{"paper_id":"2b3edd28-ef09-4642-a599-ba2fa2670a7e","body_text":"CD155/PVR as a determinant of anti-HER2 therapy sensitivity in breast cancer | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article CD155/PVR as a determinant of anti-HER2 therapy sensitivity in breast cancer Murugan Kalimutho, Haresh Shankar, Wei Shi, George Ambalathingal, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7994408/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract CD155 (poliovirus receptor, PVR) is frequently overexpressed across cancers and has been associated with tumor progression, poor prognosis, and therapy resistance. Here, we identify CD155 as a modulator of HER2-targeted monoclonal antibody efficacy. Analysis of clinical datasets revealed that high CD155 expression correlates with overall, progression-free, and disease-specific survival in breast cancer, and has potential as a predictive biomarker for anti-HER2 therapy. Mechanistically, CD155 co-localizes with HER2 at the tumor cell membrane and modulates HER2-dependent signaling, including AKT phosphorylation and receptor clustering. Genetic or antibody-mediated loss of CD155 significantly impaired trastuzumab-mediated cytotoxicity in vitro and abolished therapeutic efficacy in both primary and metastatic HER2-positive breast cancer models in vivo , without altering HER2 expression. Moreover, CD155 depletion reduced CD8⁺ T cell infiltration, highlighting its dual role in modulating HER2 receptor biology and shaping the tumor immune microenvironment. Collectively, these findings position CD155 as a determinant of trastuzumab efficacy and a promising biomarker to guide patient stratification and optimize therapeutic outcomes in HER2-positive breast cancer. Biological sciences/Cancer/Breast cancer Biological sciences/Cell biology Biological sciences/Cancer/Cancer therapy/Drug development HER2 CD155 Trastuzumab Breast Cancer Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction HER2 (Human Epidermal Growth Factor Receptor 2, also known as ErbB2) is a member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases. While HER2 lacks a known ligand, it forms stable heterodimers with other ligand-activated EGFR family members, amplifying downstream kinase-driven signaling pathways( 1 ). HER2 is overexpressed in multiple malignancies, particularly in 20–30% of invasive breast carcinomas, classified as HER2-positive breast cancers( 2 , 3 ). Trastuzumab (Herceptin), a monoclonal antibody (mAb) targeting HER2, is a cornerstone therapy for HER2-positive breast cancers. Beyond its ability to directly inhibit oncogenic HER2 signaling, the therapeutic efficacy of trastuzumab relies on engagement of the immune system. Previous studies, including our own, have demonstrated that trastuzumab-like therapies depend on both innate and adaptive immune components, such as CD11b⁺ and F4/80⁺ myeloid cells, NK cells, CD4⁺ and CD8⁺ T lymphocytes, and cytokines including type I and II interferons (IFNs) and IL-21, in HER2-positive mouse models of breast cancer( 4 – 9 ). Additionally, inhibition of immunosuppressive pathways such as CD73-adenosine and PD-1/PD-L1 has been shown to enhance trastuzumab-mediated antitumor activity( 10 ). However, despite significant survival benefits, both acquired and intrinsic resistance to trastuzumab remains a major clinical hurdle in the treatment of HER2-positive metastatic breast cancer( 11 – 14 ). Thus, elucidating both tumor-intrinsic and -extrinsic mechanisms of trastuzumab action is critical to improve patient outcomes. CD155 (also known as the poliovirus receptor, PVR, or NECL5) has emerged as a dual-function molecule with immunomodulatory and tumor-intrinsic roles. It interacts with a family of receptors including DNAM-1 (CD226), TIGIT, and CD96, which collectively modulate anti-tumor immune responses( 15 , 16 ). DNAM-1 serves as a co-stimulatory receptor, while TIGIT and CD96 function as immune checkpoints. CD155 is primarily expressed by tumor cells, endothelial cells, and myeloid cells, and it competes with CD112 (Nectin-2, PVRL2), another ligand for DNAM-1 and TIGIT, with a ubiquitous expression on normal epithelial cells, fibroblasts, neuron cells as well as on tumor and antigen-presenting cells( 17 ). The balance of activating (DNAM-1) versus inhibitory (TIGIT/CD96) signaling is influenced by ligand affinity and receptor expression on immune cells, enabling CD155 and CD112 to exert both immune-activating and immune-suppressive effects( 18 ). Beyond immune regulation, CD155 also exerts tumor cell-intrinsic functions that contribute to malignancy( 16 ). Overexpression of CD155 is associated with reduced tumor-infiltrating lymphocytes (TILs), higher relapse rates, and poorer survival across multiple cancer types, including breast cancer( 19 – 22 ). In breast tumors, CD155 correlates with increased PD-L1 expression on immune cells and a higher proportion of exhausted PD-1⁺CD4⁺ TILs, supporting its role in immunosuppression( 23 ). Experimental deletion of tumor cell CD155 has been shown to decrease cancer cell proliferation in vitro , reduce tumor growth and metastasis in several mouse models( 22 ). Moreover, CD155 and PD-L1 display distinct expression patterns and prognostic implications, suggesting they act through non-redundant immunosuppressive mechanisms, particularly in high-grade serous ovarian cancer( 24 ). In triple-negative breast cancer (TNBC), CD155 knockdown induced a mesenchymal-to-epithelial transition in tumor cells( 25 ), and treatment with adriamycin (doxorubicin) upregulated CD155 expression, with combined CD155 knockdown and chemotherapy enhancing apoptosis( 26 ). TIGIT, inhibitory receptor, is preferentially expressed on NK cells and blockade of TIGIT and/or CD112R was shown to enhance trastuzumab-triggered tumor cell killing by NK cells( 27 ). Additionally, higher CpG promoter methylation was associated with HER2 receptor expression( 28 ). These findings support the therapeutic targeting of CD155 in combination with standard treatments for invasive breast cancers. In fact, a phase I clinical trial targeting CD155 as a monotherapy and in combination with pembrolizumab in advanced solid cancer patients is already in progress (NCT05378425)( 29 ). Given the complex role of CD155 and its receptors in tumor progression and its association with resistance to chemotherapy and immune checkpoint therapies( 30 , 31 ), we sought to investigate its role in trastuzumab response in HER2-positive breast cancers. Interestingly, we demonstrate that the loss of tumor cell CD155, through CRISPR/Cas9-mediated knockout or antibody blockade, markedly impaired the efficacy of trastuzumab-like anti-HER2 therapy in both primary and metastatic models without affecting tumor cell HER2 expression. This effect was mediated by a direct CD155-HER2 interaction at the tumor cell membrane. Importantly, these findings caution against indiscriminate CD155 blockade in HER2-positive breast cancer and highlight tumor CD155 expression as a potential predictive biomarker for trastuzumab response. Results Prognostic significance of CD155 expression in breast cancer patients CD155 has garnered considerable attention in oncology for its dual role in tumour progression and immune regulation( 15 ). Previous studies have shown that CD155 overexpression promotes breast cancer growth; however, its broader clinical relevance remains incompletely understood. To assess the prognostic significance of CD155, we analyzed The Cancer Genome Atlas (TCGA) breast cancer datasets( 32 ). High CD155 expression was significantly associated with poorer outcomes across multiple survival endpoints, including overall survival (OS), progression-free survival (PFS), disease-free survival (DFS), and disease-specific survival (DSS) (Fig. 1 A). Given CD155’s function as an immune checkpoint regulator capable of modulating antitumor immunity through both inhibitory and stimulatory pathways( 33 ), we next investigated its relationship with clinical outcomes in patients treated with immune checkpoint inhibitors (ICIs) targeting PD-1 and CTLA-4 across various cancer types. Consistent with the proposed role of CD155 as a marker of therapy resistance, low CD155 expression correlated with improved OS and progression free survival in patients receiving anti-PD-1 or anti-CTLA-4 therapy (Fig. 1 B). To evaluate whether CD155 could also predict treatment response, we analyzed transcriptomic profiles from ICI-treated patients classified as responders or non-responders. Overall, non-responders exhibited higher CD155 expression compared to responders, a pattern that persisted across different checkpoint inhibitor classes ( Fig. S1A and data not shown ). We then examined the predictive capacity of CD155 as a marker of response to clinically approved chemotherapeutic agents in breast cancer, including taxanes, anthracyclines, ixabepilone, CMF (Cyclophosphamide o + Methotrexate + Fluorouracil (5-FU)), FAC (Fluorouracil (5-FU) + Adriamycin (doxorubicin) + Cyclophosphamide), and FEC (Fluorouracil (5-FU) + Epirubicin + Cyclophosphamide) regimens. No statistically significant differences in CD155 expression were observed between responders and non-responders to these agents ( Fig. S1B ). Importantly, CD155 displayed good correlation between mRNA and protein level in breast cancers ( Fig. S1C ) and ErbB2-hi CD155-hi cancers exhibited better survival compared to ErbB2-hi CD155-low cancers ( Fig. S1D ). In HER2-positive patients, CD155 expression emerged as a strong predictive biomarker for anti-HER2 therapy (Fig. 1 C). Specifically, higher CD155 levels were significantly associated with better pathological complete response to both trastuzumab and lapatinib (Fig. 1 C). Notably, similar patterns were also observed when evaluating relapse-free survival at 5 years (Fig. 1 D), reinforcing the association between elevated CD155 expression and favourable outcomes with anti-HER2 therapy. Collectively, these findings suggest that CD155 serves as a negative prognostic factor in breast cancer overall, yet its predictive value varies substantially across therapeutic contexts. While low CD155 expression favours response to PD-1 and CTLA-4 blockade, high expression may predict improved benefit from anti-HER2 therapies. CD155 knockdown reduces response to anti-HER2 therapy via regulation of AKT signalling Given that CD155 expression predicted response to anti-HER2 therapy, we sought to investigate the underlying mechanisms by which CD155 influences this sensitivity. First, we determined CD155 transcript levels across a panel of breast cancer cell lines. CD155 expression was significantly higher in TNBC compared to luminal subtypes ( Fig. S2A ). HER2-positive cell lines displayed intermediate CD155 expression relative to hormone receptor-positive lines, with SKBR3 cells exhibiting the highest CD155 levels among HER2-positive lines ( Fig. S2B). Surface flow cytometry analysis further revealed that CD155 was expressed in all three HER2-positive breast cancer cell lines tested (Fig. 2 A). Among them, SKBR3 cells showed the highest expression levels of both CD155 and HER2. Moreover, a significant positive correlation was observed between CD155 and HER2 expression levels (Fig. 2 B). As CD155 is a transmembrane protein frequently associated with intracellular signaling cascades, we hypothesized that it may physically interact with the HER2 receptor at the plasma membrane. Immunofluorescence analysis confirmed colocalization of CD155 and HER2 in both low and high CD155 expressing HER2-positive cell lines (Fig. 2 C). This membrane-level colocalization was further validated using a Bimolecular Fluorescent Complementation (BiFC) Assay, which only yields a signal when direct protein-protein interaction occurs. Using BiFC, we demonstrated that CD155 directly interacts with HER2, with the BiFC signal localized at the plasma membrane, supporting that this interaction occurs at the cell surface (Fig. 2 D). We next examined whether this interaction was exclusive to HER2 or extended to other receptor tyrosine kinases. CD155 was found to interact with all members of the HER family (EGFR, ERBB3, ERBB4), as well as IGFR and PDGFR ( Fig. S3B ). No interaction was detected with c-MET, suggesting that CD155 may function as a chaperone-like molecule for a specific subset of membrane receptors. Notably, the interaction between CD155 and HER2 persisted even after treatment with both lapatinib and trastuzumab (Fig. 2 D, Fig. S3A ), indicating that it is independent of receptor blockade. To investigate the regulatory role of CD155 in HER2 signaling, we first performed transient knockdown of CD155 in SKBR3 cells using siRNA. Acute CD155 depletion led to a reduction in both phosphorylated HER2 and total HER2 protein levels ( Fig. S4A ). Notably, when control and CD155-knockdown cells were treated with anti-HER2 agents (lapatinib or trastuzumab), HER2 levels were further reduced in control cells but remained unchanged in CD155-knockdown cells ( Fig. S4A ). To validate these findings, we generated stable CD155-knockdown SKBR3 polyclonal lines using shRNA. Two independent polyclonal populations showed effective CD155 depletion ( Fig. S4B ). However, unlike the acute siRNA knockdown, stable CD155 knockdown did not alter total HER2 protein levels (Fig. 2 E), suggesting that the reduction observed in transient knockdown was likely due to acute CD155 loss, whereas in long-term knockdown lines, compensatory signaling may mitigate HER2 downregulation. Consistent with this, flow cytometry analysis revealed no change in cell-surface HER2 expression in the stable knockdown cells (Fig. 2 F). Interestingly, CD155 knockdown in these stable lines did not result in a reduction in phosphorylated HER2 levels under basal conditions (Fig. 2 G). To assess whether CD155 influences HER2-related signaling in the context of anti-HER2 therapy, we treated control and knockdown cells with lapatinib or trastuzumab and probed key EGFR/HER2 pathway components (Fig. 2 H). Total EGFR levels were slightly reduced in knockdown cells, but the most notable effect was a clear decrease in pAKT and pERK1/2 (Fig. 2 H). Given that HER2 is an orphan receptor requiring heterodimerization with other HER family members for activation, we serum starved and stimulated cells with EGF to assess downstream signalling upon ligand stimulation (Fig. 2 I). While phosphorylation of ERK1/2 and AKT after EGF stimulation was not substantially altered, HER2 activation, marked by pHER2 itself was partially compromised in CD155-depleted cells (Fig. 2 I). Finally, to determine the functional relevance of these signaling changes, we measured trastuzumab sensitivity in CD155 parental and -knockdown cells. Trastuzumab treatment significantly reduced clonogenic growth in control cells; however, CD155-knockdown cells were still able to form colonies, suggesting that CD155 is required for optimal anti-HER2 sensitivity (Fig. 2 J) Together, these results indicate that CD155 is required to maintain active HER2-AKT signalling in HER2-positive breast cancer cells. Loss of CD155 dampens the pathway, leading cells to bypass HER2 dependency and resist trastuzumab. CD155 is required for optimal T cell-mediated killing Because loss of CD155 impaired HER2 signaling, we next examined whether this effect might be related to alterations in HER2 receptor regulation at the plasma membrane. Receptor clustering and internalization are well-established mechanisms that modulate receptor signaling output( 34 ). In parental SKBR3 cells, trastuzumab treatment led to a reduction in surface HER2 levels within 2 hours (Fig. 3 A). In contrast, CD155 surface levels remained unchanged, suggesting that CD155 does not undergo the same pattern of antibody-induced internalization as HER2. Consistent with this observation, flow cytometry analysis showed that treatment with either trastuzumab or lapatinib reduced HER2 expression to a similar extent in control and CD155-knockdown cells (Fig. 3 B). When CD155-knockdown and control cells were treated with trastuzumab (10 µg/mL) for 24 hours, most control cells exhibited extensive HER2 internalization, whereas CD155-depleted cells retained higher levels of HER2 on the plasma membrane (Fig. 3 C). Because the antibody treatment normally promotes receptor clustering, we next examined HER2 spatial distribution following 30 minutes of trastuzumab exposure. In control cells, trastuzumab induced prominent HER2 clustering at the membrane and subsequent internalization (Fig. 3 D). In contrast, CD155-knockdown cells displayed a pre-existing clustered pattern that was not further enhanced by trastuzumab (Fig. 3 D), suggesting that CD155 facilitates proper HER2 receptor organization and dynamic redistribution in response to antibody binding. To assess the functional impact of CD155 loss on HER2-mediated immune responses, we performed a HER2-specific TCR-based T cell killing assay, which relies on efficient presentation of HER2-derived peptides on the plasma membrane. CD155-knockdown SKBR3 cells were significantly less susceptible to TCR-mediated cytolysis compared with control cells (Fig. 3 E), indicating impaired T cell killing efficiency. A similar trend was observed in antibody-dependent cytotoxicity assays, in which naïve peripheral blood mononuclear cells (PBMCs) from healthy donors were co-cultured with tumor cells in the presence of trastuzumab. In this context, CD155-depleted cells showed reduced susceptibility to immune-mediated lysis, with a noticeable delay in killing kinetics relative to controls (Fig. 3 F). Collectively, these results demonstrate that CD155 supports proper HER2 receptor localization and dynamics at the plasma membrane, enhancing the effectiveness of both TCR-mediated and antibody-dependent cytotoxicity, and highlighting its role in modulating tumor cell sensitivity to immune-based therapies. CD155 is required for the therapeutic effect of anti-HER2 mAb in a murine breast cancer model Having established that CD155 regulates HER2 receptor localization, signaling, and T cell-mediated killing in human breast cancer cells, we next investigated whether these effects extend in vivo . To address this, we employed syngeneic murine models, which provide an intact immune system necessary to evaluate both tumor growth dynamics and immune-based therapeutic responses to anti-HER2 monoclonal antibody (mAb) therapy. Both H2N100 and TUBO tumor cells expressed comparable high levels of HER2 and CD155, as confirmed by flow cytometry (Fig. 4 A). Consistent with our observations in the human cell line SKBR3, CD155 expression was not significantly altered after treatment with anti-HER2 mAb therapy in murine tumour cell lines ( Fig. S5A ). Next, to evaluate the role of CD155 in anti-HER2 mAb therapy, we treated both anti-HER2 sensitive H2N100 and relatively resistant TUBO tumors( 8 ) with anti-HER2 mAb in the presence or absence of a CD155-blocking mAb. Anti-HER2 mAb therapy alone significantly inhibited primary tumor growth in both H2N100 (Fig. 4 B) and TUBO (Fig. 4 C) models. However, co-administration of the CD155-blocking mAb abolished the anti-tumor efficacy of anti-HER2 mAb therapy in both tumour types (Fig. 4 B, C). Similarly, CD155 blockade markedly reduced the therapeutic benefit of anti-HER2 mAb treatment in the H2N100 experimental metastasis model (Fig. 4 D). Anti-CD155 treatment alone has modest decrease in primary tumor growth and metastasis in line with its role in promoting tumor growth( 15 ). In agreement with previous reports( 35 ), tumor-infiltrating lymphocyte (TIL) analysis of H2N100 tumours revealed that anti-HER2 mAb therapy increased the frequency of intratumoral T cells, particularly CD8⁺ T cells in H2N100 tumours. Notably, the frequency of CD8 + T cells was significantly reduced when anti-HER2 mAb was combined with CD155 blockade (Fig. 4 E, F). Given that CD155 ligand TIGIT is predominantly expressed on NK cells and intratumoral regulatory T cells (Tregs), and has been implicated in tumour immune evasion( 36 , 37 ), we also analyzed TIGIT + Tregs populations in tumours populations across different treatment groups. However, no significant differences were observed in TIGIT + Tregs frequencies (Fig. 4 F). Collectively, these results suggest that CD155 is essential for the anti-tumour activity of anti-HER2 therapy in vivo , likely through supporting CD8⁺ T cell-mediated immune responses. Loss or blockade of CD155 function compromises both local tumour control and metastatic suppression by impairing the immune component of anti-HER2 therapeutic efficacy. Tumour cell expression of CD155 is essential for the anti-tumour efficacy of anti-HER2 mAb therapy CD155 expression is not only restricted to tumour cells but is also detected on myeloid and endothelial cells [20]. Previous studies have shown that tumor cell derived CD155 plays a particularly important role in regulating anti-tumour immune responses [24, 27, 28]. To directly evaluate the contribution of tumour cell-intrinsic CD155 to the therapeutic efficacy of anti-HER2 mAb, we used the CRISPR-Cas9 system ( 38 ) to delete CD155 in H2N100 and TUBO tumour cells. Two independent single-guide RNAs (sgRNAs; sg2 and sg6) targeting CD155 were designed ( Table S1 ), and CD155 deletion was confirmed by immunoblotting (Fig. 5 A. C ). Importantly, CD155 loss did not alter HER2 protein expression ( Fig. S5B ). In vivo , anti-HER2 mAb treatment significantly inhibited the growth of CD155-expressing (sg control) H2N100 and TUBO tumours but failed to reduce tumour growth in mice bearing CD155-deficient tumours (sg2 and sg6) (Fig. 5 B, D). Similarly, anti-HER2 mAb markedly reduced both the number of lung metastases and lung metastatic burden (lung weight) in mice harboring CD155-positive TUBO tumours, whereas no anti-metastatic effect was observed in those bearing CD155-deficient tumors (Fig. 5 E, F). We next examined whether tumor cell CD155 is required for the efficacy of other immune checkpoint-based therapies. CD155 knockout and parental tumor cells exhibited comparable levels of PD-L1 expression and there was no correlation between CD155 and PD-L1 expression in breast cancer samples ( Fig. S6A, B ). Consistent with previous findings demonstrating that anti-PD-L1 monoclonal antibody (mAb) suppresses H2N100 tumour growth ( 39 , 40 ), we observed that anti-PD-L1 treatment reduced primary tumor growth regardless of tumor cell CD155 expression levels (Fig. 5 G). Given that the successful anti-HER2 therapy relies on both tumour-intrinsic factors and host immune effector mechanisms [5, 9], we further assessed therapeutic efficacy in the absence of an intact immune system. H2N100 sg control and CD155-deficient (sg2) tumour cells were implanted into immunocompetent wild-type (WT) or immunodeficient NOD-SCID-γ (NSG) mice, which lack mature B cells, T cells, and NK cells. As expected, the number of lung metastases in NSG mice was significantly higher than the WT mice in the absence of immune control in NSG mice. In WT mice bearing CD155-positive tumors, anti-HER2 mAb reduced lung metastases by nearly 80%, whereas the reduction in NSG mice was limited to ~ 30% (Fig. 5 H, I), consistent with the loss of antibody-dependent cellular cytotoxicity (ADCC) in the absence of immune effector cells in NSG mice. In contrast, anti-HER2 therapy failed to reduce metastases in either WT or NSG mice when tumours lacked CD155 expression (Fig. 5 H, I). Collectively, these data demonstrate that tumour cell CD155 is indispensable for the full therapeutic efficacy of anti-HER2 mAb, acting cooperatively with an intact host immune system to mediate both primary tumor suppression and metastatic control. Discussion CD155 is overexpressed in many cancer types, where its high expression is generally associated with a worse prognosis due to its immunosuppressive function through interactions with TIGIT and CD96 receptors on immune cells( 41 – 43 ). Indeed, CD155 has been implicated in promoting resistance to chemotherapies and ICI-based therapies( 37 ). In line with this, a clinical trial targeting CD155 in solid cancers is currently ongoing( 29 ). Importantly, our study cautions against the indiscriminate use of CD155 blockade and instead identifies CD155 as a favourable prognostic biomarker of response to anti-HER2 therapies. We demonstrate that tumor-intrinsic CD155 is a critical determinant of HER2-targeted antibody efficacy, as anti-HER2 therapy fails to reduce tumour growth and lung metastatic burden in the absence of tumour CD155. Consistent with these findings, breast cancers patients with high HER2 and CD155 expression showed improved survival compared to patients with high HER2 but low CD155 expression. Conversely, low CD155 expression correlated with better outcomes in cancer patients receiving anti-PD-1 or anti-CTLA4 therapy, highlighting the complex context-dependent immunomodulatory role of CD155 as a predictive biomarker. While CD155’s immunosuppressive roles are well-established, its tumour intrinsic role in regulating other oncogenic signalling pathways, particularly HER2 has been largely unexplored. Our findings reveal that CD155 directly supports HER2 receptor organization and downstream signaling. Binding of anti-HER2 antibodies such as trastuzumab to HER2 receptor promotes HER2 clustering and internalization and here we demonstrate that CD155 facilitates these receptor dynamics. Reduced levels of phosphorylated Her2 (pHER2) and phosphorylated AKT (pAKT) in CD155 KO tumour cells indicate that CD155 supports downstream oncogenic HER2 signalling likely through its role in receptor clustering and internalization. These findings expand the known role of CD155 beyond immune modulation, implicating it as a structural regulator of oncogenic receptor organization and trafficking. Interestingly, we observed that in CD155-deficient cells, HER2 receptors exhibited constitutive clustering at the plasma membrane, even in the absence of antibody stimulation. In contrast, control cells displayed a diffuse HER2 distribution that underwent pronounced clustering and subsequent internalization upon trastuzumab treatment. This finding suggests that CD155 is required to maintain the dynamic organization and responsiveness of HER2 at the cell surface. Constitutive clustering in CD155-deficient cells may reflect aberrant receptor confinement within rigid membrane domains or disrupted interactions with membrane scaffolding components, resulting in reduced receptor mobility. Despite being pre-clustered, these receptors were functionally inactive, as evidenced by decreased phosphorylation of HER2 and AKT. Therefore, the inability of trastuzumab to further induce receptor clustering or internalization likely reflects a loss of receptor plasticity rather than simple loss of expression. This “locked” HER2 conformation provides a mechanistic explanation for impaired HER2 signaling and therapeutic resistance observed in the absence of CD155. In human HER2-positive SKBR3 breast cancer cells, CD155 depletion impaired T cell-mediated killing in both HER2-specific TCR assays and PBMC-trastuzumab co-cultures supporting the concept that increased HER2 clustering enhances anti-HER2 immune activity by concentrating the target antigen at the tumor cell surface. In vivo , both antibody-mediated blockade and CRISPR/Cas9-mediated deletion of CD155 in HER2-positive murine tumours abolished the anti-tumour and anti-metastatic efficacy of anti-HER2 mAb therapy, despite preserved HER2 expression. The requirement for CD155 was shown to be therapy-specific as anti-PD-L1 mAb retained full anti-tumour activity against CD155-deficient tumours, whereas anti-HER2 mAb efficacy was dependent on tumour CD155 expression. This distinction likely reflects differences in the mechanistic basis of action for each therapy. While PD-L1 blockade primarily reinvigorates exhausted T cells to increase anti-tumor T cell activity, HER2-targeted antibodies rely on both tumour cell-intrinsic HER2 receptor modulation and oncogenic signalling, and immune cell-dependent antibody-dependent cellular cytotoxicity (ADCC). Supporting this, anti-HER2 therapy reduced lung metastases less effectively in immunodeficient NSG mice compared with wild-type mice, consistent with loss of ADCC in the absence of NK and T cells. Strikingly, loss of tumor cell CD155 abolished anti-HER2 efficacy in both WT and NSG mice, suggesting that CD155’s contribution to therapeutic response is predominantly tumor intrinsic. Future studies should explore whether the mechanism by which CD155 promotes receptor clustering and enhances the efficacy of receptor-targeted therapy is unique to HER2 or extends to other receptor tyrosine kinases such as EGFR, VEGFR and c-MET. Overall, by integrating both human and murine tumour models, with clinical datasets from breast cancer patients, this work provides several key insights. First, CD155 exerts a tumor-intrinsic role essential for the in vivo efficacy of HER2-targeted monoclonal antibodies, Second, CD155 regulates HER2 receptor dynamics, linking a cell adhesion molecule to oncogenic receptor clustering and internalization. Notably, the loss of CD155 resulted in constitutive, antibody-unresponsive HER2 clustering, revealing that CD155 maintains receptor plasticity required for proper signaling and therapeutic responsiveness.Third, CD155 has context-specific prognostic implications across cancer therapies, acting as a positive predictor of anti-HER2 response but a negative indicator in settings of immune checkpoint inhibition and chemotherapy. These findings raise multiple translational opportunities. First, CD155 expression could be explored as part of a composite biomarker panel to guide patient selection for HER2-targeted therapy. Second, strategies that enhance or stabilize CD155 expression in HER2-positive tumors may potentiate trastuzumab efficacy and overcome therapeutic resistance. Third, elucidating the molecular interface between CD155 and HER2 clustering machinery may identify new druggable targets. Finally, the contrasting roles of CD155 in anti-HER2 versus ICI or chemotherapy responses highlight the need for therapy-tailored combination strategies that consider CD155 status to maximize clinical benefit while minimizing unnecessary toxicity. In conclusion, CD155 is a pivotal modulator of HER2 receptor biology and immune effector engagement acting as both a mechanistic driver of HER2-targeted antibody efficacy and a candidate biomarker for precision oncology. Declarations Acknowledgements We gratefully acknowledge the QIMR Berghofer Animal Research Facility for their support in breeding and maintaining the mice used in this study. We also thank the QIMR Berghofer and Mater Research Imaging and Flow Cytometry Facilities for their expert technical assistance, which was invaluable for the experiments. We are particularly grateful to the laboratory of Prof. Mark Smyth for generously providing key reagents and materials essential for this study. We also acknowledge the Rajiv Khanna Laboratory for their valuable contribution to the cytolysis activity experiments. D.M. acknowledges the “Walk to End Women’s Cancers” grant for their generous financial support, which made this work possible. Authors contribution: Conception and design: D. Mittal, M. Kalimutho, K.K. Khanna Acquisition of data: M. Kalimutho, D. Mittal, H. Shankar, W. Shi, G. Ambalathingal, S. Latham Analysis and interpretation of data: D. MIttal, M. Kalimutho, H. Shankar, W. Shi, G. Ambalathingal, S. Latham, K.K. Khanna Manuscript writing: D. Mittal, M. Kalimutho Study supervision: D. Mittal, M. Kalimutho, K.K. Khanna, J. Beesley, D. Croucher Consent for publication: All authors consent to this manuscript for publication. Data Availability and Material: The datasets generated and/or analyzed during the current study are included in this published article (and its supplementary information files) and all the raw data available from the corresponding author on reasonable request. Competing interests: The authors declare no potential conflicts of interest. Materials and Methods Mice Balb/c wild type (WT) mice were purchased from the Walter and Eliza Hall Institute for Medical Research or bred in house. All mice including Balb/c.NOD -/- gc -/- (NSG) mice were bred and maintained at the QIMR Berghofer Medical Research Institute and used from the age of 8 weeks. All experiments were approved by the QIMR Berghofer Medical Research Institute Animal Ethics Committee. Cell culture Mouse ErbB2-positive H2N100 and TUBO tumor cells were generated from female BALB/c MMTV-ErbB2/neu transgenic mice and cultured as described previously(44) [42]. H2N100 and H2NB2 cells were derived in the laboratory of Prof. Mark J. Smyth (QIMR Berghofer Medical Research Institute, Herston, Queensland, Australia). TUBO cells were maintained in complete DMEM supplemented with 10% heat-inactivated fetal calf serum (Thermo Scientific), 1X GlutaMAX, 50 U/mL penicillin, 100 μg/mL streptomycin, and 10 mM HEPES (Sigma-Aldrich). H2N100 and H2NB2 cells were cultured in RPMI supplemented with 10% heat-inactivated fetal calf serum, 1X GlutaMAX, 50 U/mL penicillin, 100 μg/mL streptomycin, 1 mM sodium pyruvate (Gibco-Life Technologies), 10 mM HEPES, and 1% non-essential amino acids, and incubated at 37°C in a 5% CO₂ incubator. Human breast cancer cell lines SKBR3, MDA-MB-361, and T47D were purchased from ATCC and cultured according to ATCC recommendations. All cell lines were used within fewer than 10 freeze-thaw cycles and were routinely tested for mycoplasma contamination. CD155 KO with CRISPR-Cas9 in tumor cells CD155 small guide RNAs (sgRNAs) were designed following the guidelines provided by the Broad Institute (http://www.genome-engineering.org/). Briefly, CD155 sgRNAs were cloned into the PX330 vector (Addgene, #42230). H2N100 and TUBO tumor cells were transfected with either the PX330-CD155 sgRNA plasmids or the empty PX330 vector, along with a GFP-expressing plasmid (pRp-GFP). GFP-positive cells were sorted using a FACSAria II Cell Sorter (BD Biosciences). After 7–10 days of culture, CD155-deficient tumor cells were further sorted to establish CD155 knockout (KO) cell lines. The sequences of primers used for CD155 sgRNAs are listed in Supplementary Table 1. siRNA-Mediated Knockdown and Western Blot Analysis SKBR3 cells were seeded in 10-cm dishes at a density of 5 × 10⁵ cells per well and allowed to attach overnight under standard culture conditions. Reverse transfection was performed using 20 nM of the indicated small interfering RNAs (siRNAs) (Table S1) complexed with Lipofectamine RNAiMAX according to the manufacturer’s protocol. Cells were incubated for 24 hours post-transfection to allow for efficient gene silencing. Following incubation, cells were lysed in 7M urea buffer and protein concentrations were determined using the BCA assay. Equal amounts of protein were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and probed with antibodies specific for CD155 and appropriate loading controls. Protein expression was detected using chemiluminescent substrates, confirming the efficiency of CD155 knockdown. Colony formation assays A total of 1,000 tumor cells were seeded in 24-well plates, followed by treatment with trastuzumab. The cells were incubated for an additional 14 days to assess colony survival. Colonies were then fixed with 0.05% crystal violet for 30 minutes, washed, and the stain was extracted using Sorenson buffer. Immunoblotting Immunoblotting was performed as described previously (45). Briefly, protein lysates were prepared from cells and quantified using BCA protein assay. Equal amounts of protein were resolved by SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with 5% non-fat dry milk in TBST for 1 hour at room temperature and then incubated overnight at 4°C with primary antibodies against the proteins of interest. Following washes with TBST, membranes were incubated with appropriate HRP-conjugated secondary antibodies for 1 hour at room temperature. Protein bands were visualized using the SuperSignal Chemiluminescent ECL Plus detection system (Amersham) according to the manufacturer’s instructions. ADCC and TCR-Based Killing Assays Immune-mediated tumor cell killing was assessed using antibody-dependent cellular cytotoxicity (ADCC) and T-cell receptor (TCR)-based killing assays. For the ADCC assay, peripheral blood mononuclear cells (PBMCs) were used as effector cells and co-cultured with target tumor cells at defined effector-to-target (E:T) ratios in the presence of trastuzumab. Cytotoxicity was monitored in real time using the xCELLigence system, and killing efficiency was calculated relative to untreated controls.For the TCR-based killing assay, HER2-expressing tumor cells were co-cultured with HER2-specific TCR-engineered T cells at defined E:T ratios. Cytotoxicity reflects antigen-specific, MHC-restricted T-cell killing, independent of the endogenous HER2 receptor, and was similarly measured using the xCELLigence system. Specific lysis was calculated relative to untreated controls. Immunofluorescence Assays Cells were plated on coverslips and incubated overnight. The coverslips were then fixed with 4% paraformaldehyde in PBS for 15 minutes, followed by permeabilization with 0.5% Triton X-100-PBS for 15 minutes. To block non-specific binding, the coverslips were treated with 2% filtered bovine serum albumin (BSA). Primary antibodies were diluted in the blocking solution and applied to the coverslips overnight at 4°C. Subsequently, Alexafluor conjugated secondary antibodies, diluted at 1/300 in the blocking solution, were used for staining and incubated for 45 minutes at 37°C in a humidifier chamber. After washing, the coverslips were counterstained with DAPI (diluted 1/500 in blocking buffer, stock concentration 1mg/ml) and mounted in Prolong Gold mounting medium. The slides were visualized using a GE DeltaVision Deconvolution microscope, and image analysis was performed using Image J software. Bimolecular Fluorescent Complementation (BiFC) Assay To investigate receptor interactions with HER2 using BiFC, the pDEST-ERBB2-V1 plasmid was used as the “bait.” When co-transfected with the complementary pDEST-V2 plasmid encoding CD155, a positive interaction was indicated by the appearance of a fluorescent signal from the reconstituted Venus protein, as described previously(46). T47D cells were seeded onto collagen-coated 35-mm imaging dishes at a density of 2 × 10⁵ cells per dish. Cells were then transfected with either 2 μg of Venus control plasmid or 1 μg each of PVR/CD155-V1 and ERBB2-V2, along with other receptor tyrosine kinase (RTK) constructs, using JetPrime transfection reagent, according to the manufacturer’s instructions and as previously described(47). Following transfection, samples were treated with either lapatinib (100 nM) or trastuzumab (5 μg/mL) for 1 hour or 16 hours. Nuclei were counterstained with Hoechst live-cell imaging reagent, fixed with 4% paraformaldehyde, and washed with PBS. Images were acquired using a Leica SP8 confocal microscope equipped with a 63×/1.4 NA objective lens. Scale bars represent 10 μm. In vivo studies H2N100 and TUBO (5 x 10 5 cells) tumor cells were injected subcutaneously into Balb/c WT mice. Mice were treated with four intraperitoneal injections of 10 mg anti-ErbB2/HER2 (7.16.4) or control immunoglobulin (cIg, 2A3) twice a week for two weeks after the tumors reached a size of 60-80 mm 2 (~ day 13 for TUBO and day 18 for H2N100). The anti-rat HER2 mAb (clone 7.16.4) hybridoma was generously provided by Mark I. Greene, University of Pennsylvania. A subset of mice additionally received 250 mg of anti-CD155 mAb (4.24) or anti-PD-L1 mAb (BioXCell, 10F.9G2) as indicated. Tumor growth was monitored and tumor size was recorded with a digital caliper every two to three days as the product of two perpendicular diameters. For lung metastases studies, H2N100 and TUBO sg control and sg2-CD155 KO tumor cells (2 x 10 5 cells) were injected intravenously to WT mice or NOD -/- gc -/- mice and treated on day 8 with 10 mg of control Ig (clone 2A3) or anti-HER2 (clone 7.16.4) mAbs intraperitoneally. Lungs were harvested on day 16 (TUBO) or day 20 (H2N100), washed in PBS, and fixed in Bouin's solution for 24 hours before counting tumor nodules using a dissection microscope. Flow cytometry analysis For tumor infiltrating lymphocytes analysis, H2N100 tumors were cut into small pieces and digested in medium containing RPMI with collagenase II (1 mg/mL) and DNAse (20 μg/mL) for 45 minutes. Samples were filtered through 70 μm filter, washed in PBS and lysed for red blood cells by ACK lysis buffer. After washing with PBS, single-cell suspensions were incubated for 20 minutes in Fc blocking buffer (2.4G2 antibody) and stained with viability stain Zombie Yellow (Biolegend) and following fluorescence-conjugated mAbs diluted in FACS buffer (2% FCS in PBS): anti-mouse-CD45.2 (104), TCR-β (H57-597), CD4 (RM4-5), CD8 (53.6.7) and TIGIT (1G9) for 30 minutes in ice. Samples were fixed and permeabilized using fixation permeabilization buffer (Thermo Fisher Scientific) for 20 min and stained with anti-mouse FoxP3 (FJK-16S). For flow cytometry of cultured tumor cells, cells were treated or not with 1 mg/ml of anti-ErbB2 mAb (7.16.4) or 1 mg/ml of control IgG (2A3) or were stimulated with 10 ng/ml of IFN-g (R&D Systems) for 24 h, 48 h or 72 h. Samples were collected, washed with FACS buffer and stained with viability stain Zombie Yellow (Biolegend) and following antibodies: anti-mouse-PD-L1 (MIH5), HER2 (7.16.4), CD155 (TX56) for 30 minutes in ice. All mAbs were purchased either from Thermo Fisher Scientific or Biologend. Samples were acquired on LSR Fortessa IV Flow Cytometer (BD Biosciences) and data was analyzed on FlowJo V10 (Treestar). Expression data from clinical samples GEPIA (Gene Expression Profiling Interactive Analysis) is an interactive web server for analyzing the RNA sequencing expression data of 9,736 tumors and 8,587 normal samples from the TCGA and the GTEx projects(48). cBioPortal was used to explore CD155 expression and its relation with survival analysis in HER2+ breast cancer samples(49). Briefly, breast cancer patients with high Her2+ expression were divided into CD155-low and CD155-high expression cancers to draw Kaplan–Meier based overall and disease-free survival. Kmplotter (Kaplan-Meier Plotter) is a Web-based gene-expression database that includes more than 6,000 breast cancer patients with clinical and survival data (50). Survival data was plotted using Km plotter for Her2+ and Her2- breast cancer types. cProSite (Cancer Proteogenomic Data Analysis Site) is a web-based, interactive platform for analyzing cancer data from the National Cancer Institute's Clinical Proteomic Tumor Analysis Consortium (CPTAC) Ref: Wang D et.al. Journal of Biotechnology and Biomedicine (2023). Statistical analysis All comparisons between samples were evaluated using unpaired t -tests or one-way ANOVA with Tukey’s post hoc test, unless otherwise stated in the figure legends. Statistical analyses were performed using GraphPad Prism version 8.0 (GraphPad Software, La Jolla, CA, USA). Where applicable, statistical significance is indicated as follows: P ≤ 0.05 (* ), P ≤ 0.01 (**), P ≤ 0.001 (*** ) and P ≤ 0.0001 (**** ). Data are expressed as mean ± standard error of the mean (SEM) or standard deviation (SD), as indicated. References C. Gutierrez, R. Schiff, HER2: biology, detection, and clinical implications. 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Mittal et al. , Improved Treatment of Breast Cancer with Anti-HER2 Therapy Requires Interleukin-21 Signaling in CD8+ T Cells. Cancer Res 76 , 264-274 (2016). S. Park et al. , The therapeutic effect of anti-HER2/neu antibody depends on both innate and adaptive immunity. Cancer Cell 18 , 160-170 (2010). M. Turcotte et al. , CD73 Promotes Resistance to HER2/ErbB2 Antibody Therapy. Cancer Res 77 , 5652-5663 (2017). H. Korkaya et al. , Activation of an IL6 inflammatory loop mediates trastuzumab resistance in HER2+ breast cancer by expanding the cancer stem cell population. Mol Cell 47 , 570-584 (2012). H. J. Choi et al. , CDK12 drives breast tumor initiation and trastuzumab resistance via WNT and IRS1-ErbB-PI3K signaling. EMBO Rep 20 , e48058 (2019). P. R. Pohlmann, I. A. Mayer, R. Mernaugh, Resistance to Trastuzumab in Breast Cancer. Clin Cancer Res 15 , 7479-7491 (2009). B. K. R. Chaganty et al. , Trastuzumab upregulates PD-L1 as a potential mechanism of trastuzumab resistance through engagement of immune effector cells and stimulation of IFNgamma secretion. Cancer letters 430 , 47-56 (2018). J. Gao, Q. Zheng, N. Xin, W. Wang, C. Zhao, CD155, an onco-immunologic molecule in human tumors. Cancer Sci 108 , 1934-1938 (2017). J. S. O'Donnell, J. Madore, X. Y. Li, M. J. Smyth, Tumor intrinsic and extrinsic immune functions of CD155. Semin Cancer Biol 65 , 189-196 (2020). H. Stamm, J. Wellbrock, W. Fiedler, Interaction of PVR/PVRL2 with TIGIT/DNAM-1 as a novel immune checkpoint axis and therapeutic target in cancer. Mamm Genome 29 , 694-702 (2018). S. J. Blake, W. C. Dougall, J. J. Miles, M. W. Teng, M. J. Smyth, Molecular Pathways: Targeting CD96 and TIGIT for Cancer Immunotherapy. Clin Cancer Res 22 , 5183-5188 (2016). H. Yong et al. , CD155 expression and its prognostic value in postoperative patients with breast cancer. Biomed Pharmacother 115 , 108884 (2019). K. Zhao et al. , CD155 Overexpression Correlates With Poor Prognosis in Primary Small Cell Carcinoma of the Esophagus. Front Mol Biosci 7 , 608404 (2020). A. Iguchi-Manaka et al. , High expression of soluble CD155 in estrogen receptor-negative breast cancer. Breast Cancer 27 , 92-99 (2020). Y. C. Li et al. , Overexpression of an Immune Checkpoint (CD155) in Breast Cancer Associated with Prognostic Significance and Exhausted Tumor-Infiltrating Lymphocytes: A Cohort Study. J Immunol Res 2020 , 3948928 (2020). R. B. Wang et al. , Overexpression of CD155 is associated with PD-1 and PD-L1 expression on immune cells, rather than tumor cells in the breast cancer microenvironment. World J Clin Cases 8 , 5935-5943 (2020). J. Smazynski et al. , The immune suppressive factors CD155 and PD-L1 show contrasting expression patterns and immune correlates in ovarian and other cancers. Gynecol Oncol 158 , 167-177 (2020). Q. Zheng et al. , CD155 contributes to the mesenchymal phenotype of triple-negative breast cancer. Cancer Sci 111 , 383-394 (2020). J. Gao, Q. Zheng, Y. Shao, W. Wang, C. Zhao, CD155 downregulation synergizes with adriamycin to induce breast cancer cell apoptosis. Apoptosis 23 , 512-520 (2018). F. Xu et al. , Blockade of CD112R and TIGIT signaling sensitizes human natural killer cell functions. Cancer Immunol Immunother 66 , 1367-1375 (2017). H. Triki et al. , Immune checkpoint CD155 promoter methylation profiling reveals cancer-associated behaviors within breast neoplasia. Cancer Immunol Immun 71 , 1139-1155 (2022). A. Atieh et al. , Phase I trial of first-in-class anti-PVR mAb NTX1088: Restoration of DNAM1 expression as MOA for enhanced antitumor immunity. Cancer Research 83 (2023). M. Braun et al. , CD155 on Tumor Cells Drives Resistance to Immunotherapy by Inducing the Degradation of the Activating Receptor CD226 in CD8(+) T Cells. Immunity 53 , 805-823 e815 (2020). A. Lepletier et al. , Tumor CD155 Expression Is Associated with Resistance to Anti-PD1 Immunotherapy in Metastatic Melanoma. Clin Cancer Res 26 , 3671-3681 (2020). N. Cancer Genome Atlas Research et al. , The Cancer Genome Atlas Pan-Cancer analysis project. Nat Genet 45 , 1113-1120 (2013). L. Liu et al. , CD155/TIGIT, a novel immune checkpoint in human cancers (Review). Oncol Rep 45 , 835-845 (2021). M. F. Sanchez, R. Tampe, Ligand-independent receptor clustering modulates transmembrane signaling: a new paradigm. Trends Biochem Sci 48 , 156-171 (2023). M. Luque et al. , Tumor-Infiltrating Lymphocytes and Immune Response in HER2-Positive Breast Cancer. Cancers (Basel) 14 (2022). K. Murakami, S. Ganguly, The Nectin family ligands, PVRL2 and PVR, in cancer immunology and immunotherapy. Front Immunol 15 , 1441730 (2024). R. Zhou et al. , CD155 and its receptors in cancer immune escape and immunotherapy. Cancer Lett 573 , 216381 (2023). K. L. McKinley, I. M. Cheeseman, Large-Scale Analysis of CRISPR/Cas9 Cell-Cycle Knockouts Reveals the Diversity of p53-Dependent Responses to Cell-Cycle Defects. Developmental cell 40 , 405-420.e402 (2017). D. Mittal et al. , Blockade of ErbB2 and PD-L1 using a bispecific antibody to improve targeted anti-ErbB2 therapy. Oncoimmunology 8 , e1648171 (2019). S. Rovero et al. , DNA vaccination against rat Her-2/neu p185 more effectively inhibits carcinogenesis than transplantable carcinomas in transgenic BALB/c mice. J Immunol 165 , 5133-5142 (2000). M. Zhan et al. , CD155 in tumor progression and targeted therapy. Cancer Lett 545 , 215830 (2022). D. Zhang, J. T. Liu, M. X. Zheng, C. Y. Meng, J. H. Liao, Prognostic and clinicopathological significance of CD155 expression in cancer patients: a meta-analysis. World J Surg Oncol 20 (2022). R. Paolini, R. Molfetta, CD155 and Its Receptors as Targets for Cancer Therapy. International Journal of Molecular Sciences 24 (2023). S. Rovero et al. , DNA vaccination against rat her-2/Neu p185 more effectively inhibits carcinogenesis than transplantable carcinomas in transgenic BALB/c mice. J Immunol 165 , 5133-5142 (2000). M. Kalimutho et al. , CEP55 is a determinant of cell fate during perturbed mitosis in breast cancer. Embo Molecular Medicine 10 (2018). D. R. Croucher et al. , Bimolecular complementation affinity purification (BiCAP) reveals dimer-specific protein interactions for ERBB2 dimers. Sci Signal 9 , ra69 (2016). S. P. Kennedy et al. , Targeting promiscuous heterodimerization overcomes innate resistance to ERBB2 dimerization inhibitors in breast cancer. Breast Cancer Res 21 , 43 (2019). Z. Tang et al. , GEPIA: a web server for cancer and normal gene expression profiling and interactive analyses. Nucleic Acids Res 45 , W98-W102 (2017). I. de Bruijn et al. , Analysis and Visualization of Longitudinal Genomic and Clinical Data from the AACR Project GENIE Biopharma Collaborative in cBioPortal. Cancer Res 83 , 3861-3867 (2023). B. Gyorffy et al. , An online survival analysis tool to rapidly assess the effect of 22,277 genes on breast cancer prognosis using microarray data of 1,809 patients. Breast cancer research and treatment 123 , 725-731 (2010). Additional Declarations There is no conflict of interest Supplementary Files s1.jpg Figure S1. Prognostic and predictive significance of CD155 expression across therapeutic contexts. (A) Box plots showing PVR expression levels in responders and non-responders to immune checkpoint inhibitor therapy, including anti–PD-1 ( n = 454), anti–PD-L1 ( n = 459), and anti–CTLA-4 ( n = 121) treatment cohorts. Receiver operating characteristic (ROC) curve analysis was performed to evaluate the predictive potential of PVR expression. The area under the curve (AUC = 0.574, p < 0.0001) indicates the sensitivity and specificity of PVR as a biomarker in patients receiving immunotherapy. (B) Box plots showing PVR expression levels in responders ( n = 119) and non-responders ( n = 388) to standard chemotherapeutic regimens, including taxanes, anthracyclines, ixabepilone, CMF (cyclophosphamide, methotrexate, and fluorouracil), FAC (fluorouracil, Adriamycin, and Cytoxan), and FEC (fluorouracil, epirubicin, and cyclophosphamide). ROC curve analysis was used to assess PVR’s predictive potential (AUC = 0.529, p = 0.18). (C) Correlation between CD155 mRNA and protein expression levels in breast cancer patient datasets, analyzed using the cProSite platform (mass spectrometry data). (D) Kaplan–Meier (KM) survival analysis of HER2/ErbB2-high breast cancer patients stratified by PVR/CD155 expression (low vs. high). Overall survival (OS) and disease-free survival (DFS) curves were generated using data from cBioPortal. s2.jpg Figure S2. Subtype-specific CD155 expression in breast cancer cell lines. (A)Subtype-specific CD155 mRNA expression (log₂-transformed) in breast cancer cell lines, analyzed using the GOBO software platform. Data were derived from the Neve dataset (Neve et al., 2006). The graph was generated using the GOBO online tool. (B) Comparative analysis of CD155 mRNA expression (log₂-transformed) in basal-like versus non-basal-like breast cancer cell lines. TNBC indicates triple-negative breast cancer, and HR+ indicates hormone receptor-positive lines. Analysis was performed as described in panel (A). s3.jpg Figure S3. BiFC analysis of CD155 interactions with HER family and other receptor tyrosine kinases. (A)Representative images from the Bimolecular Fluorescent Complementation (BiFC) assay showing the interaction between CD155 and HER2. T47D cells were transfected with the indicated V1 and V2 plasmid constructs. Under basal conditions, the PVR-V1 and ERBB2-V2 interaction was predominantly localized at the plasma membrane. (B) Representative BiFC images showing interactions between CD155 and other receptor tyrosine kinases, including EGFR, HER3, HER4, IGFR, PDGFR, and MET in T47D cells. s4.jpg Figure S4. CD155 knockdown in SKBR3 cells affects HER2 signaling. (A) SKBR3 cells were transfected with 20 nM CD155-targeting siRNA for 24 hours, followed by treatment with lapatinib or trastuzumab for an additional 24 hours. Immunoblot analysis was performed to assess CD155, phosphorylated HER2 (pHER2), and total HER2 levels. COX IV served as a loading control. (B) Representative flow cytometry-based histogram plots showing CD155 knockdown in SKBR3 cells, detected using an APC-conjugated anti-CD155 antibody. s5.jpg Figure S5. CD155 expression in HER2-positive tumor cells. (A) Flow cytometry-based histogram plots showing CD155 surface expression in HER2-positive H2NB2, H2N100 and TUBO tumor cells 24 hours after treatment with control IgG, 10 ng/mL IFNγ, or 1 μg/mL anti-HER2 antibody (clone 7.16.4). Grey histograms represent isotype control staining for CD155. (B) Flow cytometry-based histogram plots showing CD155 and HER2 surface expression in HER2-positive H2N100 and TUBO tumor cells. Comparisons are shown for sg Control (red), sg2 CD155 KO (purple), and sg6 CD155 KO cells. s6.jpg Figure S6. No correlation in expression between CD155 and PD-L1 in HER2-positive tumor cells. (A) Flow cytometry-based histogram plots showing PD-L1 surface expression in HER2-positive H2N100 sg Control and sg2 CD155 KO tumor cells at 24 and 72 hours following treatment with control IgG, 10 ng/mL IFNγ, TNF-α, or 1 μg/mL anti-HER2 antibody (clone 7.16.4). Grey histograms represent isotype control staining for PD-L1. (B) Gene expression correlation between CD155 and PD-L1 in breast cancer patient datasets, analyzed using GEPIA. Expression is plotted as transcripts per million (TPM) on a log₂ scale. 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07:17:46\",\"extension\":\"jpg\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":606263,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eDifferential prognostic significance of CD155.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(A) \\u003c/strong\\u003eKM plotter graphs showing overall survival, progression-free survival, disease-free survival, and disease-specific survival. These graphs were generated using the BRCA_TCGA_Pan_Cancer_Atlas_2018 dataset. The log-rank test P-value and HR are shown in the figure, with n=1084 patient samples.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(B)\\u003c/strong\\u003e KM plotter graphs showing how the expression of PVR associates with overall survival in patients treated with anti-Immunotherapeutics (anti-PD-1; n=454, and anti-CTLA-4; n=121) across different cancers (bladder, n=73; esophageal adenocarcinoma, n=103; glioblastoma, n=28; hepatocellular carcinoma, n=22; HNSCC, n=5; melanoma, n=423; NSCLC, n=21; NSLC, n=22; and urothelial, n=348). The log-rank test P-value and HR are shown in the figure.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(C, D) \\u003c/strong\\u003eBox plots depict PVR expression in responders and non-responders to anti-HER2 therapies (Trastuzumab and Lapatinib), based on pathological complete response (pCR) and 5-year relapse-free survival (RFS). Receiver operating characteristic (ROC) curve analysis was performed to evaluate the predictive potential of PVR, with the area under the curve (AUC) reported as 0.6 (p \\u0026lt; 0.044) and 0.72 (p \\u0026lt; 0.02), respectively.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7994408/v1/3c9f513ac6311f0985ee6ad6.jpg\"},{\"id\":95895986,\"identity\":\"2421c2e7-3c70-42bf-bd19-8b7cf7241673\",\"added_by\":\"auto\",\"created_at\":\"2025-11-14 07:17:46\",\"extension\":\"jpg\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":640852,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eCD155 knockdown impacts signaling cascades and reduces Trastuzumab therapeutic response.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(A)\\u003c/strong\\u003eNormalized mean fluorescence intensity (MFI) of CD155 and HER2 expression in HER2-positive breast cancer cell lines. A total of 10,000 cells were analyzed using fluorophore-conjugated HER2 and CD155 antibodies. MDA-MB-361 cells were used as the normalization reference. Data represents the mean ± SD from three independent experiments.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(B)\\u003c/strong\\u003e Linear regression analysis showing a positive correlation between HER2 and CD155 expression levels across HER2-positive breast cancer cell lines (R² = 0.7010). Expression values were obtained from the experiment shown in \\u003cstrong\\u003e(A)\\u003c/strong\\u003e.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(C)\\u003c/strong\\u003eRepresentative immunofluorescence images showing both intracellular and membrane-bound CD155 and HER2 localization in MDA-MB-361 and SKBR3 cells. HER2 is shown in red, CD155 in green, and nuclei are counterstained with DAPI (blue).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(D) \\u003c/strong\\u003eRepresentative images from the Bimolecular Fluorescent Complementation (BiFC) assay confirming the interaction between CD155 and HER2. T47D cells were transfected with the indicated V1 and V2 plasmid constructs. The PVR-V1 and ERBB2-V2 interaction was predominantly localized at the plasma membrane under basal conditions. Cells were treated with lapatinib (100 nM) or trastuzumab (5 μg/mL) for 1 hour and 16 hours to assess the surface localization of these receptor interactions.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(E)\\u003c/strong\\u003eImmunoblot analysis showing HER2 expression levels in parental and shPVR-depleted SKBR3 cells. Cell lysates were prepared and subjected to SDS-PAGE followed by immunoblotting with an anti-HER2 antibody. COX IV was used as a loading control to confirm equal protein loading across samples.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(F)\\u003c/strong\\u003eRepresentative flow cytometry-based histogram plots showing surface expression levels of CD155 and HER2 in parental and shCD155-depleted cells. Surface CD155 expression (left) and HER2 expression (right) were quantified using fluorophore-conjugated antibodies. A marked reduction in CD155 levels confirmed efficient knockdown.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(G)\\u003c/strong\\u003eImmunoblot analysis showing total and phosphorylated HER2, along with CD155 expression, in SKBR3 parental and shCD155#3 cells. Cell lysates were analyzed by SDS-PAGE followed by immunoblotting with antibodies specific for total HER2, phospho-HER2, and CD155. COX IV served as a loading control to verify equal protein loading across samples.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(H)\\u003c/strong\\u003eImmunoblot analysis of HER2-dependent downstream signaling in SKBR3 parental and shCD155#3 cells. Cells were left untreated or treated with trastuzumab (10 μg/mL) or lapatinib (100 nM) for 24 hours. The indicated phosphorylated and total signaling proteins were analyzed by Western blotting to evaluate HER2 pathway activity. COX IV served as a loading control.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(I)\\u003c/strong\\u003eImmunoblot analysis showing EGF-induced signaling in SKBR3 parental and shCD155#3 cells. After serum starvation for 16 hours, cells were stimulated with EGF (20 ng/mL) for 20 minutes. Phosphorylated and total proteins were examined by Western blotting. COX IV served as a loading control.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(J)\\u003c/strong\\u003e \\u003cem\\u003eLeft:\\u003c/em\\u003eColony formation assay of SKBR3 parental and shCD155#3 cells treated with increasing concentrations of trastuzumab for 14 days. Colonies were stained with crystal violet, solubilized using Sorenson buffer, and quantified spectrophotometrically. Data were normalized to untreated controls and are presented as mean ± SD from two independent experiments. \\u003cem\\u003eRight:\\u003c/em\\u003eRepresentative images of stained colonies corresponding to the quantified results shown on the left.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"2.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7994408/v1/0ccc3715ef5c0a83de93ba89.jpg\"},{\"id\":96243362,\"identity\":\"4a7bdd2e-a7e0-460d-b80a-725c97f8f4c4\",\"added_by\":\"auto\",\"created_at\":\"2025-11-19 07:16:10\",\"extension\":\"jpg\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":610549,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eCD155 modulates HER2 localization and immune-mediated cytotoxicity.\\u003cbr\\u003e\\n(A)\\u003c/strong\\u003e Representative immunofluorescence images showing membrane-associated CD155 and HER2 localization in SKBR3 cells treated with trastuzumab (10 μg/mL) for 2 hours. CD155 (red) and HER2 (green) were visualized using specific antibodies, and nuclei were counterstained with DAPI (blue).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(B)\\u003c/strong\\u003eRepresentative flow cytometry-based histogram plots showing surface expression of CD155 and HER2 in parental and shCD155 SKBR3 cells following treatment with lapatinib (100 nM) or trastuzumab (10 μg/mL) for 2 hours.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(C)\\u003c/strong\\u003eRepresentative immunofluorescence images showing membrane and intracellular localization of CD155 (red) and HER2 (green) in SKBR3 cells treated with trastuzumab (10 μg/mL) for 24 hours. Nuclei were counterstained with DAPI (blue).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(D)\\u003c/strong\\u003eRepresentative immunofluorescence images illustrating HER2 clustering at the plasma membrane in SKBR3 cells treated with trastuzumab (10 μg/mL) for 30 minutes. HER2 is visualized in red, and nuclei are counterstained with DAPI (blue).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(E)\\u003c/strong\\u003e SKBR3 parental and shCD155 cells were co-cultured with HER2-targeting T cells at an effector-to-target (E:T) ratio of 2:1. Cytolytic activity was monitored in real time using the xCELLigence system. Data represent the mean ± SD from two independent experiments (n = 2). Statistical significance is indicated as ***P ≤ 0.001.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(F)\\u003c/strong\\u003e SKBR3 parental and shCD155 cells were co-cultured with peripheral blood mononuclear cells (PBMCs) at an effector-to-target (E:T) ratio of 2:1 and subsequently treated with trastuzumab. Cytolytic activity was monitored over time using the xCELLigence system. Data represent the mean ± SD from two independent experiments (n = 2). Statistical significance is indicated as ***P ≤ 0.001.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"3.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7994408/v1/fbd62774fc8500c228481123.jpg\"},{\"id\":95895988,\"identity\":\"5b1bfeb1-486a-4042-94d5-f6218f2c57f2\",\"added_by\":\"auto\",\"created_at\":\"2025-11-14 07:17:46\",\"extension\":\"jpg\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":504472,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eAnti-HER2 monoclonal antibody therapy depends on CD155 signaling.\\u003cbr\\u003e\\n (A)\\u003c/strong\\u003e Flow cytometric analysis of CD155 and HER2 surface expression in H2N100 and TUBO tumor cell lines.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(B, C)\\u003c/strong\\u003e H2N100 (B) and TUBO (C) tumor cells (5 × 10⁵) were implanted subcutaneously (s.c.) into BALB/c mice. Tumors were treated with anti-HER2 monoclonal antibody (mAb; clone 7.16.4, 10 μg/mouse) or anti-CD155 blocking mAb (BioLegend, clone 4.24, 250 μg/mouse), alone or in combination. Treatments were administered intraperitoneally (i.p.) twice weekly for two weeks, beginning on day 18 for H2N100 tumors and day 13 for TUBO tumors. The anti-CD155 mAb was given one day prior to anti-HER2 mAb administration. Tumor growth was monitored by measuring two perpendicular diameters using calipers.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(D)\\u003c/strong\\u003e H2N100 tumor cells were injected intravenously (i.v.) into mice, followed by treatment with anti-HER2 mAb, anti-CD155 mAb, or the combination. Lungs were harvested on day 20 to assess metastatic burden.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(E, F)\\u003c/strong\\u003e H2N100 tumors were treated with anti-HER2 mAb, anti-CD155 mAb, or their combination, and tumors were collected 24 hours after the second treatment for tumor-infiltrating lymphocyte (TIL) analysis. Bar graphs represent the frequency of gated immune cell populations.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"4.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7994408/v1/eb4c8b594b9693d7d1067bb5.jpg\"},{\"id\":95895994,\"identity\":\"fbb714a9-5f1f-4daa-894f-6ca06a39c222\",\"added_by\":\"auto\",\"created_at\":\"2025-11-14 07:17:46\",\"extension\":\"jpg\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":575596,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eTumor cell CD155 expression is required for anti-ErbB2 monoclonal antibody therapy.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(A, C)\\u003c/strong\\u003e Immunoblot analysis of CD155 expression in H2N100 (A) and TUBO (C) tumor cells. Control cells (sg_Control) and CD155 knockout cells (sg2_CD155 KO and sg6_CD155 KO) were analyzed. β-actin served as a loading control.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(B, D)\\u003c/strong\\u003e H2N100 (B) and TUBO (D) control or CD155 KO (sg2 and sg6) tumor cells (5 × 10⁵) were implanted subcutaneously (s.c.) into BALB/c mice. Tumors were treated with anti-HER2 monoclonal antibody (mAb; clone 7.16.4) administered intraperitoneally (i.p.) twice weekly for two weeks, beginning on day 18 for H2N100 tumors and day 13 for TUBO tumors. Arrows indicate the initiation of anti-HER2 treatment.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(E, F)\\u003c/strong\\u003e TUBO control and sg2_CD155 KO tumor cells (2 × 10⁵) were injected intravenously (i.v.) into BALB/c mice. Mice were treated with anti-HER2 mAb (10 μg/mouse) on day 8. Lungs were collected on day 16 to assess metastatic burden by counting visible lung metastases (E) and measuring lung weight (F).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(G)\\u003c/strong\\u003e H2N100 control and sg2_CD155 KO tumor cells (5 × 10⁵) were implanted s.c. into BALB/c mice and treated with anti–PD-L1 mAb (clone 10F.9G2, 250 μg/mouse) on days 18, 21, 25, and 28. Tumor growth is shown as mean tumor volume calculated from two perpendicular diameters.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(H, I)\\u003c/strong\\u003eH2N100 control (sg_Control) and sg2_CD155 KO tumor cells (2 × 10⁵) were injected i.v. into BALB/c wild-type (WT) or NOD-/-γc-/- (NSG) mice. Mice were treated with anti-HER2 mAb (10 μg/mouse) on day 8, and lungs were collected on day 20 to quantify lung metastases.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"5.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7994408/v1/b6643efc95b72e7febcf96dd.jpg\"},{\"id\":98439175,\"identity\":\"74bf872d-565c-4f84-aefe-91f9d6b8b529\",\"added_by\":\"auto\",\"created_at\":\"2025-12-17 17:01:23\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":4200017,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7994408/v1/dee15e44-8822-4b7a-81db-36d269b6f489.pdf\"},{\"id\":96242735,\"identity\":\"57707fdf-1079-450c-8e0f-abb7ce8b7675\",\"added_by\":\"auto\",\"created_at\":\"2025-11-19 07:14:11\",\"extension\":\"jpg\",\"order_by\":1,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":396828,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eFigure S1. Prognostic and predictive significance of CD155 expression across therapeutic contexts.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(A)\\u003c/strong\\u003e Box plots showing PVR expression levels in responders and non-responders to immune checkpoint inhibitor therapy, including anti–PD-1 (\\u003cem\\u003en\\u003c/em\\u003e = 454), anti–PD-L1 (\\u003cem\\u003en\\u003c/em\\u003e = 459), and anti–CTLA-4 (\\u003cem\\u003en\\u003c/em\\u003e = 121) treatment cohorts. Receiver operating characteristic (ROC) curve analysis was performed to evaluate the predictive potential of PVR expression. The area under the curve (AUC = 0.574, \\u003cem\\u003ep\\u003c/em\\u003e\\u0026lt; 0.0001) indicates the sensitivity and specificity of PVR as a biomarker in patients receiving immunotherapy.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(B)\\u003c/strong\\u003e Box plots showing PVR expression levels in responders (\\u003cem\\u003en\\u003c/em\\u003e = 119) and non-responders (\\u003cem\\u003en\\u003c/em\\u003e = 388) to standard chemotherapeutic regimens, including taxanes, anthracyclines, ixabepilone, CMF (cyclophosphamide, methotrexate, and fluorouracil), FAC (fluorouracil, Adriamycin, and Cytoxan), and FEC (fluorouracil, epirubicin, and cyclophosphamide). ROC curve analysis was used to assess PVR’s predictive potential (AUC = 0.529, \\u003cem\\u003ep\\u003c/em\\u003e = 0.18).\\u003cbr\\u003e\\n \\u003cstrong\\u003e(C)\\u003c/strong\\u003e Correlation between CD155 mRNA and protein expression levels in breast cancer patient datasets, analyzed using the cProSite platform (mass spectrometry data).\\u003cbr\\u003e\\n \\u003cstrong\\u003e(D)\\u003c/strong\\u003e Kaplan–Meier (KM) survival analysis of HER2/ErbB2-high breast cancer patients stratified by PVR/CD155 expression (low vs. high). Overall survival (OS) and disease-free survival (DFS) curves were generated using data from cBioPortal.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"s1.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7994408/v1/d8535c4947c0f161c9ef1de7.jpg\"},{\"id\":96243104,\"identity\":\"1bead59c-32c1-49c5-a512-0e83fa76463d\",\"added_by\":\"auto\",\"created_at\":\"2025-11-19 07:15:32\",\"extension\":\"jpg\",\"order_by\":2,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":294319,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eFigure S2. Subtype-specific CD155 expression in breast cancer cell lines.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(A)\\u003c/strong\\u003eSubtype-specific CD155 mRNA expression (log₂-transformed) in breast cancer cell lines, analyzed using the GOBO software platform. Data were derived from the Neve dataset (Neve et al., 2006). The graph was generated using the GOBO online tool.\\u003cbr\\u003e\\n \\u003cstrong\\u003e(B)\\u003c/strong\\u003e Comparative analysis of CD155 mRNA expression (log₂-transformed) in basal-like versus non-basal-like breast cancer cell lines. TNBC indicates triple-negative breast cancer, and HR+ indicates hormone receptor-positive lines. Analysis was performed as described in panel (A).\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"s2.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7994408/v1/bd7b2de1417dc5b18597bd4d.jpg\"},{\"id\":96242755,\"identity\":\"4d4ca21a-567d-404d-805d-43d509a91847\",\"added_by\":\"auto\",\"created_at\":\"2025-11-19 07:14:13\",\"extension\":\"jpg\",\"order_by\":3,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":414747,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eFigure S3. BiFC analysis of CD155 interactions with HER family and other receptor tyrosine kinases.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(A)\\u003c/strong\\u003eRepresentative images from the Bimolecular Fluorescent Complementation (BiFC) assay showing the interaction between CD155 and HER2. T47D cells were transfected with the indicated V1 and V2 plasmid constructs. Under basal conditions, the PVR-V1 and ERBB2-V2 interaction was predominantly localized at the plasma membrane. \\u003cbr\\u003e\\n \\u003cstrong\\u003e(B)\\u003c/strong\\u003e Representative BiFC images showing interactions between CD155 and other receptor tyrosine kinases, including EGFR, HER3, HER4, IGFR, PDGFR, and MET in T47D cells.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"s3.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7994408/v1/3337c031fa710814c0f58aea.jpg\"},{\"id\":96243526,\"identity\":\"2ad41e1b-7f36-4fb5-80a9-92c3b0d137a9\",\"added_by\":\"auto\",\"created_at\":\"2025-11-19 07:16:36\",\"extension\":\"jpg\",\"order_by\":4,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":247671,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eFigure S4. CD155 knockdown in SKBR3 cells affects HER2 signaling.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(A)\\u003c/strong\\u003e SKBR3 cells were transfected with 20 nM CD155-targeting siRNA for 24 hours, followed by treatment with lapatinib or trastuzumab for an additional 24 hours. Immunoblot analysis was performed to assess CD155, phosphorylated HER2 (pHER2), and total HER2 levels. COX IV served as a loading control.\\u003cbr\\u003e\\n \\u003cstrong\\u003e(B)\\u003c/strong\\u003e Representative flow cytometry-based histogram plots showing CD155 knockdown in SKBR3 cells, detected using an APC-conjugated anti-CD155 antibody.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"s4.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7994408/v1/a3264f58952e05e394d86d7d.jpg\"},{\"id\":95895998,\"identity\":\"2ba71779-cacb-4013-b77f-7a07550a06de\",\"added_by\":\"auto\",\"created_at\":\"2025-11-14 07:17:46\",\"extension\":\"jpg\",\"order_by\":5,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":190903,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eFigure S5. CD155 expression in HER2-positive tumor cells.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(A)\\u003c/strong\\u003e Flow cytometry-based histogram plots showing CD155 surface expression in HER2-positive H2NB2, H2N100 and TUBO tumor cells 24 hours after treatment with control IgG, 10 ng/mL IFNγ, or 1 μg/mL anti-HER2 antibody (clone 7.16.4). Grey histograms represent isotype control staining for CD155.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(B)\\u003c/strong\\u003e Flow cytometry-based histogram plots showing CD155 and HER2 surface expression in HER2-positive H2N100 and TUBO tumor cells. Comparisons are shown for sg Control (red), sg2 CD155 KO (purple), and sg6 CD155 KO cells.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"s5.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7994408/v1/e28b28fb8a03c548ad462a8b.jpg\"},{\"id\":96243461,\"identity\":\"baa67c87-5c3c-4e9f-bbd4-c8da0332af43\",\"added_by\":\"auto\",\"created_at\":\"2025-11-19 07:16:26\",\"extension\":\"jpg\",\"order_by\":6,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":252959,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eFigure S6. No correlation in expression between CD155 and PD-L1 in HER2-positive tumor cells.\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e(A)\\u003c/strong\\u003e Flow cytometry-based histogram plots showing PD-L1 surface expression in HER2-positive H2N100 sg Control and sg2 CD155 KO tumor cells at 24 and 72 hours following treatment with control IgG, 10 ng/mL IFNγ, TNF-α, or 1 μg/mL anti-HER2 antibody (clone 7.16.4). Grey histograms represent isotype control staining for PD-L1.\\u003cbr\\u003e\\n \\u003cstrong\\u003e(B)\\u003c/strong\\u003e Gene expression correlation between CD155 and PD-L1 in breast cancer patient datasets, analyzed using GEPIA. Expression is plotted as transcripts per million (TPM) on a log₂ scale.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"s6.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7994408/v1/ef80db0ff1fe2a4cf53fb865.jpg\"},{\"id\":96243360,\"identity\":\"79b58467-0c1f-49d1-b6f4-73cafa4299c7\",\"added_by\":\"auto\",\"created_at\":\"2025-11-19 07:16:10\",\"extension\":\"docx\",\"order_by\":7,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":16833,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"SupplementaryTable1.docx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7994408/v1/1636567a454632058b3865f1.docx\"}],\"financialInterests\":\"There is no conflict of interest\",\"formattedTitle\":\"CD155/PVR as a determinant of anti-HER2 therapy sensitivity in breast cancer\",\"fulltext\":[{\"header\":\"Introduction\",\"content\":\"\\u003cp\\u003eHER2 (Human Epidermal Growth Factor Receptor 2, also known as ErbB2) is a member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases. While HER2 lacks a known ligand, it forms stable heterodimers with other ligand-activated EGFR family members, amplifying downstream kinase-driven signaling pathways(\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e). HER2 is overexpressed in multiple malignancies, particularly in 20\\u0026ndash;30% of invasive breast carcinomas, classified as HER2-positive breast cancers(\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003eTrastuzumab (Herceptin), a monoclonal antibody (mAb) targeting HER2, is a cornerstone therapy for HER2-positive breast cancers. Beyond its ability to directly inhibit oncogenic HER2 signaling, the therapeutic efficacy of trastuzumab relies on engagement of the immune system. Previous studies, including our own, have demonstrated that trastuzumab-like therapies depend on both innate and adaptive immune components, such as CD11b⁺ and F4/80⁺ myeloid cells, NK cells, CD4⁺ and CD8⁺ T lymphocytes, and cytokines including type I and II interferons (IFNs) and IL-21, in HER2-positive mouse models of breast cancer(\\u003cspan additionalcitationids=\\\"CR5 CR6 CR7 CR8\\\" citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e). Additionally, inhibition of immunosuppressive pathways such as CD73-adenosine and PD-1/PD-L1 has been shown to enhance trastuzumab-mediated antitumor activity(\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e). However, despite significant survival benefits, both acquired and intrinsic resistance to trastuzumab remains a major clinical hurdle in the treatment of HER2-positive metastatic breast cancer(\\u003cspan additionalcitationids=\\\"CR12 CR13\\\" citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e). Thus, elucidating both tumor-intrinsic and -extrinsic mechanisms of trastuzumab action is critical to improve patient outcomes.\\u003c/p\\u003e\\u003cp\\u003eCD155 (also known as the poliovirus receptor, PVR, or NECL5) has emerged as a dual-function molecule with immunomodulatory and tumor-intrinsic roles. It interacts with a family of receptors including DNAM-1 (CD226), TIGIT, and CD96, which collectively modulate anti-tumor immune responses(\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e). DNAM-1 serves as a co-stimulatory receptor, while TIGIT and CD96 function as immune checkpoints. CD155 is primarily expressed by tumor cells, endothelial cells, and myeloid cells, and it competes with CD112 (Nectin-2, PVRL2), another ligand for DNAM-1 and TIGIT, with a ubiquitous expression on normal epithelial cells, fibroblasts, neuron cells as well as on tumor and antigen-presenting cells(\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e). The balance of activating (DNAM-1) versus inhibitory (TIGIT/CD96) signaling is influenced by ligand affinity and receptor expression on immune cells, enabling CD155 and CD112 to exert both immune-activating and immune-suppressive effects(\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003eBeyond immune regulation, CD155 also exerts tumor cell-intrinsic functions that contribute to malignancy(\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e). Overexpression of CD155 is associated with reduced tumor-infiltrating lymphocytes (TILs), higher relapse rates, and poorer survival across multiple cancer types, including breast cancer(\\u003cspan additionalcitationids=\\\"CR20 CR21\\\" citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e). In breast tumors, CD155 correlates with increased PD-L1 expression on immune cells and a higher proportion of exhausted PD-1⁺CD4⁺ TILs, supporting its role in immunosuppression(\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e). Experimental deletion of tumor cell CD155 has been shown to decrease cancer cell proliferation \\u003cem\\u003ein vitro\\u003c/em\\u003e, reduce tumor growth and metastasis in several mouse models(\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e). Moreover, CD155 and PD-L1 display distinct expression patterns and prognostic implications, suggesting they act through non-redundant immunosuppressive mechanisms, particularly in high-grade serous ovarian cancer(\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e). In triple-negative breast cancer (TNBC), CD155 knockdown induced a mesenchymal-to-epithelial transition in tumor cells(\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e), and treatment with adriamycin (doxorubicin) upregulated CD155 expression, with combined CD155 knockdown and chemotherapy enhancing apoptosis(\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e). TIGIT, inhibitory receptor, is preferentially expressed on NK cells and blockade of TIGIT and/or CD112R was shown to enhance trastuzumab-triggered tumor cell killing by NK cells(\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e). Additionally, higher CpG promoter methylation was associated with HER2 receptor expression(\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e). These findings support the therapeutic targeting of CD155 in combination with standard treatments for invasive breast cancers. In fact, a phase I clinical trial targeting CD155 as a monotherapy and in combination with pembrolizumab in advanced solid cancer patients is already in progress (NCT05378425)(\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003eGiven the complex role of CD155 and its receptors in tumor progression and its association with resistance to chemotherapy and immune checkpoint therapies(\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e), we sought to investigate its role in trastuzumab response in HER2-positive breast cancers. Interestingly, we demonstrate that the loss of tumor cell CD155, through CRISPR/Cas9-mediated knockout or antibody blockade, markedly impaired the efficacy of trastuzumab-like anti-HER2 therapy in both primary and metastatic models without affecting tumor cell HER2 expression. This effect was mediated by a direct CD155-HER2 interaction at the tumor cell membrane. Importantly, these findings caution against indiscriminate CD155 blockade in HER2-positive breast cancer and highlight tumor CD155 expression as a potential predictive biomarker for trastuzumab response.\\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003ePrognostic significance of CD155 expression in breast cancer patients\\u003c/h2\\u003e\\u003cp\\u003eCD155 has garnered considerable attention in oncology for its dual role in tumour progression and immune regulation(\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e). Previous studies have shown that CD155 overexpression promotes breast cancer growth; however, its broader clinical relevance remains incompletely understood. To assess the prognostic significance of CD155, we analyzed The Cancer Genome Atlas (TCGA) breast cancer datasets(\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e). High CD155 expression was significantly associated with poorer outcomes across multiple survival endpoints, including overall survival (OS), progression-free survival (PFS), disease-free survival (DFS), and disease-specific survival (DSS) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eA).\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003eGiven CD155\\u0026rsquo;s function as an immune checkpoint regulator capable of modulating antitumor immunity through both inhibitory and stimulatory pathways(\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e), we next investigated its relationship with clinical outcomes in patients treated with immune checkpoint inhibitors (ICIs) targeting PD-1 and CTLA-4 across various cancer types. Consistent with the proposed role of CD155 as a marker of therapy resistance, low CD155 expression correlated with improved OS and progression free survival in patients receiving anti-PD-1 or anti-CTLA-4 therapy (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eB).\\u003c/p\\u003e\\u003cp\\u003eTo evaluate whether CD155 could also predict treatment response, we analyzed transcriptomic profiles from ICI-treated patients classified as responders or non-responders. Overall, non-responders exhibited higher CD155 expression compared to responders, a pattern that persisted across different checkpoint inhibitor classes (\\u003cb\\u003eFig. S1A and data not shown\\u003c/b\\u003e).\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003e We then examined the predictive capacity of CD155 as a marker of response to clinically approved chemotherapeutic agents in breast cancer, including taxanes, anthracyclines, ixabepilone, CMF (Cyclophosphamide o\\u0026thinsp;+\\u0026thinsp;Methotrexate\\u0026thinsp;+\\u0026thinsp;Fluorouracil (5-FU)), FAC (Fluorouracil (5-FU)\\u0026thinsp;+\\u0026thinsp;Adriamycin (doxorubicin)\\u0026thinsp;+\\u0026thinsp;Cyclophosphamide), and FEC (Fluorouracil (5-FU)\\u0026thinsp;+\\u0026thinsp;Epirubicin\\u0026thinsp;+\\u0026thinsp;Cyclophosphamide) regimens. No statistically significant differences in CD155 expression were observed between responders and non-responders to these agents (\\u003cb\\u003eFig. S1B\\u003c/b\\u003e). Importantly, CD155 displayed good correlation between mRNA and protein level in breast cancers (\\u003cb\\u003eFig. S1C\\u003c/b\\u003e) and ErbB2-hi CD155-hi cancers exhibited better survival compared to ErbB2-hi CD155-low cancers (\\u003cb\\u003eFig. S1D\\u003c/b\\u003e). In HER2-positive patients, CD155 expression emerged as a strong predictive biomarker for anti-HER2 therapy (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eC). Specifically, higher CD155 levels were significantly associated with better pathological complete response to both trastuzumab and lapatinib (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eC). Notably, similar patterns were also observed when evaluating relapse-free survival at 5 years (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eD), reinforcing the association between elevated CD155 expression and favourable outcomes with anti-HER2 therapy. Collectively, these findings suggest that CD155 serves as a negative prognostic factor in breast cancer overall, yet its predictive value varies substantially across therapeutic contexts. While low CD155 expression favours response to PD-1 and CTLA-4 blockade, high expression may predict improved benefit from anti-HER2 therapies.\\u003c/p\\u003e\\u003c/div\\u003e\\n\\u003ch3\\u003eCD155 knockdown reduces response to anti-HER2 therapy via regulation of AKT signalling\\u003c/h3\\u003e\\n\\u003cp\\u003eGiven that CD155 expression predicted response to anti-HER2 therapy, we sought to investigate the underlying mechanisms by which CD155 influences this sensitivity. First, we determined CD155 transcript levels across a panel of breast cancer cell lines. CD155 expression was significantly higher in TNBC compared to luminal subtypes (\\u003cb\\u003eFig. S2A\\u003c/b\\u003e). HER2-positive cell lines displayed intermediate CD155 expression relative to hormone receptor-positive lines, with SKBR3 cells exhibiting the highest CD155 levels among HER2-positive lines (\\u003cb\\u003eFig. S2B).\\u003c/b\\u003e Surface flow cytometry analysis further revealed that CD155 was expressed in all three HER2-positive breast cancer cell lines tested (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eA). Among them, SKBR3 cells showed the highest expression levels of both CD155 and HER2. Moreover, a significant positive correlation was observed between CD155 and HER2 expression levels (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eB).\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003eAs CD155 is a transmembrane protein frequently associated with intracellular signaling cascades, we hypothesized that it may physically interact with the HER2 receptor at the plasma membrane. Immunofluorescence analysis confirmed colocalization of CD155 and HER2 in both low and high CD155 expressing HER2-positive cell lines (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eC). This membrane-level colocalization was further validated using a Bimolecular Fluorescent Complementation (BiFC) Assay, which only yields a signal when direct protein-protein interaction occurs. Using BiFC, we demonstrated that CD155 directly interacts with HER2, with the BiFC signal localized at the plasma membrane, supporting that this interaction occurs at the cell surface (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eD).\\u003c/p\\u003e\\u003cp\\u003eWe next examined whether this interaction was exclusive to HER2 or extended to other receptor tyrosine kinases. CD155 was found to interact with all members of the HER family (EGFR, ERBB3, ERBB4), as well as IGFR and PDGFR (\\u003cb\\u003eFig. S3B\\u003c/b\\u003e). No interaction was detected with c-MET, suggesting that CD155 may function as a chaperone-like molecule for a specific subset of membrane receptors. Notably, the interaction between CD155 and HER2 persisted even after treatment with both lapatinib and trastuzumab (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eD, \\u003cb\\u003eFig. S3A\\u003c/b\\u003e), indicating that it is independent of receptor blockade.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003eTo investigate the regulatory role of CD155 in HER2 signaling, we first performed transient knockdown of CD155 in SKBR3 cells using siRNA. Acute CD155 depletion led to a reduction in both phosphorylated HER2 and total HER2 protein levels (\\u003cb\\u003eFig. S4A\\u003c/b\\u003e). Notably, when control and CD155-knockdown cells were treated with anti-HER2 agents (lapatinib or trastuzumab), HER2 levels were further reduced in control cells but remained unchanged in CD155-knockdown cells (\\u003cb\\u003eFig. S4A\\u003c/b\\u003e).\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003eTo validate these findings, we generated stable CD155-knockdown SKBR3 polyclonal lines using shRNA. Two independent polyclonal populations showed effective CD155 depletion (\\u003cb\\u003eFig. S4B\\u003c/b\\u003e). However, unlike the acute siRNA knockdown, stable CD155 knockdown did not alter total HER2 protein levels (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eE), suggesting that the reduction observed in transient knockdown was likely due to acute CD155 loss, whereas in long-term knockdown lines, compensatory signaling may mitigate HER2 downregulation. Consistent with this, flow cytometry analysis revealed no change in cell-surface HER2 expression in the stable knockdown cells (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eF).\\u003c/p\\u003e\\u003cp\\u003eInterestingly, CD155 knockdown in these stable lines did not result in a reduction in phosphorylated HER2 levels under basal conditions (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eG). To assess whether CD155 influences HER2-related signaling in the context of anti-HER2 therapy, we treated control and knockdown cells with lapatinib or trastuzumab and probed key EGFR/HER2 pathway components (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eH). Total EGFR levels were slightly reduced in knockdown cells, but the most notable effect was a clear decrease in pAKT and pERK1/2 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eH).\\u003c/p\\u003e\\u003cp\\u003eGiven that HER2 is an orphan receptor requiring heterodimerization with other HER family members for activation, we serum starved and stimulated cells with EGF to assess downstream signalling upon ligand stimulation (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eI). While phosphorylation of ERK1/2 and AKT after EGF stimulation was not substantially altered, HER2 activation, marked by pHER2 itself was partially compromised in CD155-depleted cells (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eI).\\u003c/p\\u003e\\u003cp\\u003eFinally, to determine the functional relevance of these signaling changes, we measured trastuzumab sensitivity in CD155 parental and -knockdown cells. Trastuzumab treatment significantly reduced clonogenic growth in control cells; however, CD155-knockdown cells were still able to form colonies, suggesting that CD155 is required for optimal anti-HER2 sensitivity (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eJ)\\u003c/p\\u003e\\u003cp\\u003eTogether, these results indicate that CD155 is required to maintain active HER2-AKT signalling in HER2-positive breast cancer cells. Loss of CD155 dampens the pathway, leading cells to bypass HER2 dependency and resist trastuzumab.\\u003c/p\\u003e\\n\\u003ch3\\u003eCD155 is required for optimal T cell-mediated killing\\u003c/h3\\u003e\\n\\u003cp\\u003eBecause loss of CD155 impaired HER2 signaling, we next examined whether this effect might be related to alterations in HER2 receptor regulation at the plasma membrane. Receptor clustering and internalization are well-established mechanisms that modulate receptor signaling output(\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003eIn parental SKBR3 cells, trastuzumab treatment led to a reduction in surface HER2 levels within 2 hours (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA). In contrast, CD155 surface levels remained unchanged, suggesting that CD155 does not undergo the same pattern of antibody-induced internalization as HER2. Consistent with this observation, flow cytometry analysis showed that treatment with either trastuzumab or lapatinib reduced HER2 expression to a similar extent in control and CD155-knockdown cells (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eB).\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003eWhen CD155-knockdown and control cells were treated with trastuzumab (10 \\u0026micro;g/mL) for 24 hours, most control cells exhibited extensive HER2 internalization, whereas CD155-depleted cells retained higher levels of HER2 on the plasma membrane (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eC). Because the antibody treatment normally promotes receptor clustering, we next examined HER2 spatial distribution following 30 minutes of trastuzumab exposure. In control cells, trastuzumab induced prominent HER2 clustering at the membrane and subsequent internalization (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eD). In contrast, CD155-knockdown cells displayed a pre-existing clustered pattern that was not further enhanced by trastuzumab (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eD), suggesting that CD155 facilitates proper HER2 receptor organization and dynamic redistribution in response to antibody binding.\\u003c/p\\u003e\\u003cp\\u003eTo assess the functional impact of CD155 loss on HER2-mediated immune responses, we performed a HER2-specific TCR-based T cell killing assay, which relies on efficient presentation of HER2-derived peptides on the plasma membrane. CD155-knockdown SKBR3 cells were significantly less susceptible to TCR-mediated cytolysis compared with control cells (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eE), indicating impaired T cell killing efficiency. A similar trend was observed in antibody-dependent cytotoxicity assays, in which na\\u0026iuml;ve peripheral blood mononuclear cells (PBMCs) from healthy donors were co-cultured with tumor cells in the presence of trastuzumab. In this context, CD155-depleted cells showed reduced susceptibility to immune-mediated lysis, with a noticeable delay in killing kinetics relative to controls (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig7\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eF). Collectively, these results demonstrate that CD155 supports proper HER2 receptor localization and dynamics at the plasma membrane, enhancing the effectiveness of both TCR-mediated and antibody-dependent cytotoxicity, and highlighting its role in modulating tumor cell sensitivity to immune-based therapies.\\u003c/p\\u003e\\u003cp\\u003e\\u003cb\\u003eCD155 is required for the therapeutic effect of anti-HER2 mAb in a murine breast cancer model\\u003c/b\\u003e\\u003c/p\\u003e\\u003cp\\u003eHaving established that CD155 regulates HER2 receptor localization, signaling, and T cell-mediated killing in human breast cancer cells, we next investigated whether these effects extend \\u003cem\\u003ein vivo\\u003c/em\\u003e. To address this, we employed syngeneic murine models, which provide an intact immune system necessary to evaluate both tumor growth dynamics and immune-based therapeutic responses to anti-HER2 monoclonal antibody (mAb) therapy.\\u003c/p\\u003e\\u003cp\\u003eBoth H2N100 and TUBO tumor cells expressed comparable high levels of HER2 and CD155, as confirmed by flow cytometry (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eA). Consistent with our observations in the human cell line SKBR3, CD155 expression was not significantly altered after treatment with anti-HER2 mAb therapy in murine tumour cell lines (\\u003cb\\u003eFig. S5A\\u003c/b\\u003e). Next, to evaluate the role of CD155 in anti-HER2 mAb therapy, we treated both anti-HER2 sensitive H2N100 and relatively resistant TUBO tumors(\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e) with anti-HER2 mAb in the presence or absence of a CD155-blocking mAb. Anti-HER2 mAb therapy alone significantly inhibited primary tumor growth in both H2N100 (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eB) and TUBO (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eC) models. However, co-administration of the CD155-blocking mAb abolished the anti-tumor efficacy of anti-HER2 mAb therapy in both tumour types (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eB, C). Similarly, CD155 blockade markedly reduced the therapeutic benefit of anti-HER2 mAb treatment in the H2N100 experimental metastasis model (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eD). Anti-CD155 treatment alone has modest decrease in primary tumor growth and metastasis in line with its role in promoting tumor growth(\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e).\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003eIn agreement with previous reports(\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e), tumor-infiltrating lymphocyte (TIL) analysis of H2N100 tumours revealed that anti-HER2 mAb therapy increased the frequency of intratumoral T cells, particularly CD8⁺ T cells in H2N100 tumours. Notably, the frequency of CD8\\u003csup\\u003e+\\u003c/sup\\u003e T cells was significantly reduced when anti-HER2 mAb was combined with CD155 blockade (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eE, F). Given that CD155 ligand TIGIT is predominantly expressed on NK cells and intratumoral regulatory T cells (Tregs), and has been implicated in tumour immune evasion(\\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e), we also analyzed TIGIT\\u0026thinsp;+\\u0026thinsp;Tregs populations in tumours populations across different treatment groups. However, no significant differences were observed in TIGIT\\u0026thinsp;+\\u0026thinsp;Tregs frequencies (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig8\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003eF). Collectively, these results suggest that CD155 is essential for the anti-tumour activity of anti-HER2 therapy \\u003cem\\u003ein vivo\\u003c/em\\u003e, likely through supporting CD8⁺ T cell-mediated immune responses. Loss or blockade of CD155 function compromises both local tumour control and metastatic suppression by impairing the immune component of anti-HER2 therapeutic efficacy.\\u003c/p\\u003e\\n\\u003ch3\\u003eTumour cell expression of CD155 is essential for the anti-tumour efficacy of anti-HER2 mAb therapy\\u003c/h3\\u003e\\n\\u003cp\\u003eCD155 expression is not only restricted to tumour cells but is also detected on myeloid and endothelial cells [20]. Previous studies have shown that tumor cell derived CD155 plays a particularly important role in regulating anti-tumour immune responses [24, 27, 28]. To directly evaluate the contribution of tumour cell-intrinsic CD155 to the therapeutic efficacy of anti-HER2 mAb, we used the CRISPR-Cas9 system (\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e) to delete CD155 in H2N100 and TUBO tumour cells. Two independent single-guide RNAs (sgRNAs; sg2 and sg6) targeting CD155 were designed (\\u003cb\\u003eTable S1\\u003c/b\\u003e), and CD155 deletion was confirmed by immunoblotting (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig10\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eA. \\u003cb\\u003eC\\u003c/b\\u003e). Importantly, CD155 loss did not alter HER2 protein expression (\\u003cb\\u003eFig. S5B\\u003c/b\\u003e).\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003cem\\u003eIn vivo\\u003c/em\\u003e, anti-HER2 mAb treatment significantly inhibited the growth of CD155-expressing (sg control) H2N100 and TUBO tumours but failed to reduce tumour growth in mice bearing CD155-deficient tumours (sg2 and sg6) (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig10\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eB, D). Similarly, anti-HER2 mAb markedly reduced both the number of lung metastases and lung metastatic burden (lung weight) in mice harboring CD155-positive TUBO tumours, whereas no anti-metastatic effect was observed in those bearing CD155-deficient tumors (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig10\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eE, F).\\u003c/p\\u003e\\u003cp\\u003eWe next examined whether tumor cell CD155 is required for the efficacy of other immune checkpoint-based therapies. CD155 knockout and parental tumor cells exhibited comparable levels of PD-L1 expression and there was no correlation between CD155 and PD-L1 expression in breast cancer samples (\\u003cb\\u003eFig. S6A, B\\u003c/b\\u003e). Consistent with previous findings demonstrating that anti-PD-L1 monoclonal antibody (mAb) suppresses H2N100 tumour growth (\\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e), we observed that anti-PD-L1 treatment reduced primary tumor growth regardless of tumor cell CD155 expression levels (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig10\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eG). Given that the successful anti-HER2 therapy relies on both tumour-intrinsic factors and host immune effector mechanisms [5, 9], we further assessed therapeutic efficacy in the absence of an intact immune system. H2N100 sg control and CD155-deficient (sg2) tumour cells were implanted into immunocompetent wild-type (WT) or immunodeficient NOD-SCID-γ (NSG) mice, which lack mature B cells, T cells, and NK cells. As expected, the number of lung metastases in NSG mice was significantly higher than the WT mice in the absence of immune control in NSG mice. In WT mice bearing CD155-positive tumors, anti-HER2 mAb reduced lung metastases by nearly 80%, whereas the reduction in NSG mice was limited to ~\\u0026thinsp;30% (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig10\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eH, I), consistent with the loss of antibody-dependent cellular cytotoxicity (ADCC) in the absence of immune effector cells in NSG mice. In contrast, anti-HER2 therapy failed to reduce metastases in either WT or NSG mice when tumours lacked CD155 expression (Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig10\\\" class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eH, I). Collectively, these data demonstrate that tumour cell CD155 is indispensable for the full therapeutic efficacy of anti-HER2 mAb, acting cooperatively with an intact host immune system to mediate both primary tumor suppression and metastatic control.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eCD155 is overexpressed in many cancer types, where its high expression is generally associated with a worse prognosis due to its immunosuppressive function through interactions with TIGIT and CD96 receptors on immune cells(\\u003cspan additionalcitationids=\\\"CR42\\\" citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e41\\u003c/span\\u003e\\u0026ndash;\\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e43\\u003c/span\\u003e). Indeed, CD155 has been implicated in promoting resistance to chemotherapies and ICI-based therapies(\\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e). In line with this, a clinical trial targeting CD155 in solid cancers is currently ongoing(\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e). Importantly, our study cautions against the indiscriminate use of CD155 blockade and instead identifies CD155 as a favourable prognostic biomarker of response to anti-HER2 therapies.\\u003c/p\\u003e\\u003cp\\u003eWe demonstrate that tumor-intrinsic CD155 is a critical determinant of HER2-targeted antibody efficacy, as anti-HER2 therapy fails to reduce tumour growth and lung metastatic burden in the absence of tumour CD155. Consistent with these findings, breast cancers patients with high HER2 and CD155 expression showed improved survival compared to patients with high HER2 but low CD155 expression. Conversely, low CD155 expression correlated with better outcomes in cancer patients receiving anti-PD-1 or anti-CTLA4 therapy, highlighting the complex context-dependent immunomodulatory role of CD155 as a predictive biomarker.\\u003c/p\\u003e\\u003cp\\u003eWhile CD155\\u0026rsquo;s immunosuppressive roles are well-established, its tumour intrinsic role in regulating other oncogenic signalling pathways, particularly HER2 has been largely unexplored. Our findings reveal that CD155 directly supports HER2 receptor organization and downstream signaling. Binding of anti-HER2 antibodies such as trastuzumab to HER2 receptor promotes HER2 clustering and internalization and here we demonstrate that CD155 facilitates these receptor dynamics. Reduced levels of phosphorylated Her2 (pHER2) and phosphorylated AKT (pAKT) in CD155 KO tumour cells indicate that CD155 supports downstream oncogenic HER2 signalling likely through its role in receptor clustering and internalization. These findings expand the known role of CD155 beyond immune modulation, implicating it as a structural regulator of oncogenic receptor organization and trafficking.\\u003c/p\\u003e\\u003cp\\u003eInterestingly, we observed that in CD155-deficient cells, HER2 receptors exhibited constitutive clustering at the plasma membrane, even in the absence of antibody stimulation. In contrast, control cells displayed a diffuse HER2 distribution that underwent pronounced clustering and subsequent internalization upon trastuzumab treatment. This finding suggests that CD155 is required to maintain the dynamic organization and responsiveness of HER2 at the cell surface. Constitutive clustering in CD155-deficient cells may reflect aberrant receptor confinement within rigid membrane domains or disrupted interactions with membrane scaffolding components, resulting in reduced receptor mobility. Despite being pre-clustered, these receptors were functionally inactive, as evidenced by decreased phosphorylation of HER2 and AKT. Therefore, the inability of trastuzumab to further induce receptor clustering or internalization likely reflects a loss of receptor plasticity rather than simple loss of expression. This \\u0026ldquo;locked\\u0026rdquo; HER2 conformation provides a mechanistic explanation for impaired HER2 signaling and therapeutic resistance observed in the absence of CD155.\\u003c/p\\u003e\\u003cp\\u003eIn human HER2-positive SKBR3 breast cancer cells, CD155 depletion impaired T cell-mediated killing in both HER2-specific TCR assays and PBMC-trastuzumab co-cultures supporting the concept that increased HER2 clustering enhances anti-HER2 immune activity by concentrating the target antigen at the tumor cell surface. \\u003cem\\u003eIn vivo\\u003c/em\\u003e, both antibody-mediated blockade and CRISPR/Cas9-mediated deletion of CD155 in HER2-positive murine tumours abolished the anti-tumour and anti-metastatic efficacy of anti-HER2 mAb therapy, despite preserved HER2 expression. The requirement for CD155 was shown to be therapy-specific as anti-PD-L1 mAb retained full anti-tumour activity against CD155-deficient tumours, whereas anti-HER2 mAb efficacy was dependent on tumour CD155 expression. This distinction likely reflects differences in the mechanistic basis of action for each therapy. While PD-L1 blockade primarily reinvigorates exhausted T cells to increase anti-tumor T cell activity, HER2-targeted antibodies rely on both tumour cell-intrinsic HER2 receptor modulation and oncogenic signalling, and immune cell-dependent antibody-dependent cellular cytotoxicity (ADCC). Supporting this, anti-HER2 therapy reduced lung metastases less effectively in immunodeficient NSG mice compared with wild-type mice, consistent with loss of ADCC in the absence of NK and T cells. Strikingly, loss of tumor cell CD155 abolished anti-HER2 efficacy in both WT and NSG mice, suggesting that CD155\\u0026rsquo;s contribution to therapeutic response is predominantly tumor intrinsic. Future studies should explore whether the mechanism by which CD155 promotes receptor clustering and enhances the efficacy of receptor-targeted therapy is unique to HER2 or extends to other receptor tyrosine kinases such as EGFR, VEGFR and c-MET.\\u003c/p\\u003e\\u003cp\\u003eOverall, by integrating both human and murine tumour models, with clinical datasets from breast cancer patients, this work provides several key insights. First, CD155 exerts a tumor-intrinsic role essential for the \\u003cem\\u003ein vivo\\u003c/em\\u003e efficacy of HER2-targeted monoclonal antibodies, Second, CD155 regulates HER2 receptor dynamics, linking a cell adhesion molecule to oncogenic receptor clustering and internalization. Notably, the loss of CD155 resulted in constitutive, antibody-unresponsive HER2 clustering, revealing that CD155 maintains receptor plasticity required for proper signaling and therapeutic responsiveness.Third, CD155 has context-specific prognostic implications across cancer therapies, acting as a positive predictor of anti-HER2 response but a negative indicator in settings of immune checkpoint inhibition and chemotherapy.\\u003c/p\\u003e\\u003cp\\u003eThese findings raise multiple translational opportunities. First, CD155 expression could be explored as part of a composite biomarker panel to guide patient selection for HER2-targeted therapy. Second, strategies that enhance or stabilize CD155 expression in HER2-positive tumors may potentiate trastuzumab efficacy and overcome therapeutic resistance. Third, elucidating the molecular interface between CD155 and HER2 clustering machinery may identify new druggable targets. Finally, the contrasting roles of CD155 in anti-HER2 versus ICI or chemotherapy responses highlight the need for therapy-tailored combination strategies that consider CD155 status to maximize clinical benefit while minimizing unnecessary toxicity.\\u003c/p\\u003e\\u003cp\\u003eIn conclusion, CD155 is a pivotal modulator of HER2 receptor biology and immune effector engagement acting as both a mechanistic driver of HER2-targeted antibody efficacy and a candidate biomarker for precision oncology.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgements\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eWe gratefully acknowledge the QIMR Berghofer Animal Research Facility for their support in breeding and maintaining the mice used in this study. We also thank the QIMR Berghofer and Mater Research Imaging and Flow Cytometry Facilities for their expert technical assistance, which was invaluable for the experiments. We are particularly grateful to the laboratory of Prof. Mark Smyth for generously providing key reagents and materials essential for this study. We also acknowledge the Rajiv Khanna Laboratory for their valuable contribution to the cytolysis activity experiments. D.M. acknowledges the “Walk to End Women’s Cancers” grant for their generous financial support, which made this work possible.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthors contribution:\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConception and design:\\u003c/strong\\u003e D. Mittal, M. Kalimutho, K.K. Khanna\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAcquisition of data:\\u003c/strong\\u003e M. Kalimutho, D. Mittal, H. Shankar, W. Shi, G. Ambalathingal, S. Latham\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAnalysis and interpretation of data:\\u003c/strong\\u003e D. MIttal, M. Kalimutho, H. Shankar, W. Shi, G. Ambalathingal, S. Latham, K.K. Khanna\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eManuscript writing:\\u003c/strong\\u003e D. Mittal, M. Kalimutho\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eStudy supervision:\\u003c/strong\\u003e D. Mittal, M. Kalimutho, K.K. Khanna, J. Beesley, D. Croucher\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConsent for publication:\\u003c/strong\\u003e All authors consent to this manuscript for publication.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eData Availability and Material:\\u0026nbsp;\\u003c/strong\\u003eThe datasets generated and/or analyzed during the current study are included in this published article (and its supplementary information files) and all the raw data available from the corresponding author on reasonable request.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests:\\u003c/strong\\u003e The authors declare no potential conflicts of interest.\\u003c/p\\u003e\"},{\"header\":\"Materials and Methods\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eMice\\u0026nbsp;\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eBalb/c wild type (WT) mice were purchased from the Walter and Eliza Hall Institute for Medical Research or bred in house. All mice including Balb/c.NOD\\u003csup\\u003e-/-\\u003c/sup\\u003egc\\u003csup\\u003e-/-\\u0026nbsp;\\u003c/sup\\u003e(NSG) mice were bred and maintained at the QIMR Berghofer Medical Research Institute and used from the age of 8 weeks. All experiments were approved by the QIMR Berghofer Medical Research Institute Animal Ethics Committee.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eCell culture\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eMouse ErbB2-positive H2N100 and TUBO tumor cells were generated from female BALB/c MMTV-ErbB2/neu transgenic mice and cultured as described previously(44) [42]. H2N100 and H2NB2 cells were derived in the laboratory of Prof. Mark J. Smyth (QIMR Berghofer Medical Research Institute, Herston, Queensland, Australia).\\u003c/p\\u003e\\n\\u003cp\\u003eTUBO cells were maintained in complete DMEM supplemented with 10% heat-inactivated fetal calf serum (Thermo Scientific), 1X GlutaMAX, 50 U/mL penicillin, 100 \\u0026mu;g/mL streptomycin, and 10 mM HEPES (Sigma-Aldrich). H2N100 and H2NB2 cells were cultured in RPMI supplemented with 10% heat-inactivated fetal calf serum, 1X GlutaMAX, 50 U/mL penicillin, 100 \\u0026mu;g/mL streptomycin, 1 mM sodium pyruvate (Gibco-Life Technologies), 10 mM HEPES, and 1% non-essential amino acids, and incubated at 37\\u0026deg;C in a 5% CO₂ incubator.\\u003c/p\\u003e\\n\\u003cp\\u003eHuman breast cancer cell lines SKBR3, MDA-MB-361, and T47D were purchased from ATCC and cultured according to ATCC recommendations. All cell lines were used within fewer than 10 freeze-thaw cycles and were routinely tested for mycoplasma contamination.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eCD155 KO with CRISPR-Cas9 in tumor cells\\u003c/em\\u003e\\u003c/strong\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eCD155 small guide RNAs (sgRNAs) were designed following the guidelines provided by the Broad Institute (http://www.genome-engineering.org/). Briefly, CD155 sgRNAs were cloned into the PX330 vector (Addgene, #42230). H2N100 and TUBO tumor cells were transfected with either the PX330-CD155 sgRNA plasmids or the empty PX330 vector, along with a GFP-expressing plasmid (pRp-GFP). GFP-positive cells were sorted using a FACSAria II Cell Sorter (BD Biosciences). After 7\\u0026ndash;10 days of culture, CD155-deficient tumor cells were further sorted to establish CD155 knockout (KO) cell lines. The sequences of primers used for CD155 sgRNAs are listed in Supplementary Table 1.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003esiRNA-Mediated Knockdown and Western Blot Analysis\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eSKBR3 cells were seeded in 10-cm dishes at a density of 5 \\u0026times; 10⁵ cells per well and allowed to attach overnight under standard culture conditions. Reverse transfection was performed using 20 nM of the indicated small interfering RNAs (siRNAs) (Table S1) complexed with Lipofectamine RNAiMAX according to the manufacturer\\u0026rsquo;s protocol. Cells were incubated for 24 hours post-transfection to allow for efficient gene silencing. Following incubation, cells were lysed in 7M urea buffer and protein concentrations were determined using the BCA assay. Equal amounts of protein were resolved by SDS-PAGE, transferred to nitrocellulose membranes, and probed with antibodies specific for CD155 and appropriate loading controls. Protein expression was detected using chemiluminescent substrates, confirming the efficiency of CD155 knockdown.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eColony formation assays\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eA total of 1,000 tumor cells were seeded in 24-well plates, followed by treatment with trastuzumab. The cells were incubated for an additional 14 days to assess colony survival. Colonies were then fixed with 0.05% crystal violet for 30 minutes, washed, and the stain was extracted using Sorenson buffer.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eImmunoblotting\\u0026nbsp;\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eImmunoblotting was performed as described previously\\u0026nbsp;(45). Briefly, protein lysates were prepared from cells and quantified using BCA protein assay. Equal amounts of protein were resolved by SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked with 5% non-fat dry milk in TBST for 1 hour at room temperature and then incubated overnight at 4\\u0026deg;C with primary antibodies against the proteins of interest. Following washes with TBST, membranes were incubated with appropriate HRP-conjugated secondary antibodies for 1 hour at room temperature. Protein bands were visualized using the SuperSignal Chemiluminescent ECL Plus detection system (Amersham) according to the manufacturer\\u0026rsquo;s instructions.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eADCC and TCR-Based Killing Assays\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eImmune-mediated tumor cell killing was assessed using antibody-dependent cellular cytotoxicity (ADCC) and T-cell receptor (TCR)-based killing assays. For the ADCC assay, peripheral blood mononuclear cells (PBMCs) were used as effector cells and co-cultured with target tumor cells at defined effector-to-target (E:T) ratios in the presence of trastuzumab. Cytotoxicity was monitored in real time using the xCELLigence system, and killing efficiency was calculated relative to untreated controls.For the TCR-based killing assay, HER2-expressing tumor cells were co-cultured with HER2-specific TCR-engineered T cells at defined E:T ratios. Cytotoxicity reflects antigen-specific, MHC-restricted T-cell killing, independent of the endogenous HER2 receptor, and was similarly measured using the xCELLigence system. Specific lysis was calculated relative to untreated controls.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eImmunofluorescence Assays\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eCells were plated on coverslips and incubated overnight. The coverslips were then fixed with 4% paraformaldehyde in PBS for 15 minutes, followed by permeabilization with 0.5% Triton X-100-PBS for 15 minutes. To block non-specific binding, the coverslips were treated with 2% filtered bovine serum albumin (BSA). Primary antibodies were diluted in the blocking solution and applied to the coverslips overnight at 4\\u0026deg;C. Subsequently, Alexafluor conjugated secondary antibodies, diluted at 1/300 in the blocking solution, were used for staining and incubated for 45 minutes at 37\\u0026deg;C in a humidifier chamber. After washing, the coverslips were counterstained with DAPI (diluted 1/500 in blocking buffer, stock concentration 1mg/ml) and mounted in Prolong Gold mounting medium. The slides were visualized using a GE DeltaVision Deconvolution microscope, and image analysis was performed using Image J software.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eBimolecular Fluorescent Complementation (BiFC) Assay\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTo investigate receptor interactions with HER2 using BiFC, the pDEST-ERBB2-V1 plasmid was used as the \\u0026ldquo;bait.\\u0026rdquo; When co-transfected with the complementary pDEST-V2 plasmid encoding CD155, a positive interaction was indicated by the appearance of a fluorescent signal from the reconstituted Venus protein, as described previously(46). T47D cells were seeded onto collagen-coated 35-mm imaging dishes at a density of 2 \\u0026times; 10⁵ cells per dish. Cells were then transfected with either 2 \\u0026mu;g of Venus control plasmid or 1 \\u0026mu;g each of PVR/CD155-V1 and ERBB2-V2, along with other receptor tyrosine kinase (RTK) constructs, using JetPrime transfection reagent, according to the manufacturer\\u0026rsquo;s instructions and as previously described(47). Following transfection, samples were treated with either lapatinib (100 nM) or trastuzumab (5 \\u0026mu;g/mL) for 1 hour or 16 hours. Nuclei were counterstained with Hoechst live-cell imaging reagent, fixed with 4% paraformaldehyde, and washed with PBS. Images were acquired using a Leica SP8 confocal microscope equipped with a 63\\u0026times;/1.4 NA objective lens. Scale bars represent 10 \\u0026mu;m.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eIn vivo studies\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eH2N100 and TUBO (5 x 10\\u003csup\\u003e5\\u003c/sup\\u003e cells) tumor cells were injected subcutaneously into Balb/c WT mice. Mice were treated with four intraperitoneal injections of 10 mg anti-ErbB2/HER2 (7.16.4) or control immunoglobulin (cIg, 2A3) twice a week for two weeks after the tumors reached a size of 60-80 mm\\u003csup\\u003e2\\u0026nbsp;\\u003c/sup\\u003e(~ day 13 for TUBO and day 18 for H2N100). The anti-rat HER2 mAb (clone 7.16.4) hybridoma was generously provided by Mark I. Greene, University of Pennsylvania. A subset of mice additionally received 250\\u0026nbsp;mg of anti-CD155 mAb (4.24) or anti-PD-L1 mAb (BioXCell, 10F.9G2) as indicated.\\u0026nbsp;Tumor growth was monitored and tumor size was recorded with a digital caliper every two to three days as the product of two perpendicular diameters. For lung metastases studies, H2N100 and TUBO sg control and sg2-CD155 KO tumor cells (2 x 10\\u003csup\\u003e5\\u003c/sup\\u003e cells) were injected intravenously to WT mice or NOD\\u003csup\\u003e-/-\\u003c/sup\\u003egc\\u003csup\\u003e-/-\\u003c/sup\\u003emice and treated on day 8 with 10\\u0026nbsp;mg of control Ig (clone 2A3) or anti-HER2 (clone 7.16.4) mAbs intraperitoneally. Lungs were harvested on day 16 (TUBO) or day 20 (H2N100), washed in PBS, and fixed in Bouin\\u0026apos;s solution for 24 hours before counting tumor nodules using a dissection microscope.\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eFlow cytometry analysis\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eFor tumor infiltrating lymphocytes analysis, H2N100 tumors were cut into small pieces and digested in medium containing RPMI with collagenase II (1 mg/mL) and DNAse (20 \\u0026mu;g/mL) for 45 minutes. Samples were filtered through 70 \\u0026mu;m filter, washed in PBS and lysed for red blood cells by ACK lysis buffer. After washing with PBS, single-cell suspensions were incubated for 20 minutes in Fc blocking buffer (2.4G2 antibody) and stained with viability stain Zombie Yellow (Biolegend) and following fluorescence-conjugated mAbs diluted in FACS buffer (2% FCS in PBS): anti-mouse-CD45.2 (104), TCR-\\u0026beta; (H57-597), CD4 (RM4-5), CD8 (53.6.7) and TIGIT (1G9) for 30 minutes in ice. Samples were fixed and permeabilized using fixation permeabilization buffer (Thermo Fisher Scientific) for 20 min and stained with anti-mouse FoxP3 (FJK-16S). For flow cytometry of cultured tumor cells, cells were treated or not with 1\\u0026nbsp;mg/ml of anti-ErbB2 mAb (7.16.4) or 1\\u0026nbsp;mg/ml of control IgG (2A3) or were stimulated with 10 ng/ml of IFN-g\\u0026nbsp;(R\\u0026amp;D Systems) for 24 h, 48 h or 72 h. Samples were collected, washed with FACS buffer and stained with viability stain Zombie Yellow (Biolegend) and following antibodies: anti-mouse-PD-L1 (MIH5), HER2 (7.16.4), CD155 (TX56) for 30 minutes in ice. All mAbs were purchased either from Thermo Fisher Scientific or Biologend. Samples were acquired on LSR Fortessa IV Flow Cytometer (BD Biosciences) and data was analyzed on FlowJo V10 (Treestar).\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eExpression data from clinical samples\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eGEPIA (Gene Expression Profiling Interactive Analysis) is an interactive web server for analyzing the RNA sequencing expression data of 9,736 tumors and 8,587 normal samples from the TCGA and the GTEx projects(48). \\u0026nbsp; cBioPortal was used to explore CD155 expression and its relation with survival analysis in HER2+ breast cancer samples(49). Briefly, breast cancer patients with high Her2+ expression were divided into CD155-low and CD155-high expression cancers to draw Kaplan\\u0026ndash;Meier based overall and disease-free survival. Kmplotter (Kaplan-Meier Plotter) is a Web-based gene-expression database that includes more than 6,000 breast cancer patients with clinical and survival data (50). Survival data was plotted using Km plotter for Her2+ and Her2- breast cancer types. cProSite (Cancer Proteogenomic Data Analysis Site) is a web-based, interactive platform for analyzing cancer data from the National Cancer Institute\\u0026apos;s Clinical Proteomic Tumor Analysis Consortium (CPTAC) Ref: Wang D et.al. Journal of Biotechnology and Biomedicine (2023).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003e\\u003cem\\u003eStatistical analysis\\u003c/em\\u003e\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAll comparisons between samples were evaluated using unpaired \\u003cem\\u003et\\u003c/em\\u003e-tests or one-way ANOVA with Tukey\\u0026rsquo;s post hoc test, unless otherwise stated in the figure legends. Statistical analyses were performed using GraphPad Prism version 8.0 (GraphPad Software, La Jolla, CA, USA). Where applicable, statistical significance is indicated as follows: \\u003cem\\u003eP\\u003c/em\\u003e\\u0026thinsp;\\u0026le;\\u0026thinsp;0.05 (*\\u003cem\\u003e), P\\u0026thinsp;\\u0026le;\\u0026thinsp;0.01 (**), P\\u0026thinsp;\\u0026le;\\u0026thinsp;0.001 (***\\u003c/em\\u003e) \\u003cem\\u003eand P\\u0026thinsp;\\u0026le;\\u0026thinsp;0.0001 (****\\u003c/em\\u003e). \\u0026nbsp;Data are expressed as mean\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;standard error of the mean (SEM) or standard deviation (SD), as indicated.\\u003cstrong\\u003e\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eC. Gutierrez, R. Schiff, HER2: biology, detection, and clinical implications. \\u003cem\\u003eArch Pathol Lab Med\\u003c/em\\u003e \\u003cstrong\\u003e135\\u003c/strong\\u003e, 55-62 (2011).\\u003c/li\\u003e\\n\\u003cli\\u003eG. Valabrega, F. Montemurro, M. 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Gyorffy\\u003cem\\u003e et al.\\u003c/em\\u003e, An online survival analysis tool to rapidly assess the effect of 22,277 genes on breast cancer prognosis using microarray data of 1,809 patients. \\u003cem\\u003eBreast cancer research and treatment\\u003c/em\\u003e \\u003cstrong\\u003e123\\u003c/strong\\u003e, 725-731 (2010).\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":true,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":true,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"HER2, CD155, Trastuzumab, Breast Cancer\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-7994408/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-7994408/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003eCD155 (poliovirus receptor, PVR) is frequently overexpressed across cancers and has been associated with tumor progression, poor prognosis, and therapy resistance. Here, we identify CD155 as a modulator of HER2-targeted monoclonal antibody efficacy. Analysis of clinical datasets revealed that high CD155 expression correlates with overall, progression-free, and disease-specific survival in breast cancer, and has potential as a predictive biomarker for anti-HER2 therapy. Mechanistically, CD155 co-localizes with HER2 at the tumor cell membrane and modulates HER2-dependent signaling, including AKT phosphorylation and receptor clustering. Genetic or antibody-mediated loss of CD155 significantly impaired trastuzumab-mediated cytotoxicity \\u003cem\\u003ein vitro\\u003c/em\\u003e and abolished therapeutic efficacy in both primary and metastatic HER2-positive breast cancer models \\u003cem\\u003ein vivo\\u003c/em\\u003e, without altering HER2 expression. Moreover, CD155 depletion reduced CD8⁺ T cell infiltration, highlighting its dual role in modulating HER2 receptor biology and shaping the tumor immune microenvironment. Collectively, these findings position CD155 as a determinant of trastuzumab efficacy and a promising biomarker to guide patient stratification and optimize therapeutic outcomes in HER2-positive breast cancer.\\u003c/p\\u003e\",\"manuscriptTitle\":\"CD155/PVR as a determinant of anti-HER2 therapy sensitivity in breast cancer\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-11-14 07:17:41\",\"doi\":\"10.21203/rs.3.rs-7994408/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"baeea443-f9b1-4125-8678-6d6085531054\",\"owner\":[],\"postedDate\":\"November 14th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[{\"id\":57526263,\"name\":\"Biological sciences/Cancer/Breast cancer\"},{\"id\":57526264,\"name\":\"Biological sciences/Cell biology\"},{\"id\":57526265,\"name\":\"Biological sciences/Cancer/Cancer therapy/Drug development\"}],\"tags\":[],\"updatedAt\":\"2025-12-17T02:20:59+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2025-11-14 07:17:41\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-7994408\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-7994408\",\"identity\":\"rs-7994408\",\"version\":[\"v1\"]},\"buildId\":\"XKTyCvWXoU3ODBz1xrDgd\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}