Functional HER1/HER2-Expressing Murine Tumor Models for Preclinical Evaluation of Targeted Therapies

Abstract

Background: HER1 and HER2 are critical receptors involved in tumorigenesis and the development of targeted therapies for various carcinomas. However, most antibodies and drugs currently in development do not recognize murine orthologs, which restricts their evaluation in immunocompetent models. Methods: We generated nine tumor models through the lentiviral transduction of murine prostate (RM1), lung (3LL-D122), and breast (4T1) carcinoma cell lines, subsequently validating them in immunocompetent BALB/c and C57BL/6 hosts. Receptor expression and functionality were characterized using flow cytometry, immunoblotting, proliferation assays, and therapeutic sensitivity testing. Results: Transduced cells exhibited stable membrane expression of HER1/HER2 and ligand-induced phosphorylation, confirming receptor functionality. In all three tumor models generated, the expression of HER1 and/or HER2 significantly enhanced cell proliferation compared to parental lines. Furthermore, treatment with specific monoclonal antibodies and the tyrosine kinase inhibitor markedly reduced the viability of cells expressing HER1 and/or HER2, without affecting negative controls. Conclusions: These models provide a robust and reproducible platform for the preclinical evaluation of HER1/HER2-targeted therapies in immunocompetent hosts. Although the current model relies on subcutaneous implantation and does not fully replicate the native tumor microenvironment, it represents a crucial first step toward the development of orthotopic and immunologically relevant models for translational cancer research.

1. Introduction

Overexpression or abnormal functionality of HER1 and/or HER2 is found in various solid tumors and plays a key role in tumor initiation and progression [1]. The uncontrolled activity of HER1/HER2 homodimers or heterodimers excessively activates critical intracellular pathways, leading to sustained proliferation [2]. These receptors have been the targets selected for many monoclonal antibodies, tyrosine kinase inhibitors and vaccines, which are approved or in development. However, tumors often develop resistance to HER specific targeting therapies that involve other receptors of the family. Therefore, therapies directed to more than one target in the HER family could be more potent and prevent or delay the emergence of resistance. Understanding the molecular basis of antitumor therapies and tumor cell resistance mechanisms is crucial [3]. Recent studies show that the effectiveness of treatments targeting tumoral growth and signaling pathways is influenced by the tumor’s microenvironment [4]. Thus, defining how oncogenic signaling involving HER1 and HER2 affects this microenvironment is increasingly important [5]. However, most therapeutic antibodies and vaccine candidates targeting HER1 and/or HER2 do not recognize murine counterparts (ErbB1 and ErbB2, respectively), limiting the characterization of their mechanisms in immunocompetent mice [6,7]. Creating syngeneic mouse tumor lines with human proteins via lentiviral transduction is an effective and cost-efficient approach for immunotherapy studies targeting tumor-associated antigens [8]. Lentiviruses facilitate efficient gene expression in various cell lines [9] and allow the stable integration of recombinant DNA into active chromatin sites [10], making them valuable for generating syngeneic tumor models.

HER1 and HER2 are overexpressed in solid tumors of epithelial origin, including prostate, lung, and breast carcinomas, and often associated with poor prognosis and reduced patient survival. HER1 is overexpressed in 80% of lung tumors, and some studies report that overexpression of HER1 is a predictive factor for trastuzumab response in HER2+ tumors [11]. In breast carcinoma, HER2 expression is linked to a more aggressive phenotype, where co-expression with HER1 could promote distant metastasis and is significantly associated with poor clinical outcomes in patients with breast cancer [12]. Furthermore, studies demonstrate a strong correlation between HER1 overactivation and metastatic progression in prostate tumors, emphasizing the importance of HER2 heterodimers in the development of resistance [13]. Given the significance of HER1/HER2 heterodimer formation in tumor progression [14], it is essential to develop models that express both receptors, enabling the evaluation of their combined inhibition in various cellular contexts. In the present study, we generated in vitro murine models by transducing RM1 (prostate cancer), 3LL (lung carcinoma), and 4T1 (breast carcinoma) cell lines, with lentivirus expressing HER1 and/or HER2 in using a lentiviral approach.

2. Materials and Methods

2.1. Antibodies and Reagents

HER1-specific monoclonal antibody cetuximab (Erbitux) was obtained from Roche, Basel, (Switzerland), while the HER2-specific mAb 5G4, a biosimilar to trastuzumab, was sourced from CIM, Havana, Cuba [15]. Antibodies targeting HER1 (#4267S), phosphorylated HER1 (Y1068, #2234L), phosphorylated ERK1/2 (T202/Y204, #9102), ERK1/2 (#9102), HER2 (#2242) and β-actin (#4967S) were acquired from Cell Signaling Technologies, Danvers, Massachusetts, USA. Antibodies targeting phosphorylated HER2 (Y1248, #K612) were acquired from Santa Cruz Biotechnology, Dallas, TX 75220, USA. The tyrosine kinase inhibitor AG 1478 (Tyrophostin AG-1478, #T4182-5MG) was purchased from Sigma-Aldrich, St. Louis, MO, USA, and recombinant human EGF was obtained from the Center for Genetic Engineering and Biotechnology (CIGB), Havana, Cuba.

2.2. Plasmids

The plasmids used in this work correspond to the third-generation HIV-1-based lentiviral (LV) packaging system and included three helper plasmids (Invitrogen, Waltham, Massachusetts, USA): (1) pLP1 (contains gag and pol genes), (2) pLP2 (contains rev gene), and (3) pLP VSV-G (contains VSV G glycoprotein gene). For HER1 expression, erbB1 gene encoding full-length human EGFR pLenti6.3/V5–DEST vector was cloned into pLenti6.3/V5–DEST vector (with blasticidin marker) and kindly donated to our lab by Prof. Maicol Mancini. For HER2 expression, pHAGE-ERBB2 was a gift from Gordon Mills & Kenneth Scott (Addgene plasmid #116734; http://n2t.net/addgene:116734 (accessed on 30 April 2025); RRID: Addgene_116734).

2.3. Cell Lines and Culture Conditions

The HEK293-T cell line (CRL-11268) was used as a packaging strain to produce lentiviral particles. The murine prostate carcinoma-derived cell line (RM1) was kindly donated by Dr. Andrea Alimonti, Director of the Swiss Cancer Research Institute, the breast carcinoma-derived cell line 4T1 was obtained from the ATCC Manassas, Virginia, USA as well as the lung carcinoma-derived cell line D122 clone of Lewis lung carcinoma (3LL) [16]. Cells were maintained in basal growth media (DMEM-F12) purchased from Gibco and supplemented with 10% fetal bovine serum (FBS; Gibco, Grand Island, NY, USA).

2.4. Transduction of Cells Using LV

2.4.1. Production of LV

Lentivirus were produced by transfection of HEK-293T using lineal PEI (Sigma-Aldrich, USA) as previously described [17]. The cells were transfected with the lentiviral transfer plasmids and helper plasmids: pLP1, pLP2, and pLP VSV-G at a ratio of (2:1:1:1) (w/w/w/w) for 30 μg of total DNA. After 6 h of incubation at 37 °C in the presence of 5% CO₂, FBS was added to the culture and the supernatant was harvested at 72 h post-transfection. The cell culture supernatant was centrifuged at 290× g for 5 min, filtered (0.45 mm membrane), and stored at 4 °C.

2.4.2. Transduction of Cells

The day prior to transduction, RM1, 3LL, and 4T1 cells were seeded in 6-well plate using DMEM/F12-FBS medium and incubated at 37 °C in 5% CO₂. After 16 h, transduction was performed by incubating supernatant LVs with cells in DMEM/F12 medium supplemented with 10 μg/mL of polybrene (Sigma-Aldrich, St. Louis, MI, USA). For co-transduction, the supernatant LV encoding HER1 and HER2 were used in 1:1 ratio. Eight hours post-transduction, the medium was replaced with fresh DMEM/F12-FBS medium and blasticidin for cell transduction with LV encoding HER1 or puromycin for cell transduction LV of HER2. A second round of transduction was performed in the same conditions as outlined above. Following antibiotic selection, single-cell clones were isolated by limiting dilution in 96-well plates (0.5 cells/well). Clones were expanded for 2 weeks, then screened for HER1/HER2 expression by flow cytometry using human-specific antibodies (as described in Section 2.6). The highest-expressing clones for each receptor combination were selected for subsequent in vivo passage.

2.5. Inoculation in Mice of Heterologous Syngeneic Models

Female mice, aged 8–12 weeks old, were purchased from the National Center for Laboratory Animals Production (CENPALAB, Havana, Cuba). All mice were kept under pathogen-free conditions. Animal experiments were approved by the Center of Molecular Immunology’s Institutional Animal Care and Use Committee, or CICUAL (CIM, Havana, Cuba). BALB/c and C57BL/6 mice were selected according to the syngeneic background of the tumor cell lines (4T1 in BALB/c mice; RM1 and 3LL in C57BL/6) to ensure optimal tumor engraftment and immune compatibility. For each tumor model, three serial in vivo passages (n = 5 mice/passage) were performed to counterbalance the initial heterogeneity in lentiviral transduction efficiency and selected for clones with stable HER1/HER2 membrane expression. Mice received 8 × 10⁵ cells subcutaneously in the right flank and were euthanized upon reaching endpoint criteria (tumor volume ˃1000 mm³). Excised tumors were disaggregated and either reinoculated into new mice or cultured to establish cell lines. This repeated passage strategy enhances transgene stability and tumor adaptation to the microenvironment, as established in lentiviral-based models [18].

2.6. Flow Cytometry Assays

Detection of HER1 and/or HER2 Expression at the Cell Membrane

RM1, 3LL, and 4T1 parental cells (serving as HER1/HER2-negative control) and corresponding modified cells expressing HER1 and/or HER2, were seeded in 6-well plates (10⁵ cells/well) for 24 h. Cells were detached with trypsin for 5 min, washed with PBS by centrifugation at 300× g for 5 min, and blocked in saline with 1% (w/v) albumin for 20 min at 4 °C. To assess specificity, the following was conducted: (1) HER2-expressing cells were stained with cetuximab (anti-HER1); (2) HER1-expressing cells were stained with 5G4 (anti-HER2); and (3) secondary antibody-only controls were included. Experimental samples were incubated with cetuximab or 5G4 (1 µg/mL) for 20 min at 4 °C, followed by an anti-human IgG secondary antibody conjugated to allophycocyanin (APC) (1:400) for 30 min at 4 °C. A minimum of 5 × 10³ cells were analyzed using a CyFlow flow cytometer (Partec Sysmex, Norderstedt, Germany). Three washes with blocking solution were performed during incubations, and data analysis was conducted using FlowJo 10.0.7 software (Tree Star Inc., Ashland, OR, USA).

2.7. Immunoblotting Assays

RM1, RM1-HER1, RM1-HER2, and RM1-HER1/HER2 (2.5 × 10⁵ cells/well) cells were seeded in 6-well plates for 24 h. The culture medium was removed, and cells were kept overnight in DMEM-F12 medium without FBS supplementation for 16 h. Afterwards, cells were stimulated with EGF (100 ng/mL) for 10 min at 37 °C. Cells were washed twice with cold PBS, and scraped into lysis buffer [50 mM Hepes (pH 7.5), 10% glycerol, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA,1 mM EGTA, 10 mM NaF, 0.1 mM Na₃VO₄], and complete protease inhibitor cocktail. Next, lysates were centrifuged at 14,000× g for 15 min at 4 °C and supernatants were preserved at −80 °C. After protein separation by gel electrophoresis, proteins were transferred to nitrocellulose membranes. Immunoblotting was performed according to the antibody manufacturers’ recommendation. Antibody binding to membranes was detected using horseradish peroxidase–secondary antibodies (BioRad, Hercules, CA, USA), followed by treatment with ECL Clarity detection reagents (BioRad).

2.8. Colorimetric Assays

2.8.1. AlamarBlue Assay

RM1, 3LL, and 4T1 parental cells, along with corresponding modified cells expressing HER1 and/or HER2, were plated in 96-well plates at 5 × 10³ cells per well in a medium with 10% FBS (culture condition) at 37 °C in a 5% CO₂ atmosphere. Absorbance was measured 4 h after plating (time 0 baseline). Cell proliferation was then assessed at 24, 48, and 72 h by removing the medium and adding 10 µL of AlamarBlue reagent (final concentration 10%) [19]. The plates were incubated in the dark at 37 °C for 2 h, and absorbance was measured at 540 nm and 630 nm wavelength using a spectrophotometer (Dialab, Wiener Neudorf, Austria).

2.8.2. MTT Assay

RM1, 3LL, and 4T1 parental cells and corresponding modified cells expressing HER1 and/or HER2 were cultured in 96-well plates at a density of 5 × 10³ cells per well for 24 h. After removing the medium, treatments were applied in culture medium with 1% FBS: 5G4 (10 μg/mL), Cetuximab (10 μg/mL), and TKI AG1478 (1:500). The final concentration of dimethyl sulfoxide (DMSO) used as vehicle was below 0.1%, a concentration not considered cytotoxic [20]. Monoclonal antibodies 5G4 and cetuximab were dissolved directly in culture medium with 1% FBS following reconstruction with sterile PBS. Control cells were incubated with medium and 1% FBS for maximum viability (100%). Six replicates per condition were incubated for 96 h. Afterward, the culture supernatant was removed, and MTT reagent (1 mg/mL) was added. Cells were incubated for 4 h at 37 °C in a 5% CO₂ atmosphere. Formazan crystals were solubilized with 100 µL of dimethyl sulfoxide per well. Absorbance at 540 nm and 620 nm was measured using a spectrophotometer, and the percentage of viable cells was calculated using untreated cells as the reference for maximum viability.

2.9. Statistical and Data Analyses

Graphical and statistical analysis was performed using GraphPad Prism 9.3.1 software. Figures were constructed in GIMP 2.10.8 software. Normality was evaluated by the Shapiro–Wilk test, and the Levene test was used to assess variance homogeneity. Tests used to determine statistical differences among group media are specified in the figure legends. In graphics, significant differences were highlighted with asterisks. (*) p < 0.05, (**) p < 0.01, (***) p < 0.001. All experiments were performed in independent biological duplicates, each with six technical replicates, to ensure reproducibility.

3. Results

3.1. Generation of Cellular Models from Various Tumor Types with Heterologous Expression of HER1 and/or HER2

Tumoral cell models expressing HER1 and/or HER2 receptors were created via lentiviral transduction using particles containing their full-length coding sequences. These lentiviral particles transduced tumoral cells from different origins (RM1-prostate, 3LL-lung, and 4T1-breast). The populations that emerged from antibiotic selection underwent cloning through limiting dilution, and those clones exhibiting the highest levels of membrane receptor expression were chosen for inoculation into mice (C57BL/6 for RM1 and 3LL, Balb/c for 4T1). The strategy is summarized in Figure 1A.

We assessed the membrane expression of the receptors in heterologous models within the primary culture after three inoculations in mice using flow cytometry with specific monoclonal antibodies: cetuximab for HER1 and the 5G4 biosimilar for HER2. Figure 1B–D demonstrate that HER1-expressing cells show increased mean fluorescence intensity (blue and violet histograms) after cetuximab incubation, while HER2-expressing cells (green and violet histograms) exhibit a similar shift after 5G4 incubation. Both antibodies exhibited high affinity for their respective receptors [21,22]. Notably, MFI values for 5G4 were consistently higher than those for cetuximab across all models, suggesting greater HER2 expression compared to HER1.

3.2. In Vitro Characterization of the Functionality of HER1 and/or HER2 Receptors in the Generated Models

The impact of HER1 and/or HER2 expression on cell proliferation was evaluated across all generated models (RM1, 3LL, and 4T1), using a colorimetric assay. HER1 and HER2 expression significantly enhanced proliferation in all models compared to parental cells, confirming the functional role of these receptors in promoting tumor cell growth (Figure 2A–C).

To validate the functionality of the heterologous receptors and further explore the mechanisms underlying the observed proliferative increase, we assessed HER1 and HER2 phosphorylation in RM1-derived models. The RM1 cell line was selected as a representative model to confirm receptor activation. Figure 2D shows phosphorylated HER1 (pHER1) bands at tyrosine Y1068 in RM1-HER1 and RM1-HER1/HER2 models. These bands were detected in stimulated and non-stimulated cells, indicating basal phosphorylation. Densitometry analysis revealed increased band intensity following EGF stimulation, confirming ligand-induced activation. In HER1-expressing models, a band corresponding to the receptor’s full molecular weight was observed; however, this expression decreased in the RM1-HER1/HER2 after EGF treatment. Furthermore, we assessed HER2 activation and found a phosphorylated receptor band (pHER2) in models with heterologous receptor expression. Notably, HER2 phosphorylation in the RM1-HER2 model occurs independently of EGF, indicating a basal self-activation mechanism. In contrast, densitometric analysis showed increased HER2 phosphorylation in the RM1-HER1/HER2 model after ligand treatment, suggesting HER1/HER2 heterodimer formation. Additionally, HER2 phosphorylation in the RM1-HER1 model post-EGF may indicate heterodimerization with murine ErBb2, likely due to due commercial antibody cross-reactivity.

Finally, we assessed the sensitivity of the generated cellular models to the blockade and inhibition of HER1 and/or HER2 receptors. The cells were treated with specific monoclonal antibodies (cetuximab or 5G4) and the HER1-specific tyrosine kinase inhibitor AG1478. As illustrated in Figure 3A–C, inhibition of HER2 by the 5G4 antibody resulted in a significant decrease in cell viability (40% to 60%) in the HER2+ models. Similarly, the HER1+ models exhibited a reduced cell viability after blocking this receptor with the cetuximab antibody or, more drastically, following treatment with a specific TKI. Noticeably, HER1-expressing models derived from 3LL cells demonstrated high sensitivity to treatment with the TKI, leading to a reduction in viability of up to 90% (Figure 3B). Parental cells exhibited no sensitivity to any of the treatments evaluated. The absence of therapeutic effects in antigen-negative parental cells confirms the treatment specificity for HER1/HER2-expressing cells.

In the RM1-derived models, we assessed the effect of HER1 inhibition using the TKI (AG1478) on ERK1/2 activation. As shown in Figure 3D, EGF stimulation induced an increase in ERK1/2 phosphorylation. However, this effect was reversed upon HER1 inhibition, as evidenced by the reduction in HER1 phosphorylation levels following treatment with the inhibitor.

4. Discussion

Uncontrolled HER1/HER2 homodimer and heterodimer activity leads to excessive activation of key intracellular pathways, resulting in increased proliferation, invasiveness, and resistance to apoptosis in cancer cells [1]. Both receptors are also involved in immune evasion and metabolic reprogramming, making them critical therapeutic targets [23]. Specific inhibitors, including tyrosine kinase inhibitors, monoclonal antibodies, and therapeutic vaccines, have been developed [24]. However, targeting a single receptor can induce resistance mechanisms due to the upregulation of other family members, demanding treatments that inhibit multiple receptors [25]. Understanding the impact of HER1 and HER2 inhibitors on tumor physiology and microenvironments is essential, and preclinical models effectively simulate tumor behavior for research optimization.

In vivo studies examining the antitumor effects of therapies targeting HER1 and/or HER2 receptors require immunocompetent mice, as they better represent tumor–microenvironment dynamics and enhance preclinical result predictions [26]. In this sense, the generation of syngeneic mouse models expressing human proteins is valuable for tumor biology and immunotherapy studies and is cost-effective to develop and maintain [27].

The lentiviral transduction method effectively introduces foreign DNA, allowing multiple copies of the integrated transgene per cell at active transcription sites [28]. However, lentivirus-generated models for heterologous expression face the challenge of random transgene integration, which can lead to variable expression levels and affect transgene stability and functionality [9]. Our results show that these models retained HER1 and/or HER2 expression levels on their membranes after inoculation in mice, indicating efficient integration of HER1 and/or HER2 DNA into the modified cells’ genomes [29]. The repeated cycles of tumor inoculation, disaggregation, and re-inoculation were essential to select and enrich tumor cell populations with stable and homogeneous expression of the heterologous HER1 and/or HER2 receptors [18]. This approach ensured the adaptation of the modified cells to the in vivo microenvironment and maintained receptor functionality, which is critical for the reproducibility and biological relevance of the murine models in therapeutic evaluations. In all the generated models, HER2 expression was higher than HER1, possibly due to its insertion in more active chromatin sites. However, structural differences among these protein receptors may also explain such differential expression levels. Recent studies suggest that post-translational modifications, such as N-glycosylation, can hinder the secretion of recombinant proteins, with HER2 having eight potential N-glycosylation sites and HER1 having 12–14 [30].

The functionality of heterologous receptors in RM1-derived models was confirmed by evaluating their phosphorylation status. EGF stimulation significantly enhanced HER1 phosphorylation, while HER2 activation also increased in HER1-expressing models, suggesting the formation of active heterodimeric complexes. While these observations primarily reflect human-to-human interactions, we acknowledge the potential for hetero-specific dimerization between human and murine receptors. However, the functional dominance of human receptor signaling evidenced by ligand-specific phosphorylation and inhibitor responses, suggests that human homodimers or heterodimers are the primary drivers of the observed effects. Future studies employing cross-species dimerization assays could further elucidate these interactions.

Notably, studies have shown that HER1/HER2 heterodimers exhibit stronger activation compared to homodimers [31]. Additionally, activated HER1 undergoes rapid internalization and lysosomal degradation, leading to reduced expression following EGF stimulation [32]. This may explain the observed decrease in HER1/HER2 levels in RM1 cells treated with EGF. Following ligand binding, homo- and heterodimeric interactions between HER receptors induce autophosphorylation at the intracellular tyrosine kinase domain, creating docking sites for adapter and scaffolding proteins that activate various downstream signaling pathways [33]. Key pathways include RAS-RAF-MEK-MAPK, which regulates gene transcription and cell cycle progression from the G1 to the S phase, and PI3K-Akt, which triggers anti-apoptotic signals [34]. The increased proliferation in models expressing HER1 and/or HER2 suggests that, indeed, inserted receptors can dimerize and transactivate while maintaining the necessary structure for ligand binding. However, it would be beneficial to further confirm that inserted HER1 and HER2 are able to engage cell signaling machinery and trigger previously mentioned cascades.

HER1 and HER2 receptors are central to tumor physiology, and their suppression attenuates tumor growth. In RM1-derived models, HER1 inhibition reversed ERK1/2 phosphorylation, demonstrating HER1 phosphorylation’s functional relevance within the heterologous context of these cell lines. Generated models expressing HER1 and/or HER2, when treated with specific monoclonal antibodies or TKIs, show decreased cell viability, unlike parental lines. This indicates the receptors’ functionality and the sensitivity of expressing lines to targeted therapies. Various modified cell lines, such as EL4-HER2 (lymphoma) [35], CT26-HER1 (colon) [36], and 4T1-HER2 (breast) [37], have facilitated studies on therapies like cetuximab and trastuzumab. However, murine models with both receptors have not been reported, limiting the simultaneous HER1 and HER2 blockade evaluation. Models from different cancer types would help assess the effects of inhibiting one or both receptors in parallel. For instance, previous reports suggest that HER2 expression in lung cancer may lead to HER1 mutations, and co-expression of HER1 and HER2 correlates with poor outcomes in breast carcinoma. Also, several studies describe an important relationship between the hyperactivation of HER1 and HER2 and the metastatic progression of prostate tumors [13].

Although models developed through subcutaneous implantation allow for the evaluation of the efficacy of therapies targeting HER1/HER2 and their impact on tumor growth, they do not accurately replicate the immunological and stromal microenvironment characteristic of the organ of origin for each tumor type (e.g., the lung in 3LL or the mammary gland in 4T1). Furthermore, while no obvious signs of immunological rejection, such as local inflammation or tumor regression, were observed, the potential immunogenicity of the human receptors expressed in immunocompetent mice warrants further investigation into tumor–immune system interactions.

In this context, the initial in vitro characterization was a crucial step in validating the localization, functionality, and specificity of the introduced HER1/HER2 receptors. It also aimed to establish their baseline sensitivity to specific therapies before progressing to more complex in vitro studies. Building on this foundation, future research will focus on developing orthotopic models, evaluating tumor depletion and metastatic dissemination, and analyzing the tumor microenvironment and the immune responses induced by therapies, all within an immunocompetent system. These investigations will enable us to explore the biological relevance and translational potential of the models in greater depth.

5. Conclusions

This study developed murine tumor models with a functional and stable expression of human HER1 and/or HER2 through lentiviral transduction. These models facilitate the evaluation of targeted therapies in an immunocompetent environment. They provide a robust platform for future orthotopic studies and investigations into the tumor microenvironment.