Enhanced Payload Release Enables Disitamab Vedotin to Surpass Trastuzumab Emtansine and Retain Efficacy in Acquired Resistance to Clinical Anti-HER2 Therapies

Abstract

Background: Resistance to HER2-targeted therapies remains a major limitation in the treatment of HER2-positive breast cancer, where disease progression inevitably occurs in advanced stages. Development of next-generation strategies that retain activity in resistant disease is therefore a critical priority. Disitamab vedotin (RC48) is a novel antibody–drug conjugate (ADC) targeting HER2 that couples a humanized anti-HER2 antibody to the potent microtubule-disrupting agent monomethyl auristatin E. Methods: We compared the activity and mechanism of action of RC48 with that of trastuzumab emtansine (T-DM1) across HER2-positive and HER2-low cellular models, including multiple sublines resistant to current HER2-targeted agents. Results: In HER2-overexpressing breast cancer cell lines, RC48 consistently demonstrated superior antiproliferative effect with respect to T-DM1. Treatment with RC48 induced G2/M arrest and apoptotic cell death, associated with increased pHistone-H3 and cyclin B1 and downregulation of Wee1, consistent with blockade of cell cycle progression in mitosis. Although RC48 and T-DM1 internalized similarly, RC48 displayed more efficient intracellular payload release, providing a mechanistic explanation for its enhanced efficacy. Notably, RC48 retained strong activity in BT474-derived sublines resistant to T-DM1, lapatinib, or neratinib, inducing cell cycle arrest, apoptosis, and caspase activation in all resistant models. In contrast, T-DM1 exhibited only partial effects in resistant cells and was completely ineffective in a T-DM1-refractory clone. Conclusions: Together, these findings identify disitamab vedotin as a potent next-generation HER2-targeting ADC with the unique capacity to overcome acquired resistance to HER2-directed therapies. RC48 represents a promising therapeutic strategy for patients with refractory HER2-positive breast cancer and warrants further clinical investigation.

1. Introduction

Breast cancer is a biologically heterogeneous disease and a leading cause of cancer-related death among women [1]. A significant subset of breast tumors, approximately 20%, exhibit amplification and overexpression of the human epidermal growth factor receptor 2 (HER2) [2], a member of the ERBB family of receptor tyrosine kinases [3]. HER2-positive breast cancers are characterized by rapid proliferation, increased metastatic potential, and historically poor prognosis [4]. However, the clinical introduction of HER2-targeted therapies has revolutionized the treatment landscape and markedly improved survival outcomes for patients with this aggressive disease subtype [5,6].

Over the past two decades, HER2-directed pharmacologic strategies have focused on two major drug classes: small-molecule tyrosine kinase inhibitors (TKIs) and monoclonal antibodies [6,7]. TKIs such as lapatinib, neratinib, and tucatinib function by reversibly or irreversibly binding the ATP-binding site of the HER2 kinase domain, thereby disrupting downstream oncogenic signaling cascades [8]. Concurrently, monoclonal antibodies, including trastuzumab and biosimilars, pertuzumab, and margetuximab, have been developed to target the extracellular domain of HER2 [9,10]. The latter type of drugs exerts antitumor effects through multiple mechanisms that include, among others, inhibition of receptor dimerization [11], blockade of ligand-independent signaling [12], and engagement of immune effector functions via antibody-dependent cellular cytotoxicity [13,14].

A sophisticated version of the antibodies directed to the ectodomain of HER2 is represented by antibody–drug conjugates (ADCs) [15,16]. These drugs combine the specificity of monoclonal antibodies with the potent cytotoxic activity of chemotherapeutic payloads, delivered selectively to HER2-expressing cells via an engineered linker system [9,16]. Two HER2-targeted ADCs, trastuzumab emtansine (T-DM1) and trastuzumab deruxtecan (T-DXd), are currently approved for the treatment of HER2-positive breast cancer, demonstrating improved clinical outcomes in both early and metastatic settings [16,17,18]. The cytotoxic payloads used in these ADCs, DM1 and DXd, are well-established components in antibody–drug conjugates, each possessing a distinct mechanism of action and targeting different intracellular pathways [17,18]. DM1 is a maytansinoid derivative that inhibits microtubule polymerization by binding to tubulin, leading to mitotic arrest and subsequent apoptotic cell death [17]. DXd (deruxtecan) is a derivative of exatecan, a topoisomerase I inhibitor. DXd interferes with DNA replication by stabilizing the topoisomerase I-DNA cleavage complex, causing DNA damage and cell death [18].

Despite clinical success, resistance mechanisms frequently limit the long-term effectiveness of HER2-directed therapies, particularly in the metastatic scenario [19]. Resistance can be classified into two main categories: primary (de novo) resistance and secondary resistance. Primary resistance occurs when tumors fail to respond to therapy from the outset, whereas secondary resistance develops following an initial response to treatment [20]. Although primary resistance is relatively uncommon, secondary resistance remains a significant hurdle in the management of HER2-positive cancer [21]. Once resistance emerges, treatment options become limited, and disease progression is often inevitable. Therefore, resistance to these therapies remains a critical challenge, necessitating the development of innovative treatment strategies.

Disitamab vedotin (also known as RC48) is a next-generation ADC that represents a promising therapeutic alternative for HER2-positive cancers [22]. Unlike trastuzumab-based ADCs, RC48 is constructed using hertuzumab, a distinct monoclonal antibody that binds HER2 with high affinity [23,24] (Figure S1). Additionally, it employs monomethyl auristatin E (MMAE) as its cytotoxic payload, differentiating it from currently approved HER2-targeting ADCs. However, similarly to DM1, MMAE also targets microtubules, but it is still unclear whether differences in their potency or efficacy exist, particularly in resistant scenarios. RC48 has already demonstrated promising antitumor activity in preclinical and early clinical studies, particularly in HER2-expressing gastric and urothelial cancers, and has received regulatory approval in China for the treatment of gastric cancer [25]. Emerging evidence also supports the activity of RC48 in breast cancer, including tumors exhibiting molecular signatures associated with therapeutic resistance [24]. However, comprehensive head-to-head comparisons with T-DM1 in breast cancer, particularly in the context of acquired resistance, remain limited. This knowledge gap hinders our ability to fully evaluate the clinical potential of RC48 and to define its optimal use in treatment sequences.

In this study, we aimed to investigate the antitumor efficacy of RC48 in HER2-positive breast cancer models. Specifically, we compared the cytotoxic activity of RC48 to that of T-DM1 and assessed its performance in in vitro models of acquired resistance to HER2-directed therapies. Our findings reveal that RC48 not only exhibits superior potency compared to T-DM1 but also retains efficacy in cells resistant to T-DM1, lapatinib, and neratinib. These results support the potential of RC48 as a promising alternative therapy for patients with HER2-positive breast cancer that has progressed on those currently approved treatment regimens.

2. Methods

2.1. Reagents and Immunochemicals

The sources of reagents and immunochemicals are detailed in Table S1.

2.2. Cell Culture

Cell lines were cultured in Dulbecco’s Modified Eagle Medium (BT474, SKBR3, MCF7, T47D) or RPMI-1640 (HCC1954, HCC1419), supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin. The cells were maintained at 37 °C in a humidified atmosphere consisting of 5% CO₂ and 95% air. All parental cell lines were obtained from the American Type Culture Collection (ATCC), and expanded 1:10 in 100 mm Petri dishes upon being thaw. Cells from these plates were refrozen to generate several identical vials. Cell line authentication was performed by short tandem repeat (STR) at the Hematology Service of the Salamanca University Hospital. Mycoplasma testing was carried out using the Mycoplasma PCR Detection Kit (Abcam, Fremont, CA, USA).

Resistant clones derived from BT474, including BT-TDM1R #1 (T-DM1 resistance) [26], BTRN #6 (neratinib resistance) [27] and BTRL #109 (lapatinib resistance) [28], were previously generated in our laboratory by exposing the BT474 parental line to T-DM1 (5 nM), neratinib (10 nM), or lapatinib (1 µM) for several months (Figure S6A). Cells were regularly tested for stable resistance to the drugs.

2.3. Immunoprecipitation and Western Blotting

Cells were rinsed with phosphate-buffered saline (PBS) containing 137 mM NaCl, 2.7 mM KCl, 8 mM Na₂HPO₄, and 1.5 mM KH₂PO₄, and then lysed using 1 mL of ice-cold lysis buffer consisting of 20 mM Tris-HCl (pH 7.0), 140 mM NaCl, 50 mM EDTA, 10% glycerol, 1% Nonidet P-40, 1 µM pepstatin, 1 µg/mL aprotinin, 1 µg/mL leupeptin, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, 50 mM sodium fluoride, and 25 mM β-glycerol phosphate. Plates were scraped, and lysates were centrifuged at 17,000× g for 10 min at 4 °C. The resulting supernatants were transferred to new tubes, and protein concentration was determined using the BCA assay (Thermo Fisher Scientific, Madrid, Spain). For immunoprecipitation, 1 mg of lysate was incubated with the corresponding antibody and protein A-Sepharose at 4 °C for at least 2 h. For ADC immunoprecipitation, 1.5 mg of protein extracted from drug-treated cells was incubated with protein A-Sepharose at 4 °C for 30 min. Immune complexes were isolated by brief centrifugation at 17,000× g for 30 s, followed by three washes with 1 mL of cold lysis buffer. Samples were boiled in electrophoresis sample buffer and loaded on SDS–PAGE gels. Proteins separated by electrophoresis were transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked for at least 1 h in Tris-buffered saline with Tween (TBST) (20 mM Tris, pH 7.5; 150 mM NaCl; 0.1% Tween 20) containing 1% bovine serum albumin (BSA), followed by incubation with the appropriate primary antibody dilution for a minimum of 2 h. After three 10-min washes with TBST, membranes were incubated for 30 min with HRP-conjugated anti-mouse or anti-rabbit secondary antibodies. Membranes were washed three more times with TBST, and protein bands were visualized by chemiluminescence using a ChemiDoc apparatus (Bio-Rad, Hercules, CA, USA) and home-made ECL [29]. Stain-free gels were prepared by adding 50 µL of 2,2,2-trichloroethanol to 10 mL of the SDS–PAGE gel solution, and proteins were visualized using the ChemiDoc apparatus after electrophoresis, following the manufacturer’s instruction. Band intensities were quantified using Image Lab Software 6.0.1 (Bio-Rad). For comparisons between experimental conditions, the control condition was assigned a relative intensity value of 1. In addition to Western blotting analyses, expression of ERBB2 in the breast cancer cell lines was analyzed using the DepMap (https://depmap.org/portal; accessed on 10 January 2025) open access online tool, that provides cancer data information in different models.

2.4. Cell Proliferation, Cell Cycle, and Apoptosis Analyses

Proliferation studies were performed by cell counting assays. For this purpose, cells were seeded in 6-well plates and treated 24 h later with medium containing the drug at different concentrations. Cells were then collected and counted in a Z1 Coulter Particle Counter (Beckman Coulter, Pasadena, CA, USA).

For cell cycle and apoptosis analysis, cells cultured in 60 mm plates were treated with drugs for the indicated times and concentrations. Cells were washed with PBS and detached using trypsin-EDTA. For cell cycle analysis, cells were fixed overnight in ice-cold 70% ethanol. The next day, cells were centrifuged and incubated in PBS with 200 μg/mL DNAse-free RNAse and propidium iodide (PI, 4 μg/mL) for 15 min at 37 °C. Cell cycle profiles were determined using an Accuri C6 Flow Cytometer (BD Biosciences, San Jose, CA, USA). For apoptosis assays, collected cells were resuspended in ice-cold binding buffer (10 mM HEPES pH 7.4, 140 mM NaCl, 2.5 mM CaCl₂) containing 5 μL of Annexin V-FITC and 10 μL of PI (50 μg/mL). Samples were incubated for 15 min at room temperature in the dark. After incubation, samples (50,000 cells) were acquired in an Accuri C6 Flow Cytometer (BD Biosciences), considering viable if they were negative for both Annexin V, which detects early apoptotic cells, and PI, which identifies necrotic cells.

To analyze the expression profiles of apoptosis-related proteins, the Proteome Profiler Human Apoptosis Array Kit (R&D Systems, Minneapolis, USA) was used according to the manufacturer’s instructions. Cells were treated with RC48 or T-DM1 at 10 nM for the indicated times. Protein expression was assessed by hybridizing 400 μg of cell lysate with the nitrocellulose membranes provided in the kit. This approach enabled the detection of the expression of 35 apoptosis-related proteins. Pixel intensities corresponding to the expression levels of each protein were analyzed using Image Lab™ Software Version 6.0.1 (Bio-Rad Laboratories), and normalized using internal standards.

2.5. Immunofluorescence Assay

Cells were seeded at low density on glass coverslips placed in 35 mm dishes and treated with the drug 24 h later. At the indicated time points, coverslips were washed with PBS/CM (1 mM CaCl₂, 0.5 mM MgCl₂ in PBS) and fixed with 2% paraformaldehyde for 30 min at room temperature. After fixation, cells were washed with PBS/CM, quenched for 10 min with 50 mM NH₄Cl, permeabilized for 30 min with 0.1% Triton X-100 and 0.2% BSA, and incubated for 1 h in blocking solution (PBS/CM containing 0.2% BSA). Coverslips were then incubated overnight at 4 °C with the corresponding primary antibodies diluted in blocking solution. The following day, cells were washed three times for 7 min each with blocking solution and incubated for 30 min at room temperature with fluorescent dye-conjugated secondary antibodies. After incubation, coverslips were washed three times for 7 min each with PBS containing 0.2% BSA, stained with Hoechst 33342, and mounted. Labeled cells were visualized using a Leica TCS SP8 confocal system (Leica Microsystems CMS, Wetzlar, Germany). To quantify mitotic cells, at least 500 cells were counted per condition. Phospho-histone H3 staining was used to identify mitotic cells. Percentages were calculated as: (number of mitotic cells/total number of cells) × 100 (%). Payload release from internalized ADC was quantified using ImageJ 1.54p software. The ADC backbone was detected using a Cy3-conjugated anti-human antibody, while the payload was visualized using an anti-payload primary antibody followed by a Cy2-conjugated secondary antibody. To specifically select internalized ADC and exclude plasma membrane signal, the red channel was thresholded and a size filter of 0.5–3 µm² was applied via the Analyze Particles function. The resulting regions of interest (ROIs) were applied to the thresholded payload channel, and the degree of colocalization was calculated as (Mean Intensity/255) × 100. This metric represents the percentage of payload signal remaining associated with the antibody. Four independent fields of view were analyzed per condition.

2.6. Bystander Effect

To assess the bystander effect, a direct co-culture was established using the SKBR3 (HER2-overexpressing) and MDA-MB-468 (HER2-null) breast cancer cell lines. Both cell types were seeded at a 1:1 ratio in the same well of a 6-well plate. The experiment was performed in duplicate (Figure S5C). After three days of treatment, one plate was used to determine total cell numbers using an automated cell counter, and in parallel the other was analyzed by flow cytometry (Accuri C6) to assess the relative proportion of each cell type within the co-culture. Cell populations were distinguished based on EGFR expression using an anti-EGFR antibody conjugated to a fluorescent dye (Cetuximab-DL488). Since SKBR3 cells express minimal levels of EGFR and MDA-MB-468 cells express high levels, this allowed effective discrimination between the two populations.

2.7. Statistical Analyses

Statistical analyses were performed using GraphPad Prism 8 (San Diego, CA, USA). Normality was assessed using the Shapiro–Wilk test. Comparisons between two independent groups were carried out using unpaired two-tailed t-tests with Welch’s correction. Comparisons among multiple independent groups were performed using one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test. p-values less than 0.05 were considered statistically significant. Data are presented as mean ± standard deviation (SD).

3. Results

3.1. Disitamab-Vedotin Is More Potent than Trastuzumab-Emtansine

With the aim of comparing the antiproliferative efficacy of RC48 and T-DM1, several human breast cancer cell lines expressing different amounts of HER2 were selected. Two of them, BT474 and SKBR3, are considered bona fide models of human HER2+ breast cancer [30]. In fact, Western blotting analyses confirmed the high expression levels of HER2 in these cells (Figure 1A). DepMap data showed that these cells present ERBB2 gene amplification (ERBB2 copy number relative to ploidy of 11.50 and 10.95 for BT474 and SKBR3, respectively, Figure 1B) and overexpress ERBB2 mRNA. In addition to those cell lines, and because of the reported antiproliferative effect found for some HER2-directed ADCs in tumors with a low complement of HER2 [18], in vitro models represented in this study by MCF7 and T47D cells were also selected. These two cell lines are derived from hormone receptor-positive tumors [30] and HER2 is not amplified (ERBB2 copy number relative to ploidy of 0.53 and 1.41 for MCF7 and T47D, respectively, Figure 1B). Western blotting studies showed the low levels of HER2 in MCF7 and T47D cells, when compared to BT474 and SKBR3 cells (Figure 1A).

Figure 1.

Antiproliferative efficacy of RC48 and T-DM1. (A) Western blot analysis of HER2 expression in four breast cancer cells lines. Fifty micrograms of cell lysates were loaded in SDS-PAGE gels, and Western blot analysis probed with anti-HER2 or calnexin antibodies. The latter was used as loading control. (B) DepMap plot showing the correlation between ERBB2 copy number and expression in breast cancer cell lines, highlighting BT474, SKBR3, MCF7 and T47D. (C) Response of BT474 and SKBR3 to RC48. Cells were treated with the indicated doses for 3 days and then counted. (D) Effect of T-DM1 on BT474 and SKBR3 under the same conditions as in (C). (E–H) Comparison of the antiproliferative effects of RC48 and T-DM1 in BT474, SKBR3, T47D and MCF7 cells after 3 days of treatment. Data in (C–H) represent mean ± SD of at least two independent experiments. Results were normalized to untreated controls. p-values indicate significant differences between RC48 and T-DM1 at the indicated concentrations (unpaired t-test with Welch’s correction).

To evaluate the antiproliferative action of RC48 relative to T-DM1, dose–response experiments were carried out. The selected breast cancer cell lines were treated for three days with a range of RC48 or T-DM1 concentrations, and cell counting used to assess the effect of the ADCs on cell proliferation. In the HER2+ cell lines, these dose–response studies showed that RC48 exerted a detectable antiproliferative effect at 50 pM, reaching maximum effectiveness at 1 nM (Figure 1C). The dose–response curves obtained for BT474 and SKBR3 were very similar, indicating analogous sensitivity to the antiproliferative action of RC48. In the case of T-DM1, SKBR3 cells were more sensitive to the action of the ADC than BT474 cells (Figure 1D). Replotting of the data to compare the sensitivity of each cell line to each of the ADCs, confirmed that BT474 cells were more sensitive to RC48 than to T-DM1 (Figure 1E). For SKBR3 cells, both drugs had similar dose–response curves, although RC48 showed higher potency than T-DM1 (Figure 1F). The higher sensitivity to RC48 with respect to T-DM1 was also observed in two additional HER2-positive breast cancer cell lines, HCC1954 and HCC1419 (Figure S2A,B). In HER2-low T47D and MCF7 cell lines, neither drug showed significant antiproliferative effects at tested concentrations (Figure 1G,H).

3.2. Disitamab-Vedotin Acts Faster than Trastuzumab-Emtansine

The above results showed that RC48 was more potent than T-DM1, and also suggested differences in the antitumoral action of T-DM1 in breast cancer cell lines which overexpressed similar amounts of HER2. Also, these studies indicated that at doses that are effective in HER2+ cells, the ADCs were unable to affect the proliferation of cells expressing low levels of HER2. Several studies were then planned in attempting to gain insights into the mechanisms responsible for the differences found in the antiproliferative actions of both ADCs.

Analysis of the effect of the ADCs on the amount of HER2 protein showed that RC48 decreased the levels of HER2 in BT474 and SKBR3 cells (Figure 2A,B). T-DM1 was also able to cause a decrease in the amount of HER2 in SKBR3 cells, and such effect was particularly evident after 48 h of treatment. In the case of BT474, T-DM1 did not substantially affect the amount of HER2. These differences in the action of both ADCs on the levels of HER2 fall in line with the proliferation data.

Figure 2.

Mechanism of action of RC48 and T-DM1. (A) Western blot analyses of the effect of RC48 and T-DM1 (1 and 10 nM) on the levels of HER2 in BT474 and SKBR3 cells at the indicated incubation times. Calnexin was used as a loading control. (B) Quantification of HER2 protein levels from two different experiments as the one shown in (A). Band intensity was normalized to calnexin and expressed relative to the untreated control, set to 1. Bars represent the mean, and individual dots indicate values from two independent experiments. (C) Effect of RC48 and T-DM1 on the cell cycle of BT474 and SKBR3 cells. Cells were treated with 1 or 10 nM of the drugs for the specified times, and cell cycle phases were assessed by PI staining followed by flow cytometry analysis (50,000 events per condition were analyzed). Histograms represent the percentage of cells in each phase from two independent experiments. (D) BT474 and SKBR3 cells were seeded in 100 mm plates and treated with RC48 or T-DM1 (1 and 10 nM) for the indicated times. Cells were lysed and analyzed by Western blot. Loading control: calnexin.

The decrease in cell number caused by the ADCs could be due to a decrease in cell cycle progression, increased cell death, or both. To gain insights into the participation of these cellular processes in the different antiproliferative responses to RC48 and T-DM1, cell cycle profiles of cultures treated with the ADCs were analyzed. Treatment of BT474 cells with RC48, at either 1 or 10 nM caused a significant increase in the amounts of cells accumulating in G2/M (Figure 2C). The effect was already evident at 24 h of treatment and did not substantially change by extending the incubation time to 48 h. In contrast, in BT474 cells, treatment with T-DM1 at the same incubation times and using identical doses, did not substantially affect the cell cycle profiles when compared to untreated cells. In SKBR3 cells, both drugs exerted similar effects, increasing the number of cells in the G2/M cell cycle phases.

Biochemically, RC48 treatment led to an increase in phosphorylated Histone H3 levels, a marker of cell accumulation in mitosis, in both BT474 and SKBR3 cells at concentrations of 1 or 10 nM, and at both 24 and 48 h (Figure 2D). In contrast, T-DM1 only elevated pHistone-H3 levels in BT474 cells after 48 h at 10 nM. In SKBR3 cells, both RC48 and T-DM1 increased pHistone-H3 at all tested concentrations and time points. These differences in drug sensitivity were also reflected in their effects on cyclin B1, a key regulator of the G2/M transition and mitotic progression, and BubR1/pBubR1 levels, a core component of the spindle assembly checkpoint (Figure 2D and Figure S3). RC48 increased cyclin B1 and pBubR1 at 24 h in both cell lines. T-DM1 produced similar effects in SKBR3 cells at both 24 and 48 h but required 48 h and 10 nM to induce changes in BT474 cells. Additionally, RC48 reduced the levels of Wee1 kinase, an inhibitor of mitotic entry through phosphorylation of CDK1 at tyrosine 15 [31], in both cell lines. T-DM1, however, decreased Wee1 levels only in SKBR3 cells.

The action of both drugs on apoptotic cell death was then explored by cytometrically measuring viable vs. non-viable cells at 24, 48 or 72 h of treatment. RC48 similarly affected both cell lines, either at 1 or 10 nM, inducing a clear increase in the non-viable cell fraction (Figure 3A). In the case of T-DM1, the drug had almost no detectable effect on BT474 cells, but triggered apoptotic cell death in SKBR3 cells, similar to that caused by RC48. Antibody array studies to explore the levels of 35 proteins involved in apoptotic processes, showed that RC48 caused an increase in cleaved caspase-3, a marker of apoptotic cell death, in BT474 cells (Figure S2A,B). That increase in cleaved caspase-3 was higher than that provoked by T-DM1 (Figure 3B,C). In SKBR3 cells, the effect of both drugs on the cleavage of caspase-3 was similar.

Figure 3.

Effect of RC48 and T-DM1 on cell death. (A) BT474 and SKBR3 cells were treated with RC48 or T-DM1 (1 and 10 nM) for the indicated times. Cell death was evaluated by Annexin V-FITC/PI double staining followed by flow cytometry analysis. Data are represented to show the proportion of viable (Annexin V negative/PI negative) and non-viable cells based on two independent experiments. (B) Antibody array analysis of 35 apoptosis-related proteins in BT474 and SKBR3 cells treated with RC48 or T-DM1 (10 nM) for the indicated times. Reference spots are indicated by blue boxes, and cleaved caspase-3 (which showed the greatest difference between treatments) is highlighted with a green box. (C) Quantification of pixel intensities corresponding to cleaved caspase-3 expression from the arrays shown in (B). Spot intensities were normalized using the reference spots and are expressed relative to the corresponding control. Data represent the mean ± SD of duplicate spots.

3.3. Payload Release from RC48 and T-DM1

To gain further insights into the differential effect of RC48 and T-DM1 on HER2+ cells, the internalization of both drugs was explored. BT474 cells were treated with 1 nM of the respective ADCs and their subcellular location was analyzed by immunofluorescence after several incubation times. After 15 min of treatment, both ADCs offered a staining pattern compatible with their interaction with cell surface HER2 (Figure 4A). After one hour of incubation, and in addition to the fluorescent signal that was compatible with surface staining, a dotted pattern was already observed, and this continued until the last incubation time analyzed (24 h). No major differences in the cellular distribution of the two ADCs were observed. Additional immunofluorescence microscopy studies showed that RC48 caused accumulation of cells in mitosis, as indicated by staining with anti-pHistone H3, and it was similarly observed in both BT474 and SKBR3 cells (Figure 4B,C). T-DM1 also provoked accumulation of cells in mitosis in SKBR3 cells. In the case of BT474 cells, the effect of T-DM1 on the accumulation of mitotic cells was more discrete.

Figure 4.

Internalization and mitotic arrest induced by RC48 and T-DM1. (A) Internalization of RC48 or T-DM1 (1 nM) in BT474 cells at the indicated times. Scale bar: 10 µm. Red: ADC staining; Blue: nuclei. (B) BT474 and SKBR3 cells were treated with 1 nM of RC48 or T-DM1 for 24 h, fixed, and immunostained for phospho-Histone H3 (green), a mitotic marker. The 0 h time point corresponds to 15 min of treatment with the ADCs. Red: ADC staining. Scale bar: 10 µm. (C) Quantification of mitotic BT474 or SKBR3 cells (green, as shown in (B)) after the treatment with RC48 or T-DM1 (1 nM, 24 h). Bars represent the mean ± SD of two independent experiments. Percentages were calculated as: number of mitotic cells (green, as shown in (B))/total number of cells (blue nuclei, not shown in (B)) × 100 (%). For each condition, 500 cells were counted. * p < 0.05; ** p < 0.01, calculated using an unpaired t test.

At 24 h, RC48-treated BT474 cultures exhibited a higher number of mitotic cells compared to those treated with T-DM1 (Figure 2C). This observation suggested that RC48 might release its cytotoxic payload more efficiently than T-DM1. To investigate this hypothesis, we performed co-staining experiments using anti-payload antibodies alongside anti-human IgG antibodies, which detect the antibody backbone. Preliminary Western blot analyses confirmed that the anti-payload antibodies specifically recognized the payloads of the respective ADCs, but not the antibody backbone (Figure S5A). These blots also showed that the payloads were conjugated in comparable amounts to both the heavy and light chains of the antibodies.

Cultures treated with the ADCs were immunostained with antibodies recognizing the humanized antibody backbone and the payload. These studies showed coincidence of the Ab and payload signals in cells incubated with the ADCs for 15 min on ice, before being fixed and processed for immunostaining (Figure 5A). In agreement with data presented above, during the short 15 min incubation time the ADCs offered a pattern compatible with surface staining. Both the anti-human antibody used to detect the ADC backbone, as well as the anti-payload antibodies offered a co-staining pattern, clearly evidenced in the merged images. When cells were incubated for 24 h with the ADCs, the staining observed in the case of cells treated with T-DM1 was indicative of coincidence of the signals created by the anti-human and the anti-payload antibodies, both at the cell surface and at the cell structures characterized by a dotted pattern (see magnified image in Figure 5A). In the case of cells treated with RC48, the signal was also coincident at the cell surface. However, a number of intracellular dots were only stained by the anti-human antibody and not by the anti-payload antibody. Moreover, Western studies using the anti-payload antibodies showed that MMAE was released more efficiently than DM1 from both the heavy and light chains of the Ig (Figure 5B). Together, these data indicated that while the internalization of RC48 and T-DM1 were similar, the former likely released the payload more easily than T-DM1. That fact could promote a bystander effect of RC48, which has not been found in the case of T-DM1 [32]. To explore that possibility, experiments using SKBR3 and MDA-MB-468 cells were designed. SKBR3 cells were selected because of their high content of HER2 and responsiveness to both RC48 and T-DM1 (see Figure 1). On the other hand, MDA-MB-468 cells were used as indicators of the action of the released payloads, since they do not express detectable levels of HER2 (Figure S5B). Differentiation between both cell lines was made by cytometric evaluation of their EGFR levels (Figure S5C). Incubation of SKBR3 cells with either RC48 or T-DM1 (5 nM) confirmed the sensitivity of these cells to the action of the ADCs (Figure 5C). In contrast, MDA-MB-468 cells were largely resistant to the action of the drugs. Coincubation of SKBR3 and MDA-MB-468 cells showed that the ADCs retained their effect on SKBR3 cells. RC48 decreased the number of MDA-MB-468 cells in these coculture experiments, while T-DM1 did not. These results support the notion that RC48, but not T-DM1, may exert a bystander effect capable of eliminating neighboring HER2-negative cells within a heterogeneous tumor environment.

Figure 5.

Payload release and bystander effect of RC48 and T-DM1. (A) Confocal immunofluorescence analysis of payload release. BT474 cells were treated with RC48 or T-DM1 (1 nM) for the indicated times. The ADC backbone is shown in red and the payload in green (areas of colocalization appear yellow in the merged panels). Blue: nuclei. In the merged images, the red dots represent internalized antibody devoid of the payload, indicating intracellular drug release. Scale bar: 20 µm. The lower graphs show the quantitation of the intracellular antibody–payload colocalization percentage at 1, 3, 6 and 24 h of treatment. Membrane signals were excluded from the calculations. Data represent mean ± SD. * p < 0.05, **** p < 0.0001 versus the 1 h time point (one-way ANOVA followed by Dunnett’s test); n.s., not significant. (B) Western blot analysis of the levels of payload (MMAE or DM1) bound to the ADC (heavy and light chain) at the indicated treatment time points. Band quantification relative to the maximum signal is shown in the graphs; each bar represents the mean and the individual dots or triangles indicate values from two independent experiments. (C) Evaluation of the bystander killing effect. HER2-positive SKBR3 cells (blue bars) and HER2-negative MDA-MB-468 cells (pink bars) were cultured alone or co-cultured (1:1) and treated with 5 nM of T-DM1 or RC48. Bars represent the mean percentage of viable cells relative to untreated controls (set to 100%), from two independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, n.s., not significant vs. untreated control (one-way ANOVA with Dunnett’s test).

3.4. Action of RC48 on HER2+ Cells Secondarily Resistant to Anti-HER2 Therapies

To evaluate whether RC48 was effective in secondary resistance to anti-HER2 drugs used in the breast cancer clinic, several resistant models were used. These models are based on the sensitive cell line BT474, from which resistant subpopulations can be generated upon continuous exposure to a particular anti-HER2 drug (Figure S6A). Following this protocol, we generated several subclones resistant to T-DM1 (BT-TDM1R#1), neratinib (BTRN#6) or lapatinib (BTRL#109). Clones phenotypically similar to parental cells (Figure S4B), with analogous levels of total HER2 (Figure S6C) and whose proliferation rates were analogous to the parental cell line [26,27,28] were selected for further studies.

RC48 was found to be more effective against the different cell lines than T-DM1 (Figure 6A). As expected, the latter, at the highest concentration tested (1 nM) did not have a detectable effect on the T-DM1R resistant clone. In the case of parental, BTRL#109 and BT-RN#6, T-DM1 decreased the cell number at a concentration of 1 nM.

Figure 6.

Action of RC48 or T-DM1 on various models of resistance. (A) Antiproliferative efficacy of RC48 and T-DM1 on parental BT474 cells and resistant clones: T-DM1-resistant (#1), neratinib-resistant (#6), and lapatinib-resistant (#109). Cells were treated with the indicated doses for 3 days and then counted. Cell viability was normalized to untreated controls. Data represent mean ± SD. p-values indicate significant differences between RC48 and T-DM1 treatment at the indicated concentrations (unpaired t-test with Welch’s correction). (B) Cell cycle analysis by flow cytometry. BT474 cells and resistant clones were treated with RC48 or T-DM1 (1 nM) for the indicated times. Graphs represent the percentage of cells in each phase of the cell cycle from two independent experiments (50,000 events analyzed per condition). (C) Western blot analysis of cell cycle proteins. BT474 and resistant clones were treated with RC48 or T-DM1 (1 nM) for the indicated times, lysed, and analyzed for levels of various cell cycle proteins. The bar graphs below show band quantification from two independent experiments, normalized to the untreated control.

Cell cycle profiling showed that RC48 increased the number of cells in G2/M cell cycle phases in the three resistant cell lines tested (Figure 6B and Figure S7A). Of note, these studies showed that previous resistance to T-DM1 slightly inhibited the action of RC48 on the cell cycle profile. In contrast to RC48, T-DM1 did not substantially affect the cell cycle profiles in any of the cell models analyzed. Biochemical studies showed that RC48 increased pHistone-H3 in all the cell lines (Figure 6C). At 24 h of treatment, the effect was already noticed and persisted for up to 72 h. T-DM1 also increased the levels of pHistone H3 in BT474, BTRL#109 and BT-RN#6 cells after 48 h of treatment, but did not have any effect on the BT-TDM1R#1 cell line. RC48 also increased the amount of cyclin B1 in parental BT474, T-DM1R#1 and BTRL#109 cells. T-DM1 was unable to induce a clear effect on cyclin B1 in any of the cell lines studied. The levels of Wee1 were decreased by RC48 in all the cell lines, while T-DM1 did not have a substantial effect. Analogous data with respect to the differential sensitivities to RC48 and T-DM1 were observed in their action on pBubR1 (Figure S7B).

The effects of RC48 and T-DM1 on apoptotic cell death were next evaluated in the various resistant clones. Microscopic examination revealed signs of apoptosis, such as cell rounding and the presence of cellular debris, in resistant cells treated with RC48 (Figure 7A). These morphological changes were not observed in cultures treated with T-DM1. Annexin V/PI double staining further confirmed that RC48 induced a significant increase in cell death in both naïve cells and those resistant to T-DM1, lapatinib, or neratinib (Figure 7B,C). In contrast, T-DM1 was markedly less effective at inducing cell death across all conditions.

Figure 7.

Cytotoxicity of RC48 and T-DM1 in resistant clones. (A) Morphological changes induced by RC48 or T-DM1 in parental BT474 cells and resistant clones: T-DM1-resistant (BT-TDM1R#1), neratinib-resistant (BTRN#6), and lapatinib-resistant (BTRL#109). Cells were cultured in 6-well plates and treated with 1 nM of the drug for 3 days. Phase-contrast images were taken at 10× magnification. Scale bar: 20 µm. (B) Flow cytometry analysis of cell death using Annexin V/PI staining. Cells were treated with RC48 or T-DM1 (1 nM) for 72 h. (C) Quantification of cell viability shown on (B). The graph represents the percentage of viable (Annexin V-negative/PI-negative) and non-viable cells from two independent experiments (50,000 events analyzed per condition). (D) Expression of apoptotic markers by Western blot in BT474 and resistant clones treated with RC48 or T-DM1 (1 nM) for the indicated times. Loading control: β-actin.

Biochemically, RC48 caused cleavage of PARP and caspase-3 in BT474 and BT-TDM1R#1 cells. Cleaved caspase-3 was also observed in BTRL#109 and BTRN#6, but this effect was of less magnitude than that observed in the case of BT474 or BT-TDM1R#1. T-DM1 did not exert such a clear effect on the apoptotic proteins analyzed in any of the resistant cell lines. In BT474 cells a small increase in cleaved caspase-3 was observed after 72 h of treatment with the drug.

4. Discussion

While initially developed for use in tumors other than breast cancer [25], the differential characteristics of RC48 with respect to other ADCs used in the breast cancer clinic, together with its demonstrated clinical value in the treatment of patients with HER2 tumors located in other organs, is raising interest for its potential use for the therapy of breast cancer [33]. Considering this, we decided to explore its value for the therapy of HER2+ tumors. The results presented here highlight the superior antiproliferative action of RC48 compared to T-DM1 in HER2-positive breast cancer. Importantly, this enhanced activity was also observed in cells that had developed resistance to multiple anti-HER2 therapies, suggesting that RC48 may be particularly effective in treatment-resistant breast tumors. Moreover, the fact that RC48 showed bystander properties allows it to target and eliminate adjacent HER2-negative cells in a heterogeneous tumor setting.

Cell proliferation studies indicated that RC48 was very potent and efficient on preclinical models of HER2+ breast cancer. Moreover, RC48 surpassed T-DM1. Given the use of the latter in HER2+ breast cancer [34], the superior activity of RC48 opens the possibility of using that ADC in place of T-DM1, with the purpose of exerting more efficient antitumoral action. Whether that hypothesis is realistic should be clinically assessed.

The absence of antiproliferative effects of RC48 and T-DM1 in HER2-low models (MCF7 and T47D) confirms the HER2 specificity of both ADCs, reinforcing the notion that a certain threshold of HER2 expression favors their effective cytotoxicity. However, some reports have described activity of RC48 in HER2-low metastatic breast cancer [33,35]. This is in line with trastuzumab-deruxtecan, which has shown activity against HER2-low tumors [18,36]. The fact that RC48 did not have any detectable effect on the HER2-low cells used in our study may be related to the fact that under physiological settings, microenvironment factors, e.g., secreted proteases, may contribute to the action of RC48 in HER2-low/null settings. Additional clinical and animal studies would be required to define the potential value of RC48 in HER2-low scenarios.

With respect to the clinical situations in which RC48 may be useful, the fact that RC48 retained antitumoral activity in models of resistance to conventional drugs used in the therapy of HER2-positive breast cancer, including models of resistance to T-DM1, open the possibility of considering the use of RC48 in situations of resistance to these drugs. Given the clinical efficacy of T-DXd, it will also be important to define situations in which RC48 could substitute or complement the use of T-DXd. For instance, it would be relevant to analyze the efficacy of RC48 in patients that became resistant to T-DXd. Moreover, given the fact that T-DXd may induce interstitial lung disease [18], which may provoke cessation of treatment with that drug, the possibility of using RC48 to continue the therapy represents an attractive possibility.

The differing efficacy of RC48 and T-DM1 in BT474 and SKBR3 cells suggests that, despite similar levels of HER2 expression, these cell lines respond differently to HER2-targeted antibody–drug conjugates. While both antibodies target HER2 and internalize, and contain payloads that act on microtubules, RC48 achieved maximal inhibition at 1 nM in both BT474 and SKBR3 cells, whereas T-DM1 demonstrated a reduced potency in BT474 cells. These findings point to differences in the mechanisms of action of the ADCs. Indeed, RC48 appeared to release its payload more efficiently than T-DM1. Of note, the linker in RC48 is designed to be cleaved by acidic proteases present in intracellular organelles, facilitating efficient drug release [37]. In contrast, T-DM1 contains a non-cleavable linker, requiring complete proteolytic degradation within the lysosome to release DM1 [17,26]. This fundamental difference may underlie the varying sensitivities of BT474 and SKBR3 cells to these two ADCs. While MMAE release from RC48 appears to occur efficiently in both cell lines, DM1 release from T-DM1 seems more effective in SKBR3 cells than in BT474 cells, potentially contributing to the relative resistance of the latter to T-DM1.

Mechanistic studies revealed that RC48 more effectively induced G2/M arrest and increased pHistone-H3 levels in BT474 cells compared to T-DM1, suggesting stronger mitotic disruption and antiproliferative activity. In SKBR3 cells, both ADCs produced similar effects on the cell cycle, indicating that factors beyond HER2 expression influence drug response. Apoptosis assays showed that RC48 induced caspase-3 cleavage in both cell lines, while T-DM1 triggered apoptosis only in SKBR3 cells. These findings highlight cell line–dependent differences in ADC sensitivity, with RC48 showing broader and more potent cytotoxic activity.

Studies on the bystander properties of RC48 and T-DM1 highlight a key functional difference between both ADCs in the context of heterogeneous tumors. The ability of RC48 to reduce the viability of neighboring HER2-negative MDA-MB-468 cells in coincubation experiments suggests that its payload can diffuse from targeted HER2-positive cells, supporting a bystander killing effect. While both ADCs contain microtubule-targeting payloads, the lack of such an effect with T-DM1 underscores the importance of linker design and perhaps payload properties in mediating this phenomenon. These findings are particularly relevant for the treatment of heterogeneous HER2+ tumors, which often contain a mix of HER2-expressing and non-expressing cells. The bystander effect observed with RC48 may enhance therapeutic efficacy in this setting by enabling the elimination of HER2-negative tumor cells that would otherwise evade direct targeting. This property could offer a significant advantage over T-DM1, which lacks such activity [18,32]. In addition, the effectiveness of RC48 in cells resistant to T-DM1 due to deficient lysosomal activity [26] suggests that the release of the RC48 payload is less sensitive to the acidic microenvironment required for T-DM1 processing, and this may be relevant in clinical scenarios of resistance to T-DM1. In fact, while under those circumstances the use of T-DXd is gaining acceptance, the appearance of toxicity to T-DXd [18] may preclude its use and therefore alternative therapies, such as RC48, may be considered.

The study also explored the potential of RC48 in overcoming secondary resistance to anti-HER2 therapies, an area of significant clinical interest. The generation of BT474 sublines resistant to T-DM1, neratinib, or lapatinib provided relevant in vitro models to assess the action of RC48 in the context of acquired resistance. Notably, RC48 retained its efficacy in all resistant models, causing G2/M arrest even in models that exhibited substantial resistance to T-DM1. It is relevant to mention that the model used as representative for the resistance to T-DM1 presents a deficit in lysosomal proteolytic activity [26]. The fact that RC48 also showed antiproliferative activity under those circumstances suggests that the mechanism of release of the payload is not exclusively dependent on an acidic lysosomal environment. This conclusion is also suggested by the payload release studies, which showed more efficient release of MMAE from RC48, than DM1 from T-DM1. Moreover, the effectiveness of RC48 to act in situations of resistance to T-DM1 caused by deficient proteolytic activity offers the possibility of using RC48 in those circumstances, adding value to its potential incorporation to the HER2+ breast cancer clinic for indications of resistance to ADCs actually used to target HER2. It is relevant to mention that the studies herewith reported agree with a previous report that demonstrated the effectiveness of RC48 on cells primarily resistant to anti-HER2 drugs [38]. The data presented here complement that study highlighting the action of RC48 in situations of secondary resistance, that are more common than primary resistance. Our study presents some limitations. No animal studies have been made, which will be required for assessment of the antitumoral action of RC48 in a physiological setting, especially with respect to its action on HER2-low or heterogeneous tumors.

5. Conclusions

This study demonstrates the superior efficacy of disitamab vedotin over trastuzumab emtansine in HER2-positive breast cancer models, including those with acquired resistance to multiple anti-HER2 therapies. The enhanced activity of RC48 is linked to its more efficient payload release, induction of mitotic arrest and apoptosis, and its bystander effect, allowing it to target neighboring HER2-negative cells. These findings position RC48 as a promising therapeutic alternative for treatment-resistant and heterogeneous HER2+ breast tumors, and support its potential incorporation into the clinical management of breast cancer.

Abbreviations

ADC	Antibody–drug conjugate
ATCC	American Type Culture Collection
BCA	Bicinchoninic acid
CNX	Calnexin
DMEM	Dulbecco’s Modified Eagle Medium
ECL	Enhanced Chemiluminescence
EGFR	Epidermal Growth Factor Receptor
FACS	Fluorescent-Activated Cell Sorting
FBS	Fetal Bovine Serum
FITC	Fluorescent Isothiocynate
HER2	Human Epidermal growth factor Receptor 2
HRP	Horseradish Peroxidase
MMAE	Monomethyl Auristatin E
PARP	Poly (ADP-ribose) Polymerase
PBS	Phosphate-Buffered Saline
PBS/CM	Phosphate-Buffered Saline with calcium and magnesium
PI	Propidium Iodide
PVDF	Polyvinylidene Fluoride
SDS-PAGE	Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis
TBST	Tris-buffered saline with Tween
T-DM1	Trastuzumab-emtansine
T-DXd	Trastuzumab-deruxtecan
TKIs	Tyrosine Kinase Inhibitors

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/pharmaceutics18020208/s1, Table S1. Reagents and immunochemicals; Figure S1. Schematic representation of RC48, T-DM1 and T-DXd; Figure S2. (A) Western blot analysis of the amount of HER2 in HER2-positive breast cancer cell lines. Calnexin was used as a loading control. (B) Comparison of the antiproliferative effects of RC48 or T-DM1 in HCC1951, HCC1419 cells after 3 days of treatment. p-values indicate significant differences between RC48 and T-DM1 at the indicated concentrations (unpaired t-test with Welch’s correction); Figure S3. Western blot analysis of phosphorylated BubR1 (pBubR1) levels in BT474 and SKBR3 cells after the treatment with RC48 or T-DM1 at the indicated doses and time points. A digitally stretched-up view is shown below to better visualize the increased in pBubR1; Figure S4. Quantification of apoptosis antibody array. (A) Relative expression levels of all proteins analyzed in the array. BT474 and SKBR3 cells were treated with RC48 or T-DM1 (10 nM) for 24 and 48 h. (B) Details of the quantifications of proteins mostly deregulated by the treatments are shown. Data are presented as mean ± SD of duplicate array spots, normalized to the untreated control; Figure S5. (A) Validation of payload conjugation and antibody specificity. Western blot probed with anti-MMAE and anti-DM1 antibodies reveal the coupling of the payload to both the heavy and light chains of RC48 and T-DM1, respectively. The results demonstrate that each antibody specifically recognizes its corresponding payload without cross-reactivity. Trastuzumab was included as a non-conjugated control. Stain-free gels (right) confirm equal protein loading. (B) Western blot analysis of HER2 and EGFR levels in SKBR3 and MDA-MB-468 cells. Cells were lysed and 50 µg of total protein were loaded. Calnexin was used as a loading control. (C) Experimental design of the bystander effect assay. The scheme illustrates the protocol for mono-culture and co-culture experiments treated with RC48 or T-DM1 (5 nM) for 3 days. The histogram validates the discrimination of the two cell populations by flow cytometry using Cetuximab-DyLight 488 (anti-EGFR antibody): the low-fluorescence peak corresponds to SKBR3 and the high-fluorescence peak to MDA-MB-468; Figure S6. Generation and characterization of resistant clones. (A) Schematic representation of the generation of neratinib, lapatinib and T-DM1 resistant clones. Parental BT474 cells were treated with T-DM1 (5 nM), neratinib (10 nM), or lapatinib (1 µm) for several months and resistant clones were selected. (B) Phase-contrast microscopy images show that resistant clones exhibit a morphological appearance similar to the parental BT474 cell line. Images were acquired at 10× magnification. Scale bar: 20 µm. (C) HER2 expression in BT474 parental cells and resistant clones analyzed by Western blot. Cells were lysed and 1 mg of total protein was immunoprecipitated. Numbers below the blot indicate the relative quantification of signal intensity, normalized to the BT474 parental cells (BT474 = 1); Figure S7. Cell cycle arrest and BubR1 phosphorylation in resistant clones. (A) Cell cycle profile of parental BT474 cells and resistant clones. Cells were treated with RC48 or T-DM1 (1 nM) and 24 h later were analyzed by flow cytometry upon PI staining. (B) BT474 and resistant clones were treated with RC48 or T-DM1 (1 nM) for the indicated times, lysed, and pBubR1/BubR1 expression was analyzed by western blot. The bar graphs below indicate band quantification from two independent experiments, normalized to the untreated control of each cell line.

Author Contributions

M.R.-P.: Investigation; Data curation; Visualization; Formal Analysis. M.d.C.G.-G.: Investigation; Data curation; Visualization; Formal Analysis. A.P.: Conceptualization; Supervision; Writing—Original Draft; Writing—Review and Editing; Project Administration. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The original contributions presented in this study are included in the article/Supplementary Materials. Further inquiries can be directed to the authors: Mónica Redondo-Puente (monicaredondopuente@gmail.com), María del Carmen Gómez-García (mamengomezgarcia@usal.es), Atanasio Pandiella (atanasio@usal.es).

Conflicts of Interest

A.P. received personal fees from Daiichi-Sankyo and CancerAppy in the last two years. The rest of the authors declare no competing interests.

Funding Statement

A.P.: Ministry of Economy and Competitiveness of Spain (PID2020-115605RB-I00), the Instituto de Salud Carlos III through CIBERONC, ALMOM, ACMUMA, UCCTA, the CRIS Cancer Foundation and the Regional Development Funding Program (FEDER) “A way to make Europe”. MRP was recipient of a predoctoral contract (FPU21/02155).