mmol) and CBr 4 (776 mg, 2.34 mmol) were dissolved in dry CH 2 Cl 2 (33 mL), under nitrogen atmosphere. The solution was cooled to 0°C and PPh 3 (613 mg, 2.34 mmol) was added. After stirring for 10 minutes, the reaction was allowed to warm to rt and left under stirring overnight. Solvent was then removed under reduced pressure and the residue was purified by column chromatography (SiO 2 , first CH 2 Cl 2 /heptane 1:1, then CH 2 Cl 2 ) to afford 4 (473 mg, 61%). Yellow solid; mp 172°C; 1 H NMR (400 MHz, CDCl 3 ) δ 8.51 (s, 1H), 8.10 (d, J = 9.7 Hz, 1H), 7.52 (dd, J = 9.7, 2.9 Hz, 1H), 6.80 (d, J = 2.8 Hz, 1H), 3.85 (t, J = 7.8 Hz, 2H), 3.71–3.45 (m, 4H), 1.31 (t, J = 7.1 Hz, 3H); 13 C NMR (101 MHz, CDCl 3 ) δ 145.1 (s), 144.7 (s), 143.3–140.4 (m), 141.5–138.6 (m), 138.5–137.6 (m), 135.5 (t, J = 16.3 Hz), 132.4 (d, J = 10.7 Hz), 130.8 (s), 128.2 (s), 124.8 (d, J = 6.1 Hz), 123.0 (s), 115.2 (d, J = 14.7 Hz), 101.6 (s), 52.3 (s), 45.6 (s), 27.9 (s), 12.7 (s); 19 F NMR (376 MHz, CDCl 3 ) δ -151.99 (t, J = 16.7 Hz), -152.89 (t, J = 16.0 Hz), -157.05 (t, J = 16.5 Hz), -159.16 (t, J = 17.2 Hz). ; IR (ATR) 3083, 3060, 3038, 2986, 2927, 2870, 1682, 1618, 1593, 1495, 1459, 1424, 1358, 1342, 1262, 1218, 1202, 1150, 1103, 1072, 1026, 994, 941, 904, 816, 658, 643, 541 cm − 1 ; Anal. Calcd. For C 17 H 13 BrF 4 N 2 : C, 50.89; H, 3.27; N, 6.98. Found: C, 50.82; H, 3.30; N, 7.01. (Additional file 2) 2-[ethyl(5,6,7,8-tetrafluoroacridin-2-yl)amino]- N -(2-hydroxyethyl)- N , N -dimethylethanaminium bromide ( 1 ). Dimethylethanolamine (200 mg, 2.24 mmol) was slowly added to a 0°C solution of 4 (450 mg, 1.12 mmol) in dry THF (5 mL), under nitrogen atmosphere. The mixture was allowed to reach rt and then stirred at 50°C overnight. The solid formed was filtered and washed extensively with cold diethyl ether to afford 1 (200 mg, 41%). Brown solid; 1 H NMR (400 MHz, D 2 O) δ 7.64 (s, 1H), 7.40–7.23 (m, 2H), 6.34 (d, J = 1.4 Hz, 1H), 4.10 (s, 2H), 3.88–3.81 (m, 2H), 3.66–3.54 (m, 4H), 3.40 (dd, J = 13.8, 6.7 Hz, 2H), 3.29 (s, 6H), 1.18 (t, J = 7.0 Hz, 3H); 13 C NMR (101 MHz, D 2 O) δ 144.7 (s), 141.4 (s), 141.0–138.1 (m), 139.9–137.1 (m), 139.4–136.3 (m), 136.2–133.2 (m), 128.9 (d, J = 9.0 Hz), 127.2 (s), 126.2 (s), 124.0 (s), 123.1 (s), 113.3–112.3 (m), 100.6 (s), 65.5 (s), 58.7 (s), 55.4 (s), 55.1 (s), 51.9 (s), 42.6 (s), 11.3 (s); 19 F NMR (376 MHz, D 2 O) δ -151.94 (s), -154.37 (s), -156.97 (s), -159.66 (s); IR (ATR) 3302, 3033, 2977, 1682, 1618, 1591, 1503, 1488, 1427, 1367, 1342, 1250, 1184, 1158, 1077, 1026, 997, 961, 945, 917, 825, 817, 776, 656, 603, 562 cm − 1 ; Anal. Calcd. For C 21 H 24 BrF 4 N 3 O: C, 51.44; H, 4.93; N, 8.57. Found: C, 51.33; H, 4.98; N, 8.64. (Additional file 3) Characterization of spectral properties Absorption spectra of CFA ( 1 ) in PBS (100 µM) were recorded using Agilent – Cary UV-Vis. Photoluminescence measurements were carried out on Cary Eclipse Fluorescence Spectrophotometer. Cell culture Human cervical (HeLa) cancer cells, Michigan Cancer Foundation-7 (MCF-7) cells and Human Cardiac Fibroblasts (HCF) were obtained from Kingston University. HeLa cells and MCF-7 cells were maintained in DMEM supplemented with 10% FBS and 100 µg/mL penicillin/streptomycin. HCF were maintained in FGM. All cell lines were grown at 37°C and in a 5% CO 2 humidified atmosphere. In vitro cell toxicity study HeLa and MCF-7 cells were seeded in a 96-wells plate at 1 ×10 5 cells/mL in supplemented medium (100 µL) and grown overnight. Cells were then incubated at increasing concentrations of CFA ( 1 ) (12.5, 25, 50, 100, 150, 200 and 250 µM) for 72 h. The cell viability was then tested, using the MTT assay: the water-soluble MTT is converted into water-insoluble purple formazan in metabolically active cells by the action of the mitochondrial reductase; the amount of formazan produced is directly proportional to the number of living cells [ 22 ]. Briefly, MTT (20 µL, final concentration of 0.1 mg/mL) was added into each well and incubated for 4 h at 37°C, in the dark. The medium was then discarded, cells were carefully washed with PBS to remove any residue of fluorescent compound and the formazan products were dissolved in DMSO (100 µL). The plate was then shaken for 15 min at rt in the dark, after which the absorbance was measured at 550 nm with a reference at 690 nm [ 22 ]. The experiment was run in triplicate. The cell viability was expressed as percentage (%) and calculated by dividing the absorbance of the cells treated with CFA by the absorbance of control (cells treated with medium only). The IC 50 value was extrapolated from the graph plotted using cell viability data against concentration [ 23 ]. Cellular uptake by confocal microscopy on fixed cells HeLa and MCF-7 cells were plated onto glass coverslips in 6-well plates, at a density of 0.7 × 10 6 cells/well and grown overnight. Cells were then incubated with CFA ( 1 ) at 5 µM for 8, 16 and 24 h. At each time point, cells were washed with PBS, fixed with paraformaldehyde (4%) for 20 min and rehydrated in PBS for 1 h, at rt. Specimens were then mounted and stored at 4°C until examination using a confocal microscope (Zeiss Axiovert 200M, Oberkochen, Germany) equipped with a LSM 5 Image Browser (Carl Zeiss, Oberkochen, Germany). An oil immersion objective (63x) was employed. Cellular uptake by fluorescence microscopy on living cells HCF, HeLa and MCF-7 cells were plated onto 6-wells plate, at a density of 0.7 × 10 6 cells/well and grown overnight. Cells were then incubated with CFA ( 1 ) at 5 µM for 24 h, at 37°C and in a 5% CO 2 humidified atmosphere. Cells were then washed with PBS (×3) and visualized alive on a Leica DM750 Fluorescence Microscope, using a 20× objective. Quantification of fluorescent intensity ImageJ software (NIH, USA) was used to quantify the signal intensity corresponding to CFA as described by Shihan et al .[ 24 ]. Statistical analysis Data were expressed as the mean ± standard deviation (SD). Statistical analyses were performed using one-way analysis of variance (ANOVA) with significance set at p < 0.05. A post-hoc test (Tukey multiple comparison test) was performed to determine differences between groups. All statistical analyses were carried out using GraphPad Prism 4.0 (GraphPad Software, Inc.). Results Synthesis and characterization of CFA (1) CFA ( 1 ) was synthesized starting from pentafluorobenzaldehyde ( 2 ) (Scheme 1). Tandem nucleophilic aromatic substitution/thermal promoted electrocyclization-aromatization by treatment with in-situ prepared 2‑[(4‑aminophenyl)(ethyl)amino]ethanol in decahydronaphthalene at 175 °C afforded 2-[ethyl(5,6,7,8-tetrafluoroacridin-2-yl)amino]ethanol ( 3 ) [25,26]. 3 was then converted into N -(2-bromoethyl)- N -ethyl-5,6,7,8-tetrafluoroacridin-2-amine ( 4 ) by reaction with CBr 4 and triphenylphosphine [27]. Finally, treatment of 4 with 2‑(dimethylamino)ethanol allowed the formation of 1 . Figure 2 shows the absorption – emission spectra of CFA ( 1 ), with Stokes shift (116 nm (0.57 eV) between absorption and emission maxima (450 nm and 566 nm, respectively). Cell toxicity in a dose-dependent manner The ability of CFA ( 1 ) to inhibit the metabolic activity of breast and cervical cancer cells in a dose-dependent manner was determined using the MTT assay (Figure 3). The viability of HeLa cells was above the 80% when concentration of CFA up to 50 µM were used over an incubation time of 72 h, while a sharp decrease in the cell viability was recorded upon higher concentrations. Instead, incubation of MCF-7 cells with increasing concentrations of CFA for 72 h experienced a steady reduction in the cell viability up to 40%. The IC 50 values determined for HeLa and MCF-7 cells were 200 ±18 µM and 105 ±3 µM, respectively, suggesting that MCF-7 cells were more susceptible to the cytotoxic action of CFA than HeLa cells. Cellular uptake of CFA by cancer cells over time Confocal microscopy was used to investigate the cellular uptake of CFA ( 1 ) by HeLa and MCF-7 cells, in a time-dependant manner (6, 16 and 24 h). At each time point, cells on coverslips were washed and fixed, before being mounted on a glass slide and observed using the confocal microscope. The excitation of the fluorescent dye was carried out at a wavelength falling within the absorption band of the fluorinated acridine moiety. After 6 h incubation, a low signal of CFA ( 1 ) was observed in HeLa cells (Figure 4) while appearing more evident after 24 h (p ≤ 0.01). Especially, the fluorescent signal of CFA ( 1 ) was detected in the perinuclear region of both HeLa and MCF-7 cells, with the intensity increasing at very high level in MCF-7 cells after 16 h incubation (p ≤ 0.0001), suggesting a higher uptake of CFA ( 1 ) in MCF-7 cells than HeLa cells. No signal corresponding to CFA was detected at incubation time lower than 6 h (data not shown). Cellular uptake of CFA (1) by normal and cancer living cells The cellular uptake of CFA ( 1 ) was assessed in living cells, both normal (HCF) and tumour (HeLa and MCF-7 cells) by fluorescence microscopy (Figure 5). In normal fibroblasts, a very low signal was noticed, indicating poor uptake of CFA ( 1 ) (p ≤ 0.0001). Instead, a strong fluorescence signal of CFA ( 1 ) was observed in the perinuclear region of HeLa cells. The signal appeared more pronounced and widespread in MCF-7 cells, indicating that the uptake of CFA ( 1 ) was higher in breast cancer cells compared to cervical cancer cells (p ≤ 0.001). Discussion The aim of this study was to investigate the ability of a novel choline derivative to act as a fluorescent choline tracer for tumours characterized by an increase metabolism of choline such as cervical and breast cancers. The novel fluorescent choline tracer was successfully synthesized and its optical properties characterized. As shown in Fig. 2 , the relatively large Stokes shift between absorption and emission maxima of CFA (116 nm) is evidence of the charge-transfer nature of the transition. Since the transitions responsible for the emission and absorption reside on the acridine moiety, the tertiary amine can participate in the acridine conjugated system in the excited state – consequently increasing the extent of conjugation and effectively lowering the emission transition energy. Such large Stokes Shift minimises interference between the excitation and the fluorescence emission, prevents self-quenching and is highly suited for bioimaging. Results showed in Figs. 4 and 5 suggested that CFA ( 1 ) possesses efficient optical properties that allow its intracellular detection at very low concentrations (5 µM) and incubation time (up to 6 h) with no induction of cell toxicity. Figure 5 shows the selective ability of CFA ( 1 ) to accumulate preferentially in cancer cells rather than in normal heart fibroblasts. These results correlate well with our previous study [ 16 ] where choline and phosphatidylcholine fluorescent derivatives accumulated preferentially in malignant breast cells in which choline uptake and metabolism are augmented [ 28 , 29 ]. This feature is key upon intravenous administration of the tracer, since it allows for specific accumulation in malignant cells while averting healthy ones. Moreover, since acridine derivatives generally have good inhibitory activity against Topoisomerase I/II, the cytotoxicity activity may be related to the inhibition of this enzyme. Indeed, acridine derivatives have been widely explored for their excellent anticancer activity in several studies [ 19 , 30 , 31 ]. Being an acridine derivative, CFA ( 1 ) was screened for its cytotoxicity toward tumour cells: cytotoxic results were expressed as growth inhibitory concentration (IC 50 ) values representing the compound concentration needed to achieve a 50% inhibition of cell growth after 72 h incubation compared to the untreated control [ 32 ]. IC 50 values of CFA indicated excellent growth inhibition against MCF-7 cells (100 µM), which was similar to other acridine derivatives, when compared to the reference drug doxorubicin (IC 50 = 65 µg/mL) [ 32 ]. Instead, growth inhibitory effects of CFA against HeLa cells were not significant compared to MCF-7 cells or compared to other acridine derivatives and Camptothecin [ 23 , 33 ]. This might correlate to the higher cellular uptake of CFA by MCF-7 cells compared to HeLa cells, as suggested by the fluorescent intensity data, where the fluorescent signal in MCF-7 cells was higher compared to the signal of HeLa cells. MCF-7 is a well-established cell line to study the altered phospholipid metabolism in breast cancer [ 16 , 34 , 35 ], since high choline levels were detected in these cancer cells [ 36 ]. However, previous studies also demonstrated a high choline uptake by HeLa cells [ 37 – 39 ]. By comparison, this preliminary study suggests that choline uptake is higher in breast cancer compared to cervical cancer yet further studies are needed to establish a potential correlation. Conclusion A novel fluorescent tracer based on choline was synthesised for the in vitro detection of cervical and breast cancers. Choline linked to fluorinated acridine (CFA) was tracked by its self-fluorescence and exhibited a preferential uptake by cancer cells compared to healthy ones ( p ≤ 0.0001). These results suggest that CFA ( 1 ) could be used as potential biomarker in the context of early diagnosis of cancerogenic lesions, being a highly sensitive, specific, affordable and practical screening tool that may complement already existing diagnostic techniques in the context of cervical and breast cancer. Abbreviations CFA, Choline Fluorinated Acridine HPV, Human Papilloma Virus HR, Hormone Receptor TIL, Tumour-Infiltrating Lymphocytes BCI, Breast Cancer Index GGI, Genomic Grade Index DNA, Deoxyribonucleic Acid ATR, Attenuated Total Reflectance IR, InfraRed NMR, Nuclear Magnetic Resonance DMEM, Dulbecco′s Modified Eagle′s Medium PBS, Phosphate-buffered saline MTT, 4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide DMSO, Dimethylsulfoxide DABCO, 1,4-diazabicyclo [2.2.2] octane THF, tetrahydrofuran rt, room temperature FBS, Foetal Bovine Serum FGM, Fibroblast Growth Medium UV-Vis, Ultra-violet visible MCF-7, Michigan Cancer Foundation-7 HCF, Human Cardiac Fibroblast IC 50 , Inhibitory Concentration ANOVA, Analysis of Variance Declarations Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Competing interests The authors declare that they have no competing interests Funding No external funding was received for this research. Authors' contributions AC performed and analysed data regarding cell toxicity and cellular uptake studies. LV was involved in the synthesis and characterization of the compound. PS performed the optical characterisation of the compound. AP and GC supervised the work. All authors read and approved the final manuscript. Acknowledgements Not applicable References Nair M, Sandhu SS, Sharma AK. Cancer molecular markers: A guide to cancer detection and management. Semin Cancer Biol 2018;52:39–55. https://doi.org/10.1016/j.semcancer.2018.02.002. NHS England. Screening and earlier diagnosis 2024. www.england.nhs.uk/cancer/early-diagnosis/screening-and-earlier-diagnosis/. Cheng Q, Lau WM, Chew SH, Ho TH, Tay SK, Hui KM. Identification of molecular markers for the early detection of human squamous cell carcinoma of the uterine cervix. 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Bypassing the statistical limit of singlet generation in sensitized upconversion using fluorinated conjugated systems. Photochemical and Photobiological Sciences 2022;21:913–21. https://doi.org/10.1007/s43630-022-00225-z. Mosmann T. Rapid colorimetric assay for cellular growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65:55–63. https://doi.org/10.1016/0022-1759(83)90303-4. Satheeshkumar R, Edatt L, Muthusankar A, Sameer Kumar VB, Rajendra Prasad KJ. Synthesis of Novel Quin[1,2-b]Acridines: InVitro Cytotoxicity and Molecular Docking Studies. Polycycl Aromat Compd 2021;41:1631–45. https://doi.org/10.1080/10406638.2019.1689515. Shihan MH, Novo SG, Le Marchand SJ, Wang Y, Duncan MK. A simple method for quantitating confocal fluorescent images. Biochem Biophys Rep 2021;25:100916. https://doi.org/10.1016/j.bbrep.2021.100916. Papagni A, del Buttero P, Moret M, Sassella A, Miozzo L, Ridolfi G. Synthesis and Properties of Some Derivatives of 1,2,3, 4-Tetrafluoroacridine for Solid State Emitting Systems. Chemistry of Materials 2003;15:5010–8. https://doi.org/10.1021/cm034504i. Liu K, Brivio M, Xiao T, Norwood VM, Kim YS, Jin S, et al. Modular Synthetic Routes to Fluorine-Containing Halogenated Phenazine and Acridine Agents That Induce Rapid Iron Starvation in Methicillin-Resistant Staphylococcus aureus Biofilms. ACS Infect Dis 2022;8:280–95. https://doi.org/10.1021/acsinfecdis.1c00402. Kienzler MA, Reiner A, Trautman E, Yoo S, Trauner D, Isacoff EY. A red-shifted, fast-relaxing azobenzene photoswitch for visible light control of an ionotropic glutamate receptor. J Am Chem Soc 2013;135:17683–6. https://doi.org/10.1021/ja408104w. Katz-Brull R, Seger D, Rivenson-Segal D, Rushkin E, & Degani H. Metabolic markers of breast cancer: enhanced choline metabolism and reduced choline-ether-phospholipid synthesis. Cancer res 2002; 62(7), 1966-1970. Aboagye EO, Zaver MB. Malignant transformation alters membrane choline phospholipid metabolism of human mammary epithelial cells. Cancer res 59.1 1999: 80-84. Zhang B, Dou Z, Xiong Z, Wang N, He S, Yan X, et al. Design, synthesis and biological research of novel N-phenylbenzamide-4-methylamine acridine derivatives as potential topoisomerase I/II and apoptosis-inducing agents. Bioorg Med Chem Lett 2019;29. https://doi.org/10.1016/j.bmcl.2019.126714. Kozurkova M. Acridine derivatives as inhibitors/poisons of topoisomerase II. Journal of Applied Toxicology 2022;42:544–52. https://doi.org/10.1002/jat.4238. Fahim AM, Tolan HEM, El-Sayed WA. Synthesis of novel 1,2,3-triazole based acridine and benzothiazole scaffold N-glycosides with anti-proliferative activity, docking studies, and comparative computational studies. J Mol Struct 2022;1251. https://doi.org/10.1016/j.molstruc.2021.131941. Haider MR, Ahmad K, Siddiqui N, Ali Z, Akhtar MJ, Fuloria N, et al. Novel 9-(2-(1-arylethylidene)hydrazinyl)acridine derivatives: Target Topoisomerase 1 and growth inhibition of HeLa cancer cells. Bioorg Chem 2019;88. https://doi.org/10.1016/j.bioorg.2019.102962. Adriá-Cebrián J, Guaita-Esteruelas S, Lam EWF, Rodríguez-Balada M, Capellades J, Girona J, et al. Mcf-7 drug resistant cell lines switch their lipid metabolism to triple negative breast cancer signature. Cancers (Basel) 2021;13. https://doi.org/10.3390/cancers13235871. Al-Saeedi F, Smith T, Welch A. [Methyl-3H]-choline Incorporation into MCF-7 Cells: Correlation with Proliferation, Choline Kinase and Phospholipase D Assay. Anticancer Res 2007;27:901–6. Bolan PJ. Magnetic Resonance Spectroscopy of the Breast: Current Status. Magn Reson Imaging Clin N Am 2013;21:625–39. https://doi.org/10.1016/j.mric.2013.04.008. Roppongi M, Mitsuru Izumisawa ·, Terasaki · Kazunori, Muraki · Yasushi, Shozushima · Masanori. 18F-FDG and 11C-choline uptake in proliferating tumor cells is dependent on the cell cycle in vitro 2019;33:237–43. https://doi.org/10.1007/s12149-018-01325-6. Iorio E, Mezzanzanica D, Alberti P, Spadaro F, Ramoni C, D’Ascenzo S, et al. Alterations of Choline Phospholipid Metabolism in Ovarian Tumor Progression. Cancer Res 2005;65:9369–76. https://doi.org/10.1158/0008-5472.CAN-05-1146. Guy GR, Murray2 AW. Tumor Promoter Stimulation of Phosphatidylcholine Turnover in HeLa Cells1. Cancer Res 1980;42. Scheme Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files Additionalfile1.docx Additionalfile2.docx Additionalfile3.docx Scheme1.png Cite Share Download PDF Status: Published Journal Publication published 20 Nov, 2024 Read the published version in BMC Medical Imaging → Version 1 posted Editorial decision: Revision requested 02 Aug, 2024 Reviews received at journal 29 Jul, 2024 Reviews received at journal 22 Jul, 2024 Reviewers agreed at journal 17 Jul, 2024 Reviewers agreed at journal 12 Jul, 2024 Reviewers invited by journal 12 Jul, 2024 Editor assigned by journal 03 Jul, 2024 Submission checks completed at journal 03 Jul, 2024 First submitted to journal 26 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4643928","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":330128609,"identity":"d60eefb2-b643-46db-b278-314d98f499ab","order_by":0,"name":"Anna Eleonora Caprifico","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA/ElEQVRIiWNgGAWjYFADZsYHBxgYbBgboHwDIrQwGwC1pJGihYEZpOgwYS38s3sPfmCoscszb2dmPPCj4rzs2hkJjB9+MBw2xqVF4s65ZAmGY8nFMoeZGQ72nLltvO1GArNkD8NhM5zOuZFjIMHYwJw4g5n/wGHGttuJQC0M0kAX2uDSIX8jx/gHY0M9UAsz0CNt50BamH/j02JwI8cMaMthmJYDIC1sIFtwOswQqMUi4djxYglmsF+Sjbededhm2WOQjtP7ckCH3fhQU50nwX+Y+cOPCjvZbceTD9/4UWFt2IDT/0CQAEZwAIoaIiIygaCKUTAKRsEoGLkAAIv8V5XuvxyTAAAAAElFTkSuQmCC","orcid":"","institution":"De Montfort University","correspondingAuthor":true,"prefix":"","firstName":"Anna","middleName":"Eleonora","lastName":"Caprifico","suffix":""},{"id":330128611,"identity":"92ddce1f-f745-4b99-8948-82d7f85b75c3","order_by":1,"name":"Luca Vaghi","email":"","orcid":"","institution":"University of Milano-Bicocca","correspondingAuthor":false,"prefix":"","firstName":"Luca","middleName":"","lastName":"Vaghi","suffix":""},{"id":330128615,"identity":"7306275e-e566-43ea-98f5-d27b58eae4ca","order_by":2,"name":"Peter Spearman","email":"","orcid":"","institution":"Kingston University","correspondingAuthor":false,"prefix":"","firstName":"Peter","middleName":"","lastName":"Spearman","suffix":""},{"id":330128616,"identity":"2a276c69-33c2-42f8-8924-0903888d7f53","order_by":3,"name":"Gianpiero Calabrese","email":"","orcid":"","institution":"Kingston University","correspondingAuthor":false,"prefix":"","firstName":"Gianpiero","middleName":"","lastName":"Calabrese","suffix":""},{"id":330128617,"identity":"9cdd2d04-27c6-4710-929a-f7cd8880b527","order_by":4,"name":"Antonio Papagni","email":"","orcid":"","institution":"University of Milano-Bicocca","correspondingAuthor":false,"prefix":"","firstName":"Antonio","middleName":"","lastName":"Papagni","suffix":""}],"badges":[],"createdAt":"2024-06-26 16:02:49","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4643928/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4643928/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12880-024-01488-x","type":"published","date":"2024-11-20T15:57:54+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":61000962,"identity":"bc296a42-3b97-4aaa-b938-8b004de9d0eb","added_by":"auto","created_at":"2024-07-24 13:07:13","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":7498,"visible":true,"origin":"","legend":"\u003cp\u003eChemical structure of choline-based fluorinated acridine (CFA) \u003cstrong\u003e1\u003c/strong\u003e.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4643928/v1/2aac75192fd879a4d8264609.png"},{"id":61000963,"identity":"c4696353-2d72-4057-8e82-b1cc28cb01d6","added_by":"auto","created_at":"2024-07-24 13:07:13","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":37806,"visible":true,"origin":"","legend":"\u003cp\u003eNormalized absorption and emission spectra of CFA (1) in PBS (100 µM); PL (red) @458 nm.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4643928/v1/ac28e3c4dd8480adcb3220bd.png"},{"id":61000968,"identity":"40c17eaf-67b4-4cd5-abb7-49d44b644e2c","added_by":"auto","created_at":"2024-07-24 13:07:14","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":30804,"visible":true,"origin":"","legend":"\u003cp\u003eCell viability (%) of HeLa cells and MCF-7 cells, after incubation with CFA for 72 h. Cell viability was determined using the MTT colorimetric assay. Each data is the mean of three independent experiments. The lines shown are trend lines.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4643928/v1/fffe4e87c50003c354cd1966.png"},{"id":61000964,"identity":"abd41a17-adc4-43b1-8da1-61c852ac81d5","added_by":"auto","created_at":"2024-07-24 13:07:13","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":241133,"visible":true,"origin":"","legend":"\u003cp\u003eConfocal images of HeLa cells and MCF-7 cells, incubated with CFA (5 µM) for 6, 16 and 24 h. Cells were then washed with PBS, fixed with paraformaldehyde before being mounted on a glass slide and observed under the confocal microscope. An oil immersion objective (63x) was employed. Scale bar: 10 µm. Graphs represent CFA staining intensity which could be translated to the amount of CFA taken up by the cancerous cells: the cellular uptake of CFA increased upon increasing incubation time. Asterisks indicate statistical significance: **p ≤ 0.01 and ****p ≤0.0001; ns, non-significant.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4643928/v1/54e3631b7bfbb6ebde073c18.png"},{"id":61000967,"identity":"b39d1d16-5dce-4108-a7ef-90d40292c333","added_by":"auto","created_at":"2024-07-24 13:07:14","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":167362,"visible":true,"origin":"","legend":"\u003cp\u003eFluorescence microscope images of normal HCF, and cancerous HeLa and MCF-7 cells incubated with CFA at 5 µM. After 24 h incubation, the surface of cells was washed twice to reduce unspecific signal, and fluorescent images were taken on living cells. Scale bar: 200 µm (HCF) and 100 µm (HeLa and MCF-7 cells). The graph represents CFA staining intensity which could be translated to the amount of CFA taken up by the living cells: a higher cellular uptake of CFA was recorded in cancerous cells compared to normal HCF. Asterisks indicate statistical significance: ***p ≤ 0.001 and ****p ≤ 0.0001.\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-4643928/v1/50d271a54c7c4add6e9c76c5.png"},{"id":69835190,"identity":"aa83b64d-a0ec-4d35-b996-9d2d6181d98e","added_by":"auto","created_at":"2024-11-25 16:12:55","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1119203,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4643928/v1/2c399da9-f222-4f27-9902-44205f95ce35.pdf"},{"id":61000961,"identity":"3d82f106-66b2-4ad0-bc88-ab2a55a9341a","added_by":"auto","created_at":"2024-07-24 13:07:13","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":536066,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4643928/v1/f29e6083a30685e28ca93079.docx"},{"id":61000966,"identity":"3fd104e3-0af4-4fd0-9836-e409252842ba","added_by":"auto","created_at":"2024-07-24 13:07:13","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":220938,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile2.docx","url":"https://assets-eu.researchsquare.com/files/rs-4643928/v1/d70a33959c9e07bdb6f43300.docx"},{"id":61000960,"identity":"ee80a654-a137-45f0-8bc0-a9f4f69aba19","added_by":"auto","created_at":"2024-07-24 13:07:12","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":232836,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile3.docx","url":"https://assets-eu.researchsquare.com/files/rs-4643928/v1/6f0d1edb9a833250a3a00e90.docx"},{"id":61001788,"identity":"a693fcbc-3a22-4279-803e-6e93f665a6f8","added_by":"auto","created_at":"2024-07-24 13:15:13","extension":"png","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":9429,"visible":true,"origin":"","legend":"","description":"","filename":"Scheme1.png","url":"https://assets-eu.researchsquare.com/files/rs-4643928/v1/43ef19a0adad3f40094073a7.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"In vitro detection of breast and cervical cancer cells using a novel fluorescent choline derivative","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCervical and breast cancers represent the leading causes of cancer-related deaths of women worldwide even though they are easily preventable [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]; cervical and breast screening programs are offered to all women from the age of 25 and 50, respectively [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Cervical cancer starts from the uterine cervix, mostly caused by an enduring infection with the human papillomavirus (HPVs), activating oncogenes and leading to the development of a pre-invasive cervical intra-epithelial neoplasia and invasive cervical carcinoma [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Detection of infection is considered a useful marker for its diagnosis [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Instead, breast cancer may have multitude causes: the presence of steroid hormone receptors (HRs) along with tumour-infiltrating lymphocytes (TILs) represent the main prognostic factors [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Breast cancer is currently detected in clinics using several techniques such as gene expression assays, Breast Cancer Index (BCI) [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], and Genomic Grade Index (GGI) [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In addition, mammography using low-dose x-rays is employed as a preventative measure; indeed, this technique can provide high quality images, detecting breast cancer before it is palpable and has spread to the axillary nodes [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. The early detection of cancer is crucial because the treatment of pre-invasive lesions is significantly more effective than treating an invasive disease. Yet, there is no screening method available that is both highly sensitive and specific, as well as affordable and practical. Consequently, there is a pressing need to identify markers that can complement traditional cyto/histopathological assessments and detect abnormal cells within healthy tissues [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAbnormal and uncontrolled cell proliferation are key features of cancer, mainly because of genetic and epigenetic changes regulating cell growth, differentiation and cell death [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. A commonly used biomarker for the detection of cancer is choline, due to its high metabolism in cancer cells. Choline is an essential nutrient, involved in several cellular functions besides being involved in lipid metabolism and in the composition of cellular membranes [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Morozov \u003cem\u003eet al\u003c/em\u003e.[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] provided a review on the role of choline in cell life and its potential as a biomarker of different pathologies. Indeed, in the last 20 years, much research has focused on targeting cancer cells by means of choline phospholipid metabolism [\u003cspan additionalcitationids=\"CR13\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, the main drawback of research involving choline metabolism is the predominant use of radiotracers (\u003csup\u003e11\u003c/sup\u003eC-choline or \u003csup\u003e18\u003c/sup\u003eF-fluoromethylcholine). Radiotracers are highly specific and produce real time and defined images, but these radiotracers have a short half-life and need to be stored and handled safely [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. To overcome the problems associated with radioactivity, our group previously generated a non-radioactive choline tracer, using its self-fluorescence for detection in living breast cancer cells, and demonstrating a preferential accumulation in breast cancer cells compared to normal ones [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the present study, we have synthesized a novel probe in which choline is linked to a fluorinated acridine (choline fluorinated acridine \u0026ndash; CFA (\u003cb\u003e1\u003c/b\u003e), Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e) which acts both as fluorescent dye and DNA intercalator, enabling its intracellular visualization by fluorescence means while also being a powerful and selective cytotoxic agent [\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. We selected fluorinated acridine scaffold to increase bioavailability by enhancing the hydrophobic interactions with choline transporters as well as to exploit peculiar optical properties of fluorinated acridine systems [\u003cspan additionalcitationids=\"CR20\" citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. The choline derivative tracer was tested on both cervical and breast tumour cells in terms of cellular uptake and cytotoxicity. CFA is a water-soluble molecule and results showed its cytotoxicity in a dose-dependent manner. Its emission lies in the visible region, therefore easily visualized in tumour living cells by fluorescence means. These results suggest a potential employment of CFA (\u003cb\u003e1\u003c/b\u003e) as cervical and breast cancer cells \u003cem\u003ein vitro\u003c/em\u003e tracer.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eMaterials\u003c/h2\u003e \u003cp\u003eAll reagents and solvents needed for the synthesis of CFA (\u003cb\u003e1\u003c/b\u003e) were purchased from commercial sources (Fluorochem Co.; Tokyo Chemical Industry Co. BLD Co and Aldrich Chemical Co.) and used as received. Chromatographic purifications were performed using Merck 9385 silica gel, pore size 60 \u0026Aring; (230\u0026ndash;400 mesh). Dulbecco\u0026rsquo;s Minimum Essential Medium (DMEM), penicillin/streptomycin, phosphate buffer saline (PBS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), dimethyl sulfoxide (DMSO), foetal bovine serum (FBS), were purchased from Merck Millipore (UK). Fibroblast Growth Medium (FGM) was purchased from Promocell (Heidelberg, Germany).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eChemical Characterization\u003c/h2\u003e \u003cp\u003eMelting points were measured with a Stanford Research Systems Optimelt apparatus. Infra-red (IR) spectra were recorded with a Perkin Elmer Spectrum 100 FT-IR spectrometer equipped with universal ATR sampling accessory. \u003csup\u003e1\u003c/sup\u003eH, \u003csup\u003e13\u003c/sup\u003eC and \u003csup\u003e19\u003c/sup\u003eF NMR were recorded with a Bruker AVANCE III HD 400 MHz spectrometer (\u003csup\u003e1\u003c/sup\u003eH: 400 MHz, \u003csup\u003e13\u003c/sup\u003eC: 101 MHz, \u003csup\u003e19\u003c/sup\u003eF: 376 MHz). Chemical shifts (δ) are expressed in parts per million (ppm), and coupling constants are given in Hz. Splitting patterns are indicated as follows: s\u0026thinsp;=\u0026thinsp;singlet, d\u0026thinsp;=\u0026thinsp;doublet, t\u0026thinsp;=\u0026thinsp;triplet, q\u0026thinsp;=\u0026thinsp;quartet, m\u0026thinsp;=\u0026thinsp;multiplet, br\u0026thinsp;=\u0026thinsp;broad. Elemental analyses were obtained with an Elementar vario MICRO cube instrument.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eSynthesis\u003c/h2\u003e \u003cp\u003e \u003cb\u003e2-[ethyl(5,6,7,8-tetrafluoroacridin-2-yl)amino]ethanol\u003c/b\u003e (\u003cb\u003e3\u003c/b\u003e). A solution of pentafluorobenzaldehyde (\u003cb\u003e2\u003c/b\u003e) (0.7 g, 3.6 mmol), DABCO (1,6 g, 14.4 mmol) and 2-[(4-aminophenyl)(ethyl)amino]ethanol sulfate (2.0 g, 7.2 mmol) in decahydronaphthalene (40 mL) was heated at 175\u0026deg;C for 72 h under nitrogen atmosphere. After cooling to rt, the crude mixture was purified by column chromatography (SiO\u003csub\u003e2\u003c/sub\u003e, CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e/EtOH 98:2) to afford \u003cb\u003e3\u003c/b\u003e (650 mg, 53%). Orange solid; mp 184\u0026deg;C; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) δ 8.59 (s, 1H), 8.14 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9.7 Hz, 1H), 7.64 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9.7, 2.9 Hz, 1H), 6.90 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.8 Hz, 1H), 3.94 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.8 Hz, 2H), 3.67 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;5.8 Hz, 2H), 3.61 (q, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.1 Hz, 2H), 1.84 (br, 1H), 1.29 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.1 Hz, 3H); \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) δ 146.4 (s), 144.9 (s), 143.6\u0026ndash;142.9 (m), 141.7\u0026ndash;140.3 (m), 139.1\u0026ndash;137.7 (m), 130.5 (s), 128.5 (s), 124.7 (s), 124.0 (s), 122.9\u0026ndash;122.3 (m), 115.4 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.9 Hz), 113.3 (s), 101.8 (s), 60.3 (s), 52.4 (s), 45.7 (s), 12.1 (s); \u003csup\u003e19\u003c/sup\u003eF NMR (376 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) δ -152.17 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;16.4 Hz), -153.00 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;15.8 Hz), -157.28 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;16.2 Hz), -159.35 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;17.0 Hz); IR (ATR) 3315, 2979, 2955, 2893, 1679, 1615, 1593, 1501, 1491, 1463, 1426, 1367, 1339, 1286, 1250, 1238, 1163, 1127, 1082, 1058, 1025, 998, 950, 901, 822, 806, 774, 665, 603, 575 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; Anal. Calcd. For C\u003csub\u003e17\u003c/sub\u003eH\u003csub\u003e14\u003c/sub\u003eF\u003csub\u003e4\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003eO: C, 60.36; H, 4.17; N, 8.28. Found: C, 60.58; H, 4.12; N, 8.19. (Additional file 1)\u003c/p\u003e \u003cp\u003e \u003cb\u003eN\u003c/b\u003e \u003cb\u003e-(2-bromoethyl)-\u003c/b\u003e \u003cb\u003eN\u003c/b\u003e \u003cb\u003e-ethyl-5,6,7,8-tetrafluoroacridin-2-amine\u003c/b\u003e (\u003cb\u003e4\u003c/b\u003e).2-[ethyl(5,6,7,8-tetrafluoroacridin-2-yl)amino]ethanol (\u003cb\u003e3\u003c/b\u003e) (650 mg, 1.92 mmol) and CBr\u003csub\u003e4\u003c/sub\u003e (776 mg, 2.34 mmol) were dissolved in dry CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e (33 mL), under nitrogen atmosphere. The solution was cooled to 0\u0026deg;C and PPh\u003csub\u003e3\u003c/sub\u003e (613 mg, 2.34 mmol) was added. After stirring for 10 minutes, the reaction was allowed to warm to rt and left under stirring overnight. Solvent was then removed under reduced pressure and the residue was purified by column chromatography (SiO\u003csub\u003e2\u003c/sub\u003e, first CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e/heptane 1:1, then CH\u003csub\u003e2\u003c/sub\u003eCl\u003csub\u003e2\u003c/sub\u003e) to afford \u003cb\u003e4\u003c/b\u003e (473 mg, 61%). Yellow solid; mp 172\u0026deg;C; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) δ 8.51 (s, 1H), 8.10 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9.7 Hz, 1H), 7.52 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9.7, 2.9 Hz, 1H), 6.80 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;2.8 Hz, 1H), 3.85 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.8 Hz, 2H), 3.71\u0026ndash;3.45 (m, 4H), 1.31 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.1 Hz, 3H); \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) δ 145.1 (s), 144.7 (s), 143.3\u0026ndash;140.4 (m), 141.5\u0026ndash;138.6 (m), 138.5\u0026ndash;137.6 (m), 135.5 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;16.3 Hz), 132.4 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;10.7 Hz), 130.8 (s), 128.2 (s), 124.8 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;6.1 Hz), 123.0 (s), 115.2 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;14.7 Hz), 101.6 (s), 52.3 (s), 45.6 (s), 27.9 (s), 12.7 (s); \u003csup\u003e19\u003c/sup\u003eF NMR (376 MHz, CDCl\u003csub\u003e3\u003c/sub\u003e) δ -151.99 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;16.7 Hz), -152.89 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;16.0 Hz), -157.05 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;16.5 Hz), -159.16 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;17.2 Hz). ; IR (ATR) 3083, 3060, 3038, 2986, 2927, 2870, 1682, 1618, 1593, 1495, 1459, 1424, 1358, 1342, 1262, 1218, 1202, 1150, 1103, 1072, 1026, 994, 941, 904, 816, 658, 643, 541 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; Anal. Calcd. For C\u003csub\u003e17\u003c/sub\u003eH\u003csub\u003e13\u003c/sub\u003eBrF\u003csub\u003e4\u003c/sub\u003eN\u003csub\u003e2\u003c/sub\u003e: C, 50.89; H, 3.27; N, 6.98. Found: C, 50.82; H, 3.30; N, 7.01. (Additional file 2)\u003c/p\u003e \u003cp\u003e \u003cb\u003e2-[ethyl(5,6,7,8-tetrafluoroacridin-2-yl)amino]-\u003c/b\u003e \u003cb\u003eN\u003c/b\u003e \u003cb\u003e-(2-hydroxyethyl)-\u003c/b\u003e \u003cb\u003eN\u003c/b\u003e,\u003cb\u003eN\u003c/b\u003e\u003cb\u003e-dimethylethanaminium bromide\u003c/b\u003e (\u003cb\u003e1\u003c/b\u003e). Dimethylethanolamine (200 mg, 2.24 mmol) was slowly added to a 0\u0026deg;C solution of \u003cb\u003e4\u003c/b\u003e (450 mg, 1.12 mmol) in dry THF (5 mL), under nitrogen atmosphere. The mixture was allowed to reach rt and then stirred at 50\u0026deg;C overnight. The solid formed was filtered and washed extensively with cold diethyl ether to afford \u003cb\u003e1\u003c/b\u003e (200 mg, 41%). Brown solid; \u003csup\u003e1\u003c/sup\u003eH NMR (400 MHz, D\u003csub\u003e2\u003c/sub\u003eO) δ 7.64 (s, 1H), 7.40\u0026ndash;7.23 (m, 2H), 6.34 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;1.4 Hz, 1H), 4.10 (s, 2H), 3.88\u0026ndash;3.81 (m, 2H), 3.66\u0026ndash;3.54 (m, 4H), 3.40 (dd, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;13.8, 6.7 Hz, 2H), 3.29 (s, 6H), 1.18 (t, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;7.0 Hz, 3H); \u003csup\u003e13\u003c/sup\u003eC NMR (101 MHz, D\u003csub\u003e2\u003c/sub\u003eO) δ 144.7 (s), 141.4 (s), 141.0\u0026ndash;138.1 (m), 139.9\u0026ndash;137.1 (m), 139.4\u0026ndash;136.3 (m), 136.2\u0026ndash;133.2 (m), 128.9 (d, \u003cem\u003eJ\u003c/em\u003e\u0026thinsp;=\u0026thinsp;9.0 Hz), 127.2 (s), 126.2 (s), 124.0 (s), 123.1 (s), 113.3\u0026ndash;112.3 (m), 100.6 (s), 65.5 (s), 58.7 (s), 55.4 (s), 55.1 (s), 51.9 (s), 42.6 (s), 11.3 (s); \u003csup\u003e19\u003c/sup\u003eF NMR (376 MHz, D\u003csub\u003e2\u003c/sub\u003eO) δ -151.94 (s), -154.37 (s), -156.97 (s), -159.66 (s); IR (ATR) 3302, 3033, 2977, 1682, 1618, 1591, 1503, 1488, 1427, 1367, 1342, 1250, 1184, 1158, 1077, 1026, 997, 961, 945, 917, 825, 817, 776, 656, 603, 562 cm\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e; Anal. Calcd. For C\u003csub\u003e21\u003c/sub\u003eH\u003csub\u003e24\u003c/sub\u003eBrF\u003csub\u003e4\u003c/sub\u003eN\u003csub\u003e3\u003c/sub\u003eO: C, 51.44; H, 4.93; N, 8.57. Found: C, 51.33; H, 4.98; N, 8.64. (Additional file 3)\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of spectral properties\u003c/h2\u003e \u003cp\u003eAbsorption spectra of CFA (\u003cb\u003e1\u003c/b\u003e) in PBS (100 \u0026micro;M) were recorded using Agilent \u0026ndash; Cary UV-Vis. Photoluminescence measurements were carried out on Cary Eclipse Fluorescence Spectrophotometer.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eHuman cervical (HeLa) cancer cells, Michigan Cancer Foundation-7 (MCF-7) cells and Human Cardiac Fibroblasts (HCF) were obtained from Kingston University. HeLa cells and MCF-7 cells were maintained in DMEM supplemented with 10% FBS and 100 \u0026micro;g/mL penicillin/streptomycin. HCF were maintained in FGM. All cell lines were grown at 37\u0026deg;C and in a 5% CO\u003csub\u003e2\u003c/sub\u003e humidified atmosphere.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eIn vitro cell toxicity study\u003c/h2\u003e \u003cp\u003eHeLa and MCF-7 cells were seeded in a 96-wells plate at 1 \u0026times;10\u003csup\u003e5\u003c/sup\u003e cells/mL in supplemented medium (100 \u0026micro;L) and grown overnight. Cells were then incubated at increasing concentrations of CFA (\u003cb\u003e1\u003c/b\u003e) (12.5, 25, 50, 100, 150, 200 and 250 \u0026micro;M) for 72 h. The cell viability was then tested, using the MTT assay: the water-soluble MTT is converted into water-insoluble purple formazan in metabolically active cells by the action of the mitochondrial reductase; the amount of formazan produced is directly proportional to the number of living cells [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Briefly, MTT (20 \u0026micro;L, final concentration of 0.1 mg/mL) was added into each well and incubated for 4 h at 37\u0026deg;C, in the dark. The medium was then discarded, cells were carefully washed with PBS to remove any residue of fluorescent compound and the formazan products were dissolved in DMSO (100 \u0026micro;L). The plate was then shaken for 15 min at rt in the dark, after which the absorbance was measured at 550 nm with a reference at 690 nm [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The experiment was run in triplicate. The cell viability was expressed as percentage (%) and calculated by dividing the absorbance of the cells treated with CFA by the absorbance of control (cells treated with medium only). The IC\u003csub\u003e50\u003c/sub\u003e value was extrapolated from the graph plotted using cell viability data against concentration [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003eCellular uptake by confocal microscopy on fixed cells\u003c/h2\u003e \u003cp\u003eHeLa and MCF-7 cells were plated onto glass coverslips in 6-well plates, at a density of 0.7 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/well and grown overnight. Cells were then incubated with CFA (\u003cb\u003e1\u003c/b\u003e) at 5 \u0026micro;M for 8, 16 and 24 h. At each time point, cells were washed with PBS, fixed with paraformaldehyde (4%) for 20 min and rehydrated in PBS for 1 h, at rt. Specimens were then mounted and stored at 4\u0026deg;C until examination using a confocal microscope (Zeiss Axiovert 200M, Oberkochen, Germany) equipped with a LSM 5 Image Browser (Carl Zeiss, Oberkochen, Germany). An oil immersion objective (63x) was employed.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCellular uptake by fluorescence microscopy on living cells\u003c/h2\u003e \u003cp\u003eHCF, HeLa and MCF-7 cells were plated onto 6-wells plate, at a density of 0.7 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells/well and grown overnight. Cells were then incubated with CFA (\u003cb\u003e1\u003c/b\u003e) at 5 \u0026micro;M for 24 h, at 37\u0026deg;C and in a 5% CO\u003csub\u003e2\u003c/sub\u003e humidified atmosphere. Cells were then washed with PBS (\u0026times;3) and visualized alive on a Leica DM750 Fluorescence Microscope, using a 20\u0026times; objective.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eQuantification of fluorescent intensity\u003c/h2\u003e \u003cp\u003eImageJ software (NIH, USA) was used to quantify the signal intensity corresponding to CFA as described by Shihan \u003cem\u003eet al\u003c/em\u003e.[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eData were expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical analyses were performed using one-way analysis of variance (ANOVA) with significance set at \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05. A post-hoc test (Tukey multiple comparison test) was performed to determine differences between groups. All statistical analyses were carried out using GraphPad Prism 4.0 (GraphPad Software, Inc.).\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003ch2\u003eSynthesis and characterization of CFA (1)\u003c/h2\u003e\n\u003cp\u003eCFA (\u003cstrong\u003e1\u003c/strong\u003e) was synthesized starting from pentafluorobenzaldehyde (\u003cstrong\u003e2\u003c/strong\u003e) (Scheme 1). Tandem nucleophilic aromatic substitution/thermal promoted electrocyclization-aromatization by treatment with \u003cem\u003ein-situ\u003c/em\u003e prepared 2‑[(4‑aminophenyl)(ethyl)amino]ethanol in decahydronaphthalene at 175 \u0026deg;C afforded 2-[ethyl(5,6,7,8-tetrafluoroacridin-2-yl)amino]ethanol (\u003cstrong\u003e3\u003c/strong\u003e) [25,26]. \u003cstrong\u003e3\u003c/strong\u003e was then converted into \u003cem\u003eN\u003c/em\u003e-(2-bromoethyl)-\u003cem\u003eN\u003c/em\u003e-ethyl-5,6,7,8-tetrafluoroacridin-2-amine\u0026nbsp;(\u003cstrong\u003e4\u003c/strong\u003e) by reaction with CBr\u003csub\u003e4\u003c/sub\u003e and triphenylphosphine [27]. Finally, treatment of \u003cstrong\u003e4\u003c/strong\u003e with 2‑(dimethylamino)ethanol allowed the formation of \u003cstrong\u003e1\u003c/strong\u003e.\u003c/p\u003e\n\u003cp\u003eFigure 2 shows the absorption \u0026ndash; emission spectra of CFA (\u003cstrong\u003e1\u003c/strong\u003e), with Stokes shift (116 nm (0.57 eV) between absorption and emission maxima (450 nm and 566 nm, respectively).\u003c/p\u003e\n\u003ch2\u003eCell toxicity in a dose-dependent manner\u003c/h2\u003e\n\u003cp\u003eThe ability of CFA (\u003cstrong\u003e1\u003c/strong\u003e) to inhibit the metabolic activity of breast and cervical cancer cells in a dose-dependent manner was determined using the MTT assay (Figure 3). The viability of HeLa cells was above the 80% when concentration of CFA up to 50\u0026nbsp;\u0026micro;M were used over an incubation time of 72 h, while a sharp decrease in the cell viability was recorded upon higher concentrations. Instead, incubation of MCF-7 cells with increasing concentrations of CFA for 72 h experienced a steady reduction in the cell viability up to 40%. The IC\u003csub\u003e50\u003c/sub\u003e values determined for HeLa and MCF-7 cells were 200 \u0026plusmn;18 \u0026micro;M and 105 \u0026plusmn;3 \u0026micro;M, respectively, suggesting that MCF-7 cells were more susceptible to the cytotoxic action of CFA than HeLa cells.\u003c/p\u003e\n\u003ch2\u003eCellular uptake of CFA by cancer cells over time\u003c/h2\u003e\n\u003cp\u003eConfocal microscopy was used to investigate the cellular uptake of CFA (\u003cstrong\u003e1\u003c/strong\u003e) by HeLa and MCF-7 cells, in a time-dependant manner (6, 16 and 24 h). At each time point, cells on coverslips were washed and fixed, before being mounted on a glass slide and observed using the confocal microscope. The excitation of the fluorescent dye was carried out at a wavelength falling within the absorption band of the fluorinated acridine moiety. After 6 h incubation, a low signal of CFA (\u003cstrong\u003e1\u003c/strong\u003e) was observed in HeLa cells (Figure 4) while appearing more evident after 24 h (p\u0026nbsp;\u0026le;\u0026nbsp;0.01). Especially, the fluorescent signal of CFA (\u003cstrong\u003e1\u003c/strong\u003e) was detected in the perinuclear region of both HeLa and MCF-7 cells, with the intensity increasing at very high level in MCF-7 cells after 16 h incubation (p\u0026nbsp;\u0026le;\u0026nbsp;0.0001), suggesting a higher uptake of CFA (\u003cstrong\u003e1\u003c/strong\u003e) in MCF-7 cells than HeLa cells. No signal corresponding to CFA was detected at incubation time lower than 6 h (data not shown).\u003c/p\u003e\n\u003ch2\u003eCellular uptake of CFA (1) by normal and cancer living cells\u003c/h2\u003e\n\u003cp\u003eThe cellular uptake of CFA (\u003cstrong\u003e1\u003c/strong\u003e) was assessed in living cells, both normal (HCF) and tumour (HeLa and MCF-7 cells) by fluorescence microscopy (Figure 5). In normal fibroblasts, a very low signal was noticed, indicating poor uptake of CFA (\u003cstrong\u003e1\u003c/strong\u003e) (p\u0026nbsp;\u0026le;\u0026nbsp;0.0001). Instead, a strong fluorescence signal of CFA (\u003cstrong\u003e1\u003c/strong\u003e) was observed in the perinuclear region of HeLa cells. The signal appeared more pronounced and widespread in MCF-7 cells, indicating that the uptake of CFA (\u003cstrong\u003e1\u003c/strong\u003e) was higher in breast cancer cells compared to cervical cancer cells (p \u0026le; 0.001).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThe aim of this study was to investigate the ability of a novel choline derivative to act as a fluorescent choline tracer for tumours characterized by an increase metabolism of choline such as cervical and breast cancers.\u003c/p\u003e \u003cp\u003eThe novel fluorescent choline tracer was successfully synthesized and its optical properties characterized. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, the relatively large Stokes shift between absorption and emission maxima of CFA (116 nm) is evidence of the charge-transfer nature of the transition. Since the transitions responsible for the emission and absorption reside on the acridine moiety, the tertiary amine can participate in the acridine conjugated system in the excited state \u0026ndash; consequently increasing the extent of conjugation and effectively lowering the emission transition energy. Such large Stokes Shift minimises interference between the excitation and the fluorescence emission, prevents self-quenching and is highly suited for bioimaging. Results showed in Figs.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e suggested that CFA (\u003cb\u003e1\u003c/b\u003e) possesses efficient optical properties that allow its intracellular detection at very low concentrations (5 \u0026micro;M) and incubation time (up to 6 h) with no induction of cell toxicity.\u003c/p\u003e \u003cp\u003eFigure \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e shows the selective ability of CFA (\u003cb\u003e1\u003c/b\u003e) to accumulate preferentially in cancer cells rather than in normal heart fibroblasts. These results correlate well with our previous study [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] where choline and phosphatidylcholine fluorescent derivatives accumulated preferentially in malignant breast cells in which choline uptake and metabolism are augmented [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. This feature is key upon intravenous administration of the tracer, since it allows for specific accumulation in malignant cells while averting healthy ones. Moreover, since acridine derivatives generally have good inhibitory activity against Topoisomerase I/II, the cytotoxicity activity may be related to the inhibition of this enzyme. Indeed, acridine derivatives have been widely explored for their excellent anticancer activity in several studies [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. Being an acridine derivative, CFA (\u003cb\u003e1\u003c/b\u003e) was screened for its cytotoxicity toward tumour cells: cytotoxic results were expressed as growth inhibitory concentration (IC\u003csub\u003e50\u003c/sub\u003e) values representing the compound concentration needed to achieve a 50% inhibition of cell growth after 72 h incubation compared to the untreated control [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. IC\u003csub\u003e50\u003c/sub\u003e values of CFA indicated excellent growth inhibition against MCF-7 cells (100 \u0026micro;M), which was similar to other acridine derivatives, when compared to the reference drug doxorubicin (IC\u003csub\u003e50\u003c/sub\u003e\u0026thinsp;=\u0026thinsp;65 \u0026micro;g/mL) [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Instead, growth inhibitory effects of CFA against HeLa cells were not significant compared to MCF-7 cells or compared to other acridine derivatives and Camptothecin [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. This might correlate to the higher cellular uptake of CFA by MCF-7 cells compared to HeLa cells, as suggested by the fluorescent intensity data, where the fluorescent signal in MCF-7 cells was higher compared to the signal of HeLa cells. MCF-7 is a well-established cell line to study the altered phospholipid metabolism in breast cancer [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], since high choline levels were detected in these cancer cells [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. However, previous studies also demonstrated a high choline uptake by HeLa cells [\u003cspan additionalcitationids=\"CR38\" citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. By comparison, this preliminary study suggests that choline uptake is higher in breast cancer compared to cervical cancer yet further studies are needed to establish a potential correlation.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eA novel fluorescent tracer based on choline was synthesised for the \u003cem\u003ein vitro\u003c/em\u003e detection of cervical and breast cancers. Choline linked to fluorinated acridine (CFA) was tracked by its self-fluorescence and exhibited a preferential uptake by cancer cells compared to healthy ones (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026le;\u0026thinsp;0.0001). These results suggest that CFA (\u003cb\u003e1\u003c/b\u003e) could be used as potential biomarker in the context of early diagnosis of cancerogenic lesions, being a highly sensitive, specific, affordable and practical screening tool that may complement already existing diagnostic techniques in the context of cervical and breast cancer.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCFA, Choline Fluorinated Acridine\u003c/p\u003e\n\u003cp\u003eHPV, Human Papilloma Virus\u003c/p\u003e\n\u003cp\u003eHR, Hormone Receptor\u003c/p\u003e\n\u003cp\u003eTIL, Tumour-Infiltrating Lymphocytes\u003c/p\u003e\n\u003cp\u003eBCI, Breast Cancer Index\u003c/p\u003e\n\u003cp\u003eGGI, Genomic Grade Index\u003c/p\u003e\n\u003cp\u003eDNA, Deoxyribonucleic Acid\u003c/p\u003e\n\u003cp\u003eATR, Attenuated Total Reflectance\u003c/p\u003e\n\u003cp\u003eIR, InfraRed\u003c/p\u003e\n\u003cp\u003eNMR, Nuclear Magnetic Resonance\u003c/p\u003e\n\u003cp\u003eDMEM, Dulbecco′s Modified Eagle′s Medium\u003c/p\u003e\n\u003cp\u003ePBS, Phosphate-buffered saline\u003c/p\u003e\n\u003cp\u003eMTT, 4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide\u003c/p\u003e\n\u003cp\u003eDMSO, Dimethylsulfoxide\u003c/p\u003e\n\u003cp\u003eDABCO, 1,4-diazabicyclo [2.2.2] octane\u003c/p\u003e\n\u003cp\u003eTHF, tetrahydrofuran\u003c/p\u003e\n\u003cp\u003ert, room temperature\u003c/p\u003e\n\u003cp\u003eFBS, Foetal Bovine Serum\u003c/p\u003e\n\u003cp\u003eFGM, Fibroblast Growth Medium\u003c/p\u003e\n\u003cp\u003eUV-Vis, Ultra-violet visible\u003c/p\u003e\n\u003cp\u003eMCF-7, Michigan Cancer Foundation-7\u003c/p\u003e\n\u003cp\u003eHCF, Human Cardiac Fibroblast\u003c/p\u003e\n\u003cp\u003eIC\u003csub\u003e50\u003c/sub\u003e, Inhibitory Concentration\u003c/p\u003e\n\u003cp\u003eANOVA, Analysis of Variance\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo external funding was received for this research.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors' contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAC performed and analysed data regarding cell toxicity and cellular uptake studies. LV was involved in the synthesis and characterization of the compound. PS performed the optical characterisation of the compound. AP and GC supervised the work. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eNair M, Sandhu SS, Sharma AK. Cancer molecular markers: A guide to cancer detection and management. Semin Cancer Biol 2018;52:39\u0026ndash;55. https://doi.org/10.1016/j.semcancer.2018.02.002.\u003c/li\u003e\n \u003cli\u003eNHS England. Screening and earlier diagnosis 2024. www.england.nhs.uk/cancer/early-diagnosis/screening-and-earlier-diagnosis/.\u003c/li\u003e\n \u003cli\u003eCheng Q, Lau WM, Chew SH, Ho TH, Tay SK, Hui KM. Identification of molecular markers for the early detection of human squamous cell carcinoma of the uterine cervix. 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[Methyl-3H]-choline Incorporation into MCF-7 Cells: Correlation with Proliferation, Choline Kinase and Phospholipase D Assay. Anticancer Res 2007;27:901\u0026ndash;6.\u003c/li\u003e\n \u003cli\u003eBolan PJ. Magnetic Resonance Spectroscopy of the Breast: Current Status. Magn Reson Imaging Clin N Am 2013;21:625\u0026ndash;39. https://doi.org/10.1016/j.mric.2013.04.008.\u003c/li\u003e\n \u003cli\u003eRoppongi M, Mitsuru Izumisawa \u0026middot;, Terasaki \u0026middot; Kazunori, Muraki \u0026middot; Yasushi, Shozushima \u0026middot; Masanori. 18F-FDG and 11C-choline uptake in proliferating tumor cells is dependent on the cell cycle in vitro 2019;33:237\u0026ndash;43. https://doi.org/10.1007/s12149-018-01325-6.\u003c/li\u003e\n \u003cli\u003eIorio E, Mezzanzanica D, Alberti P, Spadaro F, Ramoni C, D\u0026rsquo;Ascenzo S, et al. Alterations of Choline Phospholipid Metabolism in Ovarian Tumor Progression. Cancer Res 2005;65:9369\u0026ndash;76. https://doi.org/10.1158/0008-5472.CAN-05-1146.\u003c/li\u003e\n \u003cli\u003eGuy GR, Murray2 AW. Tumor Promoter Stimulation of Phosphatidylcholine Turnover in HeLa Cells1. Cancer Res 1980;42.\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Scheme","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-medical-imaging","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bmim","sideBox":"Learn more about [BMC Medical Imaging](http://bmcmedimaging.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bmim/default.aspx","title":"BMC Medical Imaging","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Choline, fluorescent dye, biomarker, tracer, cellular uptake, cervical cancer, breast cancer","lastPublishedDoi":"10.21203/rs.3.rs-4643928/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4643928/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eIntroduction\u003c/strong\u003e\u003c/em\u003e Cervical and breast cancers can be easily preventable but they still represent the main causes of cancer-related deaths of women worldwide. Detecting cancer at its early stages is key since treatment of pre-invasive lesions is more efficient than treating an invasive disease. However, there no screening method that is highly sensitive and specific, as well as affordable and practical is currently available. Therefore, the identification of markers complementing traditional cyto/histopathological assessments is needed. Alterations in choline metabolism represents a hallmark of many malignancies, including cervical and breast cancers. Choline radiotracers are widely used for several imaging purposes for the detection of tumours, even though there are many risks associated with the use of radioactivity. Therefore, the aim of this work was to synthesize and characterize a choline tracer based on fluorinated acridine scaffold (CFA) for the \u003cem\u003ein vitro\u003c/em\u003e detection of cervical and breast cancer cells.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eMethods \u003c/strong\u003e\u003c/em\u003eCFA was synthesized, fully characterized and tested for cytotoxicity on breast (MCF-7) and cervical (HeLa) cancer cell lines. CFA’s uptake by cancer cells was investigated by confocal microscopy and its intracellular intensity was studied by fluorescence means; a comparative uptake between living normal and cancer cells was also conducted.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eResults \u003c/strong\u003e\u003c/em\u003eAn enhanced intensity of CFA was recorded in breast cancer cells compared to cervical cancer cells in both confocal and fluorescence microscope analysis (\u003cem\u003ep\u003c/em\u003e ≤ 0.001). Weak signal intensity of CFA was recorded in normal cells (\u003cem\u003ep\u003c/em\u003e ≤ 0.0001). CFA was toxic at much higher concentrations (HeLa IC\u003csub\u003e50\u003c/sub\u003e= 200 ±18 µM and MCF-7 IC\u003csub\u003e50\u003c/sub\u003e=105 ±3 µM) than the one needed for its detection in cancer cells (5 µM).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003e\u003cstrong\u003eConclusions \u003c/strong\u003e\u003c/em\u003eResults showed that CFA preferentially accumulated in cancer cells rather than in normal ones. This suggests that CFA may be a potential diagnostic probe in discriminating healthy tissues from malignant ones, due to its specific and highly sensitive features; CFA may also represent a useful tool for \u003cem\u003ein vitro\u003c/em\u003e investigations of choline metabolism in cervical and breast cancers.\u003c/p\u003e","manuscriptTitle":"In vitro detection of breast and cervical cancer cells using a novel fluorescent choline derivative","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-24 13:06:59","doi":"10.21203/rs.3.rs-4643928/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-08-02T07:14:49+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-29T05:22:03+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-22T09:47:40+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"266298068642788003513224183695421388671","date":"2024-07-17T10:49:31+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"325056460065138440834266714341354612672","date":"2024-07-12T10:48:58+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-07-12T10:45:32+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-07-03T08:17:59+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-07-03T08:17:30+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Medical Imaging","date":"2024-06-26T16:01:28+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-medical-imaging","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"bmim","sideBox":"Learn more about [BMC Medical Imaging](http://bmcmedimaging.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/bmim/default.aspx","title":"BMC Medical Imaging","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3d093d87-183b-4945-a4ad-228bd0e71164","owner":[],"postedDate":"July 24th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-11-25T16:07:36+00:00","versionOfRecord":{"articleIdentity":"rs-4643928","link":"https://doi.org/10.1186/s12880-024-01488-x","journal":{"identity":"bmc-medical-imaging","isVorOnly":false,"title":"BMC Medical Imaging"},"publishedOn":"2024-11-20 15:57:54","publishedOnDateReadable":"November 20th, 2024"},"versionCreatedAt":"2024-07-24 13:06:59","video":"","vorDoi":"10.1186/s12880-024-01488-x","vorDoiUrl":"https://doi.org/10.1186/s12880-024-01488-x","workflowStages":[]},"version":"v1","identity":"rs-4643928","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4643928","identity":"rs-4643928","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}