Endothelial PDLIM5 promotes tip cell filopodia formation and tumor angiogenesis by regulating ACTN1/ACTN4-dependent actin bundling

Domain: neuroscience
Published: 2026-01-28
Editorial outcome: major_revision

Abstract
Filopodia formation by tumor endothelial cells (TECs) is critical for tip cell-guided sprouting angiogenesis and tumor growth. However, the cytoskeletal organization that underlies this process remains elusive. Here, we demonstrate that TECs highly express actin-binding genes, with PDZ and LIM domain 5 (PDLIM5), a cytoskeletal protein, significantly upregulated in TECs and correlated with poor patient survival. Endothelial-specific deletion of Pdlim5 inhibits sprouting angiogenesis by disrupting filopodia formation through its interaction with actinin-1/actinin-4 (ACTN1/ACTN4) via its S593/F596 residues, promoting filamentous actin (F-actin) bundling. Pdlim5 knockout not only reduces tumor growth but also normalizes tumor vasculature, alleviates hypoxia, and enhances immunotherapy and chemotherapy responses. These findings highlight the PDLIM5’s role in facilitating tumor angiogenesis via ACTN1/ACTN4-mediated F-actin bundling and tip cell filopodia formation, providing mechanistic insights that may inform future therapeutic strategies targeting this pathway.

Introduction
Neovascularization accelerates malignant tumor growth and distant metastasis1. Tumor endothelial cells (TECs) are critical stromal components of the tumor microenvironment. Abnormal tumor vasculature exacerbates intratumoral hypoxia and drives resistance to chemotherapy and immunotherapy2,3. Sprouting angiogenesis is one of the primary modes of tumor angiogenesis and is characterized by the activation, migration, and proliferation of endothelial cells (ECs)1,4. Endothelial tip cells migrate to the leading edge of nascent vessels to initiate sprouting, whereas stalk cells proliferate to elongate the vessel branches. Recent studies have shown that tip TEC signature is correlated with patient survival5,6. and tip TECs are highly sensitive to vascular endothelial growth factor (VEGF) blockade. Proteomic sequencing analysis demonstrated that TECs highly express cell proliferation- and cytoskeleton-related proteins, which are pivotal for tip cell formation and activation7. However, the functional roles and regulatory mechanisms of tip TECs in orchestrating cytoskeletal dynamics during angiogenesis remain incompletely understood, limiting their translational potential in therapeutic targeting of tumor vasculature.

Endothelial tip cells are characterized by low proliferative activity but high polarization, extending filopodia and lamellipodia to guide nascent sprouts toward hypoxic and nutrient-deprived niches8,9. These cells also express abundant cell surface receptors and matrix-degrading enzymes (e.g., matrix metalloproteinase 2 and matrix metalloproteinase 9), which facilitate extracellular matrix (ECM) degradation and basement membrane (BM) remodeling10. Following tip cells are stalk ECs, which proliferate to facilitate sprout elongation and mediate lumen formation. The spatiotemporal regulation of tip-stalk cell fate is governed by the VEGF-VEGFR-Delta-like ligand 4 (Dll4)-Jagged-Notch signaling axis, which is also modulated by ECM interactions11,12. Tip ECs (TipECs) are characterized by extensive filopodia and lamellipodia that extend into multiple clusters at the apical end for direct vascular sprouting. Cytoskeletal remodeling is highly activated during tip cell specification, with prominent filamentous actin (F-actin) bundles frequently observed at the leading edge of sprouting vessels and the base of filopodia13. Nevertheless, the molecular mechanisms underlying tip cell cytoskeletal reorganization and F-actin polymerization in filopodia during tumor angiogenesis are not fully understood.

In this work, we find that the cytoskeleton-associated protein PDLIM5 is highly expressed in TECs. Endothelial-specific Pdlim5 deletion inhibits sprouting angiogenesis and tumor growth by disrupting filopodia formation and F-actin bundling, mediated though lacking of its interacting with ACTN1/ACTN4. Pdlim5 knockout also normalizes tumor vasculature, alleviates hypoxia, and enhances responses to both immunotherapy and chemotherapy. These findings demonstrate that PDLIM5 mediates tumor angiogenesis via ACTN1/ACTN4-dependent F-actin bundling and tip cell filopodia formation, providing a mechanistic rationale for therapies targeting this pathway.

Results
Elevated actin-binding related genes promote tip cell formation and tumor neovascularization
To investigate the cytoskeletal changes in TECs, we analyzed the expression of genes encoding actin-binding proteins using publicly available single-cell RNA sequencing (scRNA-seq) data of human lung TECs and normal ECs (NECs) (E-MTAB-6308)5. Compared with NECs, 70 actin-binding-related genes were upregulated, including MYO1B, CXCR4, and SYNPO, while 11 genes were downregulated, including SLC6A4 and S100A4 (Fig. 1a, Supplementary Fig. 1a). This finding suggests abnormalities in the TEC cytoskeleton that promote cell migration. Among these genes, we found that the cytoskeleton-related PDLIM family proteins, including PDLIM1, PDLIM7, and PDLIM5, were highly expressed in TECs, indicating an important role for PDLIM family proteins in TECs. PDLIM proteins are known for their roles in the regulation of cytoskeletal organization and signal transduction, usually serving as molecular scaffolds that link signaling molecules to the cytoskeleton. Further analysis revealed that the expression of PDLIM5 in TECs was notably higher than that of other PDLIM family proteins (Supplementary Fig. 1b). PDLIM5, in particular, has been implicated in various cellular processes, including actin dynamics, cell adhesion and migration, and signal transduction14. Previous study found that high PDLIM5 expression in non-small cell lung cancer (NSCLC) cells promoted cell migration and accelerated lung cancer metastasis by regulating epithelial-mesenchymal transition (EMT)15, suggesting that PDLIM5 is an important oncogenic gene. To gain a more comprehensive understanding of PDLIM5 expression in stromal cells within the tumor microenvironment, We compared PDLIM5 levels in stromal cells from tumors versus normal tissues. Beyond tumor cells, PDLIM5 expression was higher in TECs than in normal endothelial cells NECs (Fig. 1b). Similar results were observed in TECs isolated from mammary tumor models and human COLO205 subcutaneous graft tumors (Supplementary Fig. 1c–f).

To further analyze which subtypes of TECs highly expressed PDLIM5, we collected paired cancerous and adjacent normal tissues from seven patients with lung cancer. TECs are subdivided into five subtypes based on their unique gene transcription signatures: Arterial ECs (AECs), which are responsible for the transport of oxygenated blood away from the heart; Venous ECs (VECs), involved in returning deoxygenated blood to the heart; Lymphatic ECs (LECs), which play a role in immune surveillance and fluid balance; Capillary ECs (CapECs), which facilitate the exchange of nutrients, waste, and gases between blood and tissues; and TipECs, which are involved in angiogenesis, guiding the formation of new blood vessels (Fig. 1c, Supplementary Fig. 2a–e). Our results showed that, compared with those in NECs, the proportion of TipECs in TECs increased, whereas the proportion of CapECs and AECs decreased (Fig. 1d), which was consistent with previous studies5. This shift in cell subtype proportions reflected angiogenic phenotype during tumorigenesis. Further analysis revealed that PDLIM5 was upregulated in AECs, CapECs, LECs, and TipECs (Fig. 1e, Supplementary Fig. 2f). Consistent with our data, we re-analyzed the scRNA-seq dataset E-MTAB-6308 using their published methodology5, and PDLIM5 was significantly upregulated in TECs versus NECs across multiple activated subpopulations, including tip cells, artery ECs, immature ECs, and activated postcapillary vein ECs (Supplementary Fig. 2g). Additionally, in clinical lung cancer tissues, we observed higher expression of PDLIM5 in TECs compared to adjacent normal tissue ECs. Elevated PDLIM5 expression in ECs was correlated with poorer survival outcomes (Fig. 1f–h). Beyond lung cancer, patients with high PDLIM5 expression across several other cancer types also exhibited worse survival outcomes (Supplementary Fig. 3). This suggests that PDLIM5 in TECs might play a broader role in cancer progression.

A previous study showed that patients with NSCLC expressing high angiogenic TEC gene set signatures had shorter overall survival5. We hypothesized that the highly expressed cytoskeleton-associated protein PDLIM5 in TECs causes abnormal tip cell function and promotes angiogenesis. To test this hypothesis, we constructed a Lewis lung carcinoma (LLC) subcutaneous graft tumor model in Pdlim5 global-overexpressing (CAG-Pdlim5) mice. Results showed that tumors in CAG-Pdlim5 mice exhibited more rapid growth and more abundant vessels (Fig. 1i–m). These findings suggest that PDLIM5 promotes tumor angiogenesis in TECs.

Endothelial Pdlim5 deletion impedes tumor growth and angiogenesis
To further investigate the effect of endothelial PDLIM5 on tumor angiogenesis, we engineered a conditional knockout mouse model. We crossed Pdlim5flox/flox mice with Cdh5-CreERT2 mice to generate Pdlim5flox/flox; Cdh5 CreERT2 mice (Pdlim5cKO), allowing for specific deletion of Pdlim5 in ECs upon tamoxifen treatment (Supplementary Fig. 4a, b). This strategy allowed us to systematically evaluate the endothelial-specific role of PDLIM5 in tumor angiogenesis while maintaining its physiological function in other cell types. Notably, genetic ablation of endothelial Pdlim5 did not perturb normal tissue homeostasis or vascular integrity, as demonstrated by unaltered vascular density, and preserved endothelial permeability in Pdlim5cKO mice (Supplementary Fig. 4c, d). These findings were consistent with our previously published observations16.

To assess the role of PDLIM5 in tumor growth and angiogenesis, we employed three different subcutaneous xenograft tumor models using LLC lung cancer cells, Hepa1-6 hepatocellular carcinoma cells, and MC38 intestinal cancer cells (Fig. 2a, e, i). Across all three models, the deletion of Pdlim5 in ECs significantly attenuated tumor growth (Fig. 2b, c, f, g, j, k), concurrently reducing the density of CD31-positive vessels, indicating inhibited angiogenesis (Fig. 2d, h, l). These consistent findings across different tumor types suggest that PDLIM5 plays a crucial role in promoting angiogenesis in diverse tumor microenvironments. To extend these observations to an orthotopic setting, we constructed a breast cancer model using murine mammary E0771 cancer cells. Pdlim5 deletion in ECs inhibited orthotopic tumor growth and angiogenesis (Fig. 2m–p), reinforcing the broad relevance of PDLIM5 in tumor vascularization. To further validate PDLIM5 as a potential anti-angiogenic therapeutic target, we administered tamoxifen to induce endothelial Pdlim5 knockout in mice when tumor volume reached 60 mm3. The results demonstrated that both early and late Pdlim5 ablation significantly reduced tumor growth and neovascular density compared to the control group (Fig. 2q–u). This comprehensive approach, encompassing both subcutaneous and orthotopic models, highlights the universal role of PDLIM5 in facilitating tumor growth through angiogenesis.

PDLIM5 promotes filopodia formation and vessel sprouting
PDLIM5 is highly expressed in tip ECs, which are crucial for angiogenesis, and deletion of PDLIM5 inhibits tumor angiogenesis. It is difficult to directly observe the subtle morphological and functional changes of tip cells by immunofluorescence in tumour sections. To better elucidate PDLIM5’s role in tip cell function, we performed whole-mount staining of postnatal day 7 (P7) retinas, which allows for the visualization of tip cells and filopodia formation. Pdlim5 knockout significantly delayed the expansion of the retinal vascular plexus to the periphery (Fig. 3a), indicating its crucial role in angiogenesis. Further analysis revealed that vessel coverage, branching points, and the number of tip cells and filopodia were markedly reduced (Fig. 3b–e), highlighting PDLIM5’s involvement in vascular sprouting and expansion. However, retinal vascular networks in Pdlim5 knockout mice at P14 and P17 showed no significant defects compared with the control group (Supplementary Fig. 4e, f). These results suggest that PDLIM5 deficiency does not affect the final maturation and function of retinal vessels. Second, we utilized the aortic ring assay, a method that mimics vessel sprouting in Matrigel, allowing the assessment of sprouting angiogenesis in vitro. ECs with Pdlim5 deletion displayed defects in this assay (Fig. 3f–i), reinforcing the findings from the retinal model and emphasizing PDLIM5’s role in EC behavior. Lastly, to examine tip cell filopodia formation in vitro, we employed an EC fibrin bead assay (FBA), which provides a simplified and controlled setting to observe cellular behavior. Silencing PDLIM5 in ECs with two lentiviral vectors encoding different small hairpin RNA (shRNA) of PDLIM5 (shPDLIM5) effectively reduced PDLIM5 protein levels (Supplementary Fig. 5a). This knockdown (KD) led to a significant decrease in the sprouting area, number of tip cells, and filopodia (Fig. 3j–m, Supplementary Fig. 5b), further confirming the importance of PDLIM5 in tip cell function.

In addition, TECs isolated from Pdlim5 KO mice exhibited marked down-regulation of tip cell marker genes, including CXCR4, ESM1, and DLL4, when compared with those from control tumor-bearing mice (Supplementary Fig. 5c). Conversely, TECs from Pdlim5-overexpressing mice displayed an augmented sprouting area, accompanied by a larger quantity of tip cells and filopodia, in comparison to TECs from control tumor-bearing mice (Supplementary Fig. 5d, e). Taken together, these results imply that PDLIM5 facilitates tip cell filopodia formation and vessel sprouting.

To further investigate the role of endothelial PDLIM5 in angiogenesis, we first used a tube formation assay and found that PDLIM5 KD decreased tube length (Supplementary Fig. 5f, g). Conversely, PDLIM5 overexpression using a lentiviral vector encoding PDLIM5 increased tube length (Supplementary Fig. 5h–j). We also performed RNA-seq to assess the transcriptional landscape of control and PDLIM5 KD human umbilical vein ECs (HUVECs). Gene Ontology pathway analysis of differentially expressed genes (DEGs) revealed enrichment in angiogenesis, ECM organization, cell adhesion, and response to hypoxia pathways (Supplementary Fig. 5k). Genes enriched in these pathways were downregulated in PDLIM5 KD HUVECs, including collagen type IV alpha 1 (COL4A1), collagen type IV alpha 2 (COL4A2), matrix metalloproteinase-2 (MMP2), and vasohibin-1 (VASH1) (Supplementary Fig. 5l). RT-PCR further validated the downregulation of these genes in PDLIM5 KD HUVECs (Supplementary Fig. 5m). Previous studies have reported that these cell migration-related genes are regulated by integrin/focal adhesion kinase (FAK) signaling17, and our results showed that the FAK inhibitor Y15 suppressed the expression of VASH1, VEGFR3, and MMP2 (Supplementary Fig. 5n). We then investigated whether decreased filopodia formation in PDLIM5 KD HUVECs would also inhibit integrin/FAK signaling activation by reducing the response to the ECM. We found that PDLIM5 KD inhibited FAK activation, whereas PDLIM5 overexpression promoted FAK activation (Supplementary Fig. 5o). These results indicate that PDLIM5 deletion impedes FAK activation and filopodia formation in tip cells.

PDLIM5 promotes F-actin bundling during filopodia formation by interacting with ACTN1/ACTN4
To unravel the molecular mechanisms behind the defects in endothelial filopodia formation in the absence of PDLIM5, we hypothesized that PDLIM5 plays a crucial role through interactions with cytoskeletal proteins. To test this, we overexpressed PDLIM5 with a Flag tag in HUVECs, followed by Flag immunoprecipitation (IP) and mass spectrometry (MS) analysis (Fig. 4a, b), which led to the identification of 145 PDLIM5-interacting proteins, including actin. To further validate PDLIM5’s involvement in cell migration and adhesion, we analyzed adhesion datasets and found PDLIM5 at one of the highest frequencies among cell migration-related genes (Supplementary Fig. 6a), with 13 of these proteins also identified in the actin-binding gene set and meta-adhesome dataset. Our MS analysis specifically revealed that PDLIM5 interacts with actinin-1 (ACTN1) and actinin-4 (ACTN4), proteins critical for F-actin bundling and filopodia formation18,19. These interactions were confirmed by immunoblot analysis in both HEK293T cells and HUVECs (Fig. 4c, d), where PDLIM5 showed high co-localization with ACTN1/ACTN4 in the filopodia of tip cells (Fig. 4e, f, Supplementary Fig. 6b, c). To further validate the regulatory role of ACTN1/ACTN4 in FAK signaling and filopodia formation, we employed siRNA-mediated knockdown of ACTN1/ACTN4 in endothelial cells. Results showed that knocking down either ACTN1 or ACTN4 effectively suppressed FAK activation and tip cell filopodia formation (Supplementary Fig. 7a–e).

Moreover, PDLIM5 overexpression increased the binding of actin to ACTN1/ACTN4, whereas its deletion had the opposite effect (Fig. 4g). Additionally, overexpression of ACTN1 and ACTN4 promoted FAK and its downstream signaling pathways (Fig. 4h–l), whereas PDLIM5 KD abolished FAK activation and the expression of DLL4, VASH1, and MMP2, highlighting PDLIM5’s pivotal role in regulating these pathways essential for filopodia formation and angiogenesis. In conclusion, our results demonstrate that PDLIM5 interacts with ACTN1/ACTN4 to regulate filopodia formation and angiogenesis through cytoskeletal dynamics and FAK signaling.

LIMs domain S593/F596 is critical for the interaction of PDLIM5 with ACTN1/ACTN4
PDLIM5, in addition to being a cytoskeleton-binding protein, functions as a scaffolding protein because of its unique structural domains: a PDZ domain at its N-terminus and three LIM domains at its C-terminus. To understand how PDLIM5 interacts with actin and its binding partners, ACTN1/ACTN4, we conducted a series of experiments to delineate the specific binding sites. We found that PDLIM5 binds to ACTN1/ACTN4 via its LIM domains (Fig. 5a, Supplementary Fig. 8a, b) and binds to actin through its PDZ domain (Supplementary Fig. 8c). This dual interaction suggests that PDLIM5 acts as a scaffolding protein, facilitating the binding of ACTN1/ACTN4 to actin.

To further elucidate the molecular mechanism, we performed molecular docking analysis, which revealed multiple binding sites between the LIM domains of PDLIM5 to the ACTN1/ACTN4 (Supplementary Fig. 8d, e). Sequence comparisons identified conserved sites (R240, S354, K582, S593, and F596) in PDLIM5, which were predicted to interact with both ACTN1 and ACTN4 (Fig. 5b, Supplementary Fig. 8f, g). Mutational analysis of these sites revealed that the S593A and F596L mutations significantly reduced the binding of PDLIM5 to ACTN1/ACTN4, but not F-actin (Fig. 5c, d, Supplementary Fig. 8h), with molecular docking confirming the high similarity in binding regions (Fig. 5e, f). To further validate these findings, we constructed a double-mutant PDLIM5 (S593A/F596L), which further attenuated the binding of PDLIM5 to ACTN1/ACTN4 (Fig. 5g). The binding of the mutated LIM domain to ACTN1/ACTN4 was also weakened (Supplementary Fig. 8i), demonstrating the critical role of these sites in the interaction.

Filopodia formation, which relies on the bundling of actin filaments, is regulated by the interaction of ACTN1/ACTN4 with actin. We found that this regulatory mechanism can be disrupted by specific mutations in PDLIM5, as evidenced by the significantly attenuated binding of ACTN1/ACTN4 to actin after the S593A and F596L mutations (Fig. 5h, i). These results indicate that PDLIM5 interacts with ACTN1/ACTN4 through the S593/F596 residues, and acts as a scaffolding protein by promoting F-actin binding to ACTN1/ACTN4 (Fig. 5j). The above results demonstrate that PDLIM5 plays a pivotal role in filopodia formation through its interaction with cytoskeletal proteins.

S593A/F596L is indispensable for PDLIM5-induced tip cell filopodia formation and tumor angiogenesis
To determine whether the S593/F596-mediated interaction with ACTN1/ACTN4 contributes to the biological function of PDLIM5, we first evaluated the effect of the S593A/F596L mutation on tip cell filopodia formation. Results showed that re-expression of wild-type (WT) PDLIM5, but not the S593A/F596L-mutated PDLIM5, increased the number of tip cells and filopodia in PDLIM5 KD ECs (Fig. 6a–d). Additionally, tube length was also inhibited in PDLIM5 mutated HUVECs (Supplementary Fig. 9a, b). To validate these results in vivo, we used an adeno-associated virus (AAV) carrying a Tie2 promoter to overexpress WT and S588A/F591L (mouse homolog of human PDLIM5 S593/F596)-mutated Pdlim5 in TECs by multi-point injection into the tumor (Fig. 6e). The results showed that Pdlim5 was successfully overexpressed in the tumor vessels (Supplementary Fig. 9c). WT Pdlim5 significantly enhanced tumor growth and angiogenesis compared with Pdlim5cKO mice, whereas the mutated Pdlim5 group showed no such effect, exhibiting tumor growth rates (Fig. 6f–h) and angiogenesis levels (Fig. 6i, j) comparable to those observed in the Pdlim5cKO group. Thus, the interaction of PDLIM5 with ACTN1/ACTN4 through S593/F596 is critical for promoting tumor angiogenesis and growth, as evidenced by the lack of these effects when these sites are mutated.

Deletion of endothelial Pdlim5 promotes tumor vascular normalization and T cell infiltration
The tumor microenvironment typically induces morphological and functional defects in the tumor vasculature, resulting in increased hypoxia, suppressed immune cell function, and impaired delivery of chemotherapeutic agents due to low perfusion levels and high permeability20,21. To address these issues, we investigated the effect of PDLIM5 deficiency on tumor hypoxia and vascular function. Our findings reveal that Pdlim5 deletion in TECs ameliorated intratumoral hypoxia (Fig. 7a, Supplementary Fig. 10a–d), suggesting an improvement in oxygenation within the tumor. Concurrently, Pdlim5 knockout led to increased coverage of smooth muscle actin (SMA)-positive pericytes around blood vessels (Fig. 7b, Supplementary Fig. 10e–g), which is indicative of enhanced vascular stability and maturation. Tumor vessel perfusion was also improved in Pdlim5-deficient mice, as assessed by fluorescein isothiocyanate (FITC)-lectin staining (Fig. 7b), implying better blood flow. Furthermore, we observed a reduction in tumor hemorrhage with decreased presence of Ter119-positive blood cells in the interstitial space (Supplementary Fig. 10h–k) and a decrease in tumor vessel permeability (Supplementary Fig. 10l–o). These combined effects indicate that the loss of Pdlim5 in TECs not only alleviates hypoxia but also promotes the normalization of the tumor vasculature, potentially enhancing immune cell function, thereby providing a therapeutic advantage in chemosensitization and immunotherapy.

Previous studies have shown that normalization of tumor vessels can inhibit tumor metastasis22–24. Therefore, to determine the effects of Pdlim5 knockout on tumor metastasis, we established subcutaneous LLC tumor xenografts, performed surgical resection of the primary tumors, and analyzed lung metastasis after tumor regrowth. Results showed that Pdlim5 deletion significantly inhibited LLC cell lung metastasis (Supplementary Fig. 11a–c). Further, we analyzed whether Pdlim5 knockout-induced tumor vessel normalization could enhance the efficacy of low-dose chemotherapy. Results showed that low-dose cisplatin (4 mg/kg) combined with Pdlim5 deletion further reduced tumor growth and compared to cisplatin alone (Supplementary Fig. 11d–j).

Normalized tumor vessels enhance T cell infiltration and anti-tumor immunity through structural and functional remodeling3,25–29. Therefore, we aimed to further investigate whether tumor vascular normalization induced by Pdlim5 deletion can promote T cell infiltration. We first examined the proportions of CD4+ and CD8+ T cells in tumors by using multicolor immunofluorescence (Supplementary Fig. 12a) and flow cytometry (Supplementary Fig. 13a, b). The proportions of both CD4+ and CD8+ T cells were increased in the endothelial Pdlim5 deletion group (Fig. 7c, Supplementary Fig. 12a). A leukocyte trans-endothelial migration assay was conducted to evaluate the efficiency of T-cell trans-endothelial migration, which revealed that genetic ablation of Pdlim5 significantly promoted this migratory process (Fig. 7d).

The accumulation of ICAM1 on the EC membrane facilitates T cell adhesion and transendothelial transport30–32, and flow cytometry analysis confirmed increased membrane aggregation of ICAM1 in PDLIM5-knockdown HUVECs (Fig. 7e, Supplementary Fig. 13c). This suggests that the mechanism underlying the increased T cell infiltration induced by PDLIM5 deletion includes not only vascular structural and functional normalization, but also enhanced T cell adhesion in tumor vessels through upregulation of ICAM1. But how does PDLIM5 loss promote ICAM1 membrane accumulation? Previous studies have shown that endothelial ICAM1 binds to ACTN1/ACTN4 to stabilize its membrane aggregation and enhance T-cell adhesion33,34. And our molecular docking results predicted that PDLIM5 also interacts with ACTN1/ACTN4 (Fig. 5e, f), suggesting that PDLIM5 and ICAM1 competitively bind to ACTN1 and ACTN4. Further validation revealed that PDLIM5 knockdown increased the binding of ICAM1 to ACTN4 (Fig. 7f). Atomic force microscopy (AFM) detection also showed that PDLIM5 deficiency reduced endothelial cell stiffness (Fig. 7g). Collectively, these findings demonstrate that PDLIM5 deficiency promotes structural and functional normalization of tumor vessels, increases the accumulation of ICAM1 on the endothelial cell membrane and reduces endothelial cell stiffness, thereby promoting T cell adhesion and transendothelial migration, and enhancing T cell infiltration into tumors.

Deletion of endothelial Pdlim5 improves immunotherapy response by increasing T-cell infiltration
To further elucidate the potential sensitizing effect of endothelial Pdlim5 deletion on immunotherapy, we administered an anti-PD-L1 monoclonal antibody (10 mg/kg) every three days when tumors reached 60 mm3 in tumor-bearing mice (Fig. 8a). Mice treated with the anti-PD-L1 monoclonal antibody exhibited minimal inhibitory effects on LLC tumor growth (Fig. 8b–d), consistent with previous reports indicating that LLC tumors are insensitive to anti-PD-1/PD-L1 immunotherapy35,36. However, in striking contrast, Pdlim5-knockout mice treated with the anti-PD-L1 monoclonal antibody showed a significant reduction in tumor size. This reduction in tumor growth was accompanied by a substantial increase in the infiltration of CD4+ and CD8+ T cells (Fig. 8b–h), highlighting the enhanced immune response facilitated by PDLIM5 deletion.

These results demonstrate that PDLIM5 deletion not only ameliorates the tumor vascular abnormalities but also significantly enhances the antitumor efficacy of the anti-PD-L1 monoclonal antibody. Our findings indicate that PDLIM5 deletion promotes tumor susceptibility to immunotherapy, as evidenced by the smaller tumor sizes and increased T-cell infiltration in Pdlim5-knockout mice receiving PD-L1 monoclonal antibody treatment, providing compelling evidence for targeting tumor vasculature to improve immune responses and immunotherapy outcomes (Fig. 8i).

Methods
Cell culture
Normal lung ECs and tumor ECs were isolated from mouse lung tissues and tumor tissues as previously described in ref. 5. Briefly, freshly dissected tissues were minced into small fragments and enzymatically digested using collagenase type I (1 mg/mL; Sigma-Aldrich, C0130) and DNase I (30 µg/mL; Sigma-Aldrich, DN25) at 37 °C for 45 minutes with gentle agitation. The resulting single-cell suspensions were filtered through 70 µm cell strainers and subjected to magnetic-activated cell sorting (MACS) using anti-CD31 microbeads (Miltenyi Biotec, 130-097-418) to enrich endothelial cells. The purified cells were cultured in Endothelial Cell Growth Medium-2 (EGM-2; Lonza, CC-3162) supplemented with 10% fetal bovine serum (FBS). HUVECs were obtained from the Cell Bank of the Chinese Academy of Sciences (Shanghai, China) and maintained in EGM-2 medium. HEK 293T (CRL-3216, ATCC), LLC (CRL-1642, ATCC), Hepa1-6 (CRL-1830, ATCC), MC38 (ENH204-EP, NCI/NIH), and E0771 (CRL-3461, ATCC) cells were cultured in Dulbecco’s modified Eagle’s medium (KGL1211-500, KeyGEN) supplemented with 10% FBS (F101-01, Vazyme). All cells were cultured at 37 °C and 5% CO2.

Mice
Sex was not considered as a biological variable. Both male and female mice were used in this study. Pdlim5f/f mice were generously provided by Ju Chen (Department of Medicine and Cardiology, University of California San Diego, La Jolla (CA)). Details of Pdlim5f/f mice have been described previously16. Cdh5-CreERT2 mice were purchased from GemPharmatech Co., Ltd. (Nanjing, China; strain no. T052686). Pdlim5f/f mice were mated with Cdh5-cre Ert2 mice to generate the Pdlim5f/f: Cdh5-creErt2 genotype. To induce Pdlim5 deletion in ECs, mice received intraperitoneal injections of 100 µL tamoxifen (25 mg/mL) daily for five consecutive days. CAG-Pdlim5-polyATG mice were purchased from GemPharmatech (Jiangsu, China). For all in vivo experiments, 8–10-week-old mice of both sexes were used unless otherwise specified. Mice were housed under specific pathogen-free (SPF) conditions with controlled temperature (22±2 °C), humidity (50±10%), and a 12-hour light/dark cycle, with free access to food and water. All procedures were approved by the Animal Care and Use Committee of Zhejiang University School of Medicine (ZJU20220531). The mice were maintained in the specific pathogen-free facility of the Laboratory of Animal Research at the International School of Medicine, Zhejiang University.

Proteomics analysis
HUVECs were transduced with a lentiviral vector encoding PDLIM5 with a C-terminal Flag tag for 48 h, subsequently lysed in IP buffer (50 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 1 mM ethylenediaminetetraacetic acid, and protease inhibitors) and incubated with magnetic anti-FLAG beads overnight at 4 °C. After washing, proteins were eluted, concentrated, and subjected to in-gel digestion. Gel bands or spots were cut into approximately 1 mm³ pieces, destained, dehydrated, and reduced with 10 mM dithiothreitol at 37 °C for 1 hour, followed by alkylation with 55 mM iodoacetamide in the dark for 30 minutes. After dehydration, gel pieces were washed with deionized water, rehydrated in 50 mM ammonium bicarbonate, and digested with trypsin overnight at 37 °C. Peptides were extracted, combined, and desalted using C18 columns. Liquid chromatography–tandem mass spectrometry was performed with a Q Exactive HF-X mass spectrometer (Thermo Fisher Scientific Inc) using Nanospray Flex™ ion source with a 1 µg sample. The liquid chromatography conditions included a flow rate of 600 nL/min and a gradient from 8% to 95% acetonitrile over 60 minutes. Protein identification was performed using the human database with Proteome Discoverer 2.4 software. The protein mass spectrometry sequencing data were uploaded to the iProX database (https://www.iprox.cn/page/home.html). The Project ID is IPX0010745000, and the ProteomeXchange ID is PXD059449.

RNA-sequencing analysis
Total RNA was isolated from HUVECs transfected with shPDLIM5 or shScr lentivirus for 48 h using an RNA-Quick Purification Kit (ES-RN001, Yishan Biotechnology). Subsequently, cDNA libraries were constructed and sequenced by the Beijing Genomics Institute using the BGISEQ-500 platform. The bioinformatics workflow was performed according to procedures established by Dr. TOM (BGI). A false discovery rate-corrected p-value of 0.05 was considered statistically significant. The corresponding raw data were uploaded to the Genome Sequence Archive (GSA) (Project No.: HRA009823).

In vivo xenograft assays
In subcutaneous xenograft tumor models, LLC cells, Hepa1-6 cells, and MC38 cells (2×106 cells suspended in 100 µl phosphate-buffered saline (PBS)) were injected subcutaneously into the right flank. Tumor volumes were measured every two days and calculated using the formula: length × width²/2 and was measured with a digital caliper. For orthotopic breast tumor growth, 2×106 E0771 cells were orthotopically injected into the mammary fat pads of mice. In accordance with Institutional Animal Care and Use Committee (IACUC)-approved protocols at the Fourth Affiliated Hospital, Zhejiang University School of Medicine, the maximum permitted tumor burden was defined as a longest tumor diameter of ≤2.0 cm (approximately ≤ 1500–2000 mm³). Mice were euthanized at or before this threshold, or earlier if humane endpoints were reached, including tumor ulceration or necrosis, impaired mobility or feeding, severe distress, or body-weight loss exceeding 15%.
For in vivo rescue experiments, when the tumor grew to ~60 mm3, 50 µl AAV-Pdlim5 wild type (WT) or AAV-Pdlim5 mutation (MUT) adeno-associated virus (AAV) with a titer of 1×108 viral particles/µL was injected at multiple points within the tumors for twice. Pdlim5f/f and Pdlim5f/f: Cdh5-creErt2 mice were divided into four groups (n=5 per group): Pdlim5f/f or Pdlim5f/f: Cdh5-creErt2 mice injected with AAV-vector, Pdlim5f/f: Cdh5-creErt2 mice injected with AAV-Pdlim5 WT, and Pdlim5f/f: Cdh5-creErt2 mice injected with AAV-Pdlim5 MUT. The mice were euthanized by CO2 asphyxia when the experiments concluded or when the longest dimension of the tumors no more than 2.0 cm. The excised tumors were weighed and then either fixed with 4% paraformaldehyde (PFA) or stored at −80 °C until further analysis.
To elucidate the effect of PDLIM5 deletion on tumor vessel perfusion and leakage, we conducted assays for hypoxia in the tumor microenvironment, pimonidazole staining, and vascular leakage and perfusion, as previously described. Tumor hypoxia was quantified with a Hypoxyprobe kit (HP1-100, Hypoxyprobe) in accordance with the manufacturer’s instructions. To evaluate vascular permeability, 100 µl of FITC-conjugated dextran (25 µg/mL, 70 kDa, FD70, Sigma-Aldrich) was administered intravenously to the mice, followed by perfusion with 1% PFA solution 30 min later. Subsequently, the tumors were dissected and embedded in Tissue-Tek OCT compound for subsequent analysis. To quantify vessel perfusion, we administered intravenous injections of DyLight 488-conjugated Tomato lectin (1 mg/mL, 100 µL, DL-1174-1, Vector Laboratories) 30 min before sacrificing the mice, after which the tumors were harvested.
To explore the synergistic effect of PDLIM5 deletion on cisplatin treatment, tumor-bearing mice were intraperitoneally injected with 4 mg/kg cisplatin (MCE, HY-17394) dissolved in saline every 3 days starting at tumor volume ≈60 mm3; control groups received equal-volume saline injections (n=5/group), with tumor dimensions monitored by caliper every 2 days (volume=0.5×L×W²).
In experiments utilizing immunotherapeutic checkpoint inhibitors, 200 µg (10 mg/kg) of anti-PD-L1 monoclonal antibody (BioXcell, BE0101) or isotype immunoglobulin G was administered intraperitoneally to each mouse at 3-day intervals. Mice were subsequently sacrificed at the indicated times, and tumors were collected for subsequent analysis.

Patients and samples
All human studies complied with institutional and national ethical regulations and were approved by the Medical Ethics Committee of the Fourth Affiliated Hospital, Zhejiang University School of Medicine (Approval No.: K2022179). Written informed consent was obtained from all participants prior to sample collection. For single-cell RNA sequencing, paired tumor and adjacent non-tumor lung tissues were prospectively collected from seven patients with NSCLC between November 2022 and January 2023 at the Fourth Affiliated Hospital of Zhejiang University (Yiwu, Zhejiang, China). Fresh specimens were enzymatically dissociated immediately after surgical resection to obtain single-cell suspensions for library construction and sequencing.
For immunofluorescence (IF) analysis, paired tumor and adjacent non-tumor tissues from an additional ten patients were used to compare PDLIM5 expression levels in endothelial cells (CD31+). PDLIM5 fluorescence intensity was quantified using ImageJ software and normalized to DAPI to control for cell density. For survival analysis, paraffin-embedded tumor samples from twenty NSCLC patients were subjected to IF staining for PDLIM5 and CD31 to stratify cases into PDLIM5-high and PDLIM5-low endothelial expression groups based on median signal intensity. Overall survival (OS) was calculated from diagnosis to death or last follow-up. Survival curves were analyzed by Kaplan–Meier log-rank test and multivariate Cox proportional hazards regression using GraphPad Prism (v8.0.1), with statistical significance defined as p<0.05. Comprehensive clinicopathological information of all patients is provided in Supplementary Table 6.

Single-cells sequencing and data analysis
The lung tissues were dissociated into single-cell suspensions to facilitate further analysis. Single-cell suspensions were prepared at a concentration of 1×105 cells/mL in PBS and loaded onto microfluidic devices. scRNA-seq libraries were constructed using the GEXSCOPE® Single-Cell RNA Library Kit (Singleron Biotechnologies, China). Next, the individual libraries were diluted to 4 ng/µL and subsequently pooled for sequencing. The pooled libraries were subjected to sequencing on an Illumina NovaSeq 6000 instrument with 150 bp paired-end reads. Additionally, the raw data were subjected to a series of processing steps to generate the gene expression profiles. This was achieved using CeleScope v1.14.0, a software package developed by Singleron Biotechnologies, with the default parameters retained. The Scanpy package, version 1.8.1, was utilized for quality control, dimensionality reduction, and cluster analysis in the Python 3.7 programming language. We filtered the expression matrix by the following criteria: 1) cells with gene count less than 200 or with top 2% gene count were excluded; 2) cells with the top 2% UMI count were excluded; 3) cells with mitochondrial content >20% were excluded; 4) genes expressed in less than 5 cells were excluded. The batch effect was removed using the Harmony software. Furthermore, the DEGs were identified by comparing the two groups using the Wilcoxon rank-sum test with the standard parameters applied. Genes were considered differentially expressed if they were expressed in at least 10% of the cells in either of the two groups and exhibited an adjusted p-value below a threshold of 0.05. This was calculated through the Benjamini-Hochberg correction for multiple hypothesis testing. The human single-cell transcriptomics data are deposited under the accessions: HRA012795.

Statistical analysis
The presented data are derived from a minimum of three biologically independent experiments and analyzed using GraphPad Prism 8.0 software (GraphPad Software, La Jolla, CA). Statistical significance between individual groups was evaluated using two-tailed independent Student’s t-tests. When examining multiple groups, a one-way or two-way analysis of variance was performed. Survival differences were assessed using the Kaplan-Meier method, and statistical significance was determined using the log-rank test. Statistical significance was set at p-value <0.05, and this is indicated in the figures as follows: p<0.05 (*), p<0.01 (**), p<0.001 (***), with “ns” denoting “not significant,” as indicated in the figure legends.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this study, the authors investigate the role of PDLIM5 in the tumor microenvironment, focusing on its effects on tip cells and vascular normalization, immune cell infiltration, and response to anti-PD-L1 immunotherapy. Using a combination of integrative gene expression analysis, single-cell RNA sequencing, and functional experiments, they demonstrate that PDLIM5 expression is increased in tumor endothelial cells, associated with tumor neuovascularization, and functionally validated using in vitro cell assay and in vivo tumor modeling that PDLIM5 is linked to tumor vascularization, reduces tumor hypoxia, and enhances T cell infiltration, thereby improving the efficacy of immune checkpoint therapy. Using molecular docking and co-IP, the study reported that PDLIM5 interact with the ACTIN2/4, hencing regulating filopodia formation and angiogenesis. The study spans multiple tumor models, including Hepa1-6, MC38, and LLC, to validate these findings. The study has applied a comprehensive molecular and cellular approaches to evaluate the effect of PDLIM5 on tumor vasuclar functions, the molecular interactions with ACTIN2/4, and its implications for cancer therapy.\nWhile the manuscript employs a variety of experimental approaches, the work is also hindered by several limitations, including the conclusion on Tip endothelial cells, the molecular mechanisms linking PDLIM5 to normal vascular functions in tissues and tumor neovascularization, the prognostic value of PDLIM5 expression and patient outcome, as well as critical components of the immune microenvironment. Furthermore, there are quite a number of mistakes in the figures and supplementary figures, which will need significant proof reading and checking the original results. Detial comments for these concerns are highlighted below. Carefully addressing these are required before any possible consideration of the study for publication.\n1. One major conclusion of this study is the PDLIM5 affects endothelial tip cell filopodia formation and tumor angiogenesis. Tip cells are a very small fraction of endothelial cells leading the vascular angiogenesis. While tip cells are almost absence in adult normal tissues, this endothelial phenotype is increased in tumors and essential for tumor neovascularization. Furthermore, tip cells are well characterized with the expression of classic markers such as CXCR4, ESM1, DLL4. The current study had used COL4A1, PLVAP, COL4A2 as markers for TipECs, which are not correct (Figure S2C). These are mixed markers for EndoMT EC phenotypes. These jeopardize the conclusiveness of the results and conclusion made based on Figure 1 and related figures.\nFor example, in TEC (Figure 1d), the fraction of tip ECs could not be over 20% of the entire ECs. Furthermore, the increase expression of PDLIM5 is not specific to TipECs. According to the data presented in Figure 1b, as well as checking the gene expression data provided by the protein atlas database, PDLIM5 is higher expressed in vascular smooth muscle cells, skeletal monocytes, fibroblasts. Similarly, the increased PDLIM5 was not restricted to TipECs, as noted by the authors in the manuscript (line 95-96). Thus, the annotation and interpretation of TipECs will require significant revision and correction.\n2. Regarding the association between PDLIM5 expression and prognostic outcome of cancer patients (Figure 1h, and supplementary Figure 3), I am not completely sure if these data have been correctly adjusted and calculated. First, neither the main text or supplementary methods describe how these Kaplan-Meier curve have been generated, and the data source used to analysis the probability of overall survival with PDLIM5 expression. It is presumably coming from the TCGA database. However, when checking the survival data from the TCGA (https://tcga-survival.com/data-table?view=gene) or from the HPA, the expression of PDLIM5 did not show the same trajectory as what the figures provided in Supplementary Figure 3. The authors will have to clearly documented their analysis and scripts in the methods.\n3. The selective effect of PDLIM5 on tumor vasculature formation. It is plausible that the study has used both in vitro, ex vivo and in vivo models to study the effect of PDLIM5 on tumor vasculature formation. First, it remains unclear how and why loss of PDLIM5 do not affect the development of normal tissue vasculature and functions (Supplementary Figure 4c-e) but exhibit clear effect on the tumor vasculatures with the ectopic PDX models. What is the phenotype of PDLIM5cKO mice if the induction of condition PDLIM5 was induced at birth or during fetal stages? In Figure 3, the study has already demonstrated that postnatal PDLIM5 knockout in ECs inhibit vessel sprouting in retina. This contradict with the conclusion made in part 1 (line 119-120).\nIt is unclear in the current methods provided, at what ages were the TAM induction was introduced. PDLIM5 is not EC- specifically expressed, when addressing and validating the conditional KO models, it is essential to showed in Figure 2, as in Figure 1f, that PDLIM5 is not expressed by CD31 positive endothelial cells. These difference between adult and neonatal could potentially be due to the induction time.\n4. The authors have done a great work in conducting all these experiments. However, it is very important to present the results clearly and correctly. Here I highlighted a few major mistakes in the manuscript figures and results.\nFigure 4 g-l, the IP results show that shPDLIM5 down regulates DLL4, MMP2, p-FAK Y397, and VASH1 (g-h). However, quantification of IP in Figure 4i-l, show that shPDLIM5 increase their levels. Which of these are correct?\nSize of FAK and p-FAK Y397. The protein size of FAK appears to be 100kDa in some figures (S7) and 120kDa in the other (S5, S9, Figure 4). And VEGFR3 appears to be 120kDa in S7, and 180kDa in S5.\nAll these kinds of mistakes should be carefully checked, as well as the original source data.\n5. Methods regarding PDLIM5 knockdown in HUVECs. It is unclear about the methodology used for ACTIN1, ACTIN4, and PDLIM5 knockdown in HUVECs. For ACTIN1 and ACTIN4, synthetic siRNAs should have been used according to the methods. However, PDLIM5, why shRNA expressed by lentiviral have been used instead? Any reason for choosing the difference delivery approach? Please also check the siRNA sequences for si-ACTN1-1#, as it could not be found in the human ACTN1 gene.\n6. The study has integreated public scRNA-seq data and generated new data based on their own patient cohort, but the current analysis and detial provided is not sufficient to support the reproducibility. What quality control metrics and thresholds were applied to filter low-quality cells, number of cells, number of patients and metadata, how batch effects had been handled if any, how were endothelial cell subtypes identified, marker genes used. Providing these details would strengthen the accuracy and reproducibility of the scRNA-seq analysis. Additionally, consider depositing the raw data and scripts in a public repository to enhance transparency.\n7. The methods provide useful details about AAV injections and xenograft experiments, but there are areas requiring clarification: Specify the AAV titer (viral particles/μL) used for intratumoral injections to ensure reproducibility. Provide details on time points for tumor volume measurements and angiogenesis assessments after AAV injection.\nSpecify the exact frequency of tumor volume measurements within this period (e.g., daily or every other day). Clarify whether tumor volume data were averaged across multiple tumors or cohorts. Discuss variations in tumor growth rates over time to better contextualize the presented data.\n8. The authors suggest that PDLIM5 could be a promising therapeutic target and mention small molecule inhibitors as a potential strategy. However, there is no experimental data, such as preclinical trials or efficacy studies, to back up these claims. Including some preliminary studies that explore inhibitors targeting PDLIM5 or its interaction sites would help demonstrate whether this approach could actually be effective in a therapeutic setting.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "This paper investigates the role of the cytoskeletal scaffold protein PDLIM5 in promoting angiogenesis by tumor endothelial cells (TECs). The authors show that PDLIM5 stimulates vascular sprouting in the retina of healthy mice as well as in tumor grafts. By combining KO of PDLIM5 with anti-PD-L1 treatment, they further show that this PDLIM5 function can be exploited to decrease tumor growth and promote tumor immune cell infiltration, suggesting that small molecules directed against PDLIM5 might hold promise as a new antitumor treatment. To address the mechanism underlying this PDLIM5 function and identify potential PDLIM5 surfaces to target for drug development, the authors examine the interaction between PDLIM5 and ACTN1/ACTN4, which has previously been reported in other cell types. In contrast to Nakagawa et al (2006), they find that PDLIM5 binds to ACTN1/ACTN4 through its Lim domain instead of its PDZ domain. They further narrow the PDLIM5- ACTN1/4 interaction down to two residues in the third LIM domain (S593 and F596) and present some data that suggests this interaction enhances the binding of ACTN1/ACTN4 to F-actin, thereby stabilizing and promoting filopodia formation and angiogenesis. In line with this, expression of the PDLIM5 double mutant in PDLIM KO mice hampers tumor growth, angiogenesis and immune cell invasion. The merit of this work mainly lies in the large body of data supporting PDLIM5 as a potentially interesting antitumor target for a great many different types of cancer. However, the described underlying mechanism is not fully supported by the data presented. Thus, to substantiate the claim that this study identified a druggable surface on PDLIM5, the authors will have to address the issues raised below.\nFig. 4: The authors claim that PDLIM5 recruits ACTN1/4 to actin in filopodia, however the linescans from the images shown in Fig. 4e-f do not show that higher PDLIM5 signals correspond to higher ACTN1/4 signals. In addition, the immunoprecipitation assay in Fig. 4g, shows varying levels of PDLIM5 in WCL, but does not confirm that this also corresponds to varying levels of PDLIM5 bound to actin after IP. Hence, they cannot conclude that PDLIM5 regulates the amount of ACTN1/4 bound to actin. To claim that PDLIM5 recruits ACTN1/4 to F-actin, they need to show that the level of PDLIM5 bound to F-actin after immunoprecipitation changes similarly as the level of ACTN1/4 bound to F-actin after immunoprecipitation.\nIn addition Line 193: The authors state that PDLIM5 colocalizes with ACTN1/4 in tip cells (Fig. 4e). Are the cells that are shown in the images and that are used for the linescans actual tip cells?\nFig 5: The authors should explain why Fig. 5g shows that PDLIM5 mutants bind less ACTN1/4, however the reverse IP in Fig, 5c-d does not show the same result. Is this perhaps due to the fact that these IP experiments were performed with full length PDLIM5, which can bind to F-actin and ACTN1/4. The latter implies that the interpretation of the IP results could be perturbed because there are two binding interactions to account for, especially given that the authors do not demonstrate that the LIM domain mutations do not change the overall conformation of PDLIM5 such that it affects its interaction of the PDZ domain with F-actin. To make matters worse, ACTN1/4 are bone fide F-actin binding proteins, irrespective of whether their binding is increased by PDLIM5, and thus it should be taken into account that PDLIM5 binds to F-actin bound by ACTN1/4. To work around this complex situation and avoid any confusion, the mutant PDLIM5 IP experiments should be repeated with the mutations introduced in the LIM domains only.\nThis is contrary to Fig. 5h-i. In order to conclude that these mutations fail to recruit ACTN1/4 to F-actin, the authors should confirm that full length PDLIM5 mutants bind equally well to F-actin as wild type PDLIM5.\nMinor comments:\n- Include the species of PDLIM5 that was used to clone the lentiviral and adenoviral constructs in the supplemental methods section.\n- Line 334: Nakagawa et al (2006) also found that the PDLIM5 PDZ domain mediates binding to ACTN1/4. This difference between their study and this work should be addressed.\n- It would be nice of the authors could expand the discussion a bit to also talk about how the PDLIM-actinin interaction is required for the other PDLIM family members.", "reviewer_4": "In the study entitled “Endothelial PDLIM5 promotes tip cell filopodia formation and tumor angiogenesis by regulating ACTN1/ACTN4-dependent actin bundling”, the authors investigate the role of PDZ and LIM domain 5 (PDLIM5) in tumor endothelial cell (TEC)-mediated angiogenesis. They demonstrate that PDLIM5 is highly expressed in lung cancer TECs, correlates with poor patient survival, and plays a crucial role in filopodia formation by interacting with actinin-1/actinin-4 (ACTN1/ACTN4) to facilitate filamentous actin (F-actin) bundling. Endothelial-specific deletion of PDLIM5 disrupts sprouting angiogenesis, reduces tumor growth, normalizes tumor vasculature, and enhances immunotherapy response in resistant tumors. These findings suggest that PDLIM5 may serve as a novel therapeutic target to improve cancer treatment outcomes.\nComments:\nThe results presented in the manuscript provide substantial evidence on the role of PDLIM5 in TECs and its potential impact on tumor angiogenesis. However, a number of comments should be addressed to support the claims and findings presented in the manuscript. These include:\n• Additional functional studies are needed to convincingly demonstrate that endothelial PDLIM5 regulates tip cell function. For instance, in vitro migration assays and competition sprouting assays, where a mixed population WT and PDLIM5-silenced cells compete for the tip cell position, could provide additional strenght.\nIt would be also important to investigate how the tip cell subset (as well as the additional subtypes) is affected in the endothelial-specific PDLIM5 KO in mice.\n• While the authors propose that endothelial PDLIM5 deletion induces tumor vessel normalization (TVN), Figure 2 suggests vessel pruning (reduced vessel density), which is likely responsible for the observed reduced tumor growth. This discrepancy warrants an explanation. TVN is often associated with improved metastasis outcomes rather than with an effect on primary tumor growth. It would be useful to investigate the relationship between vessel pruning and reduced tumor growth and explore whether TVN by PDLIM5 deletion affects metastasis.\n• The mechanism by which endothelial PDLIM5 regulates T cell infiltration needs further investigations. Are increased T cell infiltration levels due to enhanced delivery upon TVN, or is there an effect on adhesion molecules? ICAM-1 clustering and function has been shown to be directly linked to ACTN4 (Celli et al. J Immunol. 2006). Thus, it would be crucial to assess whether PDLIM5 deletion alters ICAM-1 clustering. Additionally, as actin-binding proteins can differentially regulate EC stiffness (Schaefer, et al. J Cell 2014), it would be valuable to investigate whether PDLIM5 deletion leads to changes in EC stiffness that might favor T cell transmigration.\n• ACTN4 is known to bind endothelial eNOS and inhibit its basal activity (Hiroi Yet al. FASEB J. 2008). Furthermore, as previoulsy shown by the authors, PDLIM5 phosphorylates eNOS to regulate NO generation, which can affect vessel permeability. It would be insightful to assess whether these mechanisms, particularly in relation to NO regulation and endothelial permeability, contribute to the in vivo phenotype observed upon PDLIM5 deletion. Could these factors also play a role in promoting T cell infiltration by modulating the permeability of tumor vessels?\n• Given the evidence for improved perfusion in tumors with normalized vasculature, it would be useful to consider the potential effects of PDLIM5 deletion on chemotherapy efficacy. Better tumor perfusion could result in more effective drug delivery, independent of whether the treatment is chemotherapy or immunotherapy, unless the central mechanism is immune-mediated.\nMinor:\nIn Figure 4I-M, the indication of sh vs. scr conditions appears to be swapped.", "reviewer_5": "(General comments)\nThis study investigates the role of endothelial PDLIM5, an actin binding cytoskeletal protein with PDZ and LIM domain 4, in tumor angiogenesis. The authors demonstrate that PDLIM5 is highly expressed in tumor endothelial cells (TECs) in lung cancer, correlating with poor patient survival. Mechanistically, PDLIM5 promotes tumor angiogenesis by interaction with ACTN1/ACTN4 via its S693/F596 residues, facilitating F-actin bundling and tip cell filopodia formation. Using EC-specific PDLIM5 KO mice, the study shows that loss of PDLIM5 not only suppresses tumor growth but also improves tumor vascular function, reduces intratumoral hypoxia, enhances T cell infiltration, and increases the efficacy of immune checkpoint inhibitors (anti-PD-LI antibody treatment). The experiments from Figure 1 to Figure 6 are well designed and demonstrate the role of PDLIM5 in promoting tip cell filopodia formation and tumor angiogenesis via interaction with ACTN1/ACTN4 via S693/F596 using PDLIM5 KD cells and KO mice knocked in with either adeno-WT or S588A/F591L mutant PDLIM5.\nHowever, there are two major concerns with this study: 1) Figure 7 does not integrate well with the rest of the study, as it shifts the focus to CD8+ and CD4+T cells in LLC tumors from WT or PDLIM5 KO mice in the context of immune checkpoint inhibitors therapy; 2) Previous study (PMID:29228678) shows that PDLIM5 promotes tumor cell proliferation, invasion and migration via binding to AMPK. However, the potential interaction between PDLIM5 and AMPK in TECs has not been investigated in this study. These issues should be addressed.\n(Major comments)\n1) Figure 5g: The IP:HA and IB: Myc/Flag blots are not convincing. Please replace these blots with better ones.\n2) Figure 7: The inclusion of Figure 7 disrupts the logical flow of this study by shifting the focus to CD8+ and CD4+T cells in LLC tumors from WT or PDLIM5 KO mice treated with anti-PD-LI antibody This section should be separate paper by adding more detailed mechanisms.\n3) Given that previous study shows that PDLIM5 promotes tumor cell proliferation, invasion and migration through its binding to AMPK (PMID:29228678), it is important to investigate whether PDLIM5 also binds to AMPK in TECs. Additionally, authors should examine the relationship between PDLIM5’s interaction with AMPK and its binding to ACTN1/ACTN4 in promoting tip cell filopodia formation."}, "author_response": "Dear Reviewers,\nWe sincerely appreciate your constructive feedback on our manuscript entitled \"Endothelial PDLIM5\npromotes tip cell filopodia formation and tumor angiogenesis by regulating ACTN1/ACTN4-dependent\nactin bundling\" (NCOMMS-24-81438). The reviewers' insightful comments have significantly enhanced\nthe scientific rigor and clarity of our work. After thorough discussion among all authors, we have\nimplemented comprehensive revisions as outlined below, with all modifications clearly highlighted in red\nwithin the manuscript.\nWe discuss each of the reviewers' comments individually in this letter. To facilitate this discussion, we first\nrestate your comments in italic font, and our responses are provided directly afterward in blue text. In this\nrevised version, changes to our manuscript within the document are highlighted using red text. We hope\nour efforts could make the revised manuscript more suitable for publication, and look forward to hearing\nfrom you soon. Our itemized responses to all comments are as follows:\nReviewers' comments:\nReviewer #1 (Remarks to the Author):\nIn this study, the authors investigate the role of PDLIM5 in the tumor microenvironment, focusing on its\neffects on tip cells and vascular normalization, immune cell infiltration, and response to anti-PD-L1\nimmunotherapy. Using a combination of integrative gene expression analysis, single-cell RNA sequencing,\nand functional experiments, they demonstrate that PDLIM5 expression is increased in tumor endothelial\ncells, associated with tumor neuovascularization, and functionally validated using in vitro cell assay and\nin vivo tumor modeling that PDLIM5 is linked to tumor vascularization, reduces tumor hypoxia, and\nenhances T cell infiltration, thereby improving the efficacy of immune checkpoint therapy. Using molecular\ndocking and co-IP, the study reported that PDLIM5 interact with the ACTIN2/4, hencing regulating filopodia\nformation and angiogenesis. The study spans multiple tumor models, including Hepa1-6, MC38, and LLC,\nto validate these findings. The study has applied a comprehensive molecular and cellular approaches to\nevaluate the effect of PDLIM5 on tumor vasuclar functions, the molecular interactions with ACTIN2/4, and\nits implications for cancer therapy.\nWhile the manuscript employs a variety of experimental approaches, the work is also hindered by several\nlimitations, including the conclusion on Tip endothelial cells, the molecular mechanisms linking PDLIM5\nto normal vascular functions in tissues and tumor neovascularization, the prognostic value of PDLIM5\nexpression and patient outcome, as well as critical components of the immune microenvironment.\nFurthermore, there are quite a number of mistakes in the figures and supplementary figures, which will\nneed significant proof reading and checking the original results. Detial comments for these concerns are\nhighlighted below. Carefully addressing these are required before any possible consideration of the study\nfor publication.\n1. One major conclusion of this study is the PDLIM5 affects endothelial tip cell filopodia formation and\ntumor angiogenesis. Tip cells are a very small fraction of endothelial cells leading the vascular\nangiogenesis. While tip cells are almost absence in adult normal tissues, this endothelial phenotype is\nincreased in tumors and essential for tumor neovascularization. Furthermore, tip cells are well\ncharacterized with the expression of classic markers such as CXCR4, ESM1, DLL4. The current study\nhad used COL4A1, PLVAP, COL4A2 as markers for TipECs, which are not correct (Figure S2C). These\nare mixed markers for EndoMT EC phenotypes. These jeopardize the conclusiveness of the results and\nconclusion made based on Figure 1 and related figures.\nFor example, in TEC (Figure 1d), the fraction of tip ECs could not be over 20% of the entire ECs.\nFurthermore, the increase expression of PDLIM5 is not specific to TipECs. According to the data\npresented in Figure 1b, as well as checking the gene expression data provided by the protein atlas\ndatabase, PDLIM5 is higher expressed in vascular smooth muscle cells, skeletal monocytes, fibroblasts.\nSimilarly, the increased PDLIM5 was not restricted to TipECs, as noted by the authors in the manuscript\n(line 95-96). Thus, the annotation and interpretation of TipECs will require significant revision and\ncorrection.\nResponse：First, there might have been some misunderstandings caused by the differential gene dot plot\nwe included in the previous version of our article in Figure S2C, which we showed the gene expression\nlevels of top-ranking genes in different EC phenotypes, and those top-ranking genes were not the used\nfor the annotation and interpretation of TipECs. In fact, we have precisely utilized classic markers such as\nCXCR4, ESM1, and DLL4 to annotate TipECs, as can be seen, these classical marker genes of TipEC\nare highly expressed in TipEC (Response Fig 1A). We have now included Response Fig 1A in Fig S2c\nof the revised manuscript.\nSecond, regarding the proportion of TipEC, we have reviewed relevant literature. The proportion of TipEC\namong endothelial cells varies across different disease types. In lung adenocarcinoma, the proportion of\nTipEC might be relatively high. For instance, in the study conducted by Jermaine Goveia and colleagues,\nthey collected cancer tissues and their adjacent tissues from seven patients and carried out single-cell\nsequencing analysis. In their Supplementary Table S3, the proportions of different types of endothelial\ncells in each patient's samples are listed in detail. It can be observed that the proportion of TipECs varies\nconsiderably among different patients, ranging from 0.75% to 37.30%, and the average proportion of\nTipECs in the samples of these seven patients is 9.44% (Reference: Jermaine Goveia et al., Cancer cell,\n2021). Moreover, similar phenomena have been reported in other related studies, the proportion of TipECs\nin lung cancer tissues shows significant differences, ranging from 20% to 50% (Zhoufeng Wang et al. Nat\nCommun. 2021; Xudong Xing et al. Sci Adv. 2021; Yi-Feng Ren et al. Mol Cancer. 2024; Yilv Yan et al.\nNat Genet. 2025). It is thus evident that there are substantial individual differences in the proportion of\nTipECs in tissues from different patients. In comparison, in our research data, the proportion of TipECs is\napproximately 23%, which falls within a relatively normal range.\nThe reviewer also pointed out that PDLIM5 is highly expressed in cells such as VSMCs, skeletal\nmonocytes, and fibroblasts. However, it should be emphasized that the objective of this research is to\nuncover the expression, potential functions, and regulatory mechanisms of cytoskeleton-related proteins\nin TECs. Therefore, we mainly analyzed the expression levels of these genes in endothelial cells. After\ndiscovering the high expression of PDLIM5 in TECs, we further analyzed it among different endothelial\ncell subsets to reveal which endothelial cell functions might be more significantly influenced by the highly\nexpressed PDLIM5. We found that PDLIM5 was upregulated in AECs, CapECs, LECs, and TipECs.\nNotably, compared with endothelial cells in normal tissues, the proportion of TipECs in tumor endothelial\ncells is significantly increased. Since TipECs are the most crucial endothelial cell subset for driving\nangiogenesis, we believe that PDLIM5, as a cytoskeleton-binding protein, is highly expressed in TipECs\nand must be essential for angiogenesis-related functions of TipECs, such as migration and sprouting. This\nalso serves as the basis for our subsequent exploration using in-vitro and in-vivo models as well as clinical\nsamples. As a cytoskeleton-associated protein, PDLIM5 indeed exhibits high expression in various cell\ntypes, including vascular smooth muscle cells (VSMCs) and pericytes, and even tumor cells. While\nincreased PDLIM5 levels are not exclusive to TipECs, we still posit that PDLIM5 deserves recognition as\na critical driver of tumor endothelial cell angiogenesis.\nAll computational scripts used to prepare and analyze the data can be found on\nhttps://github.com/superxiaozhi123333?tab=repositories.\nResponse Fig 1. (A) Marker genes used for the annotation of different endothelial cell subsets.\n2. Regarding the association between PDLIM5 expression and prognostic outcome of cancer patients\n(Figure 1h, and supplementary Figure 3), I am not completely sure if these data have been correctly\nadjusted and calculated. First, neither the main text or supplementary methods describe how these\nKaplan-Meier curve have been generated, and the data source used to analysis the probability of overall\nsurvival with PDLIM5 expression. It is presumably coming from the TCGA database. However, when\nchecking the survival data from the TCGA (https://tcga-survival.com/data-table?view=gene) or from the\nHPA, the expression of PDLIM5 did not show the same trajectory as what the figures provided in\nSupplementary Figure 3. The authors will have to clearly documented their analysis and scripts in the\nmethods.\nResponse: (a) We have supplemented the relevant procedures and steps for the staining analysis in\nFigure 1h, and provided patient’s information in Response table 1. PDLIM5 fluorescence intensity was\nquantified via Image J, normalized to DAPI for cell density. Patients were stratified into PDLIM5-high/low\ngroups based on median expression in CD31+ endothelial cells. Survival outcomes (OS) were calculated\nfrom diagnosis to follow-up, analyzed via Kaplan-Meier log-rank testing and multivariate Cox models, with\nsignificance set at p<0.05 using Graphpad (version 8.0.1, survival curve). This integrated\nimmunohistochemical and clinical analysis evaluated PDLIM5's prognostic role in tumor angiogenesis.\nWe have now included the detailed patient’s information in Supplementary Table 6, and the detailed\nmethod information in Methods section/ 6. Patients and samples.\n(b) We sincerely appreciate your attention to the consistency of our data sources. We would like to clarify\nthat for the survival analysis related to PDLIM5 expression in our study, we utilized the Kaplan-Meier\nplotter platform (https://kmplot.com/analysis/index.php?p=home) instead of TCGA (https://tcga-\nsurvival.com/data-table?view=gene) or HPA. Since its inception in 2009, this platform has been\nextensively applied in studies published in established leading journals in oncology. The Affy ID used in\nour analysis was 203242_s_at. To ensure the integrity of our findings, reviewers may validate the study's\nmethodology and results via the provided URL. The reason why the analysis results differ from those of\nthe TCGA (https://tcga-survival.com/data-table?view=gene) or HPA database is that the Kaplan-Meier\nplotter platform uses more different datasets. For example, when performing overall survival (OS) analysis\nfor lung cancer, the Kaplan-Meier plotter platform combines the following datasets (n=2166) for analysis,\nincluding TCGA (n=133), GSE8894 (n=138), GSE77803 (n=156), GSE68465 (n=462), GSE68465\n(n=462), GSE4573 (n=130), GSE43580 (n=150), GSE102287 (n=66), GSE14814 (n=90), GSE157011\n(n=235), GSE19188 (n=156), GSE29013 (n=55), GSE30219 (n=307), GSE31210 (n=246), GSE3141\n(n=111), GSE31908 (n=40), and GSE37745 (n=196). Therefore, the analysis results may differ from those\nobtained using datasets from a single database source, a situation that has also been observed in many\nprevious studies. For analyses of other cancer types, we generally combine all datasets provided by the\nplatform to derive corresponding results, and we believe this analytical approach does not involve issues\nof data screening.\nResponse table 1: Patient’s information\nCharacteristics N（%）\nAge\n≤65 13（54.23）\n＞65 7（71.86）\nGender\nMale 8（40）\nFemale 12（60）\nTNM stage\nI 10（50.00）\nII 6（30.00）\nIII 4（20.00）\nTumor size\n≤2.5 8（40.00）\n＞2.5 12（60.00）\nLymph node status\nN0 7（35.00）\nN1-3 13（65.00）\nDistant metastasis\nM0 20（100.00）\nM1 0（0）\nOS status\nDead 11（55.00）\nAlive 9（45.00）\n3. The selective effect of PDLIM5 on tumor vasculature formation. It is plausible that the study has used\nboth in vitro, ex vivo and in vivo models to study the effect of PDLIM5 on tumor vasculature formation.\nFirst, it remains unclear how and why loss of PDLIM5 do not affect the development of normal tissue\nvasculature and functions (Supplementary Figure 4c-e) but exhibit clear effect on the tumor vasculatures\nwith the ectopic PDX models. What is the phenotype of PDLIM5cKO mice if the induction of condition\nPDLIM5 was induced at birth or during fetal stages? In Figure 3, the study has already demonstrated that\npostnatal PDLIM5 knockout in ECs inhibit vessel sprouting in retina. This contradicts with the conclusion\nmade in part 1 (line 119-120).\nIt is unclear in the current methods provided, at what ages were the TAM induction was introduced.\nPDLIM5 is not EC-specifically expressed, when addressing and validating the conditional KO models, it\nis essential to showed in Figure 2, as in Figure 1f, that PDLIM5 is not expressed by CD31 positive\nendothelial cells. These difference between adult and neonatal could potentially be due to the induction\ntime.\nResponse: (a) We sincerely appreciate the reviewer’s insightful questions and concerns. For the first\nquestion, regarding the differential effects of PDLIM5 knockout (KO) on normal vascular development\nversus tumor vasculature. Normal vascular development (e.g., retinal angiogenesis) is a tightly regulated,\nprogrammed process with distinct phases of sprouting, maturation, and pruning. In the retina angiogenesis\nexperiments, Cdh5 Cre-mediated PDLIM5 deletion was induced in newborn mice by intraperitoneal (i.p.)\ninjections of 50 µl tamoxifen (1(cid:3031)mg/ml) from postnatal day 1 (P1) to P3. postnatal PDLIM5 KO in ECs\ninhibited retinal vessel sprouting at P7 (Figure 3), which is consistent with its role in active angiogenesis.\nIn mice, the period from P1 to P7 marks the formation of the retinal vascular network. During this time,\nangiogenesis proceeds continuously. Subsequently, from P7 to P14, the vascular maturation period\noccurs. The vascular walls gradually reach maturity, with the connections between endothelial cells\ntightening. After P14, the retinal blood vessels enter the remodeling stage. Here, the vessels are optimized\nin accordance with the functional requirements of the retina. Unnecessary vascular branches are pruned,\nrendering the vascular network more stable and efficient. We further examined the retinal vascular density\nin PDLIM5 knockout mice on P14 and P17. The results indicated that PDLIM5 KO had no impact on the\nvascular density of mice on these two time points (Response Fig 2A-D). We have now included Response\nFig 2 in Fig S4f-g of the revised manuscript.\nIn contrast, tumor vasculature undergoes dysregulated, persistent angiogenesis driven by pathological\nsignals (e.g., VEGF, hypoxia), prior to the construction of the tumor model, the induction of PDLIM5 KO\nby tamoxifen effectively inhibits the growth of multiple tumor types and angiogenesis (Figure 2). Based\non our finding, we believe that PDLIM5 KO delayed endothelial cell migration during angiogenesis process.\nHowever, it did not completely block the development and maturation of blood vessels. This is why the\ndeletion of PDLIM5 can inhibit the angiogenesis of the P7-day retina and tumor vessel, but has no effect\non the density, function of the mature vascular network in P14 and P17 mice, as well as the tissues and\norgans of adult mice. In addition, it is worth adding that in our previous studies, we have analyzed the\ndevelopment and vascular function of PDLIM5 global knockout or endothelial-specific spontaneous\nPDLIM5 KO mice (PDLIM5f/f: Tie2-Cre mice). The results showed that mice with endothelial deletion of\nPDLIM5 could be born and grow normally. After reaching adulthood, their arterial function was normal,\nblood pressure was normal, and the vascular densities in the heart and liver were also not different from\nthose of wild-type mice (Huang et al., Arterioscler Thromb Vasc Biol. 2020). This explains why PDLIM5\nKO selectively disrupts tumor vasculature (Figure 2) but not mature normal vessels (Supplementary\nFigure 4c-e).\n(b) Regarding the timing of PDLIM5 KO induction, in the “Retinal angiogenesis method”, we noted that\n“Cre-mediated deletion of PDLIM5 was induced in newborn mice via intraperitoneal (i.p.) injections of 50\nµl tamoxifen (1 mg/ml) from postnatal day 1 (P1) to P3. Eyes were harvested on P7”. In the “In vivo\nxenograft results” (Figure 2), it was graphically demonstrated that PDLIM5 was induced to be knocked\nout by tamoxifen injection prior to tumor cell inoculation in 8-week-old mice.\nAs for the choice of verifying the KO efficiency of mice by extracting lung endothelial cells from the induced\nKO mice rather than using the CD31 and PDLIM5 co-staining method as showed in Figure 1F, this is\nbecause PDLIM5 is ubiquitously expressed in a variety of cell types. To rule out non-specific staining in\ntissue immunofluorescence or the difficulty in directly discerning endothelial-specific PDLIM5 staining\ncaused by close cell proximity, we isolated the lung vascular endothelial cells from tamoxifen-induced\nPDLIM5 knockout mice and control mice. After protein lysis, Western blot analysis was performed for\nverification. As presented in the results of Supplemental Figure 4a, PDLIM5 in endothelial cells was\neffectively knocked out.\nFurthermore, when considering transgenic modification in endothelial cells, the CDH5 Cre transgenic mice\nutilized in our study are among the most extensively applied and well-recognized tools. We believe that\nCDH5 Cre transgenic mice are the most suitable tool for this project.\nResponse Fig 2. (A) Representative images of retinal vessels in PDLIM5f/f or PDLIM5cKO P14 mice. (B)\nStatistical analysis of vessel density in retinas stained with IB4 are shown. Scale bar: 100 µm. Unpaired\nt-test. ***, p < 0.001. Data were represented as mean ± SD. (C) Representative images of retinal vessels\nin PDLIM5f/f or PDLIM5cKO P17 mice. (D) Statistical analysis of vessel density in retinas stained with IB4\nare shown. Scale bar: 100 µm. Unpaired t-test. ***, p < 0.001. Data were represented as mean ± SD.\n4. The authors have done a great work in conducting all these experiments. However, it is very important\nto present the results clearly and correctly. Here I highlighted a few major mistakes in the manuscript\nfigures and results.\nFigure 4 g-l, the IP results show that shPDLIM5 down regulates DLL4, MMP2, p-FAK Y397, and VASH1\n(g-h). However, quantification of IP in Figure 4i-l, show that shPDLIM5 increase their levels. Which of\nthese are correct?\nSize of FAK and p-FAK Y397. The protein size of FAK appears to be 100kDa in some figures (S7) and\n120kDa in the other (S5, S9, Figure 4). And VEGFR3 appears to be 120kDa in S7, and 180kDa in S5.\nAll these kinds of mistakes should be carefully checked, as well as the original source data.\nResponse: We sincerely appreciate the reviewer's positive feedback on our experimental work and the\ncareful scrutiny of our manuscript. We acknowledge the concerns raised regarding the clarity and\naccuracy of our results presentation, and we have taken immediate steps to address these issues.\n(a) First, regarding Figure 4 g-l, there was an error in the quantification data presented in Figure 4i-l. The\ncorrect interpretation, as indicated by the IP results in Figure 4 g-h, is that shPDLIM5 downregulates DLL4,\nMMP2, p-FAK Y397, and VASH1. We deeply apologize for this oversight. We have revised all the data\nand corrected the quantification figures (Response Fig 3A-D). The corrected figures will clearly show\nthe downregulation trends consistent with the immunoprecipitation results. We have now included Response\nFig 3 in Fig 4i-j of the revised manuscript.\n(b) Second, regarding the protein sizes of FAK and p-FAK Y397, and VEGFR3. In the original figures, the\nsize labels were misinterpreted. The numbers indicated on the figures (such as in S7 where FAK seemed\nto be 100kDa and in S5, S9, Figure 4 where it seemed to be 120kDa) were the positions of protein markers\non the gel, not the actual molecular weights of FAK and p-FAK Y397. The actual molecular weight of FAK\nis approximately 125 kDa. We apologize for this confusion. We have checked all protein marker positions\nin the revised manuscript to ensure there were no incorrect labels.\nResponse Fig 3. (A-D) Quantification of relative protein levels of p-FAK Y397/total FAK (A), DLL4 (B),\nMMP2 (C), and VASH1 (D) are shown as mean ± SD. One-way ANOVA with multi-comparisons. *, p <\n0.05; **, p < 0.01; and ***, p < 0.001.\n5. Methods regarding PDLIM5 knockdown in HUVECs. It is unclear about the methodology used for\nACTIN1, ACTIN4, and PDLIM5 knockdown in HUVECs. For ACTIN1 and ACTIN4, synthetic siRNAs\nshould have been used according to the methods. However, PDLIM5, why shRNA expressed by lentiviral\nhave been used instead? Any reason for choosing the difference delivery approach? Please also check\nthe siRNA sequences for si-ACTN1-1#, as it could not be found in the human ACTN1 gene.\nResponse：We sincerely appreciate the reviewer's attention to this aspect of our methods section.\n(a) Initially, during our exploration of the role of PDLIM5 in endothelial cell function, to assess the effects\nof both transient and long-term knockdown of PDLIM5 on these cells, we designed two shRNAs targeting\nPDLIM5. These were then packaged into lentiviruses to facilitate the knockdown of PDLIM5 in endothelial\ncells. After discovering that PDLIM5 regulated endothelial cell filopodia by binding to ACTN1/ACTN4,\nwe\naimed to verify whether the individual knockdown of ACTN1/ACTN4 could recapitulate the phenotypes\nobserved upon PDLIM5 knockdown. To achieve transient and efficient knockdown of ACTN1/ACTN4, we\ntransfected siRNAs into HUVEC cells using the RNAi MAX transfection reagent. The results demonstrated\nthat, following the successful knockdown of ACTN1/ACTN4, the cell phenotypes and the activation status\nof the downstream FAK signaling pathway were identical to those seen when PDLIM5 was knocked down.\nWe did not arbitrarily choose knockdown strategies for different genes. Whether employing shRNA or\nsiRNA, our goal was simply to achieve gene knockdown and conduct subsequent phenotypic analyses,\nwe are confident that different knockdown methods will not affect the conclusions of this article.\n(b) We sincerely apologize for the error in the si-ACTN1-1# sequence. The sequence\n\"CCACAAAGTGACAGTTTACAA\" we previously provided was that of Mus musculus ACTN1, which is\nutilized for targeted knockdown of ACTN1 in mouse cells in some of our other ongoing research projects.\nIn the revised version of the manuscript, we have rectified the sequence to the accurate one\n\"CGAAGAAATCGTGGATGGGAA\" in Supplementary Table 5. We deeply regret this error and any\ninconvenience it may have caused.\n6. The study has integreated public scRNA-seq data and generated new data based on their own patient\ncohort, but the current analysis and detial provided is not sufficient to support the reproducibility. What\nquality control metrics and thresholds were applied to filter low-quality cells, number of cells, number of\npatients and metadata, how batch effects had been handled if any, how were endothelial cell subtypes\nidentified, marker genes used. Providing these details would strengthen the accuracy and reproducibility\nof the scRNA-seq analysis. Additionally, consider depositing the raw data and scripts in a public repository\nto enhance transparency.\nResponse：We sincerely appreciate your constructive feedback regarding the reproducibility and\ntransparency of our scRNA-seq analysis. Below, we provide detailed responses to address your concerns:\n(a) Samples of neoplastic and adjacent non-neoplastic tissues from 7 lung adenocarcinoma patients were\nharnessed for single-cell sequencing analysis.\n(b) we filtered expression matrix by the following criteria: 1) cells with gene count less than 200 or with\ntop 2% gene count were excluded; 2) cells with top 2% UMI count were excluded; 3) cells with\nmitochondrial content > 20% were excluded; 4) genes expressed in less than 5 cells were excluded.\n(c) The batch effect is removed using Harmony.\n(d) All the raw data were uploaded in public repository, including:\n1) The RNA-sequencing raw data have been uploaded to the GSA (https://ngdc.cncb.ac.cn/gsa/). The\naccession number is HRA009823.\n2) The protein mass spectrometry sequencing data have been uploaded to the iProX database\n(https://www.iprox.cn/page/home.html). The Project ID is IPX0010745000, and the ProteomeXchange\nID is PXD059449.\n3) The single cell sequencing raw data have been uploaded to the GSA (https://ngdc.cncb.ac.cn/gsa/),\nthe accession number is HRA009971.\nWe have added the above-mentioned content to the Method section in revised manuscript.\n7. The methods provide useful details about AAV injections and xenograft experiments, but there are\nareas requiring clarification: Specify the AAV titer (viral particles/μL) used for intratumoral injections to\nensure reproducibility. Provide details on time points for tumor volume measurements and angiogenesis\nassessments after AAV injection.\nSpecify the exact frequency of tumor volume measurements within this period (e.g., daily or every other\nday). Clarify whether tumor volume data were averaged across multiple tumors or cohorts. Discuss\nvariations in tumor growth rates over time to better contextualize the presented data.\nResponse: (a) For in vivo rescue experiments, when the tumor grew to ~60 mm3, 50 μL AAV-PDLIM5 WT\nor AAV-PDLIM5 mut AAV with a titer of 1(cid:3031)×(cid:3031)108 viral particles/μL were injected at multiple points within the\ntumors.\n(b) Tumor volumes were measured every two days and calculated using the formula: length × width²/2\nand was measured with a digital caliper. Tumor volume data were averaged across multiple tumors within\neach experimental cohort. These variations in growth rates have been incorporated into the results section\nof the manuscript, along with corresponding graphs that clearly illustrate the changing slopes of the tumor\ngrowth curves over time. Tumor angiogenesis was measured by CD31 staining after the tumor tissues\nwere removed and made into paraffin sections. We have added a concise analysis of tumor growth rate\nvariations over time in the Results section, contextualizing the data with key temporal trends and group\ncomparisons. This revision enhances the interpretive framework by highlighting dynamic growth patterns\nrelevant to our findings.\nWe have added the above-mentioned content to the Method section in revised manuscript.\n8. The authors suggest that PDLIM5 could be a promising therapeutic target and mention small molecule\ninhibitors as a potential strategy. However, there is no experimental data, such as preclinical trials or\nefficacy studies, to back up these claims. Including some preliminary studies that explore inhibitors\ntargeting PDLIM5 or its interaction sites would help demonstrate whether this approach could actually be\neffective in a therapeutic setting.\nResponse: In this study, we validated that PDLIM5 deficiency inhibits the growth of multiple tumors\n(including LLC, MC38, and Hepa1-6 subcutaneous xenografts, as well as E0771 orthotopic breast cancer)\nusing endothelial-specific PDLIM5 knockout mice. We also found that mutant PDLIM5 can suppress tumor\ngrowth. Therefore, we envision that targeting PDLIM5 may serve as a promising anti-angiogenic target.\nTo further validate PDLIM5 as a potential anti-angiogenic target, we induced genetic deletion of endothelial\nPDLIM5 at two distinct therapeutic time windows: the early tumor-bearing stage and after substantial\ntumor growth. As demonstrated in Response Figure 4, compared with control groups, both early and\nlate-stage deletion of endothelial PDLIM5 effectively suppressed the growth of E0771 orthotopic tumors.\nImportantly, tumor vascularization was significantly impaired in both groups compared with control, as\nevidenced by reduced vascular density. These findings confirm that PDLIM5 inhibition exerts anti-tumor\neffects through angiogenesis suppression, regardless of therapeutic initiation timing. We have now\nincluded Response Fig 4 in Fig 2q-u of the revised manuscript.\nNotably, our preliminary attempts to develop peptide-based inhibitors mimicking existing strategies for\nblocking protein-protein interactions faced technical challenges. Although we synthesized several short\npeptides designed to disrupt PDLIM5-ACTN1/ACTN4 binding, their efficacy proved suboptimal. This may\nstem from multiple factors including: 1) Insufficient binding specificity of initial peptide designs; 2) Inability\nto effectively access the molecular binding pocket between PDLIM5 and ACTN1/ACTN4; 3) Suboptimal\npeptide solubility and binding energy parameters. These observations highlight the necessity for\nmultidisciplinary optimization of molecular design, potentially through computational chemistry\napproaches and structural biology-guided modifications.\nIn light of these findings, we propose that PDLIM5 represents a promising therapeutic target. Future\ndevelopment of targeted nanomedicines or optimized peptide drugs based on the critical S593/F596\ninteraction interface could provide viable strategies for PDLIM5-targeted therapies. The technical\nchallenges encountered in our preliminary peptide development phase underscore the importance of\nsystematic structure-function studies and advanced drug delivery systems for translating this molecular\ntarget into clinical applications.\nConsidering that emphasizing PDLIM5 as a valid target without having actually developed small-molecule\ninhibitors targeting it could mislead readers, we have revised the relevant descriptions in the abstract and\nintroduction sections of the paper.\nResponse Fig 4. (A) Schematic illustration of E0771 orthotopic breast tumor model in PDLIM5f/f or\nPDLIM5cKO mice. After mice are injected with E0771 cells or when the tumor volume reaches 60 mm³,\ntamoxifen is intraperitoneally injected for 5 consecutive days. (B) Image of tumors. (C-D) Tumor volume\nchanges (c) and weight (d) in PDLIM5f/f or PDLIM5cKO mice (n=6). Two-tailed unpaired t-test (b) and\nunpaired t-test (c). ***, p< 0.001 and **, p< 0.01. Data were represented as mean ± SD. (E-F)\nRepresentative micrograph tumors section immunostained for CD31 in PDLIM5f/f or PDLIM5cKO mice.\nNuclei are stained with DAPI. Scale bar, 100 μm. Right: Quantification of relative vessel density. Unpaired\nt-test, ***, p< 0.001. Data are represented as mean ± SD.\nReviewer #2 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the\nNature Communications initiative to facilitate training in peer review and to provide appropriate recognition\nfor Early Career Researchers who co-review manuscripts.\nResponse: Thank you for sharing this information about the co-review initiative. We greatly appreciate the\nefforts of both the main reviewer and the Early Career Researcher involved in co-reviewing our manuscript.\nWe understand that this initiative not only promotes training in peer review but also acknowledges the\nvaluable contributions of young researchers.\nWe are committed to addressing all the points raised in the review reports with the utmost care and\nprofessionalism. We will work diligently to improve our manuscript based on the reviewers' suggestions,\nensuring that it meets the high standards of Nature Communications.\nReviewer #3 (Remarks to the Author):\nThis paper investigates the role of the cytoskeletal scaffold protein PDLIM5 in promoting angiogenesis by\ntumor endothelial cells (TECs). The authors show that PDLIM5 stimulates vascular sprouting in the retina\nof healthy mice as well as in tumor grafts. By combining KO of PDLIM5 with anti-PD-L1 treatment, they\nfurther show that this PDLIM5 function can be exploited to decrease tumor growth and promote tumor\nimmune cell infiltration, suggesting that small molecules directed against PDLIM5 might hold promise as\na new antitumor treatment. To address the mechanism underlying this PDLIM5 function and identify\npotential PDLIM5 surfaces to target for drug development, the authors examine the interaction between\nPDLIM5 and ACTN1/ACTN4, which has previously been reported in other cell types. In contrast to\nNakagawa et al (2006), they find that PDLIM5 binds to ACTN1/ACTN4 through its Lim domain instead of\nits PDZ domain. They further narrow the PDLIM5-ACTN1/4 interaction down to two residues in the third\nLIM domain (S593 and F596) and present some data that suggests this interaction enhances the binding\nof ACTN1/ACTN4 to F-actin, thereby stabilizing and promoting filopodia formation and angiogenesis. In\nline with this, expression of the PDLIM5 double mutant in PDLIM KO mice hampers tumor growth,\nangiogenesis and immune cell invasion. The merit of this work mainly lies in the large body of data\nsupporting PDLIM5 as a potentially interesting antitumor target for a great many different types of cancer.\nHowever, the described underlying mechanism is not fully supported by the data presented. Thus, to\nsubstantiate the claim that this study identified a druggable surface on PDLIM5, the authors will have to\naddress the issues raised below.\n1. Fig. 4: The authors claim that PDLIM5 recruits ACTN1/4 to actin in filopodia, however the linescans\nfrom the images shown in Fig. 4e-f do not show that higher PDLIM5 signals correspond to higher ACTN1/4\nsignals. In addition, the immunoprecipitation assay in Fig. 4g, shows varying levels of PDLIM5 in WCL,\nbut does not confirm that this also corresponds to varying levels of PDLIM5 bound to actin after IP.\nHence, they cannot conclude that PDLIM5 regulates the amount of ACTN1/4 bound to actin. To claim that PDLIM5\nrecruits ACTN1/4 to F-actin, they need to show that the level of PDLIM5 bound to F-actin after\nimmunoprecipitation changes similarly as the level of ACTN1/4 bound to F-actin after immunoprecipitation.\nIn addition Line 193: The authors state that PDLIM5 colocalizes with ACTN1/4 in tip cells (Fig. 4e). Are\nthe cells that are shown in the images and that are used for the linescans actual tip cells?\nResponse: (a) In Figures 4e-f, we performed immunofluorescence analysis of HUVECs cultured in 2D\nconfocal dishes for 24 hours. Following phalloidin staining for F-actin and immunostaining for\nACTN1/ACTN4 and PDLIM5, we analyzed the co-localization of these proteins in filopodia structures.\nWhile our initial images may not have optimally demonstrated the tripartite co-localization, higher\nmagnification revealed concurrent localization of ACTN1/ACTN4, PDLIM5, and F-actin in triangular-\nmarked protrusions (Fig. 4e-f, as showed in the response Figure 5a-b). We acknowledge that the relatively\nweak F-actin signals in some regions might have affected the visual prominence of these protrusions.\nRegarding the reviewer's question about whether these cells represent actual tip cells: While 2D-cultured\nendothelial cells cannot fully recapitulate native tip cell biology, this established model system remains\nvaluable for analyzing filopodia-associated protein dynamics. This approach has been successfully\nemployed in previous angiogenesis studies, including landmark study by De Bock et al. (Cell, 2013). In\ntheir investigation of PFKFB3's role in vessel sprouting, they utilized 2D-cultured GFP-labeled endothelial\ncells to demonstrate that PFKFB3 knockdown impaired lamellipodia/filopodia formation and subsequent\nendothelial cell sprouting functions, which were further validated through retinal angiogenesis models and\nfibrin gel bead assays.\nWe also conducted additional 3D fibrin gel bead assays using HUVECs (Response Figure 5c-d). These\nexperiments confirmed co-localization of ACTN1/ACTN4, PDLIM5, and F-actin in filopodia of sprouting tip\ncells. While we initially attempted similar experiments with TECs, limited cell yields precluded these\nstudies. The HUVEC data provide complementary evidence supporting our findings in TECs, consistent\nwith previous reports demonstrating conserved mechanisms of filopodia formation across endothelial cell\ntypes. We have now included Response Fig 5c-d in Fig S6b-c of the revised manuscript. In fact, the\ninteraction between PDLIM5 and ACTN4 has been previously reported in cardiac myocytes and skeletal\nmuscle (Nakagawa, N et al. Biochem Biophys Res Commun. 2000; Wang D, et al. J Cell Mol Med. 2019;\nNiederländer Net al. Biochem Biophys Res Commun. 2004), which is consistent with our findings.\n(b)To directly address the reviewer's concern, we conducted additional immunoprecipitation (IP)\nexperiments quantifying PDLIM5-F-actin binding dynamics. Our data demonstrate that PDLIM5\noverexpression increases the level of PDLIM5 bound to F-actin, while PDLIM5 knockdown reduces this\ninteraction compared to controls (Response Figure 5e). These IP results align with our observed changes\nin ACTN1/4-F-actin binding (Fig. 4g), showing coordinated regulation of both components. We have now\nreplaced Fig. 4g with Response Fig 5e in the revised manuscript.\nResponse Fig 5. (A-B) Representative micrograph of TECs immunostained for PDLIM5, Phalloidin, and\nACTN1 or ACTN4. Nuclei are stained with DAPI. Scale bar, 10 μm. Intensity profiles of PDLIM5, Phalloidin\nand ACTN1 or ACTN4 along the line segment were shown. (C-D) Representative micrograph of EC\nspheroids immunostained for PDLIM5, Phalloidin, and ACTN1 or ACTN4. Nuclei are stained with DAPI.\nScale bar, 10 μm. Intensity profiles of PDLIM5, Phalloidin and ACTN1 or ACTN4 along the line segment\nwere shown. (E) co-IP analysis of the interaction among ACTB, ACTN1, and ACTN4 in control, PDLIM5\noverexpression, or PDLIM5 silenced (KD) HEK293 cells.\n2. Fig 5: The authors should explain why Fig. 5g shows that PDLIM5 mutants bind less ACTN1/4, however\nthe reverse IP in Fig, 5c-d does not show the same result. Is this perhaps due to the fact that these IP\nexperiments were performed with full length PDLIM5, which can bind to F-actin and ACTN1/4. The latter\nimplies that the interpretation of the IP results could be perturbed because there are two binding\ninteractions to account for, especially given that the authors do not demonstrate that the LIM domain\nmutations do not change the overall conformation of PDLIM5 such that it affects its interaction of the\nPDZ domain with F-actin. To make matters worse, ACTN1/4 are bone fide F-actin binding proteins,\nirrespective of whether their binding is increased by PDLIM5, and thus it should be taken into account that\nPDLIM5 binds to F-actin bound by ACTN1/4. To work around this complex situation and avoid any confusion,\nthe mutant PDLIM5 IP experiments should be repeated with the mutations introduced in the LIM domains only.\nThis is contrary to Fig. 5h-i. In order to conclude that these mutations fail to recruit ACTN1/4 to F-actin,\nthe authors should confirm that full length PDLIM5 mutants bind equally well to F-actin as wild type\nPDLIM5.\nResponse：(a) In actuality, Figures 5c-d already indicate a weakened binding of PDLIM5 in the S594A or\nF596L mutants. However, the original images suffered from sub-optimal background clarity. We have\nrepeated the CO-IP experiments to rigorously validate this finding. After observing in Figures 5c-d that\nsingle mutations led to a reduction in the binding affinity between PDLIM5 and ACTN1/ACTN4, we were\nmotivated to perform double-mutation experiments and subsequent confirmatory studies. In the revised\nmanuscript, we have replaced the figures with ones that offer a more distinct presentation of the results,\nas illustrated in Response Figures 6A-B.\n(b) Simultaneously, to more comprehensively verify that PDLIM5 binds to ACTN1/ACTN4 via the\nS593/F596 sites within its LIMs domain, we engineered truncated LIMs domain with S593A and F596L\nsingle mutants, as well as the S593A/F596L double mutant. We then employed IP assays to assess the\nbinding between the LIMs mutants and ACTN1/ACTN4. The data clearly demonstrate that both single\nmutations effectively reduce the binding of the LIMs domain to ACTN1/ACTN4, and the double mutation\nfurther diminishes this binding capacity (Response Figure 6C). This strongly suggests that PDLIM5 binds\nto ACTN1/ACTN4 through the S593/F596 sites in its LIMs domain. We have now included Response Fig\n5C in Fig S8i of the revised manuscript.\n(c) Furthermore, we investigated the binding of the mutated PDLIM5 to F-actin. The results reveal that\nneither the S593A and F596L single mutations nor the S593A/F596L double mutation of PDLIM5 have a\ndiscernible impact on their binding (Response Figure 6D), and neither the S593A and F596L single\nmutations nor the S593A/F596L double mutation of LIMs domain interact with F-actin. We have now\nincluded Response Fig 5D in Fig S8h of the revised manuscript.\nResponse Fig 6. (A-B) HEK293T cells were transfected with wild-type or mutant PDLIM5 plasmids as\nindicated to detect the site between PDLIM5 and ACTN1 or ACTN4 in HEK293T cells. (C) HEK293T cells\nwere transfected with wild-type or mutant LIMs plasmids as indicated to detect the site between LIMs and\nACTN1 or ACTN4 in HEK293T cells. (D) HEK293T cells were transfected with wild-type or mutant PDLIM5\nplasmids as indicated to detect the interaction between PDLIM5 and F-actin in HEK293T cells. (E)\nHEK293T cells were transfected with wild-type or mutant LIMs plasmids as indicated to detect the\ninteraction between LIMs domain of PDLIM5 and F-actin in HEK293T cells.\nMinor comments:\n- Include the species of PDLIM5 that was used to clone the lentiviral and adenoviral constructs in the\nsupplemental methods section.\nResponse：Thank you for your comments. In our previously submitted manuscript, there were indeed\ninsufficient methodological details regarding the cloning of PDLIM5. We have now supplemented the\nmethodology section with the specific species information of the cloned PDLIM5.\n- Line 334: Nakagawa et al (2006) also found that the PDLIM5 PDZ domain mediates binding to ACTN1/4.\nThis difference between their study and this work should be addressed.\nResponse：First, methodological disparities exist between the two studies. Nakagawa et al. employed\nGST-PDZ fusion proteins in pull-down assays with rat heart lysate and C2C12 myogenic cell lysate to\nidentify PDZ-binding cytoskeletal partners. While their anti-actin immunoblotting demonstrated consistent\nF-actin binding across both lysates—aligning with our findings—their anti-α-actinin analysis revealed\ntissue-specific interaction: GST-PDZ bound α-actinin (ACTN1/ACTN4) exclusively in rat heart lysate, with\nno detectable signal at the expected molecular weight in C2C12 lysate. Notably, the authors provided no\nmechanistic rationale for this cell-type dependent discrepancy. Given the inherent challenges of achieving\ncomplete protein extraction from tissues versus cultured cells, compounded by the molecular complexity\nof tissue lysates, the methodological validity of this tissue-specific observation remains questionable. In\ncontrast, our systematic approach addressed these limitations through engineered HA-tagged PDLIM5\nvariants (full-length, PDZ/LIM domain truncations, and ΔLIM constructs) co-expressed with Myc-tagged\nACTN1 and Flag-tagged ACTN4 in 293T cells. Bidirectional co-immunoprecipitation confirmed LIM\ndomain-dependent binding, while structure-guided mutagenesis (S593A, F596L, and S593A/F596L\ndouble mutant) localized the interaction interface to residues S593/F596 within the LIM domains, as\nvalidated in Response Figure 6C. This integrative strategy—combining domain mapping with residue-\nlevel mutagenesis—provides robust structural evidence that PDLIM5 engages ACTN1/ACTN4 via\nS593/F596 residues in its LIM domains. Technical enhancements including explicit epitope tagging\n(HA/Myc/Flag), cellular system specification (293T), methodological distinction between pull-down and\nCO-IP systems, and standardized structural terminology collectively strengthen scientific rigor while\nrespectfully contextualizing prior findings.\n- It would be nice of the authors could expand the discussion a bit to also talk about how the PDLIM-\nactinin interaction is required for the other PDLIM family members.\nResponse：We sincerely thank the reviewer for this important suggestion. The interaction with α-actinin\nappears to be a conserved yet functionally diversified feature across multiple PDLIM family members,\nextending beyond the previously emphasized roles of PDLIM5/7 and PDLIM1. While PDLIM1 selectively\nbinds α-actinin4 (ACTN4) at Asn145 to suppress F-actin overgrowth and sustain Hippo signaling via\nLATS1 stabilization (Huang Z, et al. Hepatology. 2020), Pdlim2 interacts broadly with α-actinins to regulate\ncytoskeletal stability (Sistani L, et al. Kidney Int. 2011; Torrado M, et al. Invest Ophthalmol Vis Sci. 2004),\nsuggesting isoform-specific partnerships fine-tuned for distinct cellular outputs. Similarly, PDLIM5/7 not\nonly recruit YAP via their PDZ domain but also rely on direct α-actinin binding to translocate to stress\nfibers under mechanical force, thereby coupling cytoskeletal tension to YAP activation (Elbediwy A, et al.\nJ Cell Sci. 2018). This recurring theme—where LIM domains engage α-actinins to scaffold cytoskeletal\norganization, while PDZ domains either anchor to F-actin or recruit signaling effectors (e.g., YAP)—implies\nthat α-actinin interactions are integral to the PDLIM family’s dual role as mechanosensitive adaptors. Such\nfunctional conservation highlights α-actinin as a central node enabling PDLIM proteins to spatially\nintegrate cytoskeletal dynamics with pathway-specific signaling, a mechanism likely critical for all family\nmembers despite context-dependent variations in binding partners or downstream targets. We have\nadded the relevant content to the discussion section of the revised manuscript.\nReviewer #4 (Remarks to the Author):\nIn the study entitled “Endothelial PDLIM5 promotes tip cell filopodia formation and tumor angiogenesis by\nregulating ACTN1/ACTN4-dependent actin bundling”, the authors investigate the role of PDZ and LIM\ndomain 5 (PDLIM5) in tumor endothelial cell (TEC)-mediated angiogenesis. They demonstrate that\nPDLIM5 is highly expressed in lung cancer TECs, correlates with poor patient survival, and plays a crucial\nrole in filopodia formation by interacting with actinin-1/actinin-4 (ACTN1/ACTN4) to facilitate filamentous\nactin (F-actin) bundling. Endothelial-specific deletion of PDLIM5 disrupts sprouting angiogenesis, reduces\ntumor growth, normalizes tumor vasculature, and enhances immunotherapy response in resistant tumors.\nThese findings suggest that PDLIM5 may serve as a novel therapeutic target to improve cancer treatment\noutcomes.\nComments:\nThe results presented in the manuscript provide substantial evidence on the role of PDLIM5 in TECs and\nits potential impact on tumor angiogenesis. However, a number of comments should be addressed to\nsupport the claims and findings presented in the manuscript. These include:\n1. Additional functional studies are needed to convincingly demonstrate that endothelial PDLIM5 regulates\ntip cell function. For instance, in vitro migration assays and competition sprouting assays, where a mixed\npopulation WT and PDLIM5-silenced cells compete for the tip cell position, could provide additional\nstrength.\nIt would be also important to investigate how the tip cell subset (as well as the additional subtypes) is\naffected in the endothelial-specific PDLIM5 KO in mice.\nResponse：(a) We sincerely appreciate the reviewer's suggestions. The proposed in vitro migration and\ncompetitive sprouting assays are indeed pivotal for elucidating PDLIM5's role in tip cell function. Therefore,\nwe first performed scratch assays on PDLIM5 knockdown and wild-type endothelial cells. Results showed\nthat PDLIM5 knockdown significantly inhibited migration in endothelial cells in a tranwell migration model.\nAdditionally, we conducted competition sprouting assays using lenti-GFP-labeled wild-type cells and lenti-\nRFP-labeled PDLIM5 knockdown endothelial cells. Quantitative analysis revealed that wild-type cells\ndominated sprouting structures in tip cell formation compared to PDLIM5-deficient counterparts\n(Response Figure 7A-B). We have now included Response Fig 7B in Fig S8h of the revised manuscript.\n(b) We sincerely thank the reviewer for their insightful comments. Investigating the impact of PDLIM5\ndeletion on tumor endothelial subsets (e.g., tip cells) is critical for understanding its angiogenesis\nregulatory mechanisms. Given the complex experimental workflows and analysis pipelines required for\nsingle-cell sequencing, we employed flow cytometry to sort CD31+ endothelial cell populations and used\nqPCR to quantify mRNA expression levels of key tip cell markers (including CXCR4, ESM1, and DLL4).\nThese genes have been validated as specific tip cell markers in multiple studies (Zhoufeng Wang et al.\nNat Commun. 2021; Xudong Xing et al. Sci Adv. 2021; Yi-Feng Ren et al. Mol Cancer. 2024; Yilv Yan et\nal. Nat Genet. 2025), with their expression dynamics effectively reflecting tip cell activation status. We\nhave successfully established an endothelial cell sorting system, and preliminary data show significant\ndownregulation of these markers in PDLIM5 KO mice (Response Figure 7C). This result aligns with our\npreviously submitted RNA-seq data in HUVECs. While this approach has slightly lower resolution than\nsingle-cell sequencing, its targeted analysis provides direct evidence for subset functional differences.\nWe will highlight the correlation between these findings and vascular phenotypic abnormalities in the\ndiscussion section and plan to perform spatial transcriptomic analysis in future studies for further validation.\nWe have now included Response Fig 7C in Fig S5C of the revised manuscript.\nResponse Figure 7. (A) Transwell migration assay detects the migration ability of WT vs PDLIM5 KD\nHUVECs. (B) Representative image and quantification of sprouts from the fibrin gel sprouting assay with\n1:1 competitive seeding of WT (Green) and PDLIM5 KD (red) HUVECs on the beads. Scale bar, 100 μm.\nData are presented as mean with SD. Unpaired t test. ***, p< 0.001. (C) Relative mRNA expression of\nPDLIM5 and tipEC markers (CXCR4, ESM1, DLL4) and MMP2 were analyzed in TECs isolated from\nPDLIM5f/f (n=3) or PDLIM5cKO (n=3) mice. Data are presented as mean with SD. Unpaired t test. ***, p<\n0.001.\n2. While the authors propose that endothelial PDLIM5 deletion induces tumor vessel normalization (TVN),\nFigure 2 suggests vessel pruning (reduced vessel density), which is likely responsible for the observed\nreduced tumor growth. This discrepancy warrants an explanation. TVN is often associated with improved\nmetastasis outcomes rather than with an effect on primary tumor growth. It would be useful to investigate\nthe relationship between vessel pruning and reduced tumor growth and explore whether TVN by PDLIM5\ndeletion affects metastasis.\nResponse：(a) We sincerely appreciate your meticulous review and the valuable questions you've raised.\nRegarding the potential discrepancy you've pointed out between the vessel pruning (manifested as\ndecreased vessel density) shown in Figure 2 and our proposed induction of tumor vessel normalization\n(TVN) by endothelial PDLIM5 deletion, the following is our explanation.\nTumor vessel normalization is an intricate process, not a simple, single alteration. Conventionally, vessel\nnormalization has often been narrowly regarded as the optimization of vascular structure and function\nwithout significant reduction in vessel quantity. However, in the context of the tumor microenvironment,\nvessel pruning is indeed an integral part of the vessel normalization process. When endothelial PDLIM5\nis depleted, it triggers a cascade of molecular and cellular events. Initially, the functional state of vascular\nendothelial cells is altered, compelling the remodeling of the chaotic, abnormally branched, and inefficient\ntumor vascular system. During this remodeling, poorly perfused and malfunctioning vascular branches\nare gradually pruned away, which is precisely what is reflected by the reduced vessel density in Figure 2,\nFigure 7 and Supplemental Figure 10. This pruning is not an isolated phenomenon separate from vessel\nnormalization; rather, it represents a crucial manifestation of normalization. By eliminating these non-\nfunctional vascular branches, the remaining vessels can re-distribute blood flow, enhancing the delivery\nof oxygen and nutrients, which lies at the heart of the functionality of vessel normalization. Additionally,\nvessel normalization also encompasses aspects such as the improvement of pericytes coverage and the\nreconstruction of the basement membrane. Collectively, these elements form the comprehensive\nlandscape of tumor vessel normalization, and many previous studies also support this view (Tian L, et al.\nNature. 2017; Huang Y, et al. Nat Rev Immunol. 2018; Lu Z, et al. Sci Transl Med. 2024; Augustin HG,\nKoh GY. Cancer Res. 2022). The observed slowdown in tumor growth in our study is precisely the outcome\nof the optimization of the tumor microenvironment and the transformation of its supportive effect on tumor\ncells following vessel normalization, which includes this vessel-pruning step.\n(b) Regarding whether PDLIM5 knockout affects tumor metastasis, we conducted relevant investigations.\nWe established subcutaneous LLC tumor xenografts, performed surgical resection of primary tumors, and\nanalyzed lung metastasis after tumor regrowth. This method has been validated previously to successfully\nestablish lung metastasis models (Lu Z, et al. Sci Transl Med. 2024). Results showed that PDLIM5 deletion\nsignificantly inhibited LLC lung metastasis (Response Figure 8A). We have now included Response Fig\n8A-C in Fig S11A-C of the revised manuscript.\nWhile the reviewer noted that \"TVN is often associated with improved metastasis outcomes,\" conflicting\nevidence exists. For example, Elena Magrini et al. (2014) demonstrated that endothelial L1 deficiency\nreduced tumor angiogenesis and promoted vessel normalization. In Panc02 tumor-bearing L1floxed vs.\nTie2-Cre;L1floxed mice, metastasis incidence was 5/7 (71%) vs. 2/7 (29%), with fewer site-specific\nmetastases in Tie2-Cre;L1floxed mice (Magrini et al. J Clin Invest. 2014). Similarly, Anna Rita Cantelmo\nand Silvia La Porta reported that endothelial-specific deletion of PFKFB3 or Tie1 reduces B16-F10 and\nLLC lung metastasis, attributing this phenomenon to the notion that \"normalized vasculature constitutes\na tighter endothelial barrier, rendering tumor cells less prone to escape.\" (Cantelmo et al. Cancer Cell.\n2016; La Porta S, et al. J Clin Invest. 2018). These findings align with numerous studies and reviews\nsupporting the concept that vessel normalization reduces metastasis. In our study, we propose that\nPDLIM5 deletion reduces primary tumor metastasis by promoting vascular normalization.\nResponse Figure 8. (A) Schematic diagram showing the establishment of a pulmonary metastasis model\nvia subcutaneous tumor xenograft. LLC cells were injected subcutaneously, and once tumors reached 1.5\ncm in diameter, the overlying skin was incised to excise the tumor mass in day 15. After wound suturing,\nmice were allowed to recover and continue to be raised. After 3 weeks lung tissues were harvested for\nfixation and histological analysis. (B) Representative H&E staining images of mouse lung tissues, with\nyellow arrows indicating pulmonary metastatic foci. Scale bar, 1 mm. (C) Quantitative analysis of the\nnumber of pulmonary metastatic foci. Data are presented as mean with SD. Unpaired t test. ***, p< 0.001.\n3. The mechanism by which endothelial PDLIM5 regulates T cell infiltration needs further investigations.\nAre increased T cell infiltration levels due to enhanced delivery upon TVN, or is there an effect on adhesion\nmolecules? ICAM-1 clustering and function has been shown to be directly linked to ACTN4 (Celli et al. J\nImmunol. 2006). Thus, it would be crucial to assess whether PDLIM5 deletion alters ICAM-1 clustering.\nAdditionally, as actin-binding proteins can differentially regulate EC stiffness (Schaefer, et al. J Cell 2014),\nit would be valuable to investigate whether PDLIM5 deletion leads to changes in EC stiffness that might\nfavor T cell transmigration.\nResponse：In Figure 7 and Supplemental Figure 10, we mainly demonstrate that PDLIM5 deficiency\npromotes tumor vascular normalization (including increased pericyte coverage, enhanced perfusion levels,\nand reduced permeability). The normalized tumor vasculature also alleviates tumor hypoxia. Previous\nstudies have shown that normalized tumor vasculature can enhance immunotherapy efficacy, as\nevidenced by clinical observations that anti-angiogenic therapy combined with immunotherapy prolongs\nsurvival in various cancers (Schoenfeld JD, et al. Lancet Oncol. 2022; Reardon DA, et al. JAMA Oncol.\n2020; Yuan L, et al. Nat Commun. 2023; Adkins D, et al. Lancet Oncol. 2024).\nIn our previous version, we did not sufficiently explain why PDLIM5 deficiency promotes T-cell infiltration.\nWe hypothesized that vascular normalization might facilitate T-cell infiltration without exploring the specific\nmechanisms. Indeed, many previous studies have found that normalized tumor vessels enhance T-cell\ninfiltration and antitumor immunity through structural and functional remodeling (Tian L, et al. Nature. 2017;\nHuang Y, et al. Nat Rev Immunol. 2018; Lu Z, et al. Sci Transl Med. 2024; Augustin HG, Koh GY. Cancer\nRes. 2022). First, vascular normalization restores vessel architecture by stabilizing the basement\nmembrane and aligning endothelial cells, reducing vascular leakage and improving blood perfusion. This\noptimization of hemodynamics enables efficient delivery of T cells to tumors while alleviating hypoxia,\nwhich suppresses immunosuppressive factors such as HIF-1α and reduces the recruitment of regulatory\nTregulatory\nT cells (Tregs) and myeloid-derived suppressor cells (MDSCs). Second, normalized endothelial cells\nupregulate adhesion molecules (e.g., ICAM-1, VCAM-1) to facilitate T-cell adhesion and transmigration,\nguided by chemotactic gradients of CXCL9, CXCL10, and CXCL16. Collectively, these mechanisms\nsynergistically enhance T-cell trafficking and infiltration into the tumor microenvironment.\nRegarding whether PDLIM5 deficiency affects ICAM1 and cell stiffness, we also conducted analysis. The\nresults showed that PDLIM5 deficiency promotes ICAM1 aggregation on endothelial cell membranes and\nreduces cell stiffness (Response Figure 9).\nKnockdown of PDLIM5 could increase the aggregation of ICAM1 on the membrane (Response Figure\n9A). The reviewer also mentioned that Celli et al. found that ICAM-1 directly interacts with cytoplasmic α-\nactinin 1 and 4, and that this interaction is required for leukocyte extravasation, and α-actinin 4 had a\nmajor role in this phenomenon (Celli et al. J Immunol. 2006). In this study, the authors identified three\namino acids (arginine 480, 486, and lysine 481) that were critical in the ICAM-1 interaction with both α-\nactinins, suggesting that α-actinins bind to the same amino acids on ICAM-1. Actually, even earlier,\nCarpén et al. also found that the region of ICAM-1, 478-505, interacting with α-actinin was mapped to the area\nclose to the membrane-spanning region. At the same time, they found that the interaction appeared to be\nsensitive to an increase in ionic strength. When the molarity of NaCl was raised from 0.15 to 0.3 M or\nabove, the interaction was markedly reduced or totally abolished. They wrote in the discussion that “The\ninteraction was partially dependent on divalent cation concentration, which is of interest since the COOH-\nterminal region of α-actinin contains two EF hand-like calcium-binding motifs” (Carpén et al. J Cell Biol.\n1992). And these two EF hand-like calcium-binding motifs are also crucial for the binding of α-actinin to\nF-actin. In 1987, Noegel et al. found that when the calcium ion concentration increased, the two EF hand-\nlike calcium-binding motifs of α-actinin would bind to calcium ions, thus releasing the binding to F-actin\n(Noegel et al. FEBS Letters. 1987). Combining with our research findings, PDLIM5 binds to the EF hand-\nlike calcium-binding motifs of α-actinin through the LIMs domain and binds to F-actin through the PDZ\ndomain. We speculate that when the calcium ion concentration increases, the binding between PDLIM5\nand α-actinin will be blocked, thereby reducing the binding of α-actinin to F-actin. At the same time,\ncombining with the findings of Carpén et al., we hypothesis that PDLIM5 may competitively bind to the EF\nhand-like calcium-binding motifs of α-actinin with ICAM1. So, after the knockdown of PDLIM5, the binding\nbetween ICAM1 and α-actinin increases, which promotes the aggregation of ICAM1 on the cell membrane.\nTo verify this, we detected the interaction between ACTN4 and ICAM1 in PDLIM5 knockdown cells, we\nfound that after the knockdown of PDLIM5, the binding between ACTN1/ACTN4 and ICAM1 increased\n(Response Figure 9B). In addition, we isolated primary pulmonary endothelial cells from mice and CD3+\nCD8+ cytotoxic T lymphocytes (CTL) from LLC tumor-bearing mice, and used a leukocyte trans-\nendothelial migration assay to detect the level of T-cell trans-endothelial migration. The results showed\nthat the knockout of PDLIM5 could increase T-cell trans-endothelial migration (Response Figure 9C).\nknockdown of PDLIM5 also reduced cell stiffness (Response Figure 9D). Based on this, we believe that\nthe deletion of PDLIM5 promotes the clustering of ICAM1 on the cell membrane, which in turn increases\nT-cell adhesion and trans-endothelial transport. We have now included Response Fig 9A-F in Fig 7d-i of\nthe revised manuscript.\nIn summary, our latest data suggest that the deletion of PDLIM5 can increase the aggregation of ICAM1\non the endothelial cell membrane and reduce cell stiffness. These factors are all conducive to promoting\nT-cell trans-endothelial migration. Moreover, other vascular normalization phenotypes induced by PDLIM5\ndeficiency (including increased pericyte coverage, enhanced perfusion levels, and reduced permeability)\ncan further promote T-cell infiltration and enhance the efficacy of immunotherapy.\nResponse Figure 9. (A) Analysis of CD56 (ICAM1) expression in WT and PDLIM5 KD HUVECs. (B) Co-\nIP analysis of the interaction between ACTN4 and ICAM1 in WT and PDLIM5 KD HUVECs. (C) Schematic\nand analysis of the relative migration of CD3⁺CD8⁺ cytotoxic T lymphocytes (CTL) across primary\npulmonary endothelial cell (PEC) barriers in a leukocyte trans-endothelial migration assay. In this assay,\nCTL migration was examined through the barriers of wild-type (PDLIM5f/f, n = 10) and PDLIM5 KO\n(PDLIM5cko, n = 10) PECs. The schematic visually depicts the experimental setup, and the analysis\npresents the relative migration data of CTLs. (D) Representative phalloidin-stained images of F-actin\norganization in WT and PDLIM5 KD HUVECs. Scale bar: 100 μm.\n4. ACTN4 is known to bind endothelial eNOS and inhibit its basal activity (Hiroi Yet al. FASEB J. 2008).\nFurthermore, as previoulsy shown by the authors, PDLIM5 phosphorylates eNOS to regulate NO\ngeneration, which can affect vessel permeability. It would be insightful to assess whether these\nmechanisms, particularly in relation to NO regulation and endothelial permeability, contribute to the in vivo\nphenotype observed upon PDLIM5 deletion. Could these factors also play a role in promoting T cell\ninfiltration by modulating the permeability of tumor vessels?\nResponse: Regarding whether PDLIM5 promotes tumor angiogenesis through regulating NO release.\nFirst, NO release increases in endothelial cells during eNOS activation. In 2003, Jean-Philippe Gratton et\nal. found that in LLC subcutaneous tumor models, tumors in eNOS−/− mice showed reduced Evans blue\npermeability compared to wild-type mice. More importantly, tumor progression was significantly attenuated\nin eNOS−/− mice versus LLC-implanted wild-type mice, accompanied by decreased PECAM-1-positive\nvascular density (6.6% ± 0.4% vs. 3.5% ± 0.7* % PECAM-1-positive density, *p < 0.05; n = 4 per group).\nThese results indicate that inhibiting NO production suppresses tumor growth and angiogenesis. Evidence\nalso shows tumor cells induce endothelial NO release to enhance vascular permeability. VEGF-induced\nvascular leakage is abolished in eNOS-deficient mice or wild-type mice treated with NOS inhibitors like L-\nNAME (Fukumura et al., 2001; Murohara et al., 1998a). Thus, NO promotes tumor growth and increases\nvascular permeability. However, our previous studies revealed that PDLIM5 expression increases during\nmechanical vascular injury, inhibiting AKT activation and subsequent eNOS phosphorylation, thereby\nreducing NO release. Endothelial PDLIM5 knockout in endothelial cells increased eNOS phosphorylation\nand NO release, impairing vascular remodeling, suggesting PDLIM5 acts as a negative regulator of the\neNOS/NO pathway (Huang et al., Arterioscler Thromb Vasc Biol. 2020). In this study, PDLIM5 deficiency\nsuppressed tumor growth, angiogenesis, and vascular permeability while enhancing T cell infiltration. If\nPDLIM5 functions through NO regulation, PDLIM5 deletion promotes NO release (Response Figure 10),\nthereby accelerating tumor growth, angiogenesis, and vascular permeability – contradicting Gratton et\nal.'s findings in eNOS−/− mice. Therefore, we conclude that PDLIM5-mediated tumor angiogenesis and\npost-knockout tumor vessel normalization are independent of NO regulation.\nRegarding Hiroi Y et al.'s research, they identified direct interaction between α-actinin-4 and eNOS: full-\nlength α-actinin-4 (1–911 aa) and its R4 domain (619–769 aa) bound eNOS, while actin-binding domains\nR1, R2, R3 (1–618 aa), and EF domain (770–911 aa) did not. They also found α-actinin-4 directly inhibits\neNOS activity, with α-actinin-4 loss in BAECs increasing eNOS activity (Hiroi Y et al., FASEB J. 2008).\nHowever, evidence for PDLIM5-α-actinin-4 interaction regulating eNOS in tumor endothelium remains\nlimited. Our prior work demonstrated PDLIM5 primarily modulates eNOS phosphorylation through AKT\nsignaling.\nIn this study, we screened PDLIM5-binding proteins ACTN1/ACTN4 through mass spectrometry analysis\nand characterized the mechanism of PDLIM5 interaction with ACTN1/ACTN4 at the molecular and cellular\nfunctional levels , as well as its impact on filopodia formation in tip cells (Figure 4-6, Figure S6-S9). Further,\nin the in vivo rescue phenotype assay, reintroducing PDLIM5 mutation failed to rescue the inhibitory effects\nof PDLIM5 deficiency on tumor growth and angiogenesis (Figure 6e-j), strongly demonstrating that\nPDLIM5 promotes angiogenesis by binding to ACTN1/ACTN4.\nResponse Figure 10. (A) NO levels in tumor vessels were detected by DAF-FM DA in PDLIM5f/f or\nPDLIM5cKO mice. Representative images were showed. Statistical analysis of NO levels (DAF-FM DA )\nin tumor vessels stained with CD31 are shown. Scale bar: 20 µm. Unpaired t-test. ***, p < 0.001. Data\nwere represented as mean ± SD.\n• Given the evidence for improved perfusion in tumors with normalized vasculature, it would be useful to\nconsider the potential effects of PDLIM5 deletion on chemotherapy efficacy. Better tumor perfusion could\nresult in more effective drug delivery, independent of whether the treatment is chemotherapy or\nimmunotherapy, unless the central mechanism is immune-mediated.\nResponse：We appreciate the insightful comments regarding the potential interplay between vascular\nnormalization and chemotherapy efficacy. In response, we conducted additional experiments in an LLC\nsubcutaneous tumor model to investigate the chemo-sensitizing effects of endothelial-specific PDLIM5\ndeletion. Our findings demonstrate that PDLIM5 knockout significantly enhances the antitumor efficacy of\ncisplatin (Response Figure 11). Specifically, low-dose cisplatin (4 mg/kg) combined with PDLIM5 deletion\nreduced tumor volume compared to cisplatin alone (p<0.01), suggesting that vascular normalization may\novercome physical barriers to drug delivery (e.g., hypoperfused zones). Beyond improved perfusion,\nPDLIM5 deletion also suppressed pathological angiogenesis (reduction in CD31+ area), creating a dual\nmechanism of \"vascular normalization + anti-angiogenesis\" akin to the chemo-sensitizing effects of clinical\nanti-angiogenic agents like bevacizumab. Furthermore, quantitative hypoxia probe (pimonidazole)\nstaining demonstrated a reduction in hypoxic areas (p<0.001) in PDLIM5-deficient tumors compared to\ncontrols (Figure 7a, Fig S10a), suggesting that vascular normalization effectively alleviates tumor hypoxia,\nwhich may contribute to the observed enhancement of cisplatin sensitivity. These results collectively\nindicate that PDLIM5 KO-mediated vascular remodeling enhances chemotherapy efficacy through both\nimproved drug delivery and microenvironment-driven sensitization. We have now included Response Fig\n11A-G in Fig S11d-j of the revised manuscript.\nResponse Figure 11. (A) Schematic illustration of LLC xenograft mouse model in PDLIM5f/f or\nPDLIM5cKO mice. tumor-bearing mice were treated with Cisplatin (4 mg/kg, every 3 days). (B-C) Tumor volume\nchanges in each group. Two-tailed unpaired t-test. ***, p< 0.001 and **, p < 0.01. Data were represented\nas mean ± SD. (D) Representative micrographs of tumor tissue sections stained with Hematoxylin and\nEosin (H&E), immunohistochemical (IHC) staining for Ki-67 and cleaved caspase 3 (CC3), and\nimmunofluorescence (IF) staining for CD31. Nuclei are stained with DAPI. Scale bar, 100 μm.\nQuantification of Ki67+ cells, CC3+ cells and relative CD31+ vessel density. One-way ANOVA with multiple\ncomparisons. ***, p< 0.001; **, p < 0.01; *, p < 0.05. Data are represented as mean ± SD.\nMinor:\nIn Figure 4I-M, the indication of sh vs. scr conditions appears to be swapped.\nResponse: Regarding Figure 4I-M, there was an error in the quantification data presented in Figure 4i-l.\nThe correct interpretation, as indicated by the IP results in Figure 4 g-h, is that shPDLIM5 downregulates\nDLL4, MMP2, p-FAK Y397, and VASH1. We deeply apologize for this oversight. We have revised all the\ndata and corrected the quantification figures (Response Fig 3A-D). The corrected figures will clearly show\nthe downregulation trends consistent with the immunoprecipitation results. We have now included\nResponse Fig 3 in Fig 4i-j of the revised manuscript.\nReviewer #5 (Remarks to the Author):\n(General comments)\nThis study investigates the role of endothelial PDLIM5, an actin binding cytoskeletal protein with PDZ and\nLIM domain 4, in tumor angiogenesis. The authors demonstrate that PDLIM5 is highly expressed in tumor\nendothelial cells (TECs) in lung cancer, correlating with poor patient survival. Mechanistically, PDLIM5\npromotes tumor angiogenesis by interaction with ACTN1/ACTN4 via its S693/F596 residues, facilitating\nF-actin bundling and tip cell filopodia formation. Using EC-specific PDLIM5 KO mice, the study shows that\nloss of PDLIM5 not only suppresses tumor growth but also improves tumor vascular function, reduces\nintratumoral hypoxia, enhances T cell infiltration, and increases the efficacy of immune checkpoint\ninhibitors (anti-PD-LI antibody treatment). The experiments from Figure 1 to Figure 6 are well designed\nand demonstrate the role of PDLIM5 in promoting tip cell filopodia formation and tumor angiogenesis via\ninteraction with ACTN1/ACTN4 via S693/F596 using PDLIM5 KD cells and KO mice knocked in with either\nadeno-WT or S588A/F591L mutant PDLIM5.\nHowever, there are two major concerns with this study:\n1) Figure 7 does not integrate well with the rest of the study, as it shifts the focus to CD8+ and CD4+T\ncells in LLC tumors from WT or PDLIM5 KO mice in the context of immune checkpoint inhibitors therapy;\nResponse：Given that this major concern essentially aligns with Major Comment 2, I aim to offer a unified\nresponse here and earnestly hope for the reviewer's comprehension\nIn our previous submission, the description of the research purpose for Figure 7 might have given readers\nthe impression of deviating from the main research focus, with insufficient logical rigor. We have revised\nthis section accordingly.\nIt is necessary to clarify why we conducted the studies related to Figures 7 and 8. The primary objective\nof this study was to investigate the role and mechanism of PDLIM5 in tumor angiogenesis. While PDLIM5\ndeficiency inhibits tumor angiogenesis, it also exerts functional impacts on tumor vasculature. Therefore,\nin Figure 7, we explored whether PDLIM5 deficiency affects tumor vascular perfusion levels and\npermeability while blocking angiogenesis, and whether it improves the tumor microenvironment,\nparticularly in terms of its potential influence on cancer immunotherapy.\nIn Figure 7 and Supplemental Figure 10, we mainly demonstrate that PDLIM5 deficiency promotes tumor\nvascular normalization (including increased pericyte coverage, enhanced perfusion levels, and reduced\npermeability). The normalized tumor vasculature also alleviates tumor hypoxia. Previous studies have\nshown that normalized tumor vasculature can enhance immunotherapy efficacy, as evidenced by clinical\nobservations that anti-angiogenic therapy combined with immunotherapy prolongs survival in various\ncancers (Schoenfeld JD, et al. Lancet Oncol. 2022; Reardon DA, et al. JAMA Oncol. 2020; Yuan L, et al.\nNat Commun. 2023; Adkins D, et al. Lancet Oncol. 2024). Therefore, we further investigated whether\nPDLIM5, as a potential anti-angiogenic target, could sensitize tumors to immunotherapy.\nIn our previous version, we did not sufficiently explain why PDLIM5 deficiency promotes T-cell infiltration.\nWe hypothesized that vascular normalization might facilitate T-cell infiltration without exploring the specific\nmechanisms. Indeed, many previous studies have found that normalized tumor vessels enhance T-cell\ninfiltration and antitumor immunity through structural and functional remodeling (Tian L, et al. Nature. 2017;\nHuang Y, et al. Nat Rev Immunol. 2018; Lu Z, et al. Sci Transl Med. 2024; Augustin HG, Koh GY. Cancer\nRes. 2022). First, vascular normalization restores vessel architecture by stabilizing the basement\nmembrane and aligning endothelial cells, reducing vascular leakage and improving blood perfusion. This\noptimization of hemodynamics enables efficient delivery of T cells to tumors while alleviating hypoxia,\nwhich suppresses immunosuppressive factors such as HIF-1α and reduces the recruitment of regulatory\nT cells (Tregs) and myeloid-derived suppressor cells (MDSCs). Second, normalized endothelial cells\nupregulate adhesion molecules (e.g., ICAM-1, VCAM-1) to facilitate T-cell adhesion and transmigration,\nguided by chemotactic gradients of CXCL9, CXCL10, and CXCL16. Collectively, these mechanisms\nsynergistically enhance T-cell trafficking and infiltration into the tumor microenvironment.\nOther reviewers also have raised questions about potential changes in endothelial cell expression of T-\ncell adhesion molecules (e.g., ICAM1) and alterations in cellular stiffness. So, we also examined the\neffects of PDLIM5 deficiency on ICAM1 and cell stiffness. The results demonstrated that PDLIM5\ndeficiency promotes ICAM1 aggregation on endothelial cell membranes and reduces cell stiffness\n(Response Figure 9). These factors are all conducive to promoting T-cell trans-endothelial migration.\nGiven that we possess endothelial-specific PDLIM5 knockout mice, we further explored whether PDLIM5\ndeficiency could enhance tumor immunotherapy by increasing T-cell infiltration. The results in Figure 8\ndemonstrate that PDLIM5 deficiency synergizes with immune checkpoint inhibitors. We believe Figures 7\nand 8 hold significant implications, as these findings provide theoretical foundations and fundamental data\nfor future translational research on PDLIM5-targeted anti-tumor therapies.\n2) Previous study (PMID:29228678) shows that PDLIM5 promotes tumor cell proliferation, invasion and\nmigration via binding to AMPK. However, the potential interaction between PDLIM5 and AMPK in TECs\nhas not been investigated in this study. These issues should be addressed.\nResponse: Given that this major concern essentially aligns with Major Comment 3, I aim to offer a unified\nresponse here and earnestly hope for the reviewer's comprehension\nFirst, the activation of AMPK by basic fibroblast growth factor (bFGF), adiponectin, hypoxia, and VEGF is\ncritically involved in angiogenesis. Notably, dominant-negative AMPK mutants have been shown to\nsuppress endothelial cell migration toward VEGF and inhibit in vitro tube formation under hypoxic\nconditions (Fisslthaler et al., Circ Res. 2009; Levine et al., J Biol Chem. 2007), supporting the pro-\nmigratory role of AMPK in endothelial cells.\nRegarding the study cited by the reviewer (PMID: 29228678), which reported that PDLIM5 promotes tumor\ncell proliferation, invasion, and migration by binding to AMPK, we note that the authors demonstrated\nPDLIM5 interaction with AMPK reduces its ubiquitination-dependent degradation, thereby enhancing\nprostate cancer cell migration. This aligns with earlier work in mouse C2C12 myofibroblasts showing that\nPdlim5 directly binds AMPK via its LIM domain and facilitates AMPK recruitment to F-actin through α-\nactinin (Yan et al., Nat Commun. 2014).\nTo clarify potential cell type-specific mechanisms, we investigated PDLIM5-AMPK interaction and its\nfunctional consequences in HUVECs. Our data demonstrate that PDLIM5 physically interacts with AMPK\nin HUVECs. PDLIM5 knockdown Inhibits AMPK phosphorylation (activation), but does not alter total\nAMPK protein levels, suggesting that PDLIM5 does not regulate AMPK ubiquitination or stability in\nendothelial cells (Response Figure 12). In summary, our findings suggest that the reduced AMPK\nactivation upon PDLIM5 knockdown in HUVECs is secondary to filopodia dysfunction and altered energy\ndemands, rather than direct regulation of AMPK stability. This highlights context-dependent roles of\nPDLIM5-AMPK interaction across cell types.\nIn this study, we screened PDLIM5-binding proteins ACTN1/ACTN4 through mass spectrometry analysis\nin HUVECs, and AMPK are not included. Then we characterized the mechanism of PDLIM5 interaction\nwith ACTN1/ACTN4 at the molecular and cellular functional levels, as well as its impact on filopodia\nformation in tip cells (Figure 4-6, Figure S6-S9). Further, in the in vivo rescue phenotype assay,\nreintroducing PDLIM5 mutation failed to rescue the inhibitory effects of PDLIM5 deficiency on tumor\ngrowth and angiogenesis (Figure 6e-j), strongly demonstrating that PDLIM5 promotes angiogenesis by\nbinding to ACTN1/ACTN4.\nWe propose the following mechanism: 1) PDLIM5 deficiency disrupts filopodia formation, impairing cell\nmigration. This reduced migratory activity may lower cellular energy demand, thereby decreasing AMPK\nactivation. 2) Filopodia formation requires ATP-driven actin polymerization. Abnormal filopodia dynamics\n(e.g., excessive or insufficient protrusion) could perturb local ATP/AMP ratios, indirectly modulating AMPK\nsensing of energy status. Whether PDLIM5 inhibits the migration of tumor endothelial cells by regulating\nAMPK activity requires further comprehensive studies in the future.\nResponse Figure 12. (A) Co-IP analysis of the interaction between PDLIM5 and AMPK in HUVECs. (B)\nDetection of p- AMPK T172 and AMPK levels in HUVECs transfected with shScr and shPDLIM5 lenti-virus\nfor 48 h.\n(Major comments)\n1) Figure 5g: The IP: HA and IB: Myc/Flag blots are not convincing. Please replace these blots with better\nones.\nResponse：We sincerely appreciate the reviewer’s careful evaluation of our data. We acknowledge that\nthe original western blot images in Figure 5g may have been affected by suboptimal exposure conditions\nduring the initial experiment. To address this concern, we have repeated the co-immunoprecipitation (Co-\nIP) experiments under rigorously optimized conditions. The newly obtained results presented in\nResponse Figure 13 (shown below) will replace the original Figure 5g in the revised manuscript, as they\nprovide an optimized representation of the data.\nResponse Figure 13. (A) HEK293T cells were transfected with wild-type or mutant PDLIM5 as indicated\nto detect the binding site between PDLIM5 and ACTN1 or ACTN4.\n2) Figure 7: The inclusion of Figure 7 disrupts the logical flow of this study by shifting the focus to CD8+\nand CD4+T cells in LLC tumors from WT or PDLIM5 KO mice treated with anti-PD-LI antibody This section\nshould be separate paper by adding more detailed mechanisms.\nResponse: Considering that this question is largely consistent with the first major concern mentioned\nearlier, we have already provided a clear and corresponding response in the preceding content. Please\nrefer to the above reply.\n3) Given that previous study shows that PDLIM5 promotes tumor cell proliferation, invasion and migration\nthrough its binding to AMPK (PMID:29228678), it is important to investigate whether PDLIM5 also binds\nto AMPK in TECs. Additionally, authors should examine the relationship between PDLIM5’s interaction\nwith AMPK and its binding to ACTN1/ACTN4 in promoting tip cell filopodia formation.\nResponse: Considering that this question is largely consistent with the second major concern mentioned\nearlier, we have already provided a clear and corresponding response in the preceding content. Please\nrefer to the above reply.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\n1. In this revision, the authors have addressed most of my comments and corrected the errors found\nin the figures. Regarding annotating all the rest of non-arterial/venus/capillary/lymphatic ECs as\nTipECs, my concern still remains. Tip ECs are referred to a very specific type of endothelial cells.\nSince the authors have referred to reference 5, as well as reanalyzing the data from reference 5,\nthere are far more heterogeneity of EC phenotypes based on the single cell RNA sequencing profiles,\nalso found in reference 5. Tip cells are one of the tumor-enriched EC phenotypes. What about other\nEC phenotypes found in TECs, such as immature ECs, proliferating ECs? Using the term of Tip Cells\nwill substantially mislead the readers. If the study could not classify this cluster ECs into further sub-\nphenotyping, another term rather TipECs should be used, for example it would be “tumor-enriched\nECs”, which exhibit increased expression of immunomodulating, tip cells and proliferating endothelial\ncells signature.\nResponse: We sincerely appreciate the reviewer's insightful comments regarding endothelial cell\n(EC) heterogeneity within the tumor microenvironment (TME) and the specific concern about using\nthe term \"tip ECs\" to annotate the cluster of non-arterial/venous/capillary/lymphatic ECs. We fully\nagree with the reviewer that tumor endothelial cells (TECs) exhibit significant phenotypic diversity,\nencompassing not only tip cells but also immature and proliferating ECs, as elegantly demonstrated\nin reference 5 and other studies.\nAs illustrated in Response Figure 1, our initial attempt to replicate the precise 11-subcluster\nclassification from reference 5 was hindered by critical technical differences. Since our ECs were\nidentified from whole-tissue dissociations without CD31/CD45 pre-sorting, the total number was\nrelatively low (~1,900 total ECs, with 863 TECs), compared to the pre-enriched ~21,000 ECs\nanalyzed in reference 5. Applying reference 5's approach as closely as possible, we classified our\nECs into 7 subtypes: Arterial ECs, Lymphatic ECs, Activated Postcapillary Vein ECs, Postcapillary\nVenous ECs, Tip Cells, Type I Alveolar Capillary ECs, and Type II Alveolar Capillary ECs. We were\nunable to further resolve the immature ECs and proliferating ECs subtypes mentioned by the\nreviewer. Importantly, the \"Tip Cells\" cluster identified this way aligned perfectly with the \"tip ECs\"\ncluster from our original manuscript, and the observation of its increased proportion in TECs\nremained consistent.\nFaced with these practical limitations in resolving the full spectrum of EC heterogeneity, we adopted\nthe widely utilized and pragmatic five-category classification (Arterial, Venous, Capillary, Lymphatic,\nTip) prevalent in numerous published tumor scRNA-seq studies (e.g., Qiu J et al., Adv Sci 2023; Li\nJ et al., Natl Sci Rev 2024; Pu W et al., Nat Commun 2021; Zhang J et al., Comput Struct Biotechnol\nJ 2022; Xie Z et al., Cell Mol Life Sci 2024). This approach provided a robust and reproducible\nframework for analyzing PDLIM5 expression across major EC compartments within the constraints\nof our dataset.\nThe reviewer suggests that our \"tip ECs\" cluster might encompass cells like immature ECs and\nproliferating ECs due to shared biological functions (e.g., high proliferative and migratory capacity),\nand proposes renaming this group \"tumor-enriched ECs, which exhibit increased expression of\nimmunomodulating, tip cells and proliferating endothelial cells signature.\" We understand the desire\nfor a more refined and specialized description for readers. However, we are concerned that \"tumor-\nenriched ECs\" is a relatively newer and broader term that may not intuitively convey the specific\nfunctional phenotype to all readers, especially those outside the tumor vasculature field. In contrast,\nthe classification into \"Arterial (A), Venous (V), Capillary (Cap), Lymphatic (L), and Tip (T)\"\nECs\nrepresents a long-established, fundamental paradigm in vascular biology that is widely understood\nand accepted. Using \"Tip ECs\" clearly communicates the core biological characteristics of this\ncluster—enrichment for classic tip cell markers and association with migration/sprouting functions—\nto a broad audience, including non-tumor vascular biology experts.\nFurthermore, to directly address the reviewer's concerns about heterogeneity and other tumor\nendothelial phenotypes, and to validate using the gold-standard reference, we re-analyzed the public\ndataset from reference 5. As shown in Response Figure 1E (to be included as Supplementary Figure\n2G and described in the Results section), PDLIM5 expression was significantly higher in TECs vs.\nNECs specifically within the following subclusters: \"tip cell\", \"artery ECs\", \"immature ECs\", and\n\"Activated Postcapillary Vein ECs\". This result confirms PDLIM5 upregulation across multiple tumor-\nassociated activated endothelial phenotypes, supporting the concept of TEC heterogeneity. Despite\nthis heterogeneity, the \"tip cell\" subcluster exhibited one of the most significant upregulations of\nPDLIM5, and its core function (migration) is precisely the key function of PDLIM5 validated by our\nfunctional experiments. These experiments conclusively demonstrate PDLIM5's contribution to\npromoting migration—the core function of tip cells.\nRegarding immature ECs: We fully acknowledge the importance of immature ECs and other\nphenotypes highlighted by the reviewer. Indeed, our re-analysis of reference 5 data confirmed\nsignificant PDLIM5 upregulation in the \"immature ECs\" subcluster. We will incorporate a detailed\ndiscussion of this point in the manuscript. It is noteworthy that immature ECs typically display high\nproliferative and migratory potential, along with immunomodulatory properties. There is considerable\noverlap in gene expression profiles and functional characteristics (e.g., high migratory capacity)\nbetween immature ECs and tip cells. While scRNA-seq can distinguish them transcriptionally, their\nbiological functions in promoting angiogenesis are likely closely related or exist on a continuum.\nTherefore, PDLIM5 upregulation in immature ECs further underscores its role in a broader\n\"activated/pro-angiogenic endothelial state.\" Tip cells, representing the most migratory vanguard\nwithin this state, served as the ideal entry point for our functional investigation. Our functional data\ndemonstrating PDLIM5's pro-migratory role is equally relevant to understanding its function in highly\nmigratory cell types, including immature ECs.\nRegarding the proportion of tip cells: We note that in reference 5, the tip cell proportion ranged from\n0.75% to 37.30%, with an average of 9.44% across seven patients, despite their finer subclustering,\nhighlighting substantial inter-individual variability. In our samples, the proportion of tip ECs in lung\ncancer tissues also showed significant variation, ranging from ~18% to 50% (Response Figure 1F).\nIn summary: While our scRNA-seq dataset itself may lack the resolution to definitively resolve\nimmature ECs, proliferating ECs, etc., even when attempting finer classification into 7 subtypes, the\nidentity and proportion of the tip cell cluster remained unchanged. This suggests a high degree of\nsimilarity in gene expression and cellular function within this cluster, consistent with the shared high\nproliferative/migratory functions of tip cells, immature ECs, and proliferating ECs. Considering that\n\"tumor-enriched ECs\" is a relatively new and potentially less intuitive term for the specific functional\nphenotype to a broad readership, we believe retaining the established five-category classification (A,\nV, Cap, L, Tip) is preferable. We will explicitly acknowledge the limitations of this classification\napproach in our Discussion section. We hope this explanation and our planned revisions adequately\naddress the reviewer's concerns and that retaining the \"tip cell\" terminology will enhance clarity for\nreaders. We thank the reviewer again for their valuable feedback.\nResponse Figure 1. A, UMAP plots of different EC phenotypes from normal and tumor tissues. B, Subset-\nspecific marker genes were identified to facilitate the annotation of endothelial cell subsets. C. Cell\nnumbers in different EC phenotypes from normal and tumor tissues. D. Relative abundance of each\nphenotype in TECs and NECs. E, Comparison of PDLIM5 abundance between normal and tumor lung\ntissues for each cell type. F, Relative abundance of each phenotype in TECs and NECs in each patient.\nOther minor comments and corrections:\n2. Since the study has conducted an incredible amount of work using Western Blot, it is very important\nto clearly present and check all the source data to ensure its accuracy and clarity. The errors found\nin the previous version are in principle unacceptable.\nResponse: Thank you for your comment. We have carefully checked all source data and have\nuploaded all statistical data as well as uncropped versions of any gels or blots in the revised\nmanuscript.\n3. Be consistent of using the right format of human and murine gene names: PDLIM5 for human, Pdlim5\nfor murine.\nResponse: We sincerely appreciate the reviewer's careful reading. We have now standardized the\ngene nomenclature throughout the manuscript: PDLIM5 for human, Pdlim5 for murine. All instances\nhave been corrected in the text, figures, and supplementary materials.\n4. Line 81, the sentence should be rewritten.\nResponse: Thank you for your suggestion. We have revised it to \"We compared PDLIM5 levels in\nstromal cells from tumors versus normal tissues. Beyond tumor cells, PDLIM5 expression was higher\nin TECs than in NECs.\"\n5. Line 98-99, You could not conclude based on the increase expression of PDLIM5 that the gene will\ncontribute to the malignant tumor progression.\nResponse: We sincerely thank the reviewer for the valid critique. As suggested, we have removed\nthe overinterpretative statement on lines 98-99 implying a causal role of PDLIM5 in angiogenesis\nand immune regulation. The original sentence has been deleted without replacement to strictly\nadhere to the correlative nature of our expression data. This adjustment ensures our conclusions\nremain fully supported by the experimental evidence presented.\n6. Figure 4a, Lenti-virus should be lentivirus.\nResponse: We thank the reviewer for catching this typographical error. The label \"Lenti-virus\" in\nFigure 4a has been corrected to \"lentivirus\"\n7. Figure 7e, what is “mode”?\nResponse: We thank the reviewer for requesting clarification. \"Mode\" refers to the statistical mode\n(peak value) in the fluorescence intensity distribution of the cell population analyzed by flow\ncytometry. In Figure 7e, data were normalized to the mode of the control group to: (1) Account for\ntechnical variations (e.g., instrument settings, staining efficiency), (2) Enable direct comparison of\nsignal shifts between experimental conditions.\nReviewer #2 (Remarks to the Author):\n1. I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part\nof the Nature Communications initiative to facilitate training in peer review and to provide appropriate\nrecognition for Early Career Researchers who co-review manuscripts.\nResponse: We sincerely appreciate the editorial team's initiative in training early-career researchers\n(ECRs) through co-reviewing, which significantly enriches the peer review ecosystem.\nReviewer #3 (Remarks to the Author):\n1. The authors have addressed the concerns raised adequately and strengthened the conclusions of\ntheir study.\nResponse: We sincerely appreciate the reviewer's thorough assessment and endorsement of our\nrevisions. Their acknowledgment that the manuscript's conclusions have been strengthened\nconfirms that our rigorous responses to previous critiques\nReviewer #4 (Remarks to the Author):\nThe authors have attempted to address the comment to the best of their ability, yet some responses\nappear superficial and suggest a lack of careful cross-referencing between the text and figures. For\ninstance, while the response for the first comment refers to scratch assay, the data presented show\na transwell migration model. Additionally, Figure S8h in the revised manuscript does not match\nFigure 7B in the response letter, as claimed. Some statements, particularly those related to Figure\n9, are overstated and not adequately supported by the data presented as specified below. The\nMaterials and Methods section does not include a description of the newly added experiments, which\nmakes it difficult to assess their validity and reproducibility. These issues should be carefully re-\nevaluated to ensure that the interpretations are fully supported by data and clearly described\nmethods.\nResponse: We sincerely thank the reviewer for your thorough evaluation and valuable feedback. We\nacknowledge the shortcomings highlighted and have taken steps to rigorously address each point:\n(1) For the Figure/Text Inconsistencies (Scratch Assay vs. Transwell Data), we sincerely apologize\nfor the significant error in describing the methodology. The reviewer is absolutely correct: the\ndata presented in previous response figure 7A were generated using a Transwell migration\nassay, not a scratch assay, to assess the effect of PDLIM5 deletion on cell migration. We deeply\nregret this mischaracterization, which was an oversight during manuscript preparation and\nrevision. We acknowledge that this error caused confusion during the review process and\nappreciate the reviewer bringing it to our attention.\n(2) We sincerely apologize for the discrepancy regarding the citation of Figure 7B in our previous\nresponse letter. Upon re-examination, we confirm that the data referenced as Figure 7B in the\nresponse is correctly presented as Supplemental Figure 5B in the revised manuscript (not\nFigure S8h as previously stated). This inconsistency resulted from an inadvertent labeling error\nduring the figure reorganization process. We have thoroughly verified that all descriptions of this\nresult in the revised manuscript consistently refer to Supplemental Figure 5B, and regret any\nconfusion caused by this oversight. We appreciate your vigilance in identifying this issue.\n(3) We thank the reviewer for identifying overstated interpretations of Figure 9 regarding ICAM1\nmembrane clustering. We have revised all related conclusions to align strictly with the data,\nsupplemented by additional quantitative analysis of ICAM1 protein level by Western Blot. More\ndetail information is provided in the response of specific comments 3. We believe claims are\nnow fully supported by the presented evidence.\n(4) We thank the reviewer for the essential reminder to document new experimental methods.\nDetailed protocols for all added experiments — including (1) T cell endothelial migration assays,\n(2) cisplatin treatment in tumor-bearing mice, (3) atomic force microscopy (AFM) of endothelial\ncells, (4) flow cytometry analysis of ICAM1 surface expression (specifying antibody clones,\nconcentrations, and gating strategies), and associated reagents/sources — have been\ncomprehensively integrated into the Materials and Methods section and Supplementary\nMethods. This ensures complete methodological transparency, enables rigorous validation, and\nguarantees reproducibility of all findings.\nWe appreciate the reviewer’s diligence in identifying these issues, which significantly strengthened\nthe manuscript. Should any further clarifications be needed, we are pleased to provide them.\nSpecific comments:\n1. Figure 7B: If the quantification represents the percentage of PDLIM5 KD/red cells at the tip position,\nthe Y-axis label should be clarified accordingly. Additionally, these results should be discussed in the\nmain text, particularly around lines 164–166, to help contextualize their relevance.\nResponse: The Figure 7B mentioned by the reviewer corresponds to the revised Supplemental\nFigure 5B. We previously quantified the proportion of all sprouts in each bead and then statistically\nanalyzed the percentages of WT/GFP and PDLIM5 KD/red cells respectively in percentage form.\nThis reflects the proportion of tip cells in the two cell groups. Therefore, we have revised the statistical\nresults and changed the Y-axis label to \"% of tip cells/bead\". As illustrated in the figure below.\nResponse Figure 2. Representative image and quantification of tip cells from the fibrin gel sprouting\nassay with 1:1 competitive seeding of WT (Green) and PDLIM5 KD (red) HUVECs on the beads.\nScale bar, 100 μm. Data are presented as mean with SD. Unpaired t test. ***, p< 0.001.\n2. The authors mention \"a supportive effect of TME optimization on tumor cells following vessel\nnormalization\". However, this concept is unclear, how is the supportive effect exerted, and what does\nit imply? Clarification is needed.\nResponse: We sincerely appreciate the reviewer's insightful question regarding the phrase \"The\nobserved slowdown in tumor growth in our study is precisely the outcome of the optimization of the\ntumor microenvironment and the transformation of its supportive effect on tumor cells following\nvessel normalization, which includes this vessel-pruning step\" in our previous response. We\nacknowledge that this wording lacked sufficient clarity and could be misinterpreted. We apologize\nfor any confusion caused and provide the following detailed explanation: (1) By \"supportive effect of\nthe TME,\" we refer to the collective physical and biochemical conditions within the tumor\nmicroenvironment that objectively enable or facilitate tumor cell survival, proliferation, invasion, and\nmetastasis. This includes, but is not limited to, nutrient and oxygen supply, waste removal, growth\nfactor signaling, immune evasion mechanisms, and the physical scaffold provided by the\nextracellular matrix and vasculature. Crucially, this is a functional description of the TME's state, not\nimplying a beneficial outcome for the host.\nThe key point we intended to convey is that endothelial PDLIM5 deletion-induced vessel\nnormalization fundamentally alters the quality and functional consequences of this TME support.\nSpecifically, it triggers a shift from a pro-tumorigenic, pathological support to a less supportive, more\nnormalized, and ultimately anti-tumorigenic environment, including improving perfusion and\nalleviating hypoxia (removing a key driver of tumor aggression), reducing acidosis and enhancing\nimmune cell infiltration (promoting anti-tumor immunity), pruning dysfunctional vessels (diminishing\nroutes for metastasis). Collectively, these changes transform the TME from a pro-tumorigenic\nenvironment into one that is restrictive to tumor growth and dissemination.\nTherefore, the \"optimization\" and \"transformation\" we described do not imply that the normalized\nTME enhances support for tumor cells. On the contrary, by mitigating key drivers of tumor\nprogression (hypoxia, acidosis, immune evasion, chaotic vasculature), the normalized TME loses its\npathological, pro-tumorigenic characteristics and becomes a less permissive, more restrictive\nenvironment for tumor growth and dissemination. The reduced primary tumor growth and metastasis\nobserved in our models (Figs. 2, 7, S10, S11) are the direct consequences of this shift towards a\nTME that is functionally normalized and thus less supportive of malignancy.\nWe hope this detailed explanation clarifies the intended meaning behind our previous statement. We\nwill revise the relevant text in the manuscript to explicitly state that vessel normalization reconfigures\nthe TME, diminishing its pro-tumorigenic capacity and creating an environment less conducive to\ntumor progression.\n3. Figure 9: The data shown do not support the associated conclusions. The claim regarding ICAM1\naggregation on the endothelial cell membrane cannot be substantiated by increased fluorescence\nintensity measured by flow cytometry. While this may indicate increased expression, aggregation\nwould need to be demonstrated using imaging-based approaches.\nResponse: We thank the reviewer for this important comment. In our revised manuscript, the flow\ncytometry analysis of ICAM1 in HUVECs was performed on live cells without fixation and\npermeabilization, specifically detecting ICAM1 surface levels (Response Figure 3A). Regarding\nwhether PDLIM5 deletion affects ICAM1 expression, we conducted additional Western blot analyses.\nAs shown in Response Figure 3B, ICAM1 protein levels remained unchanged upon PDLIM5\nknockdown. Therefore, we conclude that the increased surface ICAM1 detected by flow cytometry\nreflects enhanced membrane clustering rather than elevated expression, supporting our model that\nPDLIM5 deletion promotes ICAM1 clustering to facilitate T-cell adhesion and trans-endothelial\ntransport.\nResponse Figure 3. A, Flow cytometry analysis of CD54 (ICAM1) expression in WT and PDLIM5 KD\nHUVECs. Data are presented as mean with SD. Unpaired t test. ***, p< 0.001. B, Western blotting\nfor ICAM1 in WT and PDLIM5 KD HUVECs.\n4. Phalloidin staining does not directly measure stiffness. It may suggest changes in cytoskeletal\norganization, but stiffness must be quantified using biophysical techniques such as atomic force\nmicroscopy (AFM) or similar methods. Without such data, any inference about stiffness remains\nspeculative.\nResponse: We thank the reviewer for correctly noting that phalloidin staining indicates cytoskeletal\nreorganization but cannot directly quantify cellular stiffness. To provide direct biophysical evidence,\nwe performed atomic force microscopy (AFM) demonstrating that PDLIM5 deletion significantly\nreduces endothelial cell stiffness (Response Figure 4). This AFM results has replaced the original\nphalloidin staining results in Figure 7G, with corresponding updates to the figure legend. Complete\nmethodological details of the AFM protocols are now included in the Supplemental Methods.\nResponse Figure 4. Representative Height image and calculated Young’s modulus image of WT and\nPDLIM5 KD HUVECs by AFM detaction. Scale bar: 20 μm.\nReviewer #4 (Remarks on code availability):\n1. The code is appropriate.\nResponse: We thank the reviewer for confirming the appropriateness of our analytical code. We have\nensured all scripts used for data processing, statistical analysis, and visualization are rigorously\ndocumented, version-controlled, and publicly accessible in the repository linked in the manuscript’s\nData Availability section.\nReviewer #5 (Remarks to the Author):\n1. Authors responded to reviewer's concern satisfactory. No further comments.\nResponse: Thank you for acknowledging our response. We will complete the follow-up work as per\nthe reviewers' comments. Thank you again for your valuable suggestions!\nREVIEWERS' COMMENTS\nReviewer #1 (Remarks to the Author):\nIn this revision, the authors have well addressed my concerns. I have no further comments and\nrecommend the revised manuscript for publication.\nResponse: We are truly grateful for your positive feedback on our revised manuscript and for\nacknowledging that our revisions have addressed your concerns. Thank you sincerely for your valuable\ntime and guidance throughout the review process; we greatly appreciate your support, which helps move\nour work forward.\nReviewer #2 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the\nNature Communications initiative to facilitate training in peer review and to provide appropriate recognition\nfor Early Career Researchers who co-review manuscripts.\nResponse: Thank you for sharing this information about the co-review process as part of the Nature\nCommunications initiative. It is valuable to learn about efforts to support Early Career Researchers in peer\nreview training and recognition, and we appreciate the collective input that has contributed to the\nevaluation of our manuscript.\nReviewer #4 (Remarks to the Author):\nAuthors' reply sufficiently addresses the issues raised. No further comments.\nResponse: We are deeply grateful for your confirmation that our reply has adequately addressed the\nissues raised. Your time and thoughtful evaluation throughout the review process have been invaluable to\nus, and we sincerely appreciate your support."}, {"round": 2, "reviewer_comments": {"reviewer_1": "In this revision, the authors have addressed most of my comments and corrected the errors found in the figures. Regarding annotating all the rest of non-arterial/venus/capillary/lymphatic ECs as TipECs, my concern still remains. Tip ECs are referred to a very specific type of endothelial cells. Since the authors have referred to reference 5, as well as reanalyzing the data from reference 5, there are far more heterogeneity of EC phenotypes based on the single cell RNA sequencing profiles, also found in reference 5. Tip cells are one of the tumor-enriched EC phenotypes. What about other EC phenotypes found in TECs, such as immature ECs, proliferating ECs? Using the term of Tip Cells will substantially mislead the readers. If the study could not classify this cluster ECs into further sub-phenotyping, another term rather TipECs should be used, for example it would be “tumor-enriched ECs”, which exhibit increased expression of immunomodulating, tip cells and proliferating endothelial cells signature.\nOther minor comments and corrections:\nSince the study has conducted an incredible amount of work using Western Blot, it is very important to clearly present and check all the source data to ensure its accuracy and clarity. The errors found in the previous version are in principle unacceptable.\nBe consistent of using the right format of human and murine gene names: PDLIM5 for human, Pdlim5 for murine.\nLine 81, the sentence should be rewritten.\nLine 98-99, You could not conclude based on the increase expression of PDLIM5 that the gene will contribute to the malignant tumor progression.\nFigure 4a, Lenti-virus should be lentivirus.\nFigure 7e, what is “mode”?", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "The authors have addressed the concerns raised adequately and strengthened the conclusions of their study.", "reviewer_4": "The authors have attempted to address the comment to the best of their ability, yet some responses appear superficial and suggest a lack of careful cross-referencing between the text and figures. For instance, while the response for the first comment refers to scratch assay, the data presented show a transwell migration model. Additionally, Figure S8h in the revised manuscript does not match Figure 7B in the response letter, as claimed. Some statements, particularly those related to Figure 9, are overstated and not adequately supported by the data presented as specified below. The Materials and Methods section does not include a description of the newly added experiments, which makes it difficult to assess their validity and reproducibility.\nThese issues should be carefully re-evaluated to ensure that the interpretations are fully supported by data and clearly described methods.\nSpecific comments:\n• Figure 7B: If the quantification represents the percentage of PDLIM5 KD/red cells at the tip position, the Y-axis label should be clarified accordingly. Additionally, these results should be discussed in the main text, particularly around lines 164–166, to help contextualize their relevance.\n• The authors mention \"a supportive effect of TME optimization on tumor cells following vessel normalization\". However, this concept is unclear, how is the supportive effect exerted, and what does it imply? Clarification is needed.\n• Figure 9: The data shown do not support the associated conclusions. The claim regarding ICAM1 aggregation on the endothelial cell membrane cannot be substantiated by increased fluorescence intensity measured by flow cytometry. While this may indicate increased expression, aggregation would need to be demonstrated using imaging-based approaches. Phalloidin staining does not directly measure stiffness. It may suggest changes in cytoskeletal organization, but stiffness must be quantified using biophysical techniques such as atomic force microscopy (AFM) or similar methods. Without such data, any inference about stiffness remains speculative.", "reviewer_5": "Authors responded to reviewer's concern satisfactory. No further comments."}, "author_response": "REVIEWERS' COMMENTS\nReviewer #1 (Remarks to the Author):\nIn this revision, the authors have well addressed my concerns. I have no further comments and\nrecommend the revised manuscript for publication.\nResponse: We are truly grateful for your positive feedback on our revised manuscript and for\nacknowledging that our revisions have addressed your concerns. Thank you sincerely for your valuable\ntime and guidance throughout the review process; we greatly appreciate your support, which helps move\nour work forward.\nReviewer #2 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition\nfor Early Career Researchers who co-review manuscripts.\nResponse: Thank you for sharing this information about the co-review process as part of the Nature\nCommunications initiative. It is valuable to learn about efforts to support Early Career Researchers in peer\nreview training and recognition, and we appreciate the collective input that has contributed to the\nevaluation of our manuscript.\nReviewer #4 (Remarks to the Author):\nAuthors' reply sufficiently addresses the issues raised. No further comments.\nResponse: We are deeply grateful for your confirmation that our reply has adequately addressed the\nissues raised. Your time and thoughtful evaluation throughout the review process have been invaluable to\nus, and we sincerely appreciate your support."}, {"round": 3, "reviewer_comments": {"reviewer_1": "In this revision, the authors have well addressed my concerns. I have no further comments and recommend the revised manuscript for publication.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_4": "Authors' reply sufficiently addresses the issues raised. No further comments."}, "author_response": ""}]

Decision letter