Patient-derived 3D nasal spheroids reveal epithelial changes following Dupilumab therapy in CRSwNP: a preliminary report

ABSTRACT

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a type 2 inflammatory disease associated with epithelial dysfunction and impaired mucosal barrier integrity. Dupilumab, an IL-4 receptor alpha antagonist, has shown clinical efficacy, but its cellular effects on nasal epithelium remain poorly understood. Advanced in vitro models such as 3D spheroid cultures may provide insight into epithelial organization under treatment. We conducted a preliminary study using nasal epithelial cells obtained from three patient groups: CRSwNP treated with Dupilumab for 16 weeks (n = 3), untreated CRSwNP (n = 3), and turbinate hypertrophy controls (n = 3). Cells were isolated by enzymatic digestion and cultured in ultra-low attachment plates using sphere-promoting medium to assess spheroid formation. Observations were performed using phase-contrast microscopy. Due to the limited sample size, data were analyzed qualitatively without statistical testing. Control cells formed compact spheroids, while untreated CRSwNP cells failed to generate structured spheroids, showing only aggregates. Cells from Dupilumab-treated patients produced well-organized spheroids, suggesting improved epithelial organization. Occasional surface movement was observed but not quantitatively assessed. No molecular or ultrastructural assays were performed to confirm mechanistic hypotheses. Our preliminary findings indicate that Dupilumab treatment may be associated with improved epithelial organization in CRSwNP, as shown by spheroid formation in 3D culture. However, these observations are preliminary and based on a small cross-sectional cohort. Future studies should include longitudinal sampling, functional assays, and molecular analyses to confirm mechanisms and validate these results.

GRAPHICAL ABSTRACT

Plain language summary

Conceptual model illustrating a potential relationship between Dupilumab treatment and improved epithelial function in CRSwNP. This schematic represents a hypothesis based on preliminary 3D spheroid observations and should be interpreted as a proposed framework for future mechanistic investigations.

Introduction

Chronic rhinosinusitis with nasal polyps (CRSwNP) is a persistent inflammatory condition affecting the nasal mucosa and paranasal sinuses. It is characterized by long-lasting symptoms, including anterior or posterior rhinorrhea, hyposmia, and facial pressure or pain, which persist for over 12 weeks and significantly impact health-related quality of life (HRQoL).¹

CRSwNP typically manifests in middle-aged individuals, with an average onset around 42 years and a common diagnosis age between 40 and 60 years. The condition is more prevalent in males, although no specific genetic or environmental factors have been definitively linked to its development.²

Nasal polyps usually present as bilateral inflammatory growths originating from the ethmoid sinuses and extending into the nasal airway beneath the middle turbinate. Endoscopic examination reveals these.^3–5

Histological reports often describe nasal polyps as benign growths with an edematous, fibrotic, or myxoid stroma covered by respiratory epithelium, predominantly showing eosinophilic inflammation.⁶ Recent studies have identified mesenchymal stem cells in biopsies from CRSwNP patients, which play a critical role in the inflammatory response and may contribute to recurrence mechanisms.⁷ However, nasal cytology reveals that these polyps also contain various other cell types, affecting their severity and likelihood of recurrence.

CRSwNP is classified as a type 2 (T2) inflammatory disease, characterized by the infiltration of eosinophils, basophils, mast cells, and other inflammatory cells, along with a strong expression of cytokines associated with Th2 effector cells, including interleukins (IL) IL-4, IL-5, and IL-13.⁸ The evaluation of nasal inflammation and comorbidities through a Clinical-Cytological Grading (CCG) system helps determine the severity of CRSwNP and predict the Prognostic Index of Recurrence (PIR).⁶

Most patients with CRSwNP who have type 2 airway inflammation often develop comorbid conditions such as asthma and respiratory diseases exacerbated by nonsteroidal anti-inflammatory drugs (NSAID-ERDs) due to the shared type 2 inflammatory pathway.⁹

The standard treatments for CRSwNP patients include systemic corticosteroids, nasal corticosteroids, antibiotics, and mucolytics. Endoscopic nasal and sinus surgery is frequently used to remove polyps, restore nasal airflow, and manage recurrences, although the risk of postoperative recurrence remains high and varies with disease severity.¹⁰

Recent advances in CRSwNP treatment involve biologics, targeted therapies designed to modulate key mediators of type 2 inflammation.¹¹ Dupilumab, a monoclonal antibody used for severe atopic dermatitis, blocks the alpha chain shared by IL-4 and IL-13 receptors, thereby downregulating the type 2 immune response. This approach has shown promise in treating severe asthma and is relevant for managing CRSwNP due to its effects on various cell types involved in inflammation.^12,13 Recent in vitro evidence also supports Dupilumab’s ability to prevent nasal epithelial dysfunction induced by IL-4, highlighting its protective effects on epithelial integrity in CRSwNP.¹⁴

While the clinical benefits of Dupilumab in CRSwNP are well established through clinical trials, direct mechanistic evidence of its impact on nasal epithelial organization remains limited.^15,16 To address this gap, we employed a patient-derived, 3D spheroid model that better recapitulates the architecture and behavior of the nasal epithelium in vivo. This approach enables a more accurate assessment of epithelial responses following Dupilumab treatment, thus providing new insights into its potential cellular mechanisms.¹⁷

Materials and methods

Sample collection from CRSwNP patients with and without Dupilumab treatment

Nasal polyp cells were collected from six patients diagnosed with Chronic Rhinosinusitis with Nasal Polyps (CRSwNP). Three of these patients (two males, aged 40–65 years) had not received biological therapy, while the other three (three males, aged 40–65 years) were undergoing treatment with Dupilumab for 16 weeks.

Three of these patients (three males, aged 40–65 years) had not received biological therapy, while the other three (three males, aged 40–65 years) were undergoing treatment with Dupilumab for 16 weeks. All patients presented with moderate asthma and a type 2 inflammation pattern.

The Dupilumab-treated group received Dupixent® (300 mg/2 mL solution; 150 mg/mL) via subcutaneous injection every two weeks, using a single-use pre-filled pen. This treatment was administered in combination with topical intranasal corticosteroid therapy – specifically, mometasone furoate nasal spray at a dose of 200 µg/day (two administrations per day).

The study inclusion criteria were: (1) age ≥18 years; (2) a confirmed diagnosis of CRSwNP with severe nasal polyposis; (3) a Nasal Polyposis Endoscopic Score (NPS) ≥ 5 and/or (4) a SNOT-22 score ≥ 50; and (5) prior treatment failure, defined as lack of efficacy or discontinuation of systemic corticosteroids and/or non-response or recurrence following surgery.^8,18

Control cells were obtained from three patients with turbinate hypertrophy, a condition characterized by localized mucosal thickening but without polypoid inflammation or previous exposure to biologic therapy. While this tissue does not represent completely healthy nasal mucosa, it served as an ethically appropriate biologic-naïve baseline for comparison, as truly healthy sinonasal tissue is rarely accessible outside of cadaveric or non-indicated surgical contexts. These patients underwent inferior turbinate reduction surgery as part of their treatment for nasal obstruction.

Importantly, turbinate hypertrophy samples retain relatively intact epithelial architecture and were free of overt polypoid changes, making them an acceptable comparative baseline for early in vitro evaluation.

All tissue samples were collected via endoscopic polypectomy. While the sample size (n = 3 per group) is limited, it was deemed sufficient for this preliminary short communication to establish proof of principle. Sample selection was based on comparable clinical severity to allow for meaningful early-stage comparisons.

Cell isolation and 3D spheroid culture from nasal polyps in CRSwNP patients and controls

Cell isolation was performed by mechanical dissociation using a scalpel blade, followed by enzymatic digestion with Collagenase IV (1 mg/mL) for 16 hours at 37°C. After digestion, the cells were washed with PBS and cultured in ultra-low attachment 6-well plates using Sphere medium, consisting of DMEM F12 (Sigma-Aldrich) supplemented with 10% FBS (Life Technologies, Milan, Italy), B27 (Life Technologies, Carlsbad, CA, USA), heparin, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) (both from Sigma-Aldrich), and 1% Penicillin-Streptomycin (Sigma-Aldrich). The cultures were maintained at 37°C in a humidified atmosphere with 5% CO₂ for 24 hours, after which spheroid production was analyzed using a phase-contrast microscope (20×) (Leica DM IL LED, Leica Microsystems, Milan, Italy).¹⁹ Samples from Dupilumab-treated and untreated CRSwNP patients were obtained from different individuals. While not longitudinal, the study groups were selected based on similar clinical profiles to allow a meaningful preliminary comparison.

Statistics

All results are presented as mean ± standard deviation (SD) from three different experiments. Due to the small sample size (n = 3 per group), no inferential statistical tests were performed. The data are described qualitatively to illustrate observed trends. The small cohort size precludes meaningful assessment of normality or variance assumptions required for parametric or non-parametric tests.

Ethical considerations

This study was conducted in accordance with Good Clinical Practice guidelines and the principles outlined in the Declaration of Helsinki. Ethical approval was obtained from the appropriate ethics committees, including the local ethical committee of the Calabria Region (Catanzaro, Italy; document 182—20 May 2021), prior to study initiation. All participants provided written informed consent before enrollment.

Results

Preliminary investigation of epithelial restoration in CRSwNP: a patient-derived 3D spheroid model following Dupilumab treatment

To evaluate the effects of biological treatment and disease state on nasal polyp cell behavior, we conducted experiments involving cells from three distinct groups: patients with turbinate hypertrophy, untreated nasal polyps, and nasal polyps treated with Dupilumab. The following results highlight the differences in spheroid formation and cellular characteristics across these groups.

Patients with turbinate hypertrophy served as the control in our experimental setup. Cells derived from the turbinate biopsies formed 3D spheroid cultures that were compact and encapsulated. However, the ciliated cells within these spheroids did not exhibit motility, likely due to the hypertrophy and associated inflammation (Figure 1A).

Figure 1.

Effect of biological treatment and disease state on nasal polyp cell behavior.

1. Turbinate Hypertrophy (Control): Cells from turbinate biopsies formed compact, encapsulated 3D spheroids. Despite structural integrity, the ciliated cells lacked motility, likely due to inflammation.
2. Untreated Nasal Polyps: Cells failed to form spheroids. Numerous red blood cells were visible, likely due to tissue trauma during biopsy collection and not indicative of any intrinsic biological phenomenon.
3. Dupilumab-Treated Nasal Polyps: Cells from nasal polyps treated with Dupilumab for 16 weeks formed well-structured, uniform-sized spheroids. (D) These spheroids showed visible peripheral movement under phase-contrast microscopy, suggestive of dynamic behavior. Time-lapse video was only recorded for the Dupilumab-treated group, as spheroids in the control and untreated groups remained static during observation and were therefore adequately represented by still images.
4. Quantitative Analysis of Spheroid Formation: Bar graph shows that control cells formed the most spheroids, reflecting relatively intact epithelial function. Dupilumab treatment appeared to restore spheroid formation, with the number of spheroids approaching control levels. Due to the small sample size, no inferential statistical testing was conducted, and observations are presented descriptively. Scale bar: 100 μm.

In contrast, three-dimensional (3D) cell culture models, such as spheroids, offer improved physiological relevance over 2D cultures by better preserving cellular architecture and cell – cell interactions. These models have previously been used in CRSwNP research, and our study builds on this approach by applying it to cells from Dupilumab-treated patients. This lack of spheroid formation may reflect epithelial dysfunction, possibly associated with barrier damage or inflammatory cytokines such as IL-4 and IL-13; however, no molecular assays were performed in this study to evaluate inflammatory cytokines or barrier integrity, and this interpretation remains speculative (Figure 1B).

For patients with nasal polyps treated with Dupilumab for 16 weeks, cells derived from nasal polyp biopsies were able to consistently generate well-structured spheroids of uniform size. Notably, these 3D spheroids displayed visible surface movement at the spheroid periphery, observed under phase-contrast microscopy. While this activity may suggest dynamic cellular behavior, no direct visualization or quantification of cilia or ciliary beating was performed (Figures 1C and D) video.

The histogram in Figure 1E provides a quantitative comparison of spheroid formation across the three experimental groups. Within each group, cellular behavior was consistent across the three patient-derived samples. In the control group (turbinate hypertrophy), all samples formed compact, non-motile spheroids, indicating relatively intact epithelial function. In the untreated CRSwNP group, none of the samples formed structured spheroids; only unorganized cellular aggregates were observed, suggesting a consistent loss of epithelial integrity. In the Dupilumab-treated group, all three samples formed well-structured, motile spheroids, indicating recovery of epithelial function. No appreciable intragroup variability was noted, although the small sample size limits statistical assessment. Future studies with larger cohorts are needed to explore potential heterogeneity within treatment groups

However, cells from Dupilumab-treated CRSwNP patients displayed a marked improvement in spheroid-forming ability. The number of spheroids formed in this group approached levels observed in the control group, indicating a potential restoration of epithelial functionality, based on qualitative in vitro observations.

All patients in the Dupilumab-treated group experienced clinical improvement, including reduction in nasal obstruction and total IgE, consistent with expected treatment response.^8,18

Discussion

The nasal mucosa, once regarded primarily as a physical barrier, is now understood to play a crucial role in both metabolic activity and immune defense. As the main interface with inhaled antigens, the nasal mucosa produces various inhibitory substances and secretory IgA, which are essential for its protective functions.¹⁷

The integrity of the mucosal barrier in the upper respiratory tract, including the nasal cavity, relies on tight junctions between epithelial cells. This epithelium, composed of pseudostratified ciliated columnar cells and specialized M cells, forms a continuous defense against external antigens. Dendritic cells within the epithelium present antigens to CD4+ T cells and help maintain the epithelial barrier. Tight junctions, made up of integral membrane proteins such as claudins, occludin, and junctional adhesion molecules (JAMs), along with peripheral membrane proteins like scaffold PDZ proteins (e.g., ZO-1, ZO-2, ZO-3) and cell polarity molecules, are vital for sustaining this barrier function.^20–23

In our study, turbinate hypertrophy samples were used as controls. Although turbinate hypertrophy does not represent a fully healthy mucosal state, as it is associated with mild, chronic inflammation, it was selected as an ethically acceptable comparator due to its preserved epithelial architecture.^24,25 Nevertheless, it is important to recognize that differences between the control and CRSwNP groups – such as genetic background, comorbidities, or environmental factors – may contribute to variability and represent potential confounders.

Despite the presence of low-grade inflammation, cells from hypertrophic turbinates retained the ability to form compact, structurally organized spheroids in vitro. While this may explain the limited ciliary motility observed in these controls, it also suggests that key epithelial functions remain largely intact. As such, these samples provide a reasonable reference point for evaluating epithelial dysfunction in CRSwNP and the potential restorative effects of Dupilumab. Future studies may further refine the choice of control tissue by incorporating cadaveric samples or mucosa from non-inflammatory resections to strengthen the comparative analysis.

Type 2 inflammatory mediators such as IL-4 and IL-13 contribute to CRSwNP pathophysiology by disrupting epithelial junctions and promoting tissue remodeling.^26–31 Dupilumab, by blocking IL-4 Rα, interferes with these pathways and reduces inflammatory signaling.^28,29

Our study builds on these advancements by focusing on Chronic Rhinosinusitis with Nasal Polyps (CRSwNP) through the lens of cellular functionality and therapeutic interventions.

By comparing spheroid formation across different patient groups – turbinate hypertrophy controls, untreated CRSwNP, and CRSwNP patients treated with Dupilumab – we gained crucial insights into the condition’s pathology and treatment efficacy. Cells from turbinate hypertrophy formed compact spheroids with limited motility, reflecting the functional limitations imposed by chronic inflammation. In contrast, untreated CRSwNP cells failed to form spheroids. We hypothesize that this impaired spheroid formation is due to disruption of epithelial integrity caused by inflammation, though this mechanism remains speculative, as we did not perform direct assessments such as cytokine quantification or tight junction staining. However, this mechanism was not directly evaluated in our study (e.g., via transepithelial resistance measurements or tight junction protein labeling), and thus remains a working hypothesis requiring further investigation. However, Dupilumab treatment was associated with a qualitative improvement in spheroid formation of nasal polyp cells, resulting in well-structured spheroids that displayed visible movement under phase-contrast microscopy. We did not perform confirmatory analyses such as ciliary beat frequency measurements or electron microscopy, which are necessary to definitively characterize ciliary structure and function. Therefore, references to motility are qualitative and should be interpreted with caution. This is further supported by the quantitative analysis shown in Figure 1D, which demonstrates the differences in spheroid formation among the experimental groups. Cells from the control group (turbinate hypertrophy) formed the highest number of spheroids, reflecting relatively intact epithelial function. Conversely, cells from untreated CRSwNP patients exhibited markedly lower spheroid counts, indicative of severe epithelial dysfunction and disrupted barrier integrity. Notably, cells from Dupilumab-treated CRSwNP patients showed an improvement in spheroid formation, with numbers approaching those seen in the control group. This in vitro improvement was paralleled by favorable clinical outcomes; however, given the preliminary nature of our design, these findings should be interpreted cautiously.^8,18 The beneficial effects of Dupilumab on nasal epithelial integrity observed in our 3D spheroid model are in agreement with recent literature suggesting a potential role for the drug in supporting mucosal recovery, though further mechanistic studies are needed to confirm this effect. Fieux et al. demonstrated that Dupilumab directly prevents IL-4-induced epithelial barrier dysfunction in vitro, further validating its protective role on nasal epithelium. Similarly, Huang et al. showed that Dupilumab enhances tight junction expression and mucosal barrier resilience by counteracting IL-13-mediated disruption.²⁸ These molecular effects complement clinical studies such as DUPIREAL, where patients receiving Dupilumab showed sustained reductions in nasal polyp scores and improvements in quality of life over time. Moreover, recent data from França et al. support the relevance of using 3D spheroid cultures in studying nasal polyps, offering a more physiologically relevant model than traditional 2D cultures.¹⁷ Taken together, these findings reinforce the relevance of our in vitro results and provide a broader context for understanding the mechanisms by which Dupilumab promotes epithelial recovery and disease control in CRSwNP. While these findings suggest a potential role of Dupilumab in improving epithelial cell behavior, it is important to emphasize that the comparison was made between different patient groups, not longitudinally within the same individuals. Therefore, our findings should be interpreted as preliminary, supporting the need for future studies using longitudinal designs and more detailed clinical characterization to validate these observations.

Conclusion

In this study, we explored the effects of Dupilumab on epithelial cells derived from nasal polyps of patients with Chronic Rhinosinusitis with Nasal Polyps (CRSwNP) using a three-dimensional (3D) spheroid culture model. Our findings indicate that Dupilumab can support the restoration of epithelial architecture, which is typically altered in CRSwNP due to chronic inflammation and elevated type 2 cytokine levels. Treatment with Dupilumab was associated with the formation of more compact and organized spheroids, suggesting a beneficial impact on epithelial structure.

By comparing cells from CRSwNP patients – both untreated and treated with Dupilumab – with controls from turbinate hypertrophy, our preliminary observations suggest that Dupilumab may be associated with reduced inflammatory markers and partial improvement in cellular functionality. These exploratory findings warrant further investigation into the potential role of Dupilumab in modulating epithelial responses in CRSwNP, particularly in patients unresponsive to standard therapies or at high risk of recurrence.

The use of a 3D spheroid model offers a physiologically relevant in vitro system that complements previous studies and enhances our understanding of epithelial responses to biologic therapies in CRSwNP.

It is important to note that our study employed a cross-sectional design, comparing distinct cohorts of patients rather than evaluating changes within the same individuals over time. This limits the ability to attribute the observed epithelial improvements directly to Dupilumab. Differences in outcomes may also reflect inter-individual variability, including factors such as genetic background, underlying inflammatory endotypes, comorbid conditions like asthma severity or atopy, and environmental or occupational exposures.

In conclusion, our results provide additional insight into the cellular mechanisms involved in CRSwNP and add to the growing body of evidence supporting the role of biologic therapies targeting type 2 inflammation. Further longitudinal studies with larger patient populations are needed to confirm these findings and guide future therapeutic strategies, including the potential use of combination therapies and assessment of patient-reported outcomes.

Study limitations

While our study provides valuable insights into the effects of Dupilumab on nasal polyp cell behavior using a 3D spheroid culture model, there are several limitations to consider.

First, the study employed a cross-sectional design comparing independent cohorts of Dupilumab-treated and untreated patients at a single time point, without longitudinal follow-up. This limits the ability to infer causality, as differences observed between groups may be influenced by preexisting biological variability, such as differences in genetic background, inflammatory endotype, disease chronicity, comorbid asthma severity, or environmental exposures. Although we attempted to minimize variability by selecting patients with similar clinical profiles, these confounders could still impact the results. A within-subject longitudinal design would be more appropriate in future studies to more accurately isolate treatment effects.

Second, while we observed qualitative differences in spheroid formation across groups, we did not perform confirmatory molecular or ultrastructural assays, such as tight junction protein analysis, transepithelial resistance measurements, or electron microscopy, to support mechanistic interpretations. Our findings therefore remain descriptive and hypothesis-generating.

Another limitation is the lack of in vivo validation of the observed effects. Although 3D spheroid models are more representative of in vivo conditions than traditional 2D cultures, they cannot fully replicate the complexity of the human nasal mucosa and its immune response. Therefore, further studies utilizing animal models or clinical trials are necessary to confirm whether these findings translate to actual therapeutic benefits in CRSwNP patients.

Lastly, the spheroid culture model does not account for the full range of interactions that occur in the human sinus environment, such as the influence of the microbiome, immune cell infiltration, and the presence of other comorbid conditions. The role of these factors in influencing the response to Dupilumab should be explored in future studies to better understand the broader therapeutic potential of biologics in CRSwNP.

In conclusion, while our findings provide promising evidence for Dupilumab’s potential in CRSwNP treatment, further research with larger, more diverse patient populations, longer treatment durations, and in vivo validation is required to fully understand its therapeutic mechanisms and optimize treatment strategies.

Funding Statement

The author(s) reported there is no funding associated with the work featured in this article.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Author contributions

Conceptualization, G.L.P., N.L. and E.C.; methodology, N.L. and E.C; formal analysis, N.L. (Nadia Lobello); investigation, E.C.; resources, G.L.P.; data curation, N.L. and E.C.; writing – original draft preparation, E.C.; writing – review and editing, E.C., N.L. (Nadia Lobello), G.L.P. and P.M.; visualization, P.M.; supervision, N.L. (Nicola Lombardo); funding acquisition, N.L. and E.C. All authors have read and agreed to the published version of the manuscript.

Ethical approval

Ethical approval was obtained from the Ethics Committee of the Calabria Region (document 182—20 May 2021).

Informed consent

Written informed consent was obtained from all participants

Supplementary Information

Supplemental data for this article can be accessed online at https://doi.org/10.1080/21688370.2025.2552004