Lung cancer stands as a leading contributor to global mortality rates and occupies a prominent position in terms of cancer occurrence and fatality in China, of which approximately 85% of patients are diagnosed with non-small cell lung cancer (NSCLC) (1–3). Among NSCLC subtypes, lung adenocarcinoma emerges as the prevailing form accounting for approximately 40% of cases, and is associated with an unfavorable prognosis (4, 5). Despite the advent of novel treatments like immunotherapy, which have demonstrated favorable outcomes in select individuals, the present median overall survival (OS) for patients with metastatic NSCLC remains below three years due to the insufficient tumor immune infiltration and suboptimal immune response (4, 6, 7). Among the signaling pathways associated with lung cancer, the excessive activation of the Wnt/β-catenin pathway has garnered significant interest. This aberrant activation leads to the up-regulation of proteins related to the cell cycle, promotion of tumor cell proliferation, enhancement of intratumoral vascular density and maintenance of tumor stem cell stemness, thereby greatly contributing to the processes of tumorigenesis, progression, and metastasis (8–11). There is a growing amount of evidence indicating that the aberrantly hyperactive Wnt/β-catenin signaling significantly impedes various steps of the tumor immune response, particularly the activation and infiltration of T cells, and hence plays a pivotal role in facilitating the immune evasion of tumor cells (12–16). Consequently, targeting the inhibition of Wnt/β-catenin signaling holds promise for yielding diverse anti-tumor effects and possesses substantial therapeutic prospects.

Encouraging progress has been made in utilizing Wnt/β-catenin pathway inhibition as a potential therapeutic approach against tumors: inhibition targets encompass both upstream components and downstream target genes; various types of agents, including small molecules, peptides, and antibodies, have been investigated; and numerous studies have been conducted, ranging from preclinical animal models to clinical trials (17–22). Nevertheless, the development of Wnt inhibitors continues to pose considerable challenges. Primarily, the selection of an appropriate target for inhibition necessitates careful consideration. The Wnt/β-catenin pathway is intricately interconnected, with a diverse array of downstream target genes and complicated crosstalk between its various components and other pathways (23–25). It means that inhibiting upstream components may result in the disruption or deregulation of other normal pathways, while solely targeting downstream ones may prove inadequate in demonstrating anti-tumor effects at the cellular or tissue level. Furthermore, the Wnt/β-catenin pathway plays a crucial role in various cellular physiological processes both in normal and tumor cells, which necessitating a concentrated effort on addressing the toxicity and adverse effects associated with inadequate tumor targeting of inhibitors (26, 27). This is also the reason why numerous candidate inhibitor molecules failed to translate their promising in vitro results to in vivo efficacy. As a result of these significant constraints, there are currently no approved drugs specifically targeting the Wnt/β-catenin pathway for safe clinical utilization.

In order to tackle the aforementioned concerns, here, we developed a tumor-specific biomimetic Wnt pathway suppressor with nano-size and a convenient preparation process, capable of targeting the Wnt/β-catenin pathway in lung adenocarcinoma cells. Our choice of β-catenin as the function target is based on its pivotal role within the signaling cascade, ensuring specific and potent suppression of the canonical Wnt pathway (28, 29). β-catenin undergoes translocation from the cytoplasm to the nucleus to initiate transcription of downstream target genes, of which the transcription activation is facilitated by the BCL9 protein through direct binding with β-catenin (30). The application of carnosic acid (CA), a promising molecule derived from natural extracts capable of directly disrupting the β-catenin/BCL9 interaction, is hindered by its limited in vivo utilization and the presence of additional toxic side effects (31, 32). To improve tumor targeting specificity and biocompatibility, cell membranes from Lewis lung carcinoma (LLC) cells were employed to encapsulate CA molecules, resulting in the formation of nano-sized biomimetic particles (named CM-CA). CM-CA is designed not only to benefit from the passive targeting advantages achieved through the enhanced permeation and retention (EPR) effect, but also to possess the ability of recognition and acceptance by syngeneic tumor cells as well as to evade immune clearance, thereby achieving tumor-specific Wnt/β-catenin pathway suppression. The results of animal experiments indicate that CM-CA possesses a greater in vivo inhibitory effect on β-catenin compared to CA. Additionally, CM-CA promotes tumor immune infiltration and enhances the immune response against tumors, resulting in a more pronounced suppression of tumor growth. ( Figure 1 ) Our work offers a novel candidate inhibitor for targeting the Wnt/β-catenin pathway in lung cancer, and presents an approach to developing Wnt inhibitors that effectively balance safety and efficacy.

Non-small cell lung cancer accounts for up to 85% of lung cancers, and its predominant subtype is lung adenocarcinoma, which constitutes approximately 40% of all the cases (4, 5). Extensive research conducted over the past three decades has emphasized the significant involvement of Wnt/β-catenin signaling in lung cancer development, leading to numerous investigations exploring the potential of Wnt inhibition as an anti-tumor strategy (23). Currently developed Wnt inhibitors, including small molecules, peptides, and antibodies, face challenges associated with the implementation: small molecules exhibit a relatively high level of toxicity; peptides are prone to degradation and present difficulties in direct cellular utilization; antibodies pose complexities in production and their in vivo retention time remains unsatisfactory (47–50). To reconcile the intended acting mechanism of inhibitors with their practical application in vivo, researchers employ synthetic constituents like polyethylene glycol (PEG) to encapsulate inhibitors, thereby rendering them inconspicuous to the immune system and enabling their targeted recognition and utilization by tumor cells (51). Nevertheless, these synthetic components yield only modest extension of blood circulation, while the packaging procedure is intricate and beset with concerns pertaining to adverse immune reactions and toxicity (52, 53). As for target selection of Wnt signaling suppression, β-catenin presents as a promising candidate in lung cancer treatment. The applicability of specific targeted drugs is restricted due to multiple oncogenic mutations present frequently in NSCLC patients, and the ones without target driven gene mutations cannot benefit from it. Conversely, β-catenin is expressed in over half of NSCLCs and possesses rare variations, making inhibitors that target intracellular β-catenin advantageous in terms of both generalizability and efficacy (7, 54, 55). The interaction between β-catenin and BCL9 plays a crucial role in β-catenin-mediated transcription of downstream target genes in the nucleus, serving as a pivotal step in the activation of the Wnt pathway. Additionally, this binding exhibits a relatively substantial binding area and a moderate binding capacity, making it inherently advantageous for the development of inhibitors (47). CA, a small molecule derived from natural substances, was discovered to exhibit binding affinity towards the H1 helix of β-catenin in close proximity to BCL9 binding site, consequently disrupting the stability of β-catenin and facilitating its oligomerization and subsequent degradation (31, 56). This compound emerged as a highly promising therapeutic candidate for impeding the interaction between β-catenin and BCL9. Nevertheless, its advancement into clinical studies has been impeded by unresolved concerns regarding systemic toxicity (32, 57). CM-CA was deliberately designed with the aforementioned considerations in order to effectively and safely suppress the central component of Wnt pathway. ( Figure 1 ) Additionally, it accomplishes tumor targeting with biosafety by encapsulating homologous tumor cell membranes. The presence of distinct antigens on the outer layer of cancer cells plays a significant role in facilitating the adherence of homotypic cancer cells to tumor tissues. The encapsulation of homotypic cancer cell membranes enables the effective enrichment of camouflaged nanoparticles at tumor sites, thereby enabling active targeted drug delivery (53, 58). This design features clear components and a straightforward synthesis process, circumventing toxicity concerns associated with superfluous modifications, as well as preventing a substantial increase in synthesis costs. Examined by LLC Lewis homograft model of LUAD, it was observed that CM-CA, possessed nearly twice the tumor growth inhibition value of CA, exhibited a greater capacity to suppress tumor growth compared to CA when administered at equal doses. ( Figure 3 ) Analyses of immunohistochemical images and RNA sequencing revealed that CM-CA not only effectively targeted β-catenin to suppress Wnt/β-catenin signaling pathway, but also impede tumor cell proliferation and enhanced tumor immune infiltration, bolstering the immune response against tumors. ( Figures 3 , 4 ) This implies that the biomimetic design strategy of Wnt suppressor, which appears to be straightforward yet efficient, is viable for in vivo implementation. In this particular context, it is important to highlight that there is a need for further reduction in the complexity of cell membrane encapsulation process, as well as an increase in the percentage of intact membrane-encapsulated particles, which is crucial for the successful identification and utilization of homologous tumors (53, 59).

Another potential application of CM-CA is to aid in addressing resistance to primary therapies for lung cancer. Among the current first-line treatments, chemotherapy and radiotherapy are widely used, and the activation of Wnt/β-catenin is associated with their resistance (27, 60). While molecularly targeted therapies that inhibit cancer driver genes have shown efficacy in patients with specific mutation types, they are often hindered by inevitable recurrence (61). Immunotherapy has emerged as a valuable treatment for patients diagnosed with driver-negative NSCLC, yet is plagued by poor response rates due to inferior immune infiltration and immunosuppressive microenvironment (62). Clinical data analysis has revealed a correlation between elevated β-catenin activity and inadequate immune infiltration in LUAD, leading to a less favorable prognosis. ( Figure 2 ) This further supports the understanding that the aberrantly activated Wnt/β-catenin pathway is an important contributor to immunosuppression (42, 63). Promisingly, researches have been undertaken to incorporate Wnt/β-catenin inhibition into the established treating approaches, investigating the potential for combination therapy (22, 32, 64, 65). Actually, in an attempt to tackle the challenging immune milieu encountered in LUAD therapy, we endeavored to devise a universally applicable approach by amalgamating the β-catenin disruptor CA with the lewis lung cancer cell membrane to accomplish active targeting by means of recognizing and adhering to tumor cell membranes of the identical cancerous cells, thereby generating bionic particle CM-CA. In this work, CM-CA not only achieves the goal of disrupting intracellular β-catenin/BCL9 interaction in tumor cells, addressing the endogenous cause of insufficient immune infiltration, but also may function as a reservoir of tumor-associated antigens owing to its tumor cell mimicking properties, eliciting a heightened anti-tumor immune response (66). Our data indicate that compared to control or CA treatment, CM-CA treatment resulted in a higher abundance of immune cell infiltration and up-regulation of immune response-related pathways in tumor tissues. ( Figure 4 ) This suggests that CM-CA has the potential to be used in conjunction with immunotherapy and other molecular targeted therapies. In recent times, there has been a surge in research focusing on the crucial regulatory function of the Wnt/β-catenin pathway in determining the fate and behavior of T cells (67–69). Consequently, it may be recommended that tumor-targeted Wnt inhibitors, such as CM-CA, be incorporated into conventional treatment protocols to manipulate the immune milieu and augment therapeutic effectiveness. However, further preclinical experiments are required to substantiate this proposition. In conclusion, this tumor-specific biomimetic Wnt pathway suppressor not only enhances the tumor immune response while ensuring biosafety, but also offers a promising strategy for prolonging the efficacy of other mainstream therapeutics.

Clinical data and mRNA expression matrix of LUAD samples analyzed in this work are obtained from TCGA database (https://portal.gdc.cancer.gov/) and the Gene Expression Omnibus (GEO, https://www.ncbi.nlm.nih.gov/geo/) database. The RNA sequencing datasets in this study are available in online repository: PRJNA1000099 [NCBI Sequence Read Archive]. All the methods and materials can be found in the Supplementary Material.

The animal study was approved by Medical Ethics Committee of Xi’an Jiaotong University. The study was conducted in accordance with the local legislation and institutional requirements.

RG: Data curation, Formal Analysis, Investigation, Writing – original draft. XZ: Conceptualization, Investigation, Software, Writing – original draft. AJ: Investigation, Methodology, Validation, Writing – original draft. WH: Conceptualization, Project administration, Supervision, Writing – review & editing. TL: Conceptualization, Funding acquisition, Resources, Supervision, Validation, Visualization, Writing – original draft.