Platelet factor-4 variant chemokine CXCL4L1 inhibits melanoma and lung carcinoma growth and metastasis by preventing angiogenesis

The platelet factor-4 variant, designated PF-4var/CXCL4L1, is a recently described natural non-allelic gene variant of the CXC chemokine platelet factor-4/CXCL4. PF-4var/CXCL4L1 was cloned, and the purified recombinant protein strongly inhibited angiogenesis. Recombinant PF-4var/CXCL4L1 was angiostatically more active (at nanomolar concentration) than PF-4/CXCL4 in various test systems, including wound-healing and migration assays for microvascular endothelial cells and the rat cornea micropocket assay for angiogenesis. Furthermore, PF-4var/CXCL4L1 more efficiently inhibited tumor growth in animal models of melanoma and lung carcinoma than PF-4/CXCL4 at an equimolar concentration. For B16 melanoma in nude mice, a significant reduction in tumor size and the number of small i.t. blood vessels was obtained with i.t. applied PF-4var/CXCL4L1. For A549 adenocarcinoma in severe combined immunodeficient mice, i.t. PF-4var/CXCL4L1 reduced tumor growth and microvasculature more efficiently than PF-4/CXCL4 and prevented metastasis to various organs better than the angiostatic IFN-inducible protein 10/CXCL10. Finally, in the syngeneic model of Lewis lung carcinoma, PF-4var/CXCL4L1 inhibited tumor growth equally well as monokine induced by IFN-gamma (Mig)/CXCL9, also known to attract effector T lymphocytes. Taken together, PF-4var/CXCL4L1 is a highly potent antitumoral chemokine preventing development and metastasis of various tumors by inhibition of angiogenesis. These data confirm the clinical potential of locally released chemokines in cancer therapy.     

Angiostatic,tumor inflammatory and anti-tumor effects of CXCL447–70 and CXCL4L147–70 in an EGF-dependent breast cancer model

CXCL4 and CXCL4L1, platelet-derived CXC chemokines, and their carboxy-terminal peptides CXCL4^47–70 and CXCL4L1^47–70 previously displayed angiostatic and anti-tumoral activity in a melanoma model. Here, we found CXCL4^47–70 and CXCL4L1^47–70 to inhibit lymphatic endothelial cell proliferation in vitro. Furthermore, the angiostatic potential of CXCL4^47–70 and CXCL4L1^47–70 was tested against different angiogenic stimuli (FGF1, FGF2, FGF8, EGF and VEGF). Besides reducing FGF2-induced vascular endothelial cell growth, CXCL4^47–70 and CXCL4L1^47–70 efficiently counteracted EGF. Consequently, we considered their anti-tumoral potential in EGF-dependent MDA-MB-231 breast tumors. In tumor-bearing mice, CXCL4^47–70 reduced tumor growth better than CXCL4L1^47–70. In CXCL4^47–70-treated tumors significantly more intratumoral monocytes/macrophages and dendritic cells were present and higher expression levels of CCL5 and IFN-γ were detected by qPCR on tumor lysates. Because neither peptide was able to specifically bind CXCR3A or CXCR3B, differential glycosaminoglycan binding and direct interaction with cytokines (EGF and CCL5) might explain any differences in anti-tumoral effects. Notably, CCL5-induced monocyte chemotaxis in vitro was increased by addition of CXCL4^47–70 or CXCL4L1^47–70. Finally, CXCL4^47–70 and CXCL4L1^47–70 inhibited proliferation of MDA-MB-231 cells. Our results suggest a tumor type-dependent responsiveness to either CXCL4^47–70 or CXCL4L1^47–70 treatment, defined by anti-proliferative, angiostatic and inflammatory actions, and substantiate their therapeutic potential.     

Production of angiogenic factors by human glioblastoma cells following activation of the G-protein coupled formylpeptide receptor FPR

Activation of the formylpeptide receptor (FPR), a G-protein-coupled receptor, by its chemotactic peptide ligand N-formylmethionyl-leucyl-phenylalanine (fMLF) promotes the directional migration and survival of human glioblastoma cells. fMLF also stimulates glioblastoma cells to produce biologically active VEGF, an important angiogenic factor involved in tumor progression. In this study, we examined the capacity of FPR to regulate the production of another angiogenic factor, the chemokine IL-8 (CXCL8), in addition to its demonstrated ability to induce VEGF secretion by malignant glioma cells. We showed that the human glioblastoma cell line U87 secreted considerable levels of IL-8 (CXCL8) upon stimulation by the FPR agonist peptide fMLF. Tumor cells transfected with small interference (si)RNA targeting FPR failed to produce IL-8 as well as VEGF in response to fMLF. Glioblastoma cells bearing FPR siRNA exhibited reduced rate of tumorigenicity in nude mice and tumors formed by such tumor cells showed less active angiogenesis and lower level expression of both IL-8 and VEGF. These results suggest that FPR plays an important role in the angiogenesis of human malignant gliomas through increasing the production of angiogenic factors by FPR positive tumor cells. Xiao-Hong Yao and Yi-Fang Ping contributed equally to the study.     

Inhibition of angiogenesis by the BTB domain of promyelocytic leukemia zinc finger protein

Promyelocytic leukemia zinc finger is a negative regulator of cell cycle progression. In this study, we showed that PLZF inhibits endothelial cell angiogenesis using a human umbilical vein endothelial cell system. We also focused on characterizing the specific function of the BTB domain of PLZF as a novel apoptotic and anti-angiogenic protein via deletion mapping analysis. The BTB domain directly inhibited tube formation, as well as the biological functions of angiostatic activity in vivo, and reduced the expression of p-Akt and p-eNOS, which play a significant role in angiogenesis when stimulated by VEGF. These results strongly suggest that the BTB domain could potentially modulate the apoptotic and anti-angiogenic effects of PLZF.     

CXCL12 and vascular endothelial growth factor synergistically induce neoangiogenesis in human ovarian cancers

Ovarian carcinomas have a poor prognosis, often associated with multifocal i.p. dissemination accompanied by intense neovascularization. To examine tumor angiogenesis in the tumor microenvironment, we studied malignant ascites and tumors of patients with untreated ovarian carcinoma. We observed that malignant ascites fluid induced potent in vivo neovascularization in Matrigel assay. We detected a sizable amount of vascular endothelial cell growth factor (VEGF) in malignant ascites. However, pathologic concentration of VEGF is insufficient to induce in vivo angiogenesis. We show that ovarian tumors strongly express CXC chemokine stromal-derived factor (SDF-1/CXCL12). High concentration of CXCL12, but not the pathologic concentration of CXCL12 induces in vivo angiogenesis. Strikingly, pathologic concentrations of VEGF and CXCL12 efficiently and synergistically induce in vivo angiogenesis. Migration, expansion, and survival of vascular endothelial cells (VEC) form the essential functional network of angiogenesis. We further provide a mechanistic basis for explaining the interaction between CXCL12 and VEGF. We show that VEGF up-regulates the receptor for CXCL12, CXCR4 expression on VECs, and synergizes CXCL12-mediated VEC migration. CXCL12 synergizes VEGF-mediated VEC expansion and synergistically protects VECs from sera starvation-induced apoptosis with VEGF. Finally, we show that hypoxia synchronously induces tumor CXCL12 and VEGF production. Therefore, hypoxia triggered tumor CXCL12 and VEGF form a synergistic angiogenic axis in vivo. Hypoxia-induced signals would be the important factor for initiating and maintaining an active synergistic angiogeneic pathway mediated by CXCL12 and VEGF. Thus, interrupting this synergistic axis, rather than VEGF alone, will be a novel efficient antiangiogenesis strategy to treat cancer.     

CXCR1/2 antagonism with CXCL8/Interleukin-8 analogue CXCL8(3–72)K11R/G31P restricts lung cancer growth by inhibiting tumor cell proliferation and suppressing angiogenesis

CXCR1 and CXCR2 together with cognate chemokines are significantly upregulated in a number of cancers, where they act as key regulators of tumor cell proliferation, metastasis, and angiogenesis. We have previously reported a mutant protein of CXCL8/Interleukin-8, CXCL8_(3–72)K11R/G31P (G31P), which can act as a selective antagonist towards CXCR1/2 with therapeutic efficacy in both inflammatory diseases and malignancies. In this study, we investigated the effect of this ELR-CXC chemokine antagonist G31P on human non-small cell lung cancer cells and lung tumor progression in an orthotopic xenograft model. We report increased mRNA levels of CXCR1 and CXCR2 in human lung cancer tissues compared to normal counterparts. Expression levels of CXCR1/2 cognate ligands was determined by ELISA. CXCR1/2 receptor antagonism via G31P leads to decreased H460 and A549 cell proliferation and migration in a dose-dependent manner. G31P also enhanced apoptosis in lung cancer cells as determined by elevated levels of cleaved PARP, Caspase-8, and Bax, together with a reduced expression of the anti-apoptotic protein Bcl-2. In an in vivo orthotopic xenograft mouse model of human lung cancer, G31P treatment suppressed tumor growth, metastasis, and angiogenesis. At the molecular level, G31P treatment was correlated with decreased expression of VEGF and NFкB-p65, in addition to reduced phosphorylation of ERK1/2 and AKT. Our results suggest that G31P blockage of CXCR1 and CXCR2 can inhibit human lung cancer cell growth and metastasis, which offers potential therapeutic opportunities.     

Prostaglandin E2 receptor EP4 as the common target on cancer cells and macrophages to abolish angiogenesis,lymphangiogenesis, metastasis,and stem‐like cell functions

We previously established that COX‐2 overexpression promotes breast cancer progression and metastasis. As long‐term use of COX‐2 inhibitors (COX‐2i) can promote thrombo‐embolic events, we tested an alternative target, prostaglandin E2 receptor EP4 subtype (EP4), downstream of COX‐2. Here we used the highly metastatic syngeneic murine C3L5 breast cancer model to test the role of EP4‐expressing macrophages in vascular endothelial growth factor (VEGF)‐C/D production, angiogenesis, and lymphangiogenesis in situ, the role of EP4 in stem‐like cell (SLC) functions of tumor cells, and therapeutic effects of an EP4 antagonist RQ‐15986 (EP4A). C3L5 cells expressed all EP receptors, produced VEGF‐C/D, and showed high clonogenic tumorsphere forming ability in vitro, functions inhibited with COX‐2i or EP4A. Treating murine macrophage RAW 264.7 cell line with COX‐2i celecoxib and EP4A significantly reduced VEGF‐A/C/D production in vitro, measured with quantitative PCR and Western blots. Orthotopic implants of C3L5 cells in C3H/HeJ mice showed rapid tumor growth, angiogenesis, lymphangiogenesis (CD31/LYVE‐1 and CD31/PROX1 immunostaining), and metastasis to lymph nodes and lungs. Tumors revealed high incidence of EP4‐expressing, VEGF‐C/D producing macrophages identified with dual immunostaining of F4/80 and EP4 or VEGF‐C/D. Celecoxib or EP4A therapy at non‐toxic doses abrogated tumor growth, lymphangiogenesis, and metastasis to lymph nodes and lungs. Residual tumors in treated mice revealed markedly reduced VEGF‐A/C/D and phosphorylated Akt/ERK proteins, VEGF‐C/D positive macrophage infiltration, and proliferative/apoptotic cell ratios. Knocking down COX‐2 or EP4 in C3L5 cells or treating cells in vitro with celecoxib or EP4A and treating tumor‐bearing mice in vivo with the same drug reduced SLC properties of tumor cells including preferential co‐expression of COX‐2 and SLC markers ALDH1A, CD44, OCT‐3/4, β‐catenin, and SOX‐2. Thus, EP4 is an excellent therapeutic target to block stem‐like properties, angiogenesis, and lymphangiogenesis induced by VEGF‐A/C/D secreted by cancer cells and tumor infiltrating macrophages.     

Bcl-2 acts in a proangiogenic signaling pathway through nuclear factor-kappaB and CXC chemokines

Vascular endothelial growth factor (VEGF) induces expression of Bcl-2 in tumor-associated microvascular endothelial cells. We have previously reported that up-regulated Bcl-2 expression in microvascular endothelial cells is sufficient to enhance intratumoral angiogenesis and to accelerate tumor growth. We initially attributed these results to Bcl-2-mediated endothelial cell survival. However, in recent experiments, we observed that conditioned medium from Bcl-2-transduced human dermal microvascular endothelial cells (HDMEC-Bcl-2) is sufficient to induce potent neovascularization in the rat corneal assay, whereas conditioned medium from empty vector controls (HDMEC-LXSN) does not induce angiogenesis. These results cannot be attributed to the role of Bcl-2 in cell survival. To understand this unexpected observation, we did gene expression arrays that revealed that the expression of the proangiogenic chemokines interleukin-8 (CXCL8) and growth-related oncogene-alpha (CXCL1) is significantly higher in HDMEC exposed to VEGF and in HDMEC-Bcl-2 than in controls. Inhibition of Bcl-2 expression with small interfering RNA-Bcl-2, or the inhibition of Bcl-2 function with small molecule inhibitor BL-193, down-regulated CXCL8 and CXCL1 expression and caused marked decrease in the angiogenic potential of endothelial cells without affecting cell viability. Nuclear factor-kappaB (NF-kappaB) is highly activated in HDMEC exposed to VEGF and HDMEC-Bcl-2 cells, and genetic and chemical approaches to block the activity of NF-kappaB down-regulated CXCL8 and CXCL1 expression levels. These results reveal a novel function for Bcl-2 as a proangiogenic signaling molecule and suggest a role for this pathway in tumor angiogenesis.     

Programmed death (PD)-1/PD-ligand 1 blockade mediates antiangiogenic effects by tumor-derived CXCL10/11 as a potential predictive biomarker

Immune checkpoint inhibitor (ICI) programmed death (PD)-1/PD-ligand 1 (PD-L1) blockade has been approved for various cancers. However, the underlying antitumor mechanisms mediated by ICIs and the predictive biomarkers remain unclear. We report the effects of anti-PD-L1/PD-1 Ab in tumor angiogenesis. In syngeneic mouse models, anti-PD-L1 Ab inhibited tumor angiogenesis and induces net-like hypoxia only in ICI-sensitive cell lines. In tumor tissue and serum of ICI-sensitive cell line-bearing mice, interferon-γ (IFN-γ) inducible angiostatic chemokines CXCL10/11 were upregulated by PD-L1 blockade. In vitro, CXCL10/11 gene upregulation by IFN-γ stimulation in tumor cell lines correlated with the sensitivity of PD-L1 blockade. The CXCL10/11 receptor CXCR3-neutralizing Ab or CXCL11 silencing in tumor cells inhibited the antiangiogenic effect of PD-L1 blockade in vivo. In pretreatment serum of lung carcinoma patients receiving anti-PD-1 Ab, the concentration of CXCL10/11 significantly correlated with the clinical outcome. Our results indicate the antiangiogenic function of PD-1/PD-L1 blockade and identify tumor-derived CXCL10/11 as a potential circulating biomarker of therapeutic sensitivity.     

Inhibiting Colorectal Carcinoma Growth and Metastasis By Blocking the Expression of VEGF Using RNA Interference

Angiogenesis plays an essential role in tumor growth and metastasis and is a promising target for cancer therapy. Vascular endothelial growth factor (VEGF) is a key regulator of angiogenesis. The present study was designed to determine the role of VEGF in tumor growth and metastasis using RNA interference (RNAi) technology. Four small interfering RNA (siRNA) sequences for the VEGF gene were cloned into expression plasmids and transfected into human colorectal carcinoma (CRC) SW620 cells. Stable transfection of these plasmids decreased VEGF protein expression, leading to the potent suppression of tumor cell proliferation, migration, invasion, and angiogenesis in vitro. Furthermore, in subcutaneous and intrasplenic/portal injection models involving athymic nude mice, the tumor growth and metastasis of SW620 cells expressing VEGF siRNA were significantly inhibited compared with untransfected cells or cells transfected with control vector alone. Immunohistochemical analyses of tumor sections revealed a decreased vessel density and decreased VEGF expression in the animals where siRNA against VEGF were expressed. These results indicate that RNAi of VEGF can be an effective antiangiogenic strategy for CRC.     

Chemokines as therapeutic targets in renal cell carcinoma

Targeting novel pathways associated with tumor angiogenesis, invasion and immunity, may lead to improvement in patient outcomes for renal cell carcinoma. Chemokines potentiate tumor growth, metastasis, angiogenesis and immune evasion through interactions with stromal cells and neoplastic cells. Further understanding of the mechanisms involved in chemokine-mediated angiogenesis and metastasis may lead to improved therapeutic strategies in this disease. Interactions between chemokine expression and signaling, and the VEGF and hypoxia-inducible factor pathways offer important opportunities to intervene in the process of renal cell carcinoma proliferation, angiogenesis and invasion. Modulation of the CXCR3/CXCR3-ligand or the CXCR4/CXCL12 biologic axis may be potential therapeutic targets for the treatment of renal cell carcinoma. Furthermore, combination treatment with agents targeting chemokine signaling with therapies directed at angiogenesis and tumor immunity may lead to improved outcomes in this disease.     

A high-throughput customized cytokinome screen of colon cancer cell responses to small-molecule oncology drugs

Inflammatory cytokines, chemokines, and growth factors are molecular messengers that circulate and have the capability to modify the tumor microenvironment and impact therapeutic response. The characterization of soluble mediators as biomarkers for diagnosis and prognosis is of interest in oncology. We utilize the cytokinome to characterize the response of colorectal tumor cell lines to selected small-molecules in oncology as a proof-of-concept dataset with immunomodulatory analyte heat map rankings for drug and cell line combinations. We observed overall trends in drug class effects with MEK-, BRAF-, PARP-inhibitors, and Imipridones in cytokine, chemokine, and growth factor responses that may help guide therapy selection. MEK-inhibitor treatment downregulated analytes VEGF, CXCL9/MIG, and IL-8/CXCL8 and upregulated CXCL14/BRAK, Prolactin, and CCL5/RANTES. BRAF-inhibitor treatment downregulated VEGF and IL-8/CXCL8, while increasing soluble TRAIL-R2. Treatment with PARP-inhibitors decreased CXCL9/MIG, IL-8/CXCL8, CCL3/MIP-1 alpha, VEGF, and CXCL14/BRAK, while treatment increased soluble TRAIL-R2 and prolactin. Treatment with Imipridones decreased CCL3/MIP-1 alpha, VEGF, CXCL14/BRAK, IL-8/CXCL8, and Prolactin and increased CXCL5/ENA-78. We also observed differential responses to therapeutics depending on the mutational profile of the cell line. In the future, a similar but larger dataset may be utilized in the clinic to aid in the prediction of patient response to immunomodulatory therapies based on tumor genotype.     

Role of MIF/CXCL8/CXCR2 signaling in the growth of nasopharyngeal carcinoma tumor spheres

Macrophage migration inhibitory factor (MIF) and CXCL8 (also named IL-8) are strongly expressed in the tissues of nasopharyngeal carcinoma (NPC). However, their role in the growth of NPC has not been fully examined. This study aims to evaluate the functions of MIF and CXCL8 on the growth of NPC tumor spheres. The elevated expression of CXCL8 in tumor over normal tissues was confirmed in 37 pairs of biopsies from NPC patients. In the in vitro study, all the poorly differentiated NPC cell lines, including the EBV-positive C666-1, and the EBV-negative CNE-1, CNE-2, SUNE-1, HNE-1 and HONE-1 cells, were found to express CXCL8 and MIF. Therefore, the EBV-positive C666-1 cell was selected to examine for the role of MIF and CXCL8 in the growth of the NPC tumor spheres. Functional study showed that the growth of C666-1 tumor spheres, under the nutrient poor or growth factor supplemented culture conditions, could be inhibited by the CXCL8 specific peptide inhibitor. The growth of the tumor spheres could also be reduced by the CXCR2 specific inhibitor SB225002 or the PI3K/AKT inhibitor LY294002, indicating that the endogenously produced CXCL8 plays an autocrine role in the growth of the tumor spheres. Further mechanistic studies revealed that the gene expression of CXCL8 could be reduced by the MIF specific small interfering RNA (siRNA) or NF-κB inhibitor parthenolide, and the growth of tumor spheres was also reduced after MIF siRNA transfection. Taken together, the present study highlights the role of MIF/CXCL8/CXCR2 axis in the growth of NPC tumor spheres. Chemotherapeutic interference of this signaling pathway may help to control the growth of the NPC tumor.     

Domain swapping in a COOH-terminal fragment of platelet factor 4 generates potent angiogenesis inhibitors

A few peptide residues in structurally important locations often determine biological functions of proteins implicated in the regulation of angiogenesis. We have shown recently that the short COOH-terminal segment PF-4(47-70) derived from platelet factor 4 (PF-4) is the smallest sequence that conserves potent antiangiogenic activity in vitro and in vivo. Here we show that modified COOH-terminal PF-4 peptides containing the sequence ELR (or related DLR), a critical domain present in proangiogenic chemokines, surprisingly elicit several times greater antiangiogenic potential than the original peptide. The modified peptides inhibit binding of iodinated vascular endothelial growth factor and fibroblast growth factor 2 to endothelial cell receptors, endothelial cell proliferation, migration, and microvessel assembly in the rat aortic ring model at lower doses than PF-4(47-70). On the differentiated chick chorioallantoic membrane, topical application of 40 micro g of modified peptides potently reduces capillary angiogenesis induced by vascular endothelial growth factor(165), a dose where peptide PF-4(47-70) was inactive. Established intracranial glioma in nude mice decreased significantly in size when treated locally with a total dose of 250 micro g of peptide PF-4(47-70)DLR (n = 10) compared with the same dose of the original PF-4(47-70) peptide (n = 10) or controls (n = 30). Tailored PF-4 peptides represent a new class of antiangiogenic agents with a defined mode of action and a strong in vivo activity.     

The CXCR4-CXCL12 axis in Ewing sarcoma: promotion of tumor growth rather than metastatic disease

Background

Chemokine receptor CXCR4, together with its ligand CXCL12, plays critical roles in cancer progression, including growth, metastasis and angiogenesis. Ewing sarcoma is a sarcoma with poor prognosis despite current therapies, particularly for patients with advanced-stage disease. Lungs and bone (marrow), organs of predilection for (primary/metastatic) Ewing sarcoma, represent predominant CXCL12 sources.

Methods

To gain insight into the role of the CXCR4-CXCL12 axis in Ewing sarcoma, CXCR4, CXCL12 and hypoxia-inducible factor-1α protein expression was studied in therapy-naïve and metastatic tumors by immunohistochemistry. CXCR4 function was assessed in vitro, by flow cytometry and proliferation/ cell viability assays, in the presence of recombinant CXCL12 and/or CXCR4-antagonist AMD3100 or under hypoxic conditions.

Results

Whereas CXCR4 was predominantly expressed by tumor cells, CXCL12 was observed in both tumor and stromal areas. Survival analysis revealed an (expression level-dependent) negative impact of CXCR4 expression (p?<?0.04). A role for the CXCR4-CXCL12 axis in Ewing sarcoma growth was suggested by our observations that i) CXCR4 expression correlated positively with tumor volume at diagnosis (p?=?0.013), ii) CXCL12 was present within the microenvironment of virtually all cases, iii) CXCL12 induced proliferation of CXCR4-positive Ewing sarcoma cell lines, which could be abrogated by AMD3100. CXCR4 expression was not correlated with occurrence of metastatic disease. Also, therapy-naïve tumors demonstrated higher CXCR4 expression as compared to metastases (p?=?0.027). Evaluation of in vivo hypoxia-inducible factor-1α expression and culture of cells under hypoxic conditions revealed no role for hypoxia in CXCR4 expression.

Conclusions

Together, our results imply a crucial role for the CXCR4-CXCL12 axis in auto- and/or paracrine growth stimulation. Integration of CXCR4-targeting strategies into first- and/or second-line treatment regimens may represent a promising treatment option for Ewing sarcoma.

     

The Proangiogenic Phenotype of Natural Killer Cells in Patients with Non-Small Cell Lung Cancer

The tumor microenvironment can polarize innate immune cells to a proangiogenic phenotype. Decidual natural killer (dNK) cells show an angiogenic phenotype, yet the role for NK innate lymphoid cells in tumor angiogenesis remains to be defined. We investigated NK cells from patients with surgically resected non-small cell lung cancer (NSCLC) and controls using flow cytometric and functional analyses. The CD56⁺CD16^- NK subset in NSCLC patients, which represents the predominant NK subset in tumors and a minor subset in adjacent lung and peripheral blood, was associated with vascular endothelial growth factor (VEGF), placental growth factor (PIGF), and interleukin-8 (IL-8)/CXCL8 production. Peripheral blood CD56⁺CD16^- NK cells from patients with the squamous cell carcinoma (SCC) subtype showed higher VEGF and PlGF production compared to those from patients with adenocarcinoma (AdC) and controls. Higher IL-8 production was found for both SCC and AdC compared to controls. Supernatants derived from NSCLC CD56⁺CD16^- NK cells induced endothelial cell chemotaxis and formation of capillary-like structures in vitro, particularly evident in SCC patients and absent from controls. Finally, exposure to transforming growth factor-β₁ (TGFβ₁), a cytokine associated with dNK polarization, upregulated VEGF and PlGF in peripheral blood CD56⁺CD16^- NK cells from healthy subjects. Our data suggest that NK cells in NSCLC act as proangiogenic cells, particularly evident for SCC and in part mediated by TGFβ₁.