Development of a biosensor based on a new marine luciferase fused to an affibody to assess Her2 expression in living cells

Analytica Chimica Acta 1221 (2022) 340084 Available online 16 June 2022 0003-2670/© 2022 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/bync-nd/4.0/). Development of a biosensor based on a new marine luciferase fused to an affibody to assess Her2 expression in living cells Laura Rodríguez de la Fuente , Irene Gol´ an Cancela, ´ Anxela M. Est´ evez-Salguero, Pablo Iglesias, Jos´ e A. Costoya * Molecular Oncology Laboratory MOL, Centro Singular de Investigaci´ on en Medicina Molecular e Enfermidades Cr´ onicas (CiMUS), Departamento de Fisioloxía, Facultade de Medicina, Universidade de Santiago de Compostela, Instituto de Investigaci´ on Sanitaria de Santiago de Compostela (IDIS), 15782, Santiago de Compostela, Spain HIGHLIGHTS GRAPHICAL ABSTRACT •Functional optical imaging allows highsensitivity detection of prognostic biomarkers in cancer. •MLuc1.1, a thermostable luciferase from Metridia lucens with high bioluminescent activity, is presented as a new diagnostic tool in optical imaging. •Development of a novel biosensor, MlLuc-aff, that recognizes overexpression of HER-2 receptor in breast cancer cell lines. ARTICLE INFO Keywords: Cancer HER2 Affibody Optical imaging Bioluminiscence ABSTRACT The development of new diagnostic tools in tumor pathology allows the optimization of individualized therapies in cancer patients. The functional optical image provides a unique opportunity to identify the pathophysiological characteristics of each tumor in a non-invasive way. Although fluorescent recombinant affibodies and nanobodies, capable of detecting certain membrane proteins present in tumor cells, has been described, the use of bioluminescent molecules is gaining a great impact in this field due to its high sensitivity. In this work, we characterize a new luciferase from the Metridia lucens copepod (MlLuc) and develop a novel bioluminescent recombinant affibody (MlLuc-aff) capable of recognizing the HER2 receptors that are overexpressed in breast cancer tumors. For this purpose, the thermostability and pH sensitivity of MlLuc1.1 were determined, showing no significant changes in the activity among temperatures between 4 and 70 ◦C, and with a maximum of brightness at pH 8.0. Furthermore, MlLuc-aff was able to accurately detect HER2 receptors expressed in the SK-BR-3 cells. Future applications of this new tracer can contribute to the early diagnosis of breast cancer patients and the assessment of the efficacy of the treatment. The epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases can activate different signaling pathways involved in the regulation of cellular proliferation and survival. Among the different members of this family of receptors, HER2 promotes oncogenesis. HER2 gene amplification or gene mutations have been detected in many types of cancers such as, colorectal, lung, bladder, stomach, brain, uterus, skin * Corresponding author. E-mail address: [email protected] (J.A. Costoya). Contents lists available at ScienceDirect Analytica Chimica Acta journal homepage: www.elsevier.com/locate/aca https://doi.org/10.1016/j.aca.2022.340084 Received 17 January 2022; Received in revised form 6 June 2022; Accepted 11 June 2022 Analytica Chimica Acta 1221 (2022) 340084 2 and breast [1]. In fact, the clinical classification of breast cancer is based on the presence of several receptors, specifically estrogen, progesterone and HER2, a receptor overexpressed or amplified in 25% of patients with breast cancer, and this correlates with poor clinical outcomes [2]. Diverse immunotherapeutic approaches based on HER2-directed monoclonal antibodies has shown an improvement in clinical outcomes of patients with HER2-positive metastatic breast cancer [3–5]. Therefore, HER2 expression is highly used as a biomarker predictive of strong correlations between improved clinical outcomes and treatments with HER2-targeted agents [6]. However, the possibility of analyzing the temporal heterogeneity of metastatic breast cancer with tissue-based biomarkers is difficult because it requires sequential biopsies. Moreover, repeated biopsies may not be informative because the expression may not be found at all metastatic sites. For this reason, molecular imaging can complement the pathological, through a noninvasive and repeatable whole-body assessment of the disease [7]. Noninvasive molecular imaging uses labeled tracers to visualize biomarkers in the whole body. Among them, affibodies meet many requirements to become a remarkable imaging tracer [8]. They have a small size, ensuring an optimal tissue penetration and fast blood clearance, additionally their target specificity has been also demonstrated in vivo [9]. These molecules have been used as tools for molecular recognition in therapeutic applications, and many preclinical studies and early clinical evidence is now founding the idea that affibody molecules could be efficacious and safe in human [10]. Small luciferases, and specifically copepod luciferases, have rapidly gained relevance as reporters in in vitro and non-invasive in vivo assays, especially after water-soluble coelenterazine (s-CTZ) was available [11]. Although, both Renilla and copepod luciferases are coelenterazine-dependent luciferases, these enzymes substantially differ in size and amino acid sequences. Moreover, these luciferases demonstrate a high stability, small size, and strong bioluminescence activity. Although, many in vivo assays have been reported during the last years, there are only a few examples of applying them in analytical assays in vitro. Considering their exceptional bioluminescent and biochemical properties of these luciferases, the number of reports on their use as labels in binding assays is still reduced. This is mainly due to the difficulty of purifying the recombinant protein in E. coli because the correctly folded copepod luciferases must contain five intramolecular disulfide bonds [12]. Although several studies have demonstrated the application of HER2-specific affibody-Alexa Fluor fluorescent conjugates as optimal probes for optical imaging of HER2 in vivo [13] and HER2-binding affibody molecule labeled with a radionuclide for PET imaging [14]. A recent study has reported the construction of a hybrid protein consisting of the smallest isoform of copepod luciferase and a murine single-chain variable fragment mini-antibody to the glycoprotein E (gpE) of tick-borne encephalitis virus (TBEV) [15]. Here, we present an innovative bioluminescent biosensor based on a fusion protein comprising a new Metridia luciferase fused to a recombinant affibody (MlLuc-aff), capable of recognizing the HER2 receptors that are overexpressed in breast cancer. 1. Materials and methods 1.1. U87MG cell culture and transfection The human Uppsala 87 Malignant Glioma cell line (U87MG) was obtained from J. Seoane (Vall d’Hebron Instituto de Oncología, Barcelona, Spain). The human glioma cell line was maintained in low glucose Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich) supplemented with 10% fetal bovine serum (FBS, Thermo Fisher). Cell cultures were maintained at 37 ◦C and 5% CO 2 . The pcDNA3.1(+) plasmid harboring a neomycin (G418) resistance was used for mammalian expression of MlLuc1.1 luciferase. The pcDNA3.1(+)-MlLuc1.1 plasmid was constructed by GenScript Biotech Corporation, subcloning the fulllength MlLuc1.1 cDNA sequence into the HindIII/EcoRI site. U87MG cells were grown to 60–70% confluence, the old medium was replaced with DMEM 0% FBS, and the recombinant plasmid called pcDNA3.1 (+)-MILuc1.1 was transfected with 15 μ g/ml of DNA:7.5 μ g/ml of branched polyethylenimine (PEI 25, Sigma-Aldrich). 1.2. Determination of the minimal lethal concentration of G418 U87MG cells were seeded in DMEM 10% FBS at a density of 1 ×10 6 cells/mL in a 6-multiwell plate and they were exposed to different concentrations of Neomycin G418 (sigma-Aldrich; 0, 50, 100, 200, 300 and 400 μ g/ml). Cells were washed with phosphate-buffered saline (PBS, Sigma-Aldrich) every two days and renewed with complete media and their corresponding G418 concentration. This experiment was performed by triplicate. The minimal lethal concentration of G418 was established as the working concentration for selection of transfected U87MG cells (U87MG-MlLuc1.1). 1.3. Evaluation of luciferase activity and stability in the U87MG- MlLuc1.1 cell line U87MG-MlLuc1.1 cells were seeded in 10 cm diameter plates at a density of 2 ×10 6 per plate. MlLuc1.1 secreted to the media was assayed at regular time intervals of 15 h until 75 h post-seed. Determination of the luminescent signal was measured with a Lumat LB9507 luminometer (Berthold Technologies) and quantified as relative light units (RLUs). Briefly, 50 μ l of coelenterazine (1 ng/ μ L) (CTZ, Sigma-Aldrich) was added into 20 μ l aliquots of U87MG-MlLuc1.1 media and the exposure time was extended to 10 s. Cell culture medium was used as negative control. Bioluminescent spectra of MlLuc1.1 was measured with a FluoroMax-3 spectrofluorometer (Horiba) after addition of CTZ (1 ng/ μ L) into U87MG-MlLuc1.1 media aliquots. DMEM media without phenol red was used in this assay. To determine MlLuc1.1 thermostability, U87MG-MlLuc1.1 media samples were diluted 10-fold with 20 mM Tris–HCl (pH 8.0), 50 mM MgCl 2 and incubated 30 min over a range of 4–100 ◦C and then, cooled on ice for 5 min. The bioluminescent activity was measured at room temperature. To determine MlLuc1.1 pH sensitivity, U87MG-MlLuc1.1 media samples were diluted 10-fold with the following buffers: 0.1 M acetate buffer (pH 4.5–5.5), 0.1 M MOPS-NaOH (pH 6.0 and 6.5), 0.1 M HEPES-NaOH (pH 7.0 and 7.5), 0.1 M Tris–HCl (pH 8.0–9.0) and 0.1 M carbonate buffer (pH 9.5 and10.0). All buffers contained 50 mM MgCl 2 . Samples were incubated for 1 h on ice before measurements. MlLuc1.1 thermostability and pH sensitivity determinations were carried out in triplicate intra and inter-assay with a Lumat LB960 luminometer (Berthold Technologies) by adding 25 μ L of CTZ (1 ng/ μ L) to 10 μ l of sample. 1.4. Synthesis and characterization of MlLuc-aff The fusion of the affibody Z HER2:4 with MlLuc1.1 luciferase was carried out by positioning a flexible sequence spacer (GGGGS) 3 between both proteins and adding a tail of six histidine (His6-tag) molecules at the C-terminal end of the recombinant protein. The SacI and EcoRI restriction sites were designed for the insertion of the coding sequence MlLuc1.1-Z HER2:4 -His6-tag in the vector pET-17b. The synthesis of the construct, finally called pET-17b-MlLuc-aff, was commissioned from GenScript Biotech Corporation. Escherichia coli BL21(DE3) cells were transformed with pET-17b-MlLuc-aff plasmid by thermal shock. BL21 (DE3)-MlLuc-aff cells were grown at 25 ◦C until they reached OD 600 of ~0,5. Then, 100 μ M IPTG was added to the culture and temperature was lowered to 23 ◦C. After 6 h, BL21(DE3)-MlLuc-aff cells were collected and centrifuged at 20,000 g, 20 min at 4 ◦C. Pellet was frozen at −80 ◦C for 24 h and then, resuspended in denaturing buffer (100 mM NaH 2 PO 4 , 10 mM Tris-Cl, 6 M Gu-HCl and 1 mM PMSF). After 1 h of incubation at room temperature, the lysate was centrifuged at 10,000 g, 30 min. The L. Rodríguez de la Fuente et al. Analytica Chimica Acta 1221 (2022) 340084 3 supernatant, containing MlLuc-aff denatured, was collected and dialyzed for refolding proteins using a Slide-A-Lyzer MINI 10 K MWCO dialysis cassette, (Thermo Fisher). Briefly, supernatants were incubated at 4 ◦C in agitation during 8–12 h with 50 mL of each the following buffers: the first buffer containing 100 mM NaH 2 PO 4 , 5 mM Tris-Cl, 2 mM imidazole, 0.1 mM DTT, 4 M Gu-HCl; the second 50 mM NaH 2 PO 4 , 5 mM Tris-Cl, 150 mM NaCl, 5 mM imidazole, 0.3 mM DTT, 2 M Gu-HCl; and the third 50 mM NaH 2 PO 4 , 5 mM Tris-Cl, 300 mM NaCl, 10 mM imidazole, 0.5 mM DTT. After the dialysis process, MlLuc-aff was purified under native conditions using the HisPur Ni-NTA, 0,2 ml kit (Thermo Fisher). To eliminate the remaining imidazole from the final eluate containing MlLuc-aff, an extra dialysis was performed against 50 mL of PBS. Finally, protein expression was monitored by Coomassie blue staining and Western blot. The final concentration of MlLuc-aff was quantified by the Bradford assay method. 1.5. Binding assay performed with SK-BR-3 and MDA-MB-231 cells Breast adenocarcinoma SK-BR-3 and triple-negative breast MDA-MB- 231 cancer cells (ATCC) were seeded at density 2.5 ×10 5 per well in a 35 mm diameter plate. Both cancer cell lines were cultured with high glucose Dulbecco’s modified Eagle’s medium (DMEM, Sigma-Aldrich) supplemented with 10% FBS, at 37 ◦C and 5% CO 2 . After 48 h, MlLucaff (0.5 μ g/ μ L) was added to each plate by dilutions of 1:10, 1:50 and 1:100. SK-BR-3 and MDA-MB-231 cells were incubated with MlLuc-aff for 2 h in the same conditions described above. Then, cells were washed twice with phosphate-buffered saline (PBS, Sigma-Aldrich) and bioluminescence data was registered with IVIS® Spectrum system after 10 s of exposure time upon addition of 250 μ L CTZ (1 ng/ μ L) to each plate. 2. Results 2.1. High and stable expression of MlLuc1.1 in U87MG-MlLuc1.1 cell line Bioluminescent spectra of MlLuc1.1, secreted to the culture media by U87MG-MlLuc1.1., was analyzed with FluoroMax-3 spectrofluorometer (Horiba) by adding CTZ into aliquots with media from U87MG- MlLuc1.1. The luciferase activity reached its maximum intensity at wavelength 475 nm (Fig. 1a), which correspond with blue light emission. MlLuc1.1 secreted to the culture media was measured using the luminometer Lumat LB9507 (Berthold Technologies). After seeding U87MG-MlLuc1.1 cells, the luciferase activity was assayed at regular time intervals of 15 h until 75 h post-seed. The bioluminescence detected increased with the time elapsed since cells were seeded (Fig. 1b). This suggests that there is a correlation between luciferase expression and the number of cells in cultured, showing a stable accumulation of MlLuc1.1 Fig. 1. Bioluminescence of MlLuc1.1. a) Bioluminescence spectra of MlLuc1.1. Luciferase activity in media from U87MG-MlLuc1.1 was measured after addition of 1 ng/ μ L CTZ. MlLuc1.1 shows its maximum intensity at 475 nm. b) Correlation of bioluminescence activity with time. c) pH sensitivity of MlLuc1.1 was measured in media from U87MG-MlLuc1.1 at different pH values upon addition of CTZ. MlLuc1.1 reaches its maximum level of bioluminescence at pH 8.0. d) Thermostability of MlLuc1.1. Media from U87MG-MlLuc1.1 was heated at different temperatures for 30 min and measured after adding 25 μ l of CTZ 1 ng/ μ l. As shown, MlLuc1.1 activity is maintained and at 80 ◦C and steeply declines at higher temperatures. L. Rodríguez de la Fuente et al. Analytica Chimica Acta 1221 (2022) 340084 4 in the media. As occurs in both luciferin-dependent and coelenterazinedependent luciferases [16], the intensity of light emitted by MlLuc1.1 varies depending on pH value, reaching its maximum activity at pH 8.0 (Fig. 1c). We also examined the thermostability of MlLuc1.1, and after 30 min of incubation at different temperatures in a range of 4–100 ◦C [17], bioluminescence of MlLuc1.1 remained constant up 90 ◦C (Fig. 1d). In fact, even at 100 ◦C the level of bioluminescence continues being detectable. Based on this data we can conclude that MlLuc1.1 shows a high level of thermostability. 2.2. Design and expression of MlLuc-aff In order to use MlLuc1.1 as a probe in cancer diagnosis, the affibody Z HER2:4 [18], against HER2 receptors, was fused to the luciferase. Expression and purification of the recombinant protein, denominated MlLuc-aff, was carried out in E. coli under denaturing conditions. The presence of our recombinant protein in the lysate was monitored by Coomassie staining and Western blot (Fig. 2C, D). Although MlLuc-aff was obtained mostly as a soluble protein, it can be also detected in the insoluble fraction (Fig. 2a and b). Lysates were dyalized prior to column purification in order to remove denaturing salts preventing the refolding of our protein. Finally, MlLuc-aff was purified using HisPur Ni-NTA columns and quantified by Bradford assay method. The presence of the bioluminescence produced by MILuc-aff was quantified in each step of the purification process (Fig. 3). One aliquot was taken from each step and measured after addition of CTZ. As shown in Fig. 3, part of the bioluminescence activity can be appreciated in the Fig. 2. Coomassie staining and Western blot to check the expression and purification of MlLuc-aff. E. coli BL21(DE3) cells were transformed with the plasmid containing MlLuc-aff and grown until they reached OD 600 of ~0,5. Then, 100 μ M IPTG was added to the culture to induce protein expression. After 6 h, BL21(DE3)- MlLuc-aff bacteria were centrifuged and resuspended in denaturing buffer. The expression of MlLuc-aff was monitored by Coomassie staining (a) and Western Blot (b). Although a small portion of MlLuc-aff remained in the insoluble fraction, MlLuc-aff was also obtained from the soluble fraction (cleared lysate). Purification of MlLuc-aff was performed under native conditions and monitored by Coomassie staining (c) and Western Blot (d). Dialysis after purification resulted in an increased concentration of MILuc-aff (appreciated in Coomassie staining, Fig. 2c). L. Rodríguez de la Fuente et al. Analytica Chimica Acta 1221 (2022) 340084 5 first flowthrough (purification step: collection) from the purification column. This bioluminescence detection can be due because a small fraction of MlLuc-aff that did not properly bind to the resin column. However, most of the bioluminescence is coming from the aliquot with MlLuc-aff already purified and dialyzed, which indicates a satisfactory purification of MlLuc-aff. The concentration of protein in the sample MlLuc-aff purified and dialyzed was quantified by Bradford assay, obtaining a total of 0.6 μ g/ μ l. 2.3. Specific binding of MILuc-aff to HER2 receptors To check the ability of MlLuc-aff to recognize the receptor on the membrane of breast cancer cells, a binding assay was performed with SK-BR-3 cell line, which overexpress the target receptor, and MDA-MB- 231 cell line with low expression of the same receptor [19]. Two hours before the binding assay was performed, MlLuc1-aff was incubated with the cells. Cells were washed with PBS twice before adding CTZ and measuring bioluminescence activity with an IVIS® Spectrum system. As indicated in Fig. 4, MILuc1.1 was retained mostly in the plates containing SK-BR-3 HER2 receptors. MlLuc-aff was able to recognize specifically the HER2 receptors expressed in both cell lines (Fig. 4). Higher levels of brightness can be appreciated in the wells containing SK-BR-3 cells, which corresponds with the overexpression of HER2 for this cell line. However, MlLuc-aff is also able to recognize the low expression of HER2 in the MDA-MB-231 cell line. This suggests a high affinity of MlLuc-aff for its target receptor HER2. The results suggest that our new molecule MlLuc-aff is able to detect specifically the targeted receptor on the surface of cells. There is more emission of light on wells containing cells that overexpress the studied receptor (Fig. 4). Additionally, the high level of brightness produced by the luciferase MlLuc1.1, together with the high affinity of the anti-HER2 affibody, allows detecting with high sensitivity the receptor HER2 even at high dilutions rates (up to 1:500). 3. Discussion Despite the confinement of functional optical imaging to preclinical settings, it already shows a great potential to have a great impact on the management of cancer patients, especially those with solid tumors. One of the reasons is that this method would facilitate the detection of the tumor mass without the need to resort to invasive techniques. This work describes the design and characterization of a new biosensor (MlLucaff), formed by a novel luciferase fused to an affibody targeting the HER2 membrane receptor. Furthermore, the high affinity of affibody for its HER2 target could be a way to improve tumor specificity and, consequently, early diagnosis. The small size of MlLuc-aff will also be a great advantage when it comes to its application as a bioluminescent tracer in vivo. Given its size, it is predictable that it will present low retention times, as other molecules that have a molecular weight less than 50 kDa. Small size molecules are rapidly eliminated from the circulatory stream through glomerular filtration of the kidneys [20]. Accordingly, lower retention times reduce the likelihood that an adverse reaction will occur [21]. Likewise, the small size of the affibodies favors extravasation into the tissues, allowing the tumor to be detected more easily [22]. Besides, the structure of affibodies is endowed with low immunogenicity [23]. It would be necessary to study the biodistribution and toxicity of MlLuc-aff, but, together with its possible thermostability and its small size, it can be proposed as an interesting tool in the field of clinical oncology. Previous studies have described molecules like MlLuc-aff, capable of detecting the HER2 receptor in the tumor cell membrane but combining affibody with radioisotopes [24] or fluorochromes [25]. None of them use bioluminescent molecules in combination with affibody. It is important to remember that, although the fluorescent signals are brighter, the bioluminescence presents lower levels of background signal [26], so the sensitivity in the detection of tumor cells will increase considerably [27]. The results of this study indicate that the bioluminescent signal of MlLuc-aff is suitable for the identification with high specificity and sensitivity of tumors positive for HER2. For this reason, among the biomedical expectations of MlLuc-aff as a bioluminescent tracer are the visualization of cancer cells and the evaluation of the progression of the disease based on the different treatments used. It would also be interesting to study the ability of MlLuc-aff to block the HER2 receptor signaling pathway. Currently, there are therapies directed against this molecule, such as Trastuzumab, in which it has been seen that there is an inhibition of the growth of breast tumors [28]. In the event that MlLuc-aff were able to act in a similar way to these drugs, the molecule may offer diagnostic and therapeutic capabilities. Fig. 3. Bioluminescence quantification of the purification process. Aliquots from each step were diluted 1:100 before analysis, and their bioluminescent activity recorded upon addition of CTZ. Highest bioluminescence values were registered in the elution and dialyzed fractions. L. Rodríguez de la Fuente et al. Analytica Chimica Acta 1221 (2022) 340084 6 4. Conclusions MlLuc1.1 is proposed as a new, small, secretable and ATP- independent luciferase with high thermostability that shows its maximum functional level at pH 8.0 (6.5–9), which is favorable for its application in vivo. The fusion of MlLuc1.1 with an affibody against HER2 resulted in a new functional bioluminescent recombinant protein capable of recognizing HER2 membrane receptors present in breast cancer tumor cells with high specificity. MlLuc-aff, as a new sensor for optical imaging, has a broad range of possible applications in diagnostic medicine, from screening and early diagnosis of cancer, disease and therapy monitoring to applications during surgery. Funding sources This work received financial support from the Ministerio de Ciencia e Innovaci´ on (SAF2009-08629; J.A.C.) and ISCIII, Ministerio de Economía y Competitividad (PI15/01129), the Consellería de Cultura, Educaci´ on e Ordenaci´ on Universitaria (GPC2014/030, INCITE08PXIB208091PR and PXIB208091PR; J.A.C.), the Axencia Galega de Innovaci´ on (Galician Agency of Innovation; 2020-PG068), the Centro Singular de Investigaci´ on de Galicia accreditation 2016–2019, ED431G/05) and the European Regional Development Fund (ERDF). CRediT authorship contribution statement Laura Rodríguez de la Fuente: Conceptualization, Methodology, Data curation, Validation, Writing – review & editing. Irene Gol´ an Cancela: Investigation, Resources. ´ Anxela M. Est´ evez-Salguero: Investigation, Resources. Pablo Iglesias: Investigation, Writing – review & editing, Resources, Data curation. Jos´ e A. Costoya: Conceptualization, Methodology, Data curation, Supervision, Writing – review & editing, Funding acquisition. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgment The authors acknowledge Miguel Gonz´ alez-Blanco and Raquel Carreira-Rodríguez (DNA Repair and Genome Integrity-CiMUS-USC), Xulio Maside-Rodríguez and Carolina Bartolom´ e-Husson (Genomics and Bioinformatics Group-CiMUS-USC) and Eugenio V´ azquez-Sentís (CIQUSUSC) for helpful discussions. Abbreviations CTZ coelenterazine DMEM Dulbecco’s modified Eagle’s medium EGFR epidermal growth factor receptor gpE glycoprotein E HER2 human epidermal growth factor receptor 2 PET positron emission tomography scan TBEV tick-borne encephalitis virus Fig. 4. Binding assay of MlLuc-aff. MlLuc-aff was incubated with the cells (seeded in in 35 mm diameter plates) after dilutions 1:50, 1:100 and 1:500. After 2 h, the cells on the wells were rinsed with PBS three times and bioluminescent activity with the IVIS® Spectrum system by adding the substrate (CTZ) immediately before the measurement. Pseudocolor representation of the bioluminescence intensity from MILuc-aff. Wells containing medium only with or without CTZ served as negative controls. Quantitation of bioluminescence signal emitted (Total Flux) is expressed in photons/second (p/s). L. Rodríguez de la Fuente et al. Analytica Chimica Acta 1221 (2022) 340084 7 References [1] C.M. Connell, G.J. Doherty, Activating HER2 mutations as emerging targets in multiple solid cancers, ESMO Open 2 (2017), e000279. [2] D.J. Slamon, G.M. Clark, S.G. Wong, W.J. Levin, A. Ullrich, W.L. McGuire, Human breast cancer: correlation of relapse and survival with amplification of the HER-2/ neu oncogene, Science 235 (1987) 177–182. [3] S.M. 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