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Introduction. Natural Killer (NK) cells are innate lymphoid cells involved in tumour recognition/elimination. NK cells are altered in their phenotype and functions in diverse tumors, including prostate cancer (PCa). We demonstrated that PCa circulating NK cells (TANKs) acquire the pro-angiogenic/decidual-like CD56brightCD9+CD49a+ phenotype, release IL-8 and MMP-9, functionally support endothelial cell activation and secrete monocyte-recruiting/M2-like macrophage-polarizing factors. Materials and methods. Here, we characterized the phenotype of tumour infiltrating NKs (TINKs) and tumour-associated (TANKs) in PCa patients and evaluated the contribution of STAT3, as possible driver of NK cell polarization. PCa TINKs and TANKs were characterized by multicolour flow cytometry (FC) for decidual-like surface markers (CD9, CD49a) and degranulation capabilities (CD107a). STAT3 activation, was investigated in circulating PCa NK cells, by FC. Using a drug-repurposing approach with the antipsychotic agent Pimozide (a chemical inhibitor of STAT3), we modulated STAT3 activation, ex vivo, in PCa TANKs and monitored their secretome changes, by commercially available protein membrane arrays together with their capability to degranulate and produce perforin/GranzymeB. Results and discussion. We observed that PCa TINKs acquire the same CD9+CD49a+ decidual-like NK cell phenotype, as found in PCa TANKs. We detected the presence of CD56brightCD9+CD49a+ decidual-like NK cell also in peripheral blood of subjects with benign prostatic hyperplasia (BPH), but in a lower frequency, compared to those from PCa TANKs. Sera from PCa patients were enriched in IL-4, IL-6, IL-8 and IL-10, all cytokines able to activate STAT3 signalling. We detected increased phosphorylation of STAT3 in PCa TANKs, compared to NK cells from healthy controls, that was reduced following 24 hours of stimulation by Pimozide. This treatment resulted in decreased capabilities of PCa TANKs to secrete pro-angiogenic factors (IL-8, IL-6), molecules involved in monocytes recruitment/M2-like macrophage polarization (CCL-2, CCL5, GM-CSF, IL-10), together with increased degranulation, augmented secretion of (IFN-γ and TNF-α) and increased production of Perforin and Granzyme. Conclusions. Our results suggest that STAT3 inhibition can be envisaged as a potential strategy to limit the generation of pro-angiogenic/decidual-like NKs, while contributing to NK cell re-education in PCa. Citation Format: Matteo Gallazzi, Maria Teresa Palano, Martina Cucchiara, Federico Dehò, Paolo Capogrosso, Francesca Franzi, Fausto Sessa, Angelo Naselli, Lorenzo Mortara, Antonino Bruno. STAT-3 chemical inhibition modulates decidual-like polarization in NK cells from PCa patients and restore their anti-tumor activities. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4595.

Simple Summary Cancer patients are characterized by NK cells with altered surface markers, such as CD56 brightness, CD9, CD49a (pro-angiogenic) and PD-1, and TIM-3 (exhaustion), that favor immune escape. Transforming growth factor-beta (TGFβ) is a major tumor-derived cytokine that favors cancer growth and supports pro-angiogenic activities in NK cells by inducing pro-angiogenic molecules. TIMP-1 and TIMP-2 play a crucial role in extracellular matrix (ECM) regulation, wound healing, pregnancy and cancer, and there is increasing evidence that they are immune-modulatory. We found that recombinant TIMP-1 and -2 can partially contrast the induction of pro-tumor/pro-angiogenic decidual-like polarization of NK cells by TGFβ. Abstract Natural Killer (NK) cells have been found to be anergic, exhausted and pro-angiogenic in cancers. NK cell from healthy donors, exposed to TGFβ, acquire the CD56brightCD9+CD49a+ decidual-like-phenotype, together with decreased levels of NKG2D activation marker, increased levels of TIM-3 exhaustion marker, similar to cancer-associated NK cells. Tissue inhibitors of metalloproteases (TIMPs) exert dual roles in cancer. The role of TIMPs in modulating immune cells is a very novel concept, and the present is the first report studying their ability to contrast TGFβ action on NK cells. Here, we investigated the effects of TIMP1 and TIMP2 recombinant proteins in hindering decidual-like markers in NK cells, generated by polarizing cytolytic NK cells with TGFβ. The effects of TIMP1 or TIMP2 on NK cell surface antigens were determined by multicolor flow cytometry. We found that TIMP1 and TIMP2 were effective in interfering with TGFβ induced NK cell polarization towards a decidual-like-phenotype. TIMP1 and TIMP2 counteracted the effect of TGFβ in increasing the percentage of CD56bright, CD16−, CD9+ and CD49a+, and restoring normal levels for TIMP 1 and 2 also inhibited decrease levels of the activation marker NKG2D induced by TGFβ and decreased the TGFβ upregulated exhaustion marker TIM-3. NK cell degranulation capabilities against K562 cells were also decreased by TGFβ and not by TIMP1 or TIMP2. TIMP1 treatment could partially restore degranulation marker CD107a expression. Treatment with recombinant TIMP-1 or TIMP-2 showed a trend, although not statistically significant, to decrease CD49a+ and TIM-3+ expression and increase NKG2D in peripheral blood NK cells exposed to conditioned media from colon cancer cell lines. Our results suggest a potential role of TIMPs in controlling the tumor-associated cytokine TGFβ-induced NK cell polarization. Given the heterogeneity of released factors within the TME, it is clear that TGFβ stimulation represents a model to prove TIMP’s new properties, but it cannot be envisaged as a soloist NK cell polarizing agent. Therefore, further studies from the scientific community will help defining TIMPs immunomodulatory activities of NK cells in cancer, and their possible future diagnostic–therapeutic roles.

Background: Tissue inhibitors of metalloproteases (TIMPs) have been reported to exert dual roles in cancer, based on the different cancer type and interactions with the tumor microenvironments. As inhibitors of Matrix metalloproteases (MMP) they downregulate invasion, metastasis and angiogenesis. However, TIMP1 levels have been found increased in patients with different types of cancer. Natural Killer cells have been found to be phenotypically and functionally compromised in several solid and hematological cancers. We were the first in characterizing NK pro-angiogenic phenotype in the peripheral blood and tumor tissues of patients with Non-small Cell Lung cancer, colorectal cancers, prostate of patients. Also, we found that pro-angiogenic NK cells in the peripheral blood of colorectal cancer patients release of TIMP-1 and TIMP-2, in a STAT3/STAT5-independent manner. Here, we investigated the effects of TIMP-1 and TIMP-2 recombinant proteins, using a TGFβ-mediated NK cell polarization model, in vitro. Methods: We investigated the effects of TIMP1 and TIMP2 recombinant proteins in hindering decidual-like markers in NK cells, generated by polarizing cytolytic NK cells with TGFβ. The effects of TIMP1 or TIMP2 on NK cell surface antigens were determined by multicolor flow cytometry. Results: We found that TIMP1 and TIMP2 were effective in interfering with TGFβ induced NK cell polarization towards a decidual-like-phenotype. TIMP1 and TIMP2 counteracted the effect of TGFβ in increasing the percentage of CD56bright, CD16-, CD9+ and CD49a+, and restoring normal levels for TIMP 1 and 2 also inhibited decrease levels of the activation marker NKG2D induced by TGFβ and decreased the TGFβ upregulated exhaustion marker TIM-3. NK cell degranulation capabilities against K562 cells were also decreased by TGFβ and not by TIMP1 or TIMP2. TIMP1 treatment could partially restore degranulation marker CD107a expression. Conclusions: Our results suggest a potential role of TIMPs in controlling the tumor-associated cytokine TGFβ-induced NK cell polarization. TGFβ stimulation represents a model to prove TIMP's new properties, further studies will help defining TIMPs immunomodulatory activities of NK cells in cancer, and their possible future diagnostic-therapeutic roles. Citation Format: Adriana Albini, Matteo Gallazzi, Maria Teresa Palano, Valentina Carlini, Luana Calabrone, Riccardo Ricotta, William G. Stetler-Stevenson, Douglas M. Noonan. TIMP1 and TIMP2 re-educate TGFβ induced pro-inflammatory/pro angiogenic decidual-like NK cells phenotype [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 3843.

BACKGROUND The novel coronavirus has a high mortality rate (over 1% for patients older than 50 years). This can only be partially ascribed to other comorbidities. A possible explanation is a factor that assures a prompt response to SARS-CoV-2 in younger people, independent from the novelty of the virus itself. A factor is believed to stimulate the immune system and provide immunity against more antigens. The only external stimulation received by healthy people is vaccination (eg, the diphtheria, tetanus, and pertussis [DTP] vaccine). One hypothesis is that vaccination helps develop specific immunity but generates sprouting immunity against antigens in transit. The underlying immunological phenomena are the “bystander effect” and “trained immunity.” The developed immunity gives protection for years until it naturally fades out. After the fifth decade of life, the immune system is almost incompetent when a viral infection occurs, and thus, at this stage, the novel coronavirus can enter the body and cause acute respiratory distress syndrome. OBJECTIVE The initial aim is to demonstrate that blood monocytes and natural killer cells show overpowering hyperactivity, while CD4+ and CD8+ T cells experience impediments to their defensive functions in patients with severe SARS-CoV-2 infection. The secondary objectives are to correlate clinical data and vaccination history with laboratory immune patterns in order to identify protective factors. Subsequently, we are also interested in characterizing the phenotypes and state of the degree of activation of peripheral blood mononuclear cells, including monocytes, natural killer cells, and CD4+ and CD8+ T cells, in healthy subjects vaccinated with the Pfizer vaccine. METHODS Data will be collected using the following 3 approaches: (1) an experimental analysis to study the innate immune response and to identify genetic profiles; (2) an epidemiological analysis to identify the patients’ vaccination history; and (3) a clinical analysis to detect the immunological profile. RESULTS The protocol was approved by the Ethics Committee on April 16, 2020, and the study started on April 27, 2020. As of February 2021, enrollment has been completed. Immunological analysis is ongoing, and we expect to complete this analysis by December 2022. CONCLUSIONS We will recognize different populations of patients, each one with a specific immunological pattern in terms of cytokines, soluble factor serum levels, and immune cell activity. Anamnestic data, such as preceding vaccinations and comorbidities, biochemical findings like lymphocyte immunophenotyping, and pre-existing persistent cytomegalovirus infection, allow depicting the risk profile of severe COVID-19. Proof of the roles of these immunological phenomena in the development of COVID-19 can be the basis for the implementation of therapeutic immunomodulatory treatments. CLINICALTRIAL ClinicalTrials.gov NCT04375176; https://clinicaltrials.gov/ct2/show/NCT04375176 INTERNATIONAL REGISTERED REPORT DERR1-10.2196/29892

Leukocytes often undergo rapid changes in cell phenotype, for example, from a resting to an activated state, which places significant metabolic demands on the cell. These rapid changes in metabolic demand need to be tightly regulated to support immune cell effector functions during the initiation and downregulation of an immune response. Prospects for implementing cancer immunotherapy also rest on the idea of optimizing the metabolic profile of immune cell effectors. Here, we examine this issue by focusing on neutrophils and NK cells as cells of increasing interest in cancer immunology and tumor immunometabolism, because they can be targeted or, in the case of NK, used as effectors in immunotherapy. In addition, neutrophils and NK cells have been shown to functionally interact. In the case of neutrophils, we also extended our interest to polymorphonuclear MDSC (PMN-MDSCs), since the granulocytic subset of MDSCs share many phenotypes and are functionally similar to pro-tumor neutrophils. Finally, we reviewed relevant strategies to target tumor metabolism, focusing on neutrophils and NK cells.

Ovarian cancer (OvCA) accounts for one of the leading causes of death from gynecologic malignancy. Despite progress in therapy improvements in OvCA, most patients develop a recurrence after first-line treatments, dependent on the tumor and non-tumor complexity/heterogeneity of the neoplasm and its surrounding tumor microenvironment (TME). The TME has gained greater attention in the design of specific therapies within the new era of immunotherapy. It is now clear that the immune contexture in OvCA, here referred as tumor immune microenvironment (TIME), acts as a crucial orchestrator of OvCA progression, thus representing a necessary target for combined therapies. Currently, several advancements of antitumor immune responses in OvCA are based on the characterization of tumor-infiltrating lymphocytes, which have been shown to correlate with a significantly improved clinical outcome. Here, we reviewed the literature on selected TIME components of OvCA, such as macrophages, neutrophils, γδ T lymphocytes, and natural killer (NK) cells; these cells can have a role in either supporting or limiting OvCA, depending on the TIME stimuli. We also reviewed and discussed the major (immune)-therapeutic approaches currently employed to target and/or potentiate macrophages, neutrophils, γδ T lymphocytes, and NK cells in the OvCA context.

Graves' disease (GD) is a common autoimmune cause of hyperthyroidism, which is eventually related to the generation of IgG antibodies stimulating the thyrotropin receptor. Clinical manifestations of the disease reflect hyperstimulation of the gland, causing thyrocyte hyperplasia (goiter) and excessive thyroid hormone synthesis (hyperthyroidism). The above clinical manifestations are preceded by still partially unraveled pathogenic actions governed by the induction of aberrant phenotype/functions of immune cells. In this review article we investigated the potential contribution of natural killer (NK) cells, based on literature analysis, to discuss the bidirectional interplay with thyroid hormones (TH) in GD progression. We analyzed cellular and molecular NK-cell associated mechanisms potentially impacting on GD, in a view of identification of the main NK-cell subset with highest immunoregulatory role.

Background The novel coronavirus has a high mortality rate (over 1% for patients older than 50 years). This can only be partially ascribed to other comorbidities. A possible explanation is a factor that assures a prompt response to SARS-CoV-2 in younger people, independent from the novelty of the virus itself. A factor is believed to stimulate the immune system and provide immunity against more antigens. The only external stimulation received by healthy people is vaccination (eg, the diphtheria, tetanus, and pertussis [DTP] vaccine). One hypothesis is that vaccination helps develop specific immunity but generates sprouting immunity against antigens in transit. The underlying immunological phenomena are the “bystander effect” and “trained immunity.” The developed immunity gives protection for years until it naturally fades out. After the fifth decade of life, the immune system is almost incompetent when a viral infection occurs, and thus, at this stage, the novel coronavirus can enter the body and cause acute respiratory distress syndrome. Objective The initial aim is to demonstrate that blood monocytes and natural killer cells show overpowering hyperactivity, while CD4+ and CD8+ T cells experience impediments to their defensive functions in patients with severe SARS-CoV-2 infection. The secondary objectives are to correlate clinical data and vaccination history with laboratory immune patterns in order to identify protective factors. Subsequently, we are also interested in characterizing the phenotypes and state of the degree of activation of peripheral blood mononuclear cells, including monocytes, natural killer cells, and CD4+ and CD8+ T cells, in healthy subjects vaccinated with the Pfizer vaccine. Methods Data will be collected using the following 3 approaches: (1) an experimental analysis to study the innate immune response and to identify genetic profiles; (2) an epidemiological analysis to identify the patients’ vaccination history; and (3) a clinical analysis to detect the immunological profile. Results The protocol was approved by the Ethics Committee on April 16, 2020, and the study started on April 27, 2020. As of February 2021, enrollment has been completed. Immunological analysis is ongoing, and we expect to complete this analysis by December 2022. Conclusions We will recognize different populations of patients, each one with a specific immunological pattern in terms of cytokines, soluble factor serum levels, and immune cell activity. Anamnestic data, such as preceding vaccinations and comorbidities, biochemical findings like lymphocyte immunophenotyping, and pre-existing persistent cytomegalovirus infection, allow depicting the risk profile of severe COVID-19. Proof of the roles of these immunological phenomena in the development of COVID-19 can be the basis for the implementation of therapeutic immunomodulatory treatments. Trial Registration ClinicalTrials.gov NCT04375176; https://clinicaltrials.gov/ct2/show/NCT04375176 International Registered Report Identifier (IRRID) DERR1-10.2196/29892

Tissue inhibitors of metalloproteases (TIMPs) have been reported to exert dual roles in cancer, based on the different cancer type and interactions with the tumor microenvironments. As inhibitors of Matrix metalloproteases (MMP) they downregulate invasion, metastasis and angiogenesis. However, TIMP1 levels have been found increased in patients with different types of cancer. Natural Killer cells have been found to be phenotypically and functionally compromised in several solid and hematological cancers. We were the first in characterizing NK pro-angiogenic phenotype in the peripheral blood and tumor tissues of patients with Non-small Cell Lung cancer (NSCLC), colorectal cancers (CRC) and in pleural effusion (PE) of patients with metastatic cancers. Also, we found that pro-angiogenic NK cells in the peripheral blood of colorectal cancer patients release of TIMP-1 and TIMP-2, in a STAT3/STAT5-independent manner. Here, we investigated the effects of TIMP-1 and TIMP-2 recombinant proteins, using a TGFβ-mediated NK cell polarization model, in vitro. The effects of TGFβ, alone or in combination with TIMP-1 or TIMP-2 peptides, was determined by multicolor flow cytometry for surface antigens and cytokine productions, on cytolytic NK cells isolated from healthy subjects. Commercially available antibody membrane arrays was used to determine the modulation of the secretome, on FACS-sorted NK cells, from healthy subjects, following 72 hours of exposure to TGFβ, alone or in combination with TIMP-1 or TIMP-2 peptide. We found that NK cell from healthy donors, exposed to TGFβ, acquire the CD56brightCD9+CD49a+ decidual-like phenotype, together with decreased levels of the activation marker NKG2D. Administration of TIMP1 or TIMP2 peptides was effective in interfering with TGFβ-induced NK cell polarization towards the CD56brightCD9+CD49a+ decidual-like phenotype and in restoration of the levels of the NKG2D activation marker. We also observed that TGFβ-polarized NK cell, when co-treated with TIMP1 or TIMP2, exhibit a trend toward decreased production of VEGF and increased production of the anti-tumor cytokines IFNγ and TNFα Our results suggest a potential role of TIMPs in contrasting the angiogenic switch in NK cells and suggest a possible use of TIMPs peptides in the re-education of anergic/pro-angiogenic NK cells in cancer patients. Citation Format: Matteo Gallazzi, Antonino Bruno, Douglas M. Noonan, William G. Stetler-Stevenson, Adriana Albini. Targeting the TGFΒ/TIMP-1/2 axes to re-educate pro-inflammatory/pro angiogenic NK cells in cancer patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 3159.

Immune cells, as a consequence of their plasticity, can acquire altered phenotype/functions within the tumor microenvironment (TME). Some of these aberrant functions include attenuation of targeting and killing of tumor cells, tolerogenic/immunosuppressive behavior and acquisition of pro-angiogenic activities. Natural killer (NK) cells are effector lymphocytes involved in tumor immunosurveillance. In solid malignancies, tumor-associated NK cells (TANK cells) in peripheral blood and tumor-infiltrating NK (TINK) cells show altered phenotypes and are characterized by either anergy or reduced cytotoxicity. Here, we aim at discussing how NK cells can support tumor progression and how induction of angiogenesis, due to TME stimuli, can be a relevant part on the NK cell-associated tumor supporting activities. We will review and discuss the contribution of the TME in shaping NK cell response favoring cancer progression. We will focus on TME-derived set of factors such as TGF-β, soluble HLA-G, prostaglandin E2, adenosine, extracellular vesicles, and miRNAs, which can exhibit a dual function. On one hand, these factors can suppress NK cell-mediated activities but, on the other hand, they can induce a pro-angiogenic polarization in NK cells. Also, we will analyze the impact on cancer progression of the interaction of NK cells with several TME-associated cells, including macrophages, neutrophils, mast cells, cancer-associated fibroblasts, and endothelial cells. Then, we will discuss the most relevant therapeutic approaches aimed at potentiating/restoring NK cell activities against tumors. Finally, supported by the literature revision and our new findings on NK cell pro-angiogenic activities, we uphold NK cells to a key host cellular paradigm in controlling tumor progression and angiogenesis; thus, we should bear in mind NK cells like a TME-associated target for anti-tumor therapeutic approaches.

Background: Chronic, degenerative diseases are often characterized by inflammation and aberrant angiogenesis. For these pathologies, including rheumatoid arthritis, cardiovascular and autoimmune diseases, cancer, diabetes, and obesity, current therapies have limited efficacy. Objectives: The validation of novel (chemo)preventive and interceptive approaches, and the use of new or repurposed agents, alone or in combination with registered drugs, are urgently required. Results: Phytochemicals (triterpenoids, flavonoids, retinoids) and their derivatives, nonsteroidal anti-inflammatory drugs (aspirin) as well as biguanides (metformin and phenformin) originally developed from phytochemical backbones, are multi-target agents showing antiangiogenic and anti-anti-inflammatory proprieties. Many of them target AMPK and metabolic pathways such as the mTOR axis. We summarize the beneficial effects of several compounds in conferring protection and supporting therapy, and as a paradigm, we present data on terpenoids & biquanides on beer hop xanthohumol and hydroxytryrosol from olive mill waste waters. Conclusions: These molecules could be employed for combinatorial chemoprevention and interception approaches or chemoprevention/therapy regimens for cancer and other chronic complex diseases.

Angiogenesis has been shown to play a crucial role in prostate cancer (PCa) progression in several preclinical models, in the clinic, pro-angiogenic factors have been shown to correlate with, Gleason score, metastasis and prognosis in PCa. Plasma levels of VEGF were shown to be higher in patients with metastatic PCa than those with localized disease. The natural killer (NK) cells have been found to be non cytolytic, immunosuppressed in solid cancers. Our group and others demonstrated that tumor infiltrating NK cells in lung and colorectal cancer acquire a pro-angiogenic phenotype. These cells are similar to NK cell within the decidua (dNK), and functionally support angiogenesis in a TGFb and Stat3-dependent manner. A similar subset is present in the peripheral blood of oncology patients. We characterized systemic peripheral blood NK cells (pNK) from PCa patients and their interaction with endothelial cells and macrophages. NK cell subset distribution was investigated by multicolor flow cytometry (FC) for surface antigens on peripheral blood samples PCa patients. Conditioned media (CM) of three different PCa cell lines were used to polarize the healthy donors pNK cells. CM from FACS-sorted PCa pNKs were used for functional studies of angiogenesis, on human umbilical-vein endothelial cells (HUVEC), studies for macrophage recruitment (migration assay on Boyden chambers) and polarization. Molecular studies were performed by real time PCR (qPCR) on HUCECs and macrophages exposed to CM of pNKs. Protein arrays were performed to characterize the secretome on FACS-sorted pNKs. We found that PCa pNKs acquire a pro-angiogenic/decidual-like CD56brightCD9+CD49a+CXCR4+ phenotype. The same phenotype was observed from healthy donors pNK cells exposed to CMs of three different PCa cell lines. These results were confirmed also exposing heathy-donor derived NK cells to conditioned media of 3 different prostate cancer cell lines (PC-3, DU-145, LNCaP), together with increased production of CXCL8, Angiogenin, Angiopoietin1 and reduced production of TNFa, IFNg and GranzymeA. CMs from pNK cells support the formation on capillary-like structures on HUVEC, together with increased expression of VEGF, VEGR-2, CXCL8, ICAM-1 and VCAM-1. Although in male patients, PCa NK cells resemble decidual-like ones. Finding these cells polarized in PB suggests that they could be used as non-invasive marker of inflammation in prostate cancer. Secretome analysis revealed the ability of pNK cells release pro-angiogenic factors (CLXL8, MMP-1, MMP-9; uPAR) and cytokines/chemokines involved in macrophage recruitment (CCL1, CCL2, CCL5, CCL7,CCL13, CXCL1, CXCL11) and M2-like polarization (IL-10). CMs from pNK can recruiting THP-1 monocyte and polarize THP-1 macrophage towards CD206, Arginase1, CXCL8-expressing M2-like/TAM phenotype. Our results place PCa pNK cells interaction with endothelial cells and via macrophage polarization able in supporting angiogenesis in PCa patients. Citation Format: Adriana Albini, Denisa Baci, Matteo Gallazzi, Federico Dehò, Angelo Naselli, Andrea Guernieri, Lorenzo Mortara, Douglas M. Noonan, Antonino Bruno. NK cells from prostate cancer patients acquire a pro-angiogenic phenotype and polarize macrophages towards a M2-like/TAM subset [abstract]. In: Proceedings of the AACR Virtual Special Conference on the Evolving Tumor Microenvironment in Cancer Progression: Mechanisms and Emerging Therapeutic Opportunities; in association with the Tumor Microenvironment (TME) Working Group; 2021 Jan 11-12. Philadelphia (PA): AACR; Cancer Res 2021;81(5 Suppl):Abstract nr LT006.

Lung cancer is the leading cause of cancer-related death worldwide and the only curative treatment remains surgery. Recurrence rates in resected patients remain high: from 30% for stage I to 80% for stage III. We previously demonstrated the role of 24 microRNAs in the pathogenesis and aggressiveness of lung tumors. Natural Killer (NK) cells, cellular mediators involved in tumor immunosurveillance are functionally compromised in cancer patients. We discovered that tumor infiltrating NK cells (TINKs) in non-small cell lung cancer (NSCLC) acquire the proangiogenic CD56brightCD16-VEGF+PlGF+ phenotype and functions and that TGFβ is involved the NK cell angiogenic switch. Tumor cells can release large amounts of extracellular vesicles (EVs), that have been reported to be endowed with potent immunosuppressive activities, also in NK cells. Here, we investigate the abilities of EVs from NSCLC cell lines and patients to induce a pro-angiogenic polarization in NK cells. EVs were isolated from conditioned media of NSCLC primary tumor (A549, LT73) and metastatic (H460) cell lines, and from plasma of NSCLC metastatic patients or heavy smoker individuals (EV-donor), by ultra-centrifugation and their purity were confirmed by transmission electron microscopy (TEM), Nanosight and western blot. NK cells, FACS sorted from healthy controls were exposed for 72 hours to 15 μg of EVs, then characterized for surface antigen expression, by multicolour flow cytometry. miRNA-profiling was performed on NK cells polarized for 72 hours with 15 μg of EVs isolated from A549 or LT73 NSCLC cells lines, or from plasma of patients with metastatic NSCLC or heavy smoker individuals. NanoSight analysis revealed that the EVs-Patients and EVs-Donors had a similar median size and morphology. We found that EVs from NSCLC cell lines and NSCLC patients induce the CD56brightCD9+CD49a+ NK cell phenotype, together with upregulation of CXCR4 expression and decreased levels of the NKG2D activation marker. Interestingly, the metastatic H460 cell line exhibited stronger NK cell polarization effects as compared to those from A549 and LT73 cells. Strong pro-angiogenic NK cell polarization was also observed in cytolytic NKs, exposed to EVs from metastatic NSCLC patients, as compared to those exposed to EVs-donors Finally, miRNA profiling on NK cells polarized with EVs, showed up-regulation of miR-133a and miR-221 suggesting a possible role of these miRNAs in NK angiogenic switch. Our study demonstrates that NSCLC EVs can support lung cancer progression acting on the NK cell angiogenic switch. Citation Format: Matteo Gallazzi, Orazio Fortunato, Ugo Pastorino, Gabriella Sozzi, Douglas Noonan, Adriana Albini, Antonino Bruno. Extracellular vesicles from metastatic non-small cell lung cancer induce the angiogenic switch in natural killer cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1605.

The olive mill wastewater polyphenol extracts exhibit chemopreventive properties in lung cancer cell lines, acting on the CXCR4/CXCL12 axis. Lung cancer still is a leading cause of cancer death worldwide. Although avoidance of tobacco use and smoking cessation will have the greatest impact on lung cancer development, (chemo)prevention could prove to be very effective, particularly in high risk individuals. Diverse diet-derived compounds have been reported to exert antioxidant, anti-proliferative and anti-angiogenic effects. Olive oil production involves the generation of an aqueous phase, defined as olive mill wastewater (OMWW), a polluting effluent rich in soluble polyphenols. We established a polyphenol rich, purified extract, from OMWW, that we termed A009. The effect of A009 on cell proliferation was determined by the crystal violet assay, while induction of apoptosis was measured by flow cytometry. The ability of the A009 to regulate the cell migration/invasion axis CXCR4/CXL12 and the production of pro angiogenic factors (VEGF, CXCL8, CCL2), was investigated by flow cytometry. The sprouting assay on Matrigel was used to functional verify the ability of A009 to limit invasive properties. Hydroxytyrosol, the most abundant polyphenols present in our A009, was used as reference molecules in all the assays performed. We found that the A009 limit A549 and H1560 cell proliferation, in a dose and time dependent manner. These effects were associated with induction of apoptosis at 24 and 48 hours of treatment, that was higher in H1650, as compared to A549 cells. The A009 extracts were effective in inhibiting the production of CXCR4 and CXCL12 in both the A549 and H1650 cells, accompanied with reduction in H1650 cell capability to generate invasive sprouts. in a Matrigel layer. Finally, A009 interfere with the production of VEGF, CXCL8 and CCL2 in both the A549 and H1650 cells. Our preliminary results suggest that A009 exhibit promising preventing and interceptive properties on lung cancer cell lines in vitro. Citation Format: Matteo Gallazzi, Marco Festa, Clementina Sansone, Paola Corradino, Douglas Noonan, Adriana Albini. The CXCR4/CXCL12 axis is a target of a polyphenol extract from olive oil processing with potential cancer prevention and interception activities [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 18.

Prostate cancer (PCa) remains the second most common cancer worldwide in men. Angiogenesis has been shown to play a crucial role in PCa progression in several preclinical models. Production of pro-angiogenic factors have been shown to correlate with metastasis, Gleason score and prognosis in PCa and plasma levels of VEGF were shown to be higher in patients with metastatic PCa than those with localized disease Natural killer (NK) cells, effector lymphocytes of the innate immunity, have been found to be immunosuppressed in solid cancers, including PCa. We demonstrated that NK cells in lung and colorectal cancer, acquire a pro-angiogenic phenotype, similar to NK cell within the decidua (dNK), and functionally support angiogenesis, in a TGFβ and Stat3-dependent manner. Here, we phenotypically and functionally characterized circulating NK cells from PCa patients and their interaction with endothelial cells and macrophages. NK cell subset distribution was investigated by multicolor flow cytometry (FC) for surface antigens on peripheral blood samples PCa patients. Conditioned media (CM) from FACS-sorted PTANKs were used for functional studies of angiogenesis, on human umbilical-vein endothelial cells (HUVEC), studies for macrophage recruitment (migration assay on Boyden chambers) and polarization. Molecular studies were performed by real time PCR (qPCR) on HUCECs and macrophages exposed to CM of PTANKs. Protein arrays were performed to characterize the secretome on FACS-sorted PTANKs We found that PTANKs acquire the pro-angiogenic/ decidual-likeCD56brightCD9+CD49a+CXCR4+ phenotype. The same phenotype was observed on cytolytic NK cells, from healthy donors, exposed to CMs of three different PCa cell lines. These results were confirmed also exposing heathy-donor derived NKs to CMs of 3 different prostate cancer cell lines (PC-3, DU-145, LNCaP), together with increased production of CXCL8, Angiogenin, Angiopoietin1 and reduced production of TNFα, IFNγ and GranzymeA. CMs from PTANKs support the formation on capillary-like structures on HUVEC, together with increased expression of VEGF, VEGR-2, CXCL8, ICAM-1 and VCAM-1. Secretome analysis revealed the ability of PTANKs release pro-angiogenic factors (CLXL8, MMP-1, MMP-9; uPAR) and cytokines/chemokines involved in macrophage recruitment (CCL1, CCL2, CCL5, CCL7, CCL13, CXCL1, CXCL11) and M2-like polarization (IL-10). Finally, CMs from PTANKs can recruit THP-1 monocyte and polarize THP-1 macrophage towards CD206, Arginase1, CXCL8-expressing M2-like/TAM phenotype. Our results place PTANKS as effector cells able in supporting angiogenesis in PCa by directly interaction with endothelial cells and via macrophage polarization. Citation Format: Antonino Bruno, Denisa Baci, Matteo Gallazzi, Annalisa Bosi, Angelo Naselli, Andrea Guarneri, Lorenzo Mortara, Douglas Noonan, Adriana Albini. Prostate tumor associated NK cells (PTANKs) acquire the decidual-like/pro-angiogenic phenotype and polarize macrophages towards the M2-like/TAM subset [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1581.

Prostate cancer (PCa) represents the second cause of male cancer death worldwide. Immune cells can acquire pro-tumor phenotype and functions as consequence of their plasticity. Natural killer (NK) cells are cellular mediators of the innate immunity, primarily involved in tumor recognition and elimination. Altered NK phenotype and functions have been observed in different tumors including PCa. Here, we provide new insights on phenotype and functional characterization of peripheral blood tumor-associated NK cells (TANKs) from benign prostatic hyperplasia (BPH) and prostatic adenocarcinoma (ADK) patients. We also investigated NK interaction with other components of the tumor microenvironment. NK cell phenotype and functional characterization was performed by multicolour flow cytometry for surface antigens on BPH and PCa TANKs. Interactions of TANKs with other TME components (endothelial cells, macrophages) were investigated using released products from BPH and PCa TANKs for functional studies of angiogenesis, macrophage recruitments and polarization. The effects of TANKS on macrophage polarization state were evaluated by gene expression analysis RT-PCR (qPCR).We found that NK cells from peripheral blood of BPH and PCa patients acquire a pro-angiogenic/ decidual-like phenotype, identified as CD56+CD9+CD49a+CD69+ NKs. These results were confirmed also exposing heathy-donor derived NKs to conditioned media of 3 different prostate cancer cell lines (PC-3, DU-145, LNCaP). In addition, polarization of NKs with the PCa cell line conditioned media resulted in downregulation of IFNγ, TNFα and Granzyme A and increased production of pro-angiogenic factors, such CXCL8, Angiopoietin1 (Angiop1) and Angiogenin (ANG). Secretome analysis revealed the ability of NKs from BPH and PCa patients to release higher level of pro-angiogenic molecules (CXCL8, MMP-1, MMP-9 and uPAR) and cytokines/chemokines involved in macrophage recruitment, CCL1, CCL2, CCL5, CCL13, CXCL1, CXCL11 and GM-CSF. Conditioned media from BPH and PCa NKs were able to promote angiogenesis in vitro, by inducing the formation of capillary-like structures on human umbilical-vein endothelial cells (HUVEC), recruit the THP-1 monocyte cell line and polarize THP-1 differentiated macrophage towards M2-like tumor associated macrophages (TAM).Our data demonstrate that NK cells from PCa patients are switched toward a pro-angiogenic/pro-tumor phenotype and function. Therefore, we propose tumor associated NKs as a new hallmark and potential target in prostate cancer. Citation Format: Denisa Baci, Matteo Gallazzi, Lorenzo Mortara, Douglas M. Noonan, Antonino Bruno. Pro-inflammatory and pro-angiogenic properties of tumor associated natural killer cells in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4571.

Many efforts have been addressed on the identification of novel active compounds from natural sources, endowed with anti-proliferative, anti-oxidant, chemopreventive properties, and their ability to target the tumour microenvironment (TME). Through metabolomics approaches we previously found that in serum samples from prostate cancer (PCa) patients, three carnitine family members were significantly decreased, suggesting a potential protective role of carnitine against PCa. Acetyl-L-carnitine(ALCAR) is an aceticacid ester of carnitine with high bioavailability and is involved in the transportof fatty acids across the inner mitochondrial membrane. We have showed that ALCAR was able reduce angiogenesis in vitro, acting on the VEGF/VEGFR2 and CXCR4/CXCL12 axes. We also found that ALCAR inhibits inflammatory angiogenesis in vivo, by reducing endothelial cells and macrophage recruitment in the matrigel plugs. Here we investigated the ability of ALCAR to interfere with key functional steps of prostate carcinogenesis and identified the molecular mechanisms involved. The effects of ALCAR on PCa cells were investigated in vitro by functional assays (adhesion, migration and invasion assays), molecular and biochemical approaches (RT-PCR, FACS, Byoplex and western blot). We found that ALCAR reduces apoptosis on PCa cells (PC3, Du-145, LNcap) and inhibits crucial tumorigenic steps such as adhesion, migration and invasion. We than confirmed the results from functional assays at molecular levels, and we found that ALCAR blocks CXCR4/CXCL12 axes, a key regulator of malignant migratory/aggressive phenotype. In accordance with our published paper, we confirmed the anti-angiogenic and ant-inflammatory properties of ALCAR, that we found to lower VEGF and CXCL8 production in PCa cell lines. Furthermore, we found a significantly reduced expression of inflammatory-related chemokines/cytokines involved macrophage recruitment such as CCL2, IL-6 and TNFα in PCa cells. Our results highlight the angio/chemopreventive and anti-inflammatory properties of ALCAR and allow the identification of multiple and overlapping mechanisms of action through which ALCAR inhibits PCa progression / metastasis. Our findings provide the rational for the employment of ALCAR, as a possible supplement for approaches of chemoprevention in subjects at high risk to develop cancer. Citation Format: Adriana Albini, Antonino Bruno, Denisa Baci, Matteo Gallazzi, Matilde Tramacere. Acetyl-L-carnitine (ALCAR) inhibits angiogenesis, migration and macrophage recruitment in prostatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 5086.

e17544 Background: Prostate cancer (PCa) represents the second most common cancer worldwide in men. Angiogenesis has been shown to play a crucial role in PCa progression in several preclinical models. Production of pro-angiogenic factors can correlate with metastasis, Gleason score and prognosis in PCa and plasma levels of VEGF were shown to be higher in patients with metastatic PCa than those with localized disease. Natural killer (NK) cells, effector lymphocytes of the innate immunity, have been found to be immunosuppressed in solid cancers, including PCa. Methods: Here, we phenotypically and functionally characterize circulating NK cells from PCa patients and their interaction with endothelial cells and macrophages. NK cell subset distribution has been investigated by multicolor flow cytometry (FACS) for surface antigens on peripheral blood samples PCa patients (TANKs). Conditioned media (CM) from FACS-sorted TANKs were used for functional studies of angiogenesis, on human umbilical-vein endothelial cells (HUVEC), studies for macrophage recruitment (migration assay on Boyden chambers) and polarization. Molecular studies were performed by real time PCR (qPCR) on HUCECs and macrophages exposed to CM of TANKs. Protein arrays were performed to characterize the secretome on FACS-sorted TANKs. Results: We found that TANKs acquire a pro-angiogenic/ dNK-like CD56brightCD9+CD49a+CXCR4+ phenotype. The same phenotype was observed on cytolytic NK cells, from healthy donors, exposed to CMs of three different PCa cell lines (PC-3, DU-145, LNCaP), together with increased production of CXCL8, Angiogenin, Angiopoietin1 and reduced production of TNFa, IFNg and GranzymeA. CMs from TANKs support the formation on capillary-like structures on HUVEC, together with increased expression of VEGF, VEGR-2, CXCL8, ICAM-1 and VCAM-1. Secretome analysis revealed the ability of TANKs release pro-angiogenic pro-invasive factors (CLXL8, MMP-1, MMP-9; uPAR) and cytokines/chemokines involved in macrophage recruitment (CCL1, CCL2, CCL5, CCL7, CCL13, CXCL1, CXCL11) and M2-like polarization (IL-10). Finally, CMs from TANKs can recruit THP-1 monocyte and polarize THP-1 macrophage towards CD206, Arginase1, CXCL8-expressing M2-like/TAM phenotype. Conclusions: Our results place PCa TANKs as effector cells able in supporting angiogenesis in PCa by directly interaction with endothelial cells and via macrophage polarization.