177Lu-Trastuzumab Radionuclide Therapy: an Effective Approach for Resistant Brain Metastases in HER2+ Breast Cancer

177Lu-Trastuzumab Radionuclide Therapy: an Effective Approach for Resistant Brain Metastases in HER2+ Breast Cancer | 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 177Lu-Trastuzumab Radionuclide Therapy: an Effective Approach for Resistant Brain Metastases in HER2+ Breast Cancer Liliana Santos, Ivanna Hrynchak, José Sereno, Hugo Ferreira, Magda Silva, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6180100/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose Breast cancer (BC) is the most common malignancy in women, with HER2 amplification present in 25–30% of metastatic cases. Although HER2-targeted therapies like trastuzumab have significantly improved patient outcomes, their efficacy in HER2 + brain metastases (BrM) is hindered by the emergence of resistance mechanisms. This study explores the therapeutic potential of trastuzumab radiolabeled with the β⁻-emitting radionuclide ¹⁷⁷Lu as a strategy to overcome resistance in HER2 + BrM. Material and methods HER2 + BC cell lines and their brain-tropic derivatives were assessed for HER2 expression and sensitivity to trastuzumab and [ 177 Lu]Lu-DOTA-Trastuzumab. In vivo models were established by orthotopic implantation of HER2 + BC cells for primary tumor formation or intracardiac injection to induce BrM. Once tumors were established, the therapeutic efficacy of trastuzumab and [¹⁷⁷Lu]Lu-DOTA-Trastuzumab was evaluated by monitoring tumor progression via magnetic resonance imaging (MRI). [⁸⁹Zr]Zr-DFO-Trastuzumab PET imaging was performed to assess HER2 expression, while blood-brain barrier (BBB) permeability was evaluated using dynamic contrast-enhanced MRI. Results Brain-tropic HER2 + cells exhibited trastuzumab resistance despite maintaining HER2 expression. In contrast, [ 177 Lu]Lu-DOTA-trastuzumab induced significant DNA damage and cytotoxicity. PET imaging confirmed specific radiotracer uptake in HER2 + primary tumors and BrM. A single dose of [ 177 Lu]Lu-DOTA-trastuzumab effectively suppressed primary tumor growth and achieved complete BrM remission in 40% of treated animals. Heterogeneous BBB permeability was observed across metastatic lesions, potentially influencing radiotracer uptake and therapeutic efficacy. Conclusion These findings underscore [¹⁷⁷Lu]Lu-DOTA-trastuzumab as a novel therapeutic strategy to overcome trastuzumab resistance in HER2 + BrM, offering a promising approach to improve outcomes in metastatic BC. HER2 + breast cancer brain metastasis trastuzumab resistance targeted radionuclide therapy lutetium-177 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Background Breast cancer (BC) remains the most common malignancy in women worldwide. Approximately 25 to 30% of metastatic BC cases exhibit amplification of the human epidermal growth factor receptor 2 (HER2), which is associated with a more aggressive clinical phenotype and poorer prognosis [1]. The anti-HER2 monoclonal antibody (mAb) trastuzumab has been the mainstay of systemic therapy for HER2 + metastatic BC, significantly improving patient survival over the past decade [2]. However, despite advancements in controlling extracranial metastases, the risk of central nervous system (CNS) recurrence remains disproportionately high [3]. Brain metastases (BrM) occur in up to 50% of patients with HER2 + metastatic BC, leading to increased morbidity and reduced survival [4]. This presents a critical challenge, as systemic HER2-directed therapies exhibit limited efficacy against BrM. The ineffectiveness of targeted drugs such as trastuzumab against BrM is often attributed to inadequate penetration of the blood-brain barrier (BBB), a protective barrier of the brain [5]. However, evidence suggests that BBB permeability is significantly altered in the presence of metastatic tumors, allowing macromolecules such as mAbs to penetrate [6, 7]. Despite adequate delivery of cytotoxic agents, BrM can persist due to the development of drug-resistance mechanisms [8]. Specifically, the activation of the PI3K/AKT pathway represents a resistance mechanism underlying the limited efficacy of HER2-directed therapy against BrM and other metastatic sites [9–11]. These insights emphasize the complexity of treating BrM and highlight the urgent need for alternative therapeutic strategies that circumvent trastuzumab resistance mechanisms and improve treatment outcomes. A promising approach involves mAb conjugates carrying cytotoxic payloads, such as radionuclides, chemotherapeutics, or toxins [12]. Targeted radionuclide therapy (TRNT) employs radiolabeled molecules like mAbs to selectively deliver β⁻- or α-emitting radionuclides to cancer cells, inducing DNA damage and cell death. TRNT has demonstrated potent anti-tumor effects and has emerged as a strategy to overcome resistance to conventional targeted therapies [13]. In metastatic BC, TRNT has shown clinical promise, particularly with β⁻-emitting agents such as [¹⁷⁷Lu]Lu-DOTA-trastuzumab [14] and [¹⁷⁷Lu]Lu-DOTA-ABY-027 (a second-generation HER2-targeting affibody) [15]. The therapeutic potential of lutetium-177 (¹⁷⁷Lu) is attributed to its favorable decay properties (t₁/₂ = 6.65 days, Eβ⁻ mean = 134 keV, Eβ⁻ max = 497 keV) and tissue penetration range (~ 2 mm), which allows localized radiation delivery while sparing healthy tissue [16]. Additionally, nuclear imaging studies using trastuzumab radiolabeled with zirconium-89 (⁸⁹Zr), indium-111 (¹¹¹In), and copper-64 (⁶⁴Cu) have demonstrated trastuzumab passage across the BBB, enabling tumor uptake and visualization of HER2 + BrM in BC patients [17–21]. However, the potential of trastuzumab-based TRNT using β⁻- or α-emitting radionuclides to selectively eradicate HER2 + BrM remains unexplored. In this study, we demonstrate that brain metastatic HER2 + cells exhibit resistance to pharmacological trastuzumab but remain susceptible to [ 177 Lu]Lu-DOTA-trastuzumab. Furthermore, our findings reveal that [ 177 Lu]Lu-DOTA-trastuzumab outperforms standard trastuzumab therapy in treating both HER2 + primary tumors and BrM, offering a promising therapeutic avenue to overcome resistance and improve overall survival. Results I. Brain-tropic breast cancer cells exhibit reduced sensitivity to trastuzumab while maintaining HER2 expression Trastuzumab resistance is a major challenge in the treatment of metastatic HER2 + BC, impacting both extracranial and intracranial metastases. To assess whether brain metastatic cells acquire resistance mechanisms during the in vivo selection process for brain tropism, we compared the chemosensitivity of parental BC cells and their brain metastatic derivatives to trastuzumab. HER2 receptor density was analyzed by western blot (Fig. 1 A) and flow cytometry (Fig. 1 B). SUM190 cells exhibited higher HER2 expression levels than JIMT-1 cells. However, no significant differences in HER2 expression were observed between brain-tropic cells and their parental counterparts, indicating that brain organotropism does not inherently alter HER2 receptor density. Despite maintaining HER2 expression, brain-tropic cells displayed significantly reduced sensitivity to trastuzumab compared to their parental counterparts (Fig. 1 C-D). This was evidenced by a consistently higher percentage of viable cells across all drug concentrations tested, highlighting the limited therapeutic efficacy of trastuzumab against brain metastatic cells. These findings suggest that metastatic cells acquire specific resistance mechanisms driven by the brain microenvironment through successive adaptations independent of HER2 expression changes. This underscores the urgent need for alternative therapeutic strategies to overcome trastuzumab resistance in BrM. II. Radiolabeling of trastuzumab with Zirconium-89 and Lutetium-177 To evaluate whether TRNT could serve as an alternative for eradicating trastuzumab-resistant brain metastatic cells, we radiolabeled the mAb with Zirconium-89 ( 89 Zr) for immunoreactivity assessment and with the β − emitter 177 Lu for TRNT evaluation. Trastuzumab was conjugated to p -SCN-Bn-deferoxamine (DFO) for 89 Zr radiolabeling and p -SCN-Bn-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid (DOTA) for 177 Lu radiolabeling. After purification of the reaction mixture to remove unbound DFO and DOTA moieties, the average number of chelators attached per trastuzumab molecule was determined using matrix-assisted laser desorption-ionization time of flight (MALDI-TOF) mass spectrometry (MS). The mass peaks for DFO-Trastuzumab and DOTA-Trastuzumab conjugates were observed at 151214.0 and 149341.6 g/mol, respectively. By dividing the mass difference of the conjugate and trastuzumab by the molecular mass of a single DFO (752.94 g/mol) or DOTA (687.8 g/mol), the average number of DFO and DOTA molecules attached per trastuzumab moiety was found to be 7.5 and 6.3, respectively. Radiolabeling of DFO-Trastuzumab conjugates with [ 89 Zr]Zr-oxalate achieved a radiochemical purity (RCP) of 100%, as confirmed by radio-thin layer chromatography (radio-TLC) and size exclusion high-performance liquid chromatography (SEC-HPLC). Similarly, radiolabeling of DOTA-Trastuzumab conjugates with [ 177 Lu]LuCl 3 also yielded an RCP of 100%, as validated by radio-TLC and SEC-HPLC (Figure S1A-B). The stability of [ 89 Zr]Zr-DFO-Trastuzumab and [ 177 Lu]Lu-DOTA-Trastuzumab was assessed in PBS and mouse serum, demonstrating sustained RCP for up to seven days (Figure S1C). These results confirm the stability of the radiolabeled complexes, supporting their suitability for in vitro and in vivo studies. III. [Lu]Lu-DOTA-Trastuzumab induces cytotoxicity and DNA damage in trastuzumab-resistant brain metastatic cells Following radiolabeling, a cell binding assay was performed to evaluate the affinity and specificity of the radioimmunoconjugates to the HER2 antigen. This assay was conducted exclusively in brain-tropic cells, as they exhibit HER2 receptor densities comparable to their parental counterparts. The radioimmunoassay showed a maximum uptake of [ 89 Zr]Zr-DFO-Trastuzumab of approximately 10% in JIMT-1-BR and 20% in SUM190-BR cells, which was reached 2 h after incubation (Figure S2). The higher uptake observed in SUM190-BR cells is due to the higher density of HER2 receptors in these cells, which is substantially higher than that of the former, as shown previously. The residual uptake (~ 1.4%) detected in HER2-negative MDA-MB-231 cells confirmed the specificity of the radioimmunoconjugate for HER2, with preservation of antigen-binding capacity. Next, the cytotoxicity effects of [ 177 Lu]Lu-DOTA-Trastuzumab were evaluated in both parental and brain-tropic cells. Cells were incubated for 2 h with increasing activities of [ 177 Lu]Lu-DOTA-Trastuzumab, followed by a culture period of 3, 5, and 7 days, after which cell viability was assessed. The results revealed dose- and time-dependent reduction in cell viability, with the most pronounced effects observed on day 7 (Fig. 2A). Notably, no significant differences in responsiveness were observed between the parental and the matched brain metastatic cells, indicating similar sensitivity to the β − -particles emitted by 177 Lu. We also evaluated the radiosensitivity of BC cells to [ 177 Lu]Lu-DOTA-Trastuzumab through a survival clonogenic assay. As shown in Fig. 2B, there was a progressive decrease in the number of surviving colonies with increasing [ 177 Lu]Lu-DOTA-Trastuzumab activity in both parental and brain-tropic BC cells. The extent of DNA double-strand breaks (DSBs) induced by β⁻-particles was subsequently assessed 5 days post-treatment. The comet assay revealed longer tail lengths in cells treated with [ 177 Lu]Lu-DOTA-Trastuzumab, indicating extensive DNA damage (Fig. 2C). Consistently, quantification of the integrated density of γ-H2AX foci per nucleus similarly showed a marked increase in DNA DSBs in treated BC cells compared to untreated controls (Fig. 2D). Importantly, no significant differences in DNA damage levels were observed between parental and brain-tropic cells, further supporting the potential of [ 177 Lu]Lu-DOTA-Trastuzumab as an alternative strategy to target trastuzumab-resistant cells. IV. [Lu]Lu-DOTA-Trastuzumab inhibits the growth of trastuzumab-resistant tumors After confirming the therapeutic efficacy of [ 177 Lu]Lu-DOTA-trastuzumab against both parental and trastuzumab-resistant brain metastatic cells in vitro , we further evaluated its in vivo effectiveness in HER2 + BC xenograft models. Given the consistent in vitro results across HER2 + cell lines, we conducted in vivo experiments exclusively using JIMT-1 and JIMT-1-BR cells. Primary BC models were established by orthotopic injection of these cells into the fourth mammary fat pad. Both cell lines formed HER2 + breast tumors within 15–20 days post-injection, as confirmed by histopathological analysis (Figure S3A). To non-invasively assess HER2 expression in mammary tumors, we performed positron emission tomography (PET) imaging using [ 89 Zr]Zr-DFO-Trastuzumab. Images acquired five days post-injection revealed significant radiotracer accumulation in both JIMT-1 and JIMT-1-BR tumors (Figure S3B), confirming the specificity of the radiolabeled antibody for HER2. Biodistribution studies at seven days post-injection demonstrated preferential uptake of [ 89 Zr]Zr-DFO-Trastuzumab in BC lesions (Figure S3C), with notable radioactivity detected in the liver and spleen, suggesting hepatobiliary clearance. Additionally, bone accumulation was observed, likely due to free 89 Zr binding to bone tissue following radiolysis or metabolic degradation, as previously described in preclinical studies [22]. Following confirmation of HER2 expression in both JIMT-1 and JIMT-1-BR tumors by PET, animals were subjected to trastuzumab-targeted therapy according to a previously described protocol [23]. Treatment was initiated when tumor xenografts reached a mean volume of 45 mm³. Mice were randomized into two groups (n = 3 per group; Fig. 3 A) and received either trastuzumab or saline. In mice bearing JIMT-1 tumors, trastuzumab treatment significantly delayed tumor growth by 33 days compared to saline-treated controls (p < 0.01). In the saline group, tumors exceeded 250 mm³ by day 23, whereas trastuzumab-treated tumors remained below 170 mm³ throughout the 56-day follow-up period, as confirmed by magnetic resonance imaging (MRI) (Fig. 3 B). In contrast, trastuzumab had no significant effect on JIMT-1-BR tumor growth compared to saline-treated controls, with tumor volumes in both groups exceeding 250 mm³ between days 23 and 31 (Fig. 3 C). These findings confirm trastuzumab resistance in JIMT-1-BR tumors, consistent with in vitro observations. We next evaluated the therapeutic efficacy of [ 177 Lu]Lu-DOTA-Trastuzumab in trastuzumab-resistant JIMT-1-BR tumors. Mice received a single dose (~ 8 MBq) of [ 177 Lu]Lu-DOTA-Trastuzumab (Fig. 3 D). Unlike trastuzumab, which failed to inhibit JIMT-1-BR tumor growth, [ 177 Lu]Lu-DOTA-Trastuzumab treatment significantly delayed tumor progression by 23 days compared to the saline group (p < 0.001; Fig. 3 E). Although tumor growth resumed in some mice around day 35, it subsequently stabilized, with final tumor volumes ranging from 65 to 95 mm³. As shown in Fig. 3 F, [ 177 Lu]Lu-DOTA-Trastuzumab significantly improved overall survival compared to saline-treated controls (p < 0.05). No significant changes in body weight were observed throughout treatment, indicating a favorable safety profile. V. Efficacy of [Lu]Lu-DOTA-Trastuzumab in treating HER2 + brain metastases Although the BBB restricts the penetration of most CNS-targeted drugs [24], the newly formed vasculature in BrM is structurally compromised, leading to a defective and heterogeneous endothelial barrier that may enhance drug delivery [25]. Based on this, we investigated the therapeutic efficacy of [ 177 Lu]Lu-DOTA-trastuzumab against BrM. Mice with MRI-detected brain metastatic lesions were treated with [ 177 Lu]Lu-DOTA-Trastuzumab, trastuzumab, or saline and monitored by MRI every four days (Fig. 4 A). Consistent with findings in the primary mammary tumor, trastuzumab monotherapy had no significant effect on JIMT-1-BR BrM growth compared to saline, as evidenced by the development of multiple metastatic foci in both groups. In contrast, a single dose of [ 177 Lu]Lu-DOTA-Trastuzumab led to complete remission in 2 of 5 mice (40%), as confirmed by MRI and histopathological analysis, with no detectable malignant lesions (Fig. 4 B-C). The remaining three treated animals exhibited disease progression comparable to those receiving trastuzumab. Nevertheless, treatment with [ 177 Lu]Lu-DOTA-Trastuzumab was associated with improved survival compared to the saline-treated group (Fig. 4 D). These results support the potential of TRNT with trastuzumab as a promising strategy for treating BrM, particularly in trastuzumab-resistant cases. VI. Brain metastatic lesions exhibit heterogeneous BBB permeability To explore the factors underlying differential responses to [ 177 Lu]Lu-DOTA-Trastuzumab, we assessed BBB permeability within metastatic lesions using dynamic contrast-enhanced (DCE)-MRI with gadolinium. In this model, intracardiac tumor cell injection requires BBB traversal for successful brain colonization, closely mimicking the metastatic cascade. Using this approach, BrM formed within 20–30 days post-injection, as detected by MRI. DCE-MRI analysis revealed gadolinium extravasation into metastatic lesions, indicating increased BBB permeability. However, signal intensity varied among lesions, with some exhibiting high gadolinium accumulation (orange/red) and others showing lower uptake (green/blue), reflecting intertumoral heterogeneity in BBB integrity (Fig. 5A-B). No gadolinium enhancement was observed in healthy brain regions, confirming an intact BBB. Accordingly, PET imaging with [ 89 Zr]Zr-DFO-Trastuzumab demonstrated radiotracer accumulation only in a subset of metastatic brain lesions (Fig. 5C-D). These findings suggest that the degree of BBB disruption directly affects trastuzumab penetration and, consequently, the therapeutic efficacy of [ 177 Lu]Lu-DOTA-Trastuzumab. Given trastuzumab’s high molecular weight (150 kDa), its permeability may remain limited even in regions with BBB leakage, potentially contributing to suboptimal distribution and therapeutic response. Discussion Despite significant advances in targeted therapies, patients with metastatic HER2 + BC remain at high risk of developing BrM, for which therapeutic options are limited and often ineffective. The inadequate penetration of trastuzumab and other HER2-targeted therapies into the CNS remains a major concern but is not the only limiting factor [9]. The emergence of brain-specific drug resistance mechanisms further adds another layer of complexity to treatment, underscoring the urgent need for the development of effective anti-metastatic therapies. Our findings confirm that brain-tropic cells acquire resistance to trastuzumab driven by brain microenvironmental signals during the in vivo selective pressure, which is not related to HER2 receptor density but rather to other mechanisms. For instance, neuregulin (NRG), secreted by neurons and astrocytes, promotes HER2 heterodimerization with HER3 and HER4, activating downstream PI3K/AKT and MAPK/ERK signaling pathways, which contribute to trastuzumab resistance [26]. Although not the primary focus of this study, we observed increased phospho-AKT and phospho-ERK levels in brain-tropic cells, supporting the involvement of these survival pathways (data not shown). Similar findings have been reported in other studies, which showed that BrM from HER2 + BC frequently exhibits PI3K/AKT activation due to PTEN loss and HER3 activation, influenced by molecular cues from the brain microenvironment [27–29]. The trastuzumab-resistant phenotype of brain-tropic cells observed in vitro was confirmed in vivo , where both primary mammary tumors and BrM xenografts remained refractory to trastuzumab, regardless of HER2 status, as verified by immunohistochemistry and PET imaging with [ 89 Zr]Zr-DFO-Trastuzumab. In contrast, conjugating trastuzumab with the β-emitting radionuclide 177 Lu significantly enhanced therapeutic efficacy, overcoming resistance mechanisms in brain metastatic cells by inducing DNA damage in vitro . In vivo , a single dose of [ 177 Lu]Lu-DOTA-Trastuzumab significantly inhibited tumor growth in mice xenografted with brain-tropic cells in the mammary fat pad, leading to improved overall survival compared to trastuzumab-treated animals, which remained largely unresponsive. These results align with previous preclinical studies demonstrating the superior cytotoxic effects of [ 177 Lu]Lu-DOTA-Trastuzumab compared to its non-radioactive counterpart, with minimal off-target uptake [30, 31]. Moreover, clinical studies have established the safety of [ 177 Lu]Lu-DOTA-Trastuzumab in patients with HER2 + primary and metastatic breast lesions [14, 32]. The selective uptake of [ 177 Lu]Lu-DOTA-Trastuzumab in BC lesions, coupled with its low toxicity profile, supports its potential as a viable alternative approach for treating HER2 + trastuzumab-resistant tumors. In the context of BrM, although [ 177 Lu]Lu-DOTA-Trastuzumab did not effectively reduce the metastatic burden in all treated animals, 40% achieved complete remission, as confirmed by MRI and histopathology, and experienced a significant survival benefit compared to saline-treated controls. The variable therapeutic response suggests that [ 177 Lu]Lu-DOTA-Trastuzumab may not consistently reach BrM, preventing the delivery of a lethal dose of cytotoxic radiation to all metastatic lesions. While BBB permeability is increased in BrM, it remains highly heterogeneous, potentially limiting trastuzumab penetration in certain areas. This was supported by DCE-MRI imaging with gadolinium, which revealed intertumoral heterogeneity in BBB leakage across metastatic lesions. Additionally, a PET scan with [ 89 Zr]Zr-DFO-Trastuzumab confirmed radiotracer accumulation in some HER2 + BrM, validating vascular permeability to the antibody. However, we cannot be certain that this increased permeability was consistent in all cases, as not all treated animals underwent PET imaging before receiving [ 177 Lu]Lu-DOTA-Trastuzumab, representing a limitation of our study. Previous PET/computed tomography (CT) studies have also reported heterogeneous trastuzumab accumulation in BrM. Terrell-Hal et al. demonstrated that [ 125 I]I-trastuzumab crosses the disrupted BBB and reaches HER2 + BrM [33]. Similarly, Puttemans et al. observed a high uptake of [ 111 In]In-trastuzumab in BrM derived from the highly invasive 231Br cell line, but not in SKOV3 brain tumors, which exhibit low vascularization and minimal BBB disruption [23]. In a clinical study, Kurihara et al. found that some MRI-detected BrM were not visible on [ 64 Cu]Cu-trastuzumab PET/CT [18]. This disparity highlights the heterogeneous permeability among metastatic lesions, which can trigger drug resistance mechanisms, contributing to unsatisfactory responses to TRNT with trastuzumab. However, it is worth mentioning that animals in our study received only a single dose of [ 177 Lu]Lu-DOTA-Trastuzumab, and higher or repeated dosing may improve therapeutic outcomes. As an alternative to trastuzumab, nanobodies are being explored for their ability to cross the BBB more efficiently due to their small size – approximately one-tenth that of conventional antibodies – while retaining HER2 immunoreactivity [34]. A recent study demonstrated the potential of radiolabeled anti-HER2 nanobody 2Rs15d in treating HER2 + BrM, where administration of [ 131 I]I-2Rs15d or [ 225 Ac]Ac-2Rs15d, alone or in combination with trastuzumab, significantly improved survival in two tumor models resistant to trastuzumab monotherapy [23]. However, despite their advantages in BBB penetration, nanobodies have a shorter half-life in circulation, requiring frequent or higher dosing to achieve a sustained therapeutic effect. In summary, we have demonstrated that [ 177 Lu]Lu-DOTA-Trastuzumab is highly effective in inhibiting the growth of trastuzumab-resistant breast tumors. Although its response rate in BrM was not 100%, 40% of treated animals achieved complete remission, which is a notable result. To our knowledge, this represents the first preclinical evidence of [ 177 Lu]Lu-DOTA-Trastuzumab’s efficacy against HER2 + BrM. While preclinical, these findings mark an important step toward improving treatment options for BrM, where therapeutic choices remain limited. From a clinical perspective, since the BBB might still hamper the penetrance of [ 177 Lu]Lu-DOTA-Trastuzumab and limit its efficacy, a [ 89 Zr]Zr-DFO-Trastuzumab PET imaging before treatment could help identify patients, most likely to benefit from radionuclide therapy. Materials and methods Cell culture Two human HER2 + BC cell lines, JIMT-1 and SUM190, along with their respective brain metastatic variants, JIMT-1-BR and SUM190-BR, were generously provided by Dr. Patricia Steeg from the National Cancer Institute (Bethesda, USA). These brain metastatic sublines were generated from the parental cell lines by repeated intracardiac injections and subsequent harvesting of metastatic cells from the brains of mouse models, as previously described by Palmieri et al. for JIMT-1-BR [35] and Lyle et al. for SUM190-BR [7]. JIMT-1 cells were cultured in DMEM (Sigma-Aldrich) with 10% FBS (Gibco) and 1% antibiotic-antimycotic (Gibco). SUM190 cells were cultured in Ham's F-12 medium (Sigma-Aldrich) supplemented with 1 µg/ml hydrocortisone (Sigma-Aldrich), 10 mM HEPES (Sigma-Aldrich), insulin-transferrin-selenium-ethanolamine (Gibco), 10 nM triiodothyronine (Sigma-Aldrich), 1 g/L BSA (Sigma-Aldrich), and 1% antibiotic-antimycotic (Gibco). Colony formation assay JIMT-1 and SUM190 cells and brain metastatic derivatives were seeded in 12-well plates at densities of 600 and 4000 cells per well, respectively, and allowed to adhere overnight. JIMT-1 cells were treated with 0.5, 1, 2, and 5 MBq/mL of [ 177 Lu]Lu-DOTA-Trastuzumab, while SUM190 cells received 1, 5, 7.5, or 10 MBq/mL, in 1 mL of medium and incubated at 37°C for 2 h. Untreated cells served as controls. Subsequently, the cell culture medium was replaced with fresh medium, and plates were incubated at 37°C to allow colony formation. After 10–14 days of incubation, colonies were gently washed with PBS, fixed with 1% formaldehyde for 15 min, and stained with 0.1% crystal violet in ethanol for another 15 min. The assay was performed in duplicate for each cell line across three independent experiments. γ-H2AX assay JIMT-1 and SUM190 cells and brain metastatic derivatives were seeded in glass coverslips in a 24-well plate at a density of 4.5x10 4 and 1x10 5 cells per well, respectively, and allowed to attach overnight. JIMT-1 cells were treated with 2 or 5 MBq/mL of [ 177 Lu]Lu-DOTA-Trastuzumab, while SUM190 cells received 5 or 10 MBq/mL, in 500 µL of medium for 2 h at 37°C. Untreated cells served as controls. Subsequently, the medium was replaced with fresh culture medium, and cells were maintained at 37°C for 5 days. After three PBS washes, cells were fixed with 4% PFA for 15 min, permeabilized with 0.5% Triton X-100 at RT for 15 min, followed by two additional PBS washes. The cells were then incubated overnight with a primary antibody against γ-H2AX (ser139; Abcam, ref. ab81299) at a 1:250 dilution. After two PBS washes, cells were treated with a FITC-conjugated anti-rabbit secondary antibody (Invitrogen) at a 1:200 dilution for 1 h, followed by three PBS washes. Cell nuclei were stained with Hoechst 33342 (Sigma-Aldrich) at 5 µg/ml for 5 min at RT in the dark and then mounted in Vectashield (Vector Laboratories). Images were captured using a Carl Zeiss Axio Observer Z1 inverted microscope at 20x magnification. Six randomly selected images were captured per slide. Quantitative analysis of γ-H2AX foci was performed using ImageJ (National Institutes of Health), with a minimum of 20 nuclei analyzed per experiment, performed in duplicate. Three independent experiments were carried out for each cell line. Comet assay JIMT-1 and SUM190 cells and brain-metastatic derivatives were seeded in 6-well plates at densities of 1x10 5 and 3x10 5 cells per well, respectively, and allowed to adhere overnight. JIMT-1 cells were treated with 2 or 5 MBq/mL of [ 177 Lu]Lu-DOTA-Trastuzumab, while SUM190 cells were treated with 5 or 10 MBq/mL in 2 mL of medium and incubated at 37°C for 2 h. Untreated cells served as controls. After treatment, the medium was replaced with fresh culture medium, and cells were incubated at 37°C for 5 days. To assess genotoxicity, cells were gently scraped and processed using the comet assay kit (Enzo Life Sciences; ref. ADI-900-166) according to the manufacturer’s instructions. Comet tails were visualized with a Carl Zeiss Axio Observer Z1 inverted microscope and analyzed by measuring the length of the comet tail using ImageJ software (National Institutes of Health). Eight images were randomly collected from each slide. Three independent experiments were conducted for each cell line. Animal studies All animal experiments were approved by the Animal Welfare Committee of the Institute for Nuclear Sciences Applied to Health (ICNAS) of the University of Coimbra (ORBEA 04-2021) and by the Portuguese National Authority for Animal Health (DGAV). Female athymic Swiss FoxnI nu/nu mice aged 10–12 weeks (20–25 g) were purchased from ICNAS and housed under pathogen-free conditions in individually ventilated cages. For the BC model, mice were orthotopically injected with 2x10 6 cells of JIMT-1 or JIMT-1-BR suspended in a 50% PBS and 50% Matrigel mixture (total volume of 50 µL) into the 4th mammary fat pad. Tumor growth was monitored twice weekly with a digital caliper. Tumour volumes were calculated using the modified ellipsoid formula V = A × B 2 /2 (A length; B width). For experimental BrM formation, mice were intracardially injected with 1.75x10 5 brain-tropic JIMT-1-BR cells in 100 µL of PBS into the left ventricle as previously described [36]. BrM formation was monitored twice a week by MRI in a BioSpect 9.4T MRI scanner (Bruker Biospin, Ettlingen) under anesthesia (2% isoflurane). The animals' breathing rate and body temperature were monitored throughout the imaging procedures (SA Instruments SA). Morphological brain images were acquired on the T2-RurboRARE sequence in coronal orientation with the following parameters: TE/TR = 33.01/2500 ms, FOV = 20.0*20.0 mm, acquisition matrix = 256*256, averages = 5,18 continuous slices with 0.4 mm thick, and acquisition time of 6m40s. Therapeutic evaluation of trastuzumab and [ 177 Lu]Lu-DOTA-Trastuzumab Animals bearing a primary breast tumor or BrM were treated with trastuzumab (Evidentic GmbH) or [ 177 Lu]Lu-DOTA-Trastuzumab. In the BC model, treatments were initiated when tumors reached an average volume of 35 to 60 mm³. Mice were randomized into groups of 3 per group and received either (i) trastuzumab (loading dose 7.5 mg/kg, D0; maintenance dose 3.5 mg/kg, biweekly on D3-7-10-14), (ii) [ 177 Lu]Lu-DOTA-Trastuzumab (8 ± 1.20 MBq; 60 µg Trastuzumab), or (iii) vehicle buffer (0.9% NaCl). All treatments were administered i.v. in a total volume of 100 µL. Tumors were monitored twice weekly by MRI for up to 56 days. The study was ended when tumor volume reached ≥ 250 mm 3 or when humane endpoints were reached. For the experimental brain metastatic model, trastuzumab or [ 177 Lu]Lu-DOTA-Trastuzumab therapy was initiated when BrM were detected by MRI. At this time, mice received saline (n = 3), trastuzumab (loading dose 7.5 mg/kg, D0; maintenance dose 3.5 mg/kg, biweekly on D3-7-10-14; n = 3), [ 177 Lu]Lu-DOTA-Trastuzumab (8 ± 0.81 MBq; 60 µg Trastuzumab; n = 5). All treatments were administered i.v. in a total volume of 100 µL. Animals were weighed biweekly and checked daily for general health and well-being. Animals were sacrificed when one of the following endpoints was reached: weight loss > 20% of original body weight, immobility, or unresponsiveness to external stimuli. In both models, mice that reached the humane endpoint or completed the observation period were euthanized by cervical dislocation. Survival was analyzed by the Kaplan-Meier survival curve. Afterward, tumor lesions and the whole brain were fixed in PFA 4% and embedded in paraffin wax for histopathological analysis. Dynamic contrast-enhanced-magnetic resonance imaging (DCE-MRI) To evaluate BBB permeability alterations in vivo , DCE-MRI was performed after the intraperitoneal injection of a gadolinium-based contrast agent in healthy mice and mice bearing BrM, as previously described [37]. Briefly, dynamic contrast-enhanced images were acquired with a DCE_FLASH sequence with the following parameters: TE/TR = 2.5/100 ms, FA = 70°, FOV = 20*20 mm, acquisition matrix = 156*5, 18 coronal slices with 0.4 mm thick, 60 dynamics acquired, 7 averages, scan time per dynamic = 49s700ms, total scan time = 49m42s. The gadolinium-based contrast agent Gadobutrol (Gadovist®, LUSAL) was administered intraperitoneally after the acquisition of 5 baseline scans, with 60 dynamic scans acquired following the injection. A region of interest (ROI) was drawn aroufnd each BrM using a semiautomatic procedure. In healthy animals, a corresponding ROI was drawn in the same region. The mean variation of signal intensity as a function of time was then quantified in the predefined ROI to evaluate perfusion and vascular permeability. Statistical analysis All data are expressed as mean ± standard error of the mean (SEM). Graphics and statistical analysis were performed using GraphPad Prism software. Data were analyzed using two-way ANOVA analysis followed by Turkey’s post-hoc test, as indicated in figure legends. For the Kaplan-Meier analysis, statistical differences in survival curves were calculated by log-rank (Mantel-Cox) test. Statistical significance was set at the level of p < 0.05, and the ‘n’ represents the total number of experiments. Figure illustrations were created with Bio-Render.com. Declarations Research involving animals All animal experiments were approved by the Animal Welfare Committee of the Institute for Nuclear Sciences Applied to Health (ICNAS) of the University of Coimbra (ORBEA 04-2021) and by the Portuguese National Authority for Animal Health (DGAV). Conflicts of interest No potential conflicts of interest relevant to this article exist. Funding This work was supported by the Portuguese Foundation for Science and Technology (FCT) through the project PTDC/BTM-SAL/4451/2020 and STRATEGIC PROJECTS (UIDB/04539/2020 and UIDP/04539/2020). L.S. is a Ph.D. fellow of the FCT (PD/BDE/150707/2020). Author Contributions LS, IH, JS and HF performed experiments, acquisition, analysis, and interpretation of data. PT and RA performed histopathological analysis and immunostaining. MS performed the production of zirconium-89. LS wrote the manuscript. CG and AJA designed and supervised the study, revised the manuscript, and obtained financial support. All authors reviewed the manuscript and approved its content. Data availability All data generated and analyzed during this study are included in this article and its supplementary information files. References Goddard K, Weinmann S, Richert-Boe K, Chen C, Bulkley J, Wax C. HER2 evaluation and its impact on breast cancer treatment decisions. Public Health Genomics. 2011;15:1–10. Arteaga CL, Sliwkowski MX, Osborne CK, Perez EA, Puglisi F, Gianni L. Treatment of HER2-positive breast cancer: current status and future perspectives. Nature reviews Clinical oncology. 2012;9:16–32. 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Ni J, Ramkissoon SH, Xie S, Goel S, Stover DG, Guo H, et al. Combination inhibition of PI3K and mTORC1 yields durable remissions in mice bearing orthotopic patient-derived xenografts of HER2-positive breast cancer brain metastases. Nature medicine. 2016;22:723-6. Kodack DP, Askoxylakis V, Ferraro GB, Sheng Q, Badeaux M, Goel S, et al. The brain microenvironment mediates resistance in luminal breast cancer to PI3K inhibition through HER3 activation. Science translational medicine. 2017;9:eaal4682. Zhang L, Zhang S, Yao J, Lowery FJ, Zhang Q, Huang W-C, et al. Microenvironment-induced PTEN loss by exosomal microRNA primes brain metastasis outgrowth. Nature. 2015;527:100-4. Ramli M, Hidayat B, AGUSWARINI S, KARYADI K, ARDIYATNO CN, SUBUR H, et al. Preclinical study of 177Lu-DOTA-trastuzumab: A potential radiopharmaceutical for therapy of breast cancer positive HER-2. Jurnal Ilmu Kefarmasian Indonesia. 2013;11:116 − 22. Ray GL, Baidoo KE, Keller LM, Albert PS, Brechbiel MW, Milenic DE. Pre-Clinical Assessment of Lu-Labeled Trastuzumab Targeting HER2 for Treatment and Management of Cancer Patients with Disseminated Intraperitoneal Disease. Pharmaceuticals (Basel). 2011;5:1–15. doi:10.3390/ph5010001. Nautiyal A, Jha AK, Mithun S, Shetye B, Kameswaran M, Shah S, et al. Analysis of absorbed dose in radioimmunotherapy with 177Lu-trastuzumab using two different imaging scenarios: a pilot study. Nucl Med Commun. 2021;42:1382-95. doi:10.1097/mnm.0000000000001472. Terrell-Hall TB, Nounou MI, El-Amrawy F, Griffith JI, Lockman PR. Trastuzumab distribution in an in-vivo and in-vitro model of brain metastases of breast cancer. Oncotarget. 2017;8:83734. Hrynchak I, Santos L, Falcão A, Gomes CM, Abrunhosa AJ. Nanobody-based theranostic agents for HER2-positive breast cancer: radiolabeling strategies. International journal of molecular sciences. 2021;22:10745. Palmieri D, Duchnowska R, Woditschka S, Hua E, Qian Y, Biernat W, et al. Profound prevention of experimental brain metastases of breast cancer by temozolomide in an MGMT-dependent manner. Clinical Cancer Research. 2014;20:2727-39. Santos L, Tomatis F, Ferreira HR, Almeida SF, Ciputra E, Sereno J, et al. ENPP1 induces blood–brain barrier dysfunction and promotes brain metastasis formation in human epidermal growth factor receptor 2-positive breast cancer. Neuro-Oncology. 2025;27:167 − 83. Duarte Lobo D, Nobre RJ, Oliveira Miranda C, Pereira D, Castelhano J, Sereno J, et al. The blood-brain barrier is disrupted in Machado-Joseph disease/spinocerebellar ataxia type 3: evidence from transgenic mice and human post-mortem samples. Acta Neuropathologica Communications. 2020;8:1–19. Additional Declarations The authors declare no competing interests. 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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-6180100","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":425738228,"identity":"d320ec30-e2a5-419f-8181-147e028d3792","order_by":0,"name":"Liliana Santos","email":"","orcid":"https://orcid.org/0000-0003-2525-9572","institution":"Institute for Nuclear Sciences Applied to Health (ICNAS) and Coimbra Institute for Biomedical Imaging and Translational Research (CIBIT), University of Coimbra, Coimbra, 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Clinical and Biomedical Research (iCBR), University of Coimbra, Coimbra, Portugal","correspondingAuthor":true,"prefix":"","firstName":"Celia","middleName":"","lastName":"Gomes","suffix":""}],"badges":[],"createdAt":"2025-03-07 17:31:00","currentVersionCode":1,"declarations":{"humanSubjects":false,"vertebrateSubjects":true,"conflictsOfInterestStatement":false,"humanSubjectEthicalGuidelines":false,"humanSubjectConsent":false,"humanSubjectClinicalTrial":false,"humanSubjectCaseReport":false,"vertebrateSubjectEthicalGuidelines":true},"doi":"10.21203/rs.3.rs-6180100/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6180100/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":78328563,"identity":"6aed5d8b-eebd-4458-80e1-6b51d1769b0c","added_by":"auto","created_at":"2025-03-12 06:50:26","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":64823,"visible":true,"origin":"","legend":"\u003cp\u003eHER2+ brain metastatic breast cancer cells exhibit reduced sensitivity to trastuzumab while maintaining HER2 expression. (A) Western blot and (B) flow cytometry analysis of HER2 receptor density in JIMT-1-BR and SUM190-BR cells compared to their respective parental cell lines. Concentration-response curves showing the viability of (C) JIMT-1-BR and (D) SUM190-BR cells and their respective parental counterparts following treatment with increasing concentrations of trastuzumab. \u003csup\u003e*\u003c/sup\u003ep\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003ep\u0026lt;0.01 when compared to the parental cell line. Statistical significance was assessed using two-way ANOVA followed by Turkey's multiple comparison test\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-6180100/v1/7b4c0fd06a152afc59368967.png"},{"id":78328588,"identity":"9338e218-1b90-4769-b98d-8f690a2396cf","added_by":"auto","created_at":"2025-03-12 06:50:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":366076,"visible":true,"origin":"","legend":"\u003cp\u003eParental and brain metastatic HER2+ breast cancer cells exhibit comparable sensitivity to [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-Trastuzumab. (A) Cell viability of parental and brain metastatic cells following treatment with increasing activity of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-Trastuzumab, assessed after 3, 5, and 7 days of culture. (B) Representative images of surviving cell colonies 10-14 days after treatment with increasing activity of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-Trastuzumab. (C) Representative images of comet tail lengths in parental and brain metastatic cells 5 days after treatment with different activities of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-trastuzumab. (D) Representative images and (E) quantification of γ-H2AX foci per nucleus in parental and brain metastatic cells 5 days after treatment with different activities of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-trastuzumab. \u003csup\u003e*\u003c/sup\u003ep\u0026lt;0.05, \u003csup\u003e**\u003c/sup\u003ep\u0026lt;0.01, \u003csup\u003e***\u003c/sup\u003ep\u0026lt;0.001, \u003csup\u003e****\u003c/sup\u003ep\u0026lt;0.0001, when compared to untreated cells (\u003cem\u003edashed line\u003c/em\u003e). Statistical significance was assessed using two-way ANOVA followed by Turkey's multiple comparison test. Nuclei were stained with Hoechst 33342 (blue). Scale Bar: 20 µm\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-6180100/v1/9b1b57b34180ba93e72b483f.png"},{"id":78329607,"identity":"fb3c1649-7ef9-40d6-88a3-c5be6f2310a1","added_by":"auto","created_at":"2025-03-12 06:58:27","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":172501,"visible":true,"origin":"","legend":"\u003cp\u003eJIMT-1-BR trastuzumab-resistant tumors respond to [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-Trastuzumab. (A) Schematic representation of the treatment regimen for mice bearing JIMT-1 and JIMT-1-BR tumors. Mice received either saline or trastuzumab (loading dose 7.5 mg/kg on Day 0, followed by maintenance dose 3.5 mg/kg biweekly on Days 3, 7, 10 and 14), with MRI imaging performed throughout the study. Tumor volume over time (left) and representative MRI images (right) of mice bearing (B) JIMT-1 and (C) JIMT-1-BR tumors following trastuzumab treatment. (D) Schematic of the treatment regimen for mice bearing JIMT-1-BR tumors treated with a single dose of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-Trastuzumab (~8MBq) or saline, with MRI imaging performed throughout the study. (E) Tumor volume over time (left) and representative MRI images (right) in JIMT-1-BR tumor-bearing mice. p\u0026lt;0.01, p\u0026lt;0.001, when compared to the saline-treated mice group. Statistical significance was assessed using two-way ANOVA followed by Turkey's multiple comparison test. Yellow arrows indicate the locations of breast tumors. (F) Kaplan–Meier survival analysis showing a significantly shortened overall survival in mice treated with [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-Trastuzumab. \u003csup\u003e*\u003c/sup\u003ep\u0026lt;0.05, when compared to the saline-treated group. Statistical significance was assessed using log-rank test; n=3 per group\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-6180100/v1/574100497359063f70b9710a.png"},{"id":78329604,"identity":"a318db8f-d66f-489b-9efb-8a273573d19d","added_by":"auto","created_at":"2025-03-12 06:58:26","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":405142,"visible":true,"origin":"","legend":"\u003cp\u003eEfficacy of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-Trastuzumab in treating HER2+ brain metastases. (A) Schematic representation of the treatment regimen for mice with JIMT-1-BR brain metastases. Mice received saline (n=3), trastuzumab (loading dose 7.5 mg/kg on Day 0, followed by maintenance dose 3.5 mg/kg biweekly on Days 3, 7, 10 and 14; n=3), or a single injection of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-Trastuzumab (~8MBq; n=5), with MRI imaging conducted throughout the study. (B) Representative MRI images showing brain metastases progression over time in the different treatment groups. Yellow arrows indicate the locations of brain metastases. (C) Representative H\u0026amp;E-stained whole-brain sections showing the presence or absence of brain metastatic lesions. Scale bars: 1 mm. (D) Kaplan–Meier survival analysis showing a significantly prolonged survival in mice treated with [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-Trastuzumab. \u003csup\u003e*\u003c/sup\u003ep\u0026lt;0.05, when compared to the saline-treated group. Statistical significance was assessed using log-rank test\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-6180100/v1/3c3daa8582a65cbdee38f365.png"},{"id":78328565,"identity":"e892cbb1-de9f-4fc2-abf2-804c7d4de6cf","added_by":"auto","created_at":"2025-03-12 06:50:26","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":276154,"visible":true,"origin":"","legend":"\u003cp\u003eBrain metastatic lesions exhibit heterogeneous BBB permeability. (A) Representative MRI (left) and DCE-MRI (right) images of mice bearing brain metastases (top) and healthy controls (bottom). (B) Quantification of gadolinium leakage into the brain over time in both groups. Orange and red circles highlight brain lesions with high gadolinium accumulation, while green and blue circles indicate lower uptake, demonstrating inter-tumor heterogeneity in BBB permeability. (C) PET/MRI scan showing [\u003csup\u003e89\u003c/sup\u003eZr]Zr-DFO-Trastuzumab accumulation in a brain metastatic lesion. Yellow arrows indicate the locations of brain metastases. The scale bar represents low (black) to high (red) standardized uptake values (SUV). (D) Representative immunohistochemical images of HER2 expression in JIMT-1-BR brain metastases. Scale bars: 1 mm, in inserts: 100 µm\u003c/p\u003e","description":"","filename":"5.png","url":"https://assets-eu.researchsquare.com/files/rs-6180100/v1/0c1c310c91c5ece41edb3873.png"},{"id":78329612,"identity":"bc274e15-f0ea-4b52-ad4c-1db54924fa81","added_by":"auto","created_at":"2025-03-12 06:58:33","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2280029,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6180100/v1/e8fc67d2-3cc2-49da-acf0-38e39cb957c9.pdf"},{"id":78328575,"identity":"91af2e06-67be-40af-abfc-eb44ab84c522","added_by":"auto","created_at":"2025-03-12 06:50:27","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":33254351,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryMaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-6180100/v1/9e3474830233801444523203.docx"}],"financialInterests":"The authors declare no competing interests.","formattedTitle":"\u003cp\u003e\u003cstrong\u003e177Lu-Trastuzumab Radionuclide Therapy: an Effective Approach for Resistant Brain Metastases in HER2+ Breast Cancer\u003c/strong\u003e\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003eBreast cancer (BC) remains the most common malignancy in women worldwide. Approximately 25 to 30% of metastatic BC cases exhibit amplification of the human epidermal growth factor receptor 2 (HER2), which is associated with a more aggressive clinical phenotype and poorer prognosis [1]. The anti-HER2 monoclonal antibody (mAb) trastuzumab has been the mainstay of systemic therapy for HER2\u0026thinsp;+\u0026thinsp;metastatic BC, significantly improving patient survival over the past decade [2]. However, despite advancements in controlling extracranial metastases, the risk of central nervous system (CNS) recurrence remains disproportionately high [3].\u003c/p\u003e \u003cp\u003eBrain metastases (BrM) occur in up to 50% of patients with HER2\u0026thinsp;+\u0026thinsp;metastatic BC, leading to increased morbidity and reduced survival [4]. This presents a critical challenge, as systemic HER2-directed therapies exhibit limited efficacy against BrM. The ineffectiveness of targeted drugs such as trastuzumab against BrM is often attributed to inadequate penetration of the blood-brain barrier (BBB), a protective barrier of the brain [5]. However, evidence suggests that BBB permeability is significantly altered in the presence of metastatic tumors, allowing macromolecules such as mAbs to penetrate [6, 7]. Despite adequate delivery of cytotoxic agents, BrM can persist due to the development of drug-resistance mechanisms [8]. Specifically, the activation of the PI3K/AKT pathway represents a resistance mechanism underlying the limited efficacy of HER2-directed therapy against BrM and other metastatic sites [9\u0026ndash;11]. These insights emphasize the complexity of treating BrM and highlight the urgent need for alternative therapeutic strategies that circumvent trastuzumab resistance mechanisms and improve treatment outcomes.\u003c/p\u003e \u003cp\u003eA promising approach involves mAb conjugates carrying cytotoxic payloads, such as radionuclides, chemotherapeutics, or toxins [12]. Targeted radionuclide therapy (TRNT) employs radiolabeled molecules like mAbs to selectively deliver β⁻- or α-emitting radionuclides to cancer cells, inducing DNA damage and cell death. TRNT has demonstrated potent anti-tumor effects and has emerged as a strategy to overcome resistance to conventional targeted therapies [13]. In metastatic BC, TRNT has shown clinical promise, particularly with β⁻-emitting agents such as [\u0026sup1;⁷⁷Lu]Lu-DOTA-trastuzumab [14] and [\u0026sup1;⁷⁷Lu]Lu-DOTA-ABY-027 (a second-generation HER2-targeting affibody) [15]. The therapeutic potential of lutetium-177 (\u0026sup1;⁷⁷Lu) is attributed to its favorable decay properties (t₁/₂ = 6.65 days, Eβ⁻ mean\u0026thinsp;=\u0026thinsp;134 keV, Eβ⁻ max\u0026thinsp;=\u0026thinsp;497 keV) and tissue penetration range (~\u0026thinsp;2 mm), which allows localized radiation delivery while sparing healthy tissue [16].\u003c/p\u003e \u003cp\u003eAdditionally, nuclear imaging studies using trastuzumab radiolabeled with zirconium-89 (⁸⁹Zr), indium-111 (\u0026sup1;\u0026sup1;\u0026sup1;In), and copper-64 (⁶⁴Cu) have demonstrated trastuzumab passage across the BBB, enabling tumor uptake and visualization of HER2\u0026thinsp;+\u0026thinsp;BrM in BC patients [17\u0026ndash;21]. However, the potential of trastuzumab-based TRNT using β⁻- or α-emitting radionuclides to selectively eradicate HER2\u0026thinsp;+\u0026thinsp;BrM remains unexplored.\u003c/p\u003e \u003cp\u003eIn this study, we demonstrate that brain metastatic HER2\u0026thinsp;+\u0026thinsp;cells exhibit resistance to pharmacological trastuzumab but remain susceptible to [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-trastuzumab. Furthermore, our findings reveal that [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-trastuzumab outperforms standard trastuzumab therapy in treating both HER2\u0026thinsp;+\u0026thinsp;primary tumors and BrM, offering a promising therapeutic avenue to overcome resistance and improve overall survival.\u003c/p\u003e "},{"header":"Results","content":"\u003ch3\u003eI. Brain-tropic breast cancer cells exhibit reduced sensitivity to trastuzumab while maintaining HER2 expression\u003c/h3\u003e\n\u003cp\u003eTrastuzumab resistance is a major challenge in the treatment of metastatic HER2\u0026thinsp;+\u0026thinsp;BC, impacting both extracranial and intracranial metastases. To assess whether brain metastatic cells acquire resistance mechanisms during the \u003cem\u003ein vivo\u003c/em\u003e selection process for brain tropism, we compared the chemosensitivity of parental BC cells and their brain metastatic derivatives to trastuzumab.\u003c/p\u003e\n\u003cp\u003eHER2 receptor density was analyzed by western blot (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eA) and flow cytometry (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eB). SUM190 cells exhibited higher HER2 expression levels than JIMT-1 cells. However, no significant differences in HER2 expression were observed between brain-tropic cells and their parental counterparts, indicating that brain organotropism does not inherently alter HER2 receptor density.\u003c/p\u003e\n\u003cp\u003eDespite maintaining HER2 expression, brain-tropic cells displayed significantly reduced sensitivity to trastuzumab compared to their parental counterparts (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003eC-D). This was evidenced by a consistently higher percentage of viable cells across all drug concentrations tested, highlighting the limited therapeutic efficacy of trastuzumab against brain metastatic cells. These findings suggest that metastatic cells acquire specific resistance mechanisms driven by the brain microenvironment through successive adaptations independent of HER2 expression changes. This underscores the urgent need for alternative therapeutic strategies to overcome trastuzumab resistance in BrM.\u003c/p\u003e\n\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003eII. Radiolabeling of trastuzumab with Zirconium-89 and Lutetium-177\u003c/h2\u003e\n\u003cp\u003eTo evaluate whether TRNT could serve as an alternative for eradicating trastuzumab-resistant brain metastatic cells, we radiolabeled the mAb with Zirconium-89 (\u003csup\u003e89\u003c/sup\u003eZr) for immunoreactivity assessment and with the \u0026beta;\u003csup\u003e\u0026minus;\u003c/sup\u003e emitter \u003csup\u003e177\u003c/sup\u003eLu for TRNT evaluation.\u003c/p\u003e\n\u003cp\u003eTrastuzumab was conjugated to \u003cem\u003ep\u003c/em\u003e-SCN-Bn-deferoxamine (DFO) for \u003csup\u003e89\u003c/sup\u003eZr radiolabeling and \u003cem\u003ep\u003c/em\u003e-SCN-Bn-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetracetic acid (DOTA) for \u003csup\u003e177\u003c/sup\u003eLu radiolabeling. After purification of the reaction mixture to remove unbound DFO and DOTA moieties, the average number of chelators attached per trastuzumab molecule was determined using matrix-assisted laser desorption-ionization time of flight (MALDI-TOF) mass spectrometry (MS). The mass peaks for DFO-Trastuzumab and DOTA-Trastuzumab conjugates were observed at 151214.0 and 149341.6 g/mol, respectively. By dividing the mass difference of the conjugate and trastuzumab by the molecular mass of a single DFO (752.94 g/mol) or DOTA (687.8 g/mol), the average number of DFO and DOTA molecules attached per trastuzumab moiety was found to be 7.5 and 6.3, respectively.\u003c/p\u003e\n\u003cp\u003eRadiolabeling of DFO-Trastuzumab conjugates with [\u003csup\u003e89\u003c/sup\u003eZr]Zr-oxalate achieved a radiochemical purity (RCP) of 100%, as confirmed by radio-thin layer chromatography (radio-TLC) and size exclusion high-performance liquid chromatography (SEC-HPLC). Similarly, radiolabeling of DOTA-Trastuzumab conjugates with [\u003csup\u003e177\u003c/sup\u003eLu]LuCl\u003csub\u003e3\u003c/sub\u003e also yielded an RCP of 100%, as validated by radio-TLC and SEC-HPLC (Figure S1A-B).\u003c/p\u003e\n\u003cp\u003eThe stability of [\u003csup\u003e89\u003c/sup\u003eZr]Zr-DFO-Trastuzumab and [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-Trastuzumab was assessed in PBS and mouse serum, demonstrating sustained RCP for up to seven days (Figure S1C). These results confirm the stability of the radiolabeled complexes, supporting their suitability for \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e studies.\u003c/p\u003e\n\u003c/div\u003e\n\u003ch3\u003eIII. [Lu]Lu-DOTA-Trastuzumab induces cytotoxicity and DNA damage in trastuzumab-resistant brain metastatic cells\u003c/h3\u003e\n\u003cp\u003eFollowing radiolabeling, a cell binding assay was performed to evaluate the affinity and specificity of the radioimmunoconjugates to the HER2 antigen. This assay was conducted exclusively in brain-tropic cells, as they exhibit HER2 receptor densities comparable to their parental counterparts. The radioimmunoassay showed a maximum uptake of [\u003csup\u003e89\u003c/sup\u003eZr]Zr-DFO-Trastuzumab of approximately 10% in JIMT-1-BR and 20% in SUM190-BR cells, which was reached 2 h after incubation (Figure S2). The higher uptake observed in SUM190-BR cells is due to the higher density of HER2 receptors in these cells, which is substantially higher than that of the former, as shown previously. The residual uptake (~\u0026thinsp;1.4%) detected in HER2-negative MDA-MB-231 cells confirmed the specificity of the radioimmunoconjugate for HER2, with preservation of antigen-binding capacity.\u003c/p\u003e\n\u003cp\u003eNext, the cytotoxicity effects of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-Trastuzumab were evaluated in both parental and brain-tropic cells. Cells were incubated for 2 h with increasing activities of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-Trastuzumab, followed by a culture period of 3, 5, and 7 days, after which cell viability was assessed. The results revealed dose- and time-dependent reduction in cell viability, with the most pronounced effects observed on day 7 (Fig.\u0026nbsp;2A). Notably, no significant differences in responsiveness were observed between the parental and the matched brain metastatic cells, indicating similar sensitivity to the \u0026beta;\u003csup\u003e\u0026minus;\u003c/sup\u003e-particles emitted by \u003csup\u003e177\u003c/sup\u003eLu.\u003c/p\u003e\n\u003cp\u003eWe also evaluated the radiosensitivity of BC cells to [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-Trastuzumab through a survival clonogenic assay. As shown in Fig.\u0026nbsp;2B, there was a progressive decrease in the number of surviving colonies with increasing [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-Trastuzumab activity in both parental and brain-tropic BC cells.\u003c/p\u003e\n\u003cp\u003eThe extent of DNA double-strand breaks (DSBs) induced by \u0026beta;⁻-particles was subsequently assessed 5 days post-treatment. The comet assay revealed longer tail lengths in cells treated with [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-Trastuzumab, indicating extensive DNA damage (Fig.\u0026nbsp;2C). Consistently, quantification of the integrated density of \u0026gamma;-H2AX foci per nucleus similarly showed a marked increase in DNA DSBs in treated BC cells compared to untreated controls (Fig.\u0026nbsp;2D). Importantly, no significant differences in DNA damage levels were observed between parental and brain-tropic cells, further supporting the potential of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-Trastuzumab as an alternative strategy to target trastuzumab-resistant cells.\u003c/p\u003e\n\u003ch3\u003eIV. [Lu]Lu-DOTA-Trastuzumab inhibits the growth of trastuzumab-resistant tumors\u003c/h3\u003e\n\u003cp\u003eAfter confirming the therapeutic efficacy of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-trastuzumab against both parental and trastuzumab-resistant brain metastatic cells \u003cem\u003ein vitro\u003c/em\u003e, we further evaluated its \u003cem\u003ein vivo\u003c/em\u003e effectiveness in HER2\u0026thinsp;+\u0026thinsp;BC xenograft models. Given the consistent \u003cem\u003ein vitro\u003c/em\u003e results across HER2\u0026thinsp;+\u0026thinsp;cell lines, we conducted \u003cem\u003ein vivo\u003c/em\u003e experiments exclusively using JIMT-1 and JIMT-1-BR cells. Primary BC models were established by orthotopic injection of these cells into the fourth mammary fat pad. Both cell lines formed HER2\u0026thinsp;+\u0026thinsp;breast tumors within 15\u0026ndash;20 days post-injection, as confirmed by histopathological analysis (Figure S3A).\u003c/p\u003e\n\u003cp\u003eTo non-invasively assess HER2 expression in mammary tumors, we performed positron emission tomography (PET) imaging using [\u003csup\u003e89\u003c/sup\u003eZr]Zr-DFO-Trastuzumab. Images acquired five days post-injection revealed significant radiotracer accumulation in both JIMT-1 and JIMT-1-BR tumors (Figure S3B), confirming the specificity of the radiolabeled antibody for HER2. Biodistribution studies at seven days post-injection demonstrated preferential uptake of [\u003csup\u003e89\u003c/sup\u003eZr]Zr-DFO-Trastuzumab in BC lesions (Figure S3C), with notable radioactivity detected in the liver and spleen, suggesting hepatobiliary clearance. Additionally, bone accumulation was observed, likely due to free \u003csup\u003e89\u003c/sup\u003eZr binding to bone tissue following radiolysis or metabolic degradation, as previously described in preclinical studies [22].\u003c/p\u003e\n\u003cp\u003eFollowing confirmation of HER2 expression in both JIMT-1 and JIMT-1-BR tumors by PET, animals were subjected to trastuzumab-targeted therapy according to a previously described protocol [23]. Treatment was initiated when tumor xenografts reached a mean volume of 45 mm\u0026sup3;. Mice were randomized into two groups (n\u0026thinsp;=\u0026thinsp;3 per group; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eA) and received either trastuzumab or saline. In mice bearing JIMT-1 tumors, trastuzumab treatment significantly delayed tumor growth by 33 days compared to saline-treated controls (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01). In the saline group, tumors exceeded 250 mm\u0026sup3; by day 23, whereas trastuzumab-treated tumors remained below 170 mm\u0026sup3; throughout the 56-day follow-up period, as confirmed by magnetic resonance imaging (MRI) (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eB). In contrast, trastuzumab had no significant effect on JIMT-1-BR tumor growth compared to saline-treated controls, with tumor volumes in both groups exceeding 250 mm\u0026sup3; between days 23 and 31 (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eC). These findings confirm trastuzumab resistance in JIMT-1-BR tumors, consistent with \u003cem\u003ein vitro\u003c/em\u003e observations.\u003c/p\u003e\n\u003cp\u003eWe next evaluated the therapeutic efficacy of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-Trastuzumab in trastuzumab-resistant JIMT-1-BR tumors. Mice received a single dose (~\u0026thinsp;8 MBq) of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-Trastuzumab (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eD). Unlike trastuzumab, which failed to inhibit JIMT-1-BR tumor growth, [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-Trastuzumab treatment significantly delayed tumor progression by 23 days compared to the saline group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eE). Although tumor growth resumed in some mice around day 35, it subsequently stabilized, with final tumor volumes ranging from 65 to 95 mm\u0026sup3;. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eF, [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-Trastuzumab significantly improved overall survival compared to saline-treated controls (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). No significant changes in body weight were observed throughout treatment, indicating a favorable safety profile.\u003c/p\u003e\n\u003ch3\u003eV. Efficacy of [Lu]Lu-DOTA-Trastuzumab in treating HER2\u0026thinsp;+\u0026thinsp;brain metastases\u003c/h3\u003e\n\u003cp\u003eAlthough the BBB restricts the penetration of most CNS-targeted drugs [24], the newly formed vasculature in BrM is structurally compromised, leading to a defective and heterogeneous endothelial barrier that may enhance drug delivery [25]. Based on this, we investigated the therapeutic efficacy of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-trastuzumab against BrM. Mice with MRI-detected brain metastatic lesions were treated with [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-Trastuzumab, trastuzumab, or saline and monitored by MRI every four days (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eA).\u003c/p\u003e\n\u003cp\u003eConsistent with findings in the primary mammary tumor, trastuzumab monotherapy had no significant effect on JIMT-1-BR BrM growth compared to saline, as evidenced by the development of multiple metastatic foci in both groups. In contrast, a single dose of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-Trastuzumab led to complete remission in 2 of 5 mice (40%), as confirmed by MRI and histopathological analysis, with no detectable malignant lesions (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eB-C). The remaining three treated animals exhibited disease progression comparable to those receiving trastuzumab. Nevertheless, treatment with [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-Trastuzumab was associated with improved survival compared to the saline-treated group (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eD). These results support the potential of TRNT with trastuzumab as a promising strategy for treating BrM, particularly in trastuzumab-resistant cases.\u003c/p\u003e\n\u003ch3\u003eVI. Brain metastatic lesions exhibit heterogeneous BBB permeability\u003c/h3\u003e\n\u003cp\u003eTo explore the factors underlying differential responses to [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-Trastuzumab, we assessed BBB permeability within metastatic lesions using dynamic contrast-enhanced (DCE)-MRI with gadolinium. In this model, intracardiac tumor cell injection requires BBB traversal for successful brain colonization, closely mimicking the metastatic cascade. Using this approach, BrM formed within 20\u0026ndash;30 days post-injection, as detected by MRI. DCE-MRI analysis revealed gadolinium extravasation into metastatic lesions, indicating increased BBB permeability. However, signal intensity varied among lesions, with some exhibiting high gadolinium accumulation (orange/red) and others showing lower uptake (green/blue), reflecting intertumoral heterogeneity in BBB integrity (Fig.\u0026nbsp;5A-B). No gadolinium enhancement was observed in healthy brain regions, confirming an intact BBB.\u003c/p\u003e\n\u003cp\u003eAccordingly, PET imaging with [\u003csup\u003e89\u003c/sup\u003eZr]Zr-DFO-Trastuzumab demonstrated radiotracer accumulation only in a subset of metastatic brain lesions (Fig.\u0026nbsp;5C-D). These findings suggest that the degree of BBB disruption directly affects trastuzumab penetration and, consequently, the therapeutic efficacy of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-Trastuzumab. Given trastuzumab\u0026rsquo;s high molecular weight (150 kDa), its permeability may remain limited even in regions with BBB leakage, potentially contributing to suboptimal distribution and therapeutic response.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eDespite significant advances in targeted therapies, patients with metastatic HER2\u0026thinsp;+\u0026thinsp;BC remain at high risk of developing BrM, for which therapeutic options are limited and often ineffective. The inadequate penetration of trastuzumab and other HER2-targeted therapies into the CNS remains a major concern but is not the only limiting factor [9]. The emergence of brain-specific drug resistance mechanisms further adds another layer of complexity to treatment, underscoring the urgent need for the development of effective anti-metastatic therapies.\u003c/p\u003e \u003cp\u003eOur findings confirm that brain-tropic cells acquire resistance to trastuzumab driven by brain microenvironmental signals during the \u003cem\u003ein vivo\u003c/em\u003e selective pressure, which is not related to HER2 receptor density but rather to other mechanisms. For instance, neuregulin (NRG), secreted by neurons and astrocytes, promotes HER2 heterodimerization with HER3 and HER4, activating downstream PI3K/AKT and MAPK/ERK signaling pathways, which contribute to trastuzumab resistance [26]. Although not the primary focus of this study, we observed increased phospho-AKT and phospho-ERK levels in brain-tropic cells, supporting the involvement of these survival pathways (data not shown). Similar findings have been reported in other studies, which showed that BrM from HER2\u0026thinsp;+\u0026thinsp;BC frequently exhibits PI3K/AKT activation due to PTEN loss and HER3 activation, influenced by molecular cues from the brain microenvironment [27\u0026ndash;29].\u003c/p\u003e \u003cp\u003eThe trastuzumab-resistant phenotype of brain-tropic cells observed \u003cem\u003ein vitro\u003c/em\u003e was confirmed \u003cem\u003ein vivo\u003c/em\u003e, where both primary mammary tumors and BrM xenografts remained refractory to trastuzumab, regardless of HER2 status, as verified by immunohistochemistry and PET imaging with [\u003csup\u003e89\u003c/sup\u003eZr]Zr-DFO-Trastuzumab.\u003c/p\u003e \u003cp\u003eIn contrast, conjugating trastuzumab with the β-emitting radionuclide \u003csup\u003e177\u003c/sup\u003eLu significantly enhanced therapeutic efficacy, overcoming resistance mechanisms in brain metastatic cells by inducing DNA damage \u003cem\u003ein vitro\u003c/em\u003e. \u003cem\u003eIn vivo\u003c/em\u003e, a single dose of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-Trastuzumab significantly inhibited tumor growth in mice xenografted with brain-tropic cells in the mammary fat pad, leading to improved overall survival compared to trastuzumab-treated animals, which remained largely unresponsive. These results align with previous preclinical studies demonstrating the superior cytotoxic effects of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-Trastuzumab compared to its non-radioactive counterpart, with minimal off-target uptake [30, 31]. Moreover, clinical studies have established the safety of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-Trastuzumab in patients with HER2\u0026thinsp;+\u0026thinsp;primary and metastatic breast lesions [14, 32]. The selective uptake of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-Trastuzumab in BC lesions, coupled with its low toxicity profile, supports its potential as a viable alternative approach for treating HER2\u0026thinsp;+\u0026thinsp;trastuzumab-resistant tumors.\u003c/p\u003e \u003cp\u003eIn the context of BrM, although [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-Trastuzumab did not effectively reduce the metastatic burden in all treated animals, 40% achieved complete remission, as confirmed by MRI and histopathology, and experienced a significant survival benefit compared to saline-treated controls. The variable therapeutic response suggests that [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-Trastuzumab may not consistently reach BrM, preventing the delivery of a lethal dose of cytotoxic radiation to all metastatic lesions. While BBB permeability is increased in BrM, it remains highly heterogeneous, potentially limiting trastuzumab penetration in certain areas. This was supported by DCE-MRI imaging with gadolinium, which revealed intertumoral heterogeneity in BBB leakage across metastatic lesions. Additionally, a PET scan with [\u003csup\u003e89\u003c/sup\u003eZr]Zr-DFO-Trastuzumab confirmed radiotracer accumulation in some HER2\u0026thinsp;+\u0026thinsp;BrM, validating vascular permeability to the antibody. However, we cannot be certain that this increased permeability was consistent in all cases, as not all treated animals underwent PET imaging before receiving [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-Trastuzumab, representing a limitation of our study.\u003c/p\u003e \u003cp\u003ePrevious PET/computed tomography (CT) studies have also reported heterogeneous trastuzumab accumulation in BrM. Terrell-Hal \u003cem\u003eet al.\u003c/em\u003e demonstrated that [\u003csup\u003e125\u003c/sup\u003eI]I-trastuzumab crosses the disrupted BBB and reaches HER2\u0026thinsp;+\u0026thinsp;BrM [33]. Similarly, Puttemans \u003cem\u003eet al.\u003c/em\u003e observed a high uptake of [\u003csup\u003e111\u003c/sup\u003eIn]In-trastuzumab in BrM derived from the highly invasive 231Br cell line, but not in SKOV3 brain tumors, which exhibit low vascularization and minimal BBB disruption [23]. In a clinical study, Kurihara \u003cem\u003eet al.\u003c/em\u003e found that some MRI-detected BrM were not visible on [\u003csup\u003e64\u003c/sup\u003eCu]Cu-trastuzumab PET/CT [18]. This disparity highlights the heterogeneous permeability among metastatic lesions, which can trigger drug resistance mechanisms, contributing to unsatisfactory responses to TRNT with trastuzumab. However, it is worth mentioning that animals in our study received only a single dose of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-Trastuzumab, and higher or repeated dosing may improve therapeutic outcomes.\u003c/p\u003e \u003cp\u003eAs an alternative to trastuzumab, nanobodies are being explored for their ability to cross the BBB more efficiently due to their small size \u0026ndash; approximately one-tenth that of conventional antibodies \u0026ndash; while retaining HER2 immunoreactivity [34]. A recent study demonstrated the potential of radiolabeled anti-HER2 nanobody 2Rs15d in treating HER2\u0026thinsp;+\u0026thinsp;BrM, where administration of [\u003csup\u003e131\u003c/sup\u003eI]I-2Rs15d or [\u003csup\u003e225\u003c/sup\u003eAc]Ac-2Rs15d, alone or in combination with trastuzumab, significantly improved survival in two tumor models resistant to trastuzumab monotherapy [23]. However, despite their advantages in BBB penetration, nanobodies have a shorter half-life in circulation, requiring frequent or higher dosing to achieve a sustained therapeutic effect.\u003c/p\u003e \u003cp\u003eIn summary, we have demonstrated that [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-Trastuzumab is highly effective in inhibiting the growth of trastuzumab-resistant breast tumors. Although its response rate in BrM was not 100%, 40% of treated animals achieved complete remission, which is a notable result. To our knowledge, this represents the first preclinical evidence of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-Trastuzumab\u0026rsquo;s efficacy against HER2\u0026thinsp;+\u0026thinsp;BrM. While preclinical, these findings mark an important step toward improving treatment options for BrM, where therapeutic choices remain limited. From a clinical perspective, since the BBB might still hamper the penetrance of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-Trastuzumab and limit its efficacy, a [\u003csup\u003e89\u003c/sup\u003eZr]Zr-DFO-Trastuzumab PET imaging before treatment could help identify patients, most likely to benefit from radionuclide therapy.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eCell culture\u003c/h2\u003e \u003cp\u003eTwo human HER2\u0026thinsp;+\u0026thinsp;BC cell lines, JIMT-1 and SUM190, along with their respective brain metastatic variants, JIMT-1-BR and SUM190-BR, were generously provided by Dr. Patricia Steeg from the National Cancer Institute (Bethesda, USA). These brain metastatic sublines were generated from the parental cell lines by repeated intracardiac injections and subsequent harvesting of metastatic cells from the brains of mouse models, as previously described by Palmieri \u003cem\u003eet al.\u003c/em\u003e for JIMT-1-BR [35] and Lyle \u003cem\u003eet al.\u003c/em\u003e for SUM190-BR [7].\u003c/p\u003e \u003cp\u003eJIMT-1 cells were cultured in DMEM (Sigma-Aldrich) with 10% FBS (Gibco) and 1% antibiotic-antimycotic (Gibco). SUM190 cells were cultured in Ham's F-12 medium (Sigma-Aldrich) supplemented with 1 \u0026micro;g/ml hydrocortisone (Sigma-Aldrich), 10 mM HEPES (Sigma-Aldrich), insulin-transferrin-selenium-ethanolamine (Gibco), 10 nM triiodothyronine (Sigma-Aldrich), 1 g/L BSA (Sigma-Aldrich), and 1% antibiotic-antimycotic (Gibco).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eColony formation assay\u003c/h2\u003e \u003cp\u003eJIMT-1 and SUM190 cells and brain metastatic derivatives were seeded in 12-well plates at densities of 600 and 4000 cells per well, respectively, and allowed to adhere overnight. JIMT-1 cells were treated with 0.5, 1, 2, and 5 MBq/mL of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-Trastuzumab, while SUM190 cells received 1, 5, 7.5, or 10 MBq/mL, in 1 mL of medium and incubated at 37\u0026deg;C for 2 h. Untreated cells served as controls. Subsequently, the cell culture medium was replaced with fresh medium, and plates were incubated at 37\u0026deg;C to allow colony formation. After 10\u0026ndash;14 days of incubation, colonies were gently washed with PBS, fixed with 1% formaldehyde for 15 min, and stained with 0.1% crystal violet in ethanol for another 15 min. The assay was performed in duplicate for each cell line across three independent experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eγ-H2AX assay\u003c/h2\u003e \u003cp\u003eJIMT-1 and SUM190 cells and brain metastatic derivatives were seeded in glass coverslips in a 24-well plate at a density of 4.5x10\u003csup\u003e4\u003c/sup\u003e and 1x10\u003csup\u003e5\u003c/sup\u003e cells per well, respectively, and allowed to attach overnight. JIMT-1 cells were treated with 2 or 5 MBq/mL of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-Trastuzumab, while SUM190 cells received 5 or 10 MBq/mL, in 500 \u0026micro;L of medium for 2 h at 37\u0026deg;C. Untreated cells served as controls. Subsequently, the medium was replaced with fresh culture medium, and cells were maintained at 37\u0026deg;C for 5 days. After three PBS washes, cells were fixed with 4% PFA for 15 min, permeabilized with 0.5% Triton X-100 at RT for 15 min, followed by two additional PBS washes. The cells were then incubated overnight with a primary antibody against γ-H2AX (ser139; Abcam, ref. ab81299) at a 1:250 dilution. After two PBS washes, cells were treated with a FITC-conjugated anti-rabbit secondary antibody (Invitrogen) at a 1:200 dilution for 1 h, followed by three PBS washes. Cell nuclei were stained with Hoechst 33342 (Sigma-Aldrich) at 5 \u0026micro;g/ml for 5 min at RT in the dark and then mounted in Vectashield (Vector Laboratories). Images were captured using a Carl Zeiss Axio Observer Z1 inverted microscope at 20x magnification. Six randomly selected images were captured per slide. Quantitative analysis of γ-H2AX foci was performed using ImageJ (National Institutes of Health), with a minimum of 20 nuclei analyzed per experiment, performed in duplicate. Three independent experiments were carried out for each cell line.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eComet assay\u003c/h2\u003e \u003cp\u003eJIMT-1 and SUM190 cells and brain-metastatic derivatives were seeded in 6-well plates at densities of 1x10\u003csup\u003e5\u003c/sup\u003e and 3x10\u003csup\u003e5\u003c/sup\u003e cells per well, respectively, and allowed to adhere overnight. JIMT-1 cells were treated with 2 or 5 MBq/mL of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-Trastuzumab, while SUM190 cells were treated with 5 or 10 MBq/mL in 2 mL of medium and incubated at 37\u0026deg;C for 2 h. Untreated cells served as controls. After treatment, the medium was replaced with fresh culture medium, and cells were incubated at 37\u0026deg;C for 5 days. To assess genotoxicity, cells were gently scraped and processed using the comet assay kit (Enzo Life Sciences; ref. ADI-900-166) according to the manufacturer\u0026rsquo;s instructions. Comet tails were visualized with a Carl Zeiss Axio Observer Z1 inverted microscope and analyzed by measuring the length of the comet tail using ImageJ software (National Institutes of Health). Eight images were randomly collected from each slide. Three independent experiments were conducted for each cell line.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAnimal studies\u003c/h2\u003e \u003cp\u003e All animal experiments were approved by the Animal Welfare Committee of the Institute for Nuclear Sciences Applied to Health (ICNAS) of the University of Coimbra (ORBEA 04-2021) and by the Portuguese National Authority for Animal Health (DGAV). Female athymic Swiss FoxnI nu/nu mice aged 10\u0026ndash;12 weeks (20\u0026ndash;25 g) were purchased from ICNAS and housed under pathogen-free conditions in individually ventilated cages. For the BC model, mice were orthotopically injected with 2x10\u003csup\u003e6\u003c/sup\u003e cells of JIMT-1 or JIMT-1-BR suspended in a 50% PBS and 50% Matrigel mixture (total volume of 50 \u0026micro;L) into the 4th mammary fat pad. Tumor growth was monitored twice weekly with a digital caliper. Tumour volumes were calculated using the modified ellipsoid formula V\u0026thinsp;=\u0026thinsp;A \u0026times; B\u003csup\u003e2\u003c/sup\u003e/2 (A length; B width).\u003c/p\u003e \u003cp\u003eFor experimental BrM formation, mice were intracardially injected with 1.75x10\u003csup\u003e5\u003c/sup\u003e brain-tropic JIMT-1-BR cells in 100 \u0026micro;L of PBS into the left ventricle as previously described [36]. BrM formation was monitored twice a week by MRI in a BioSpect 9.4T MRI scanner (Bruker Biospin, Ettlingen) under anesthesia (2% isoflurane). The animals' breathing rate and body temperature were monitored throughout the imaging procedures (SA Instruments SA). Morphological brain images were acquired on the T2-RurboRARE sequence in coronal orientation with the following parameters: TE/TR\u0026thinsp;=\u0026thinsp;33.01/2500 ms, FOV\u0026thinsp;=\u0026thinsp;20.0*20.0 mm, acquisition matrix\u0026thinsp;=\u0026thinsp;256*256, averages\u0026thinsp;=\u0026thinsp;5,18 continuous slices with 0.4 mm thick, and acquisition time of 6m40s.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eTherapeutic evaluation of trastuzumab and [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-Trastuzumab\u003c/h2\u003e \u003cp\u003eAnimals bearing a primary breast tumor or BrM were treated with trastuzumab (Evidentic GmbH) or [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-Trastuzumab. In the BC model, treatments were initiated when tumors reached an average volume of 35 to 60 mm\u0026sup3;. Mice were randomized into groups of 3 per group and received either (i) trastuzumab (loading dose 7.5 mg/kg, D0; maintenance dose 3.5 mg/kg, biweekly on D3-7-10-14), (ii) [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-Trastuzumab (8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.20 MBq; 60 \u0026micro;g Trastuzumab), or (iii) vehicle buffer (0.9% NaCl). All treatments were administered \u003cem\u003ei.v.\u003c/em\u003e in a total volume of 100 \u0026micro;L. Tumors were monitored twice weekly by MRI for up to 56 days. The study was ended when tumor volume reached\u0026thinsp;\u0026ge;\u0026thinsp;250 mm\u003csup\u003e3\u003c/sup\u003e or when humane endpoints were reached.\u003c/p\u003e \u003cp\u003eFor the experimental brain metastatic model, trastuzumab or [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-Trastuzumab therapy was initiated when BrM were detected by MRI. At this time, mice received saline (n\u0026thinsp;=\u0026thinsp;3), trastuzumab (loading dose 7.5 mg/kg, D0; maintenance dose 3.5 mg/kg, biweekly on D3-7-10-14; n\u0026thinsp;=\u0026thinsp;3), [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-Trastuzumab (8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.81 MBq; 60 \u0026micro;g Trastuzumab; n\u0026thinsp;=\u0026thinsp;5). All treatments were administered \u003cem\u003ei.v.\u003c/em\u003e in a total volume of 100 \u0026micro;L. Animals were weighed biweekly and checked daily for general health and well-being. Animals were sacrificed when one of the following endpoints was reached: weight loss\u0026thinsp;\u0026gt;\u0026thinsp;20% of original body weight, immobility, or unresponsiveness to external stimuli.\u003c/p\u003e \u003cp\u003eIn both models, mice that reached the humane endpoint or completed the observation period were euthanized by cervical dislocation. Survival was analyzed by the Kaplan-Meier survival curve. Afterward, tumor lesions and the whole brain were fixed in PFA 4% and embedded in paraffin wax for histopathological analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eDynamic contrast-enhanced-magnetic resonance imaging (DCE-MRI)\u003c/h2\u003e \u003cp\u003eTo evaluate BBB permeability alterations \u003cem\u003ein vivo\u003c/em\u003e, DCE-MRI was performed after the intraperitoneal injection of a gadolinium-based contrast agent in healthy mice and mice bearing BrM, as previously described [37]. Briefly, dynamic contrast-enhanced images were acquired with a DCE_FLASH sequence with the following parameters: TE/TR\u0026thinsp;=\u0026thinsp;2.5/100 ms, FA\u0026thinsp;=\u0026thinsp;70\u0026deg;, FOV\u0026thinsp;=\u0026thinsp;20*20 mm, acquisition matrix\u0026thinsp;=\u0026thinsp;156*5, 18 coronal slices with 0.4 mm thick, 60 dynamics acquired, 7 averages, scan time per dynamic\u0026thinsp;=\u0026thinsp;49s700ms, total scan time\u0026thinsp;=\u0026thinsp;49m42s. The gadolinium-based contrast agent Gadobutrol (Gadovist\u0026reg;, LUSAL) was administered intraperitoneally after the acquisition of 5 baseline scans, with 60 dynamic scans acquired following the injection. A region of interest (ROI) was drawn aroufnd each BrM using a semiautomatic procedure. In healthy animals, a corresponding ROI was drawn in the same region. The mean variation of signal intensity as a function of time was then quantified in the predefined ROI to evaluate perfusion and vascular permeability.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eAll data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard error of the mean (SEM). Graphics and statistical analysis were performed using GraphPad Prism software. Data were analyzed using two-way ANOVA analysis followed by Turkey\u0026rsquo;s post-hoc test, as indicated in figure legends. For the Kaplan-Meier analysis, statistical differences in survival curves were calculated by log-rank (Mantel-Cox) test. Statistical significance was set at the level of p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, and the \u0026lsquo;n\u0026rsquo; represents the total number of experiments. Figure illustrations were created with Bio-Render.com.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cul\u003e\n \u003cli\u003e\u003cstrong\u003eResearch involving animals\u003c/strong\u003e\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eAll animal experiments were approved by the Animal Welfare Committee of the Institute for Nuclear Sciences Applied to Health (ICNAS) of the University of Coimbra (ORBEA 04-2021) and by the Portuguese National Authority for Animal Health (DGAV).\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003e\u003cstrong\u003eConflicts of interest\u003c/strong\u003e\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eNo potential conflicts of interest relevant to this article exist.\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eThis work was supported by the Portuguese Foundation for Science and Technology (FCT) through the project PTDC/BTM-SAL/4451/2020 and STRATEGIC PROJECTS (UIDB/04539/2020 and UIDP/04539/2020). L.S. is a Ph.D. fellow of the FCT (PD/BDE/150707/2020).\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eLS, IH, JS and HF performed experiments, acquisition, analysis, and interpretation of data. PT and RA performed histopathological analysis and immunostaining. MS performed the production of zirconium-89. LS wrote the manuscript. CG and AJA designed and supervised the study, revised the manuscript, and obtained financial support. All authors reviewed the manuscript and approved its content.\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u0026nbsp;\u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eAll data generated and analyzed during this study are included in this article and its supplementary information files.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003e Goddard K, Weinmann S, Richert-Boe K, Chen C, Bulkley J, Wax C. HER2 evaluation and its impact on breast cancer treatment decisions. Public Health Genomics. 2011;15:1\u0026ndash;10.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Arteaga CL, Sliwkowski MX, Osborne CK, Perez EA, Puglisi F, Gianni L. Treatment of HER2-positive breast cancer: current status and future perspectives. Nature reviews Clinical oncology. 2012;9:16\u0026ndash;32.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003e Koo T, Kim IA. 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Acta Neuropathologica Communications. 2020;8:1\u0026ndash;19.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"Institute for Nuclear Sciences Applied to Health (ICNAS) and Coimbra Institute for Biomedical Imaging and Translational Research (CIBIT), University of Coimbra, Coimbra, Portugal","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"HER2 + breast cancer, brain metastasis, trastuzumab resistance, targeted radionuclide therapy, lutetium-177","lastPublishedDoi":"10.21203/rs.3.rs-6180100/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6180100/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eBreast cancer (BC) is the most common malignancy in women, with HER2 amplification present in 25\u0026ndash;30% of metastatic cases. Although HER2-targeted therapies like trastuzumab have significantly improved patient outcomes, their efficacy in HER2\u0026thinsp;+\u0026thinsp;brain metastases (BrM) is hindered by the emergence of resistance mechanisms. This study explores the therapeutic potential of trastuzumab radiolabeled with the β⁻-emitting radionuclide \u0026sup1;⁷⁷Lu as a strategy to overcome resistance in HER2\u0026thinsp;+\u0026thinsp;BrM.\u003c/p\u003e\u003ch2\u003eMaterial and methods\u003c/h2\u003e \u003cp\u003eHER2\u0026thinsp;+\u0026thinsp;BC cell lines and their brain-tropic derivatives were assessed for HER2 expression and sensitivity to trastuzumab and [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-Trastuzumab. \u003cem\u003eIn vivo\u003c/em\u003e models were established by orthotopic implantation of HER2\u0026thinsp;+\u0026thinsp;BC cells for primary tumor formation or intracardiac injection to induce BrM. Once tumors were established, the therapeutic efficacy of trastuzumab and [\u0026sup1;⁷⁷Lu]Lu-DOTA-Trastuzumab was evaluated by monitoring tumor progression via magnetic resonance imaging (MRI). [⁸⁹Zr]Zr-DFO-Trastuzumab PET imaging was performed to assess HER2 expression, while blood-brain barrier (BBB) permeability was evaluated using dynamic contrast-enhanced MRI.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eBrain-tropic HER2\u0026thinsp;+\u0026thinsp;cells exhibited trastuzumab resistance despite maintaining HER2 expression. In contrast, [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-trastuzumab induced significant DNA damage and cytotoxicity. PET imaging confirmed specific radiotracer uptake in HER2\u0026thinsp;+\u0026thinsp;primary tumors and BrM. A single dose of [\u003csup\u003e177\u003c/sup\u003eLu]Lu-DOTA-trastuzumab effectively suppressed primary tumor growth and achieved complete BrM remission in 40% of treated animals. Heterogeneous BBB permeability was observed across metastatic lesions, potentially influencing radiotracer uptake and therapeutic efficacy.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eThese findings underscore [\u0026sup1;⁷⁷Lu]Lu-DOTA-trastuzumab as a novel therapeutic strategy to overcome trastuzumab resistance in HER2\u0026thinsp;+\u0026thinsp;BrM, offering a promising approach to improve outcomes in metastatic BC.\u003c/p\u003e","manuscriptTitle":"177Lu-Trastuzumab Radionuclide Therapy: an Effective Approach for Resistant Brain Metastases in HER2+ Breast Cancer","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-03-12 06:50:10","doi":"10.21203/rs.3.rs-6180100/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d10a4510-0d18-4152-b8d2-9cda127e07f6","owner":[],"postedDate":"March 12th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-03-12T06:50:10+00:00","versionOfRecord":[],"versionCreatedAt":"2025-03-12 06:50:10","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6180100","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6180100","identity":"rs-6180100","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}