Tumor-infiltrating Natural Killer Cells Research Articles

Differences in tolerogenic status of NK cells between luminal A type, luminal B type, and triple-negative breast cancer.

Globally, breast cancer is the main cause of death among female cancer patients. The tumor-infiltrating lymphocytes (TILs) in breast cancer are associated with a more favorable outcome of a disease. Natural killer (NK) cells are important cytotoxic cells involved in tumor immunosurveillance, causing the direct killing of tumor cells. In solid tumors, peripheral NK cells and tumor-infiltrating NK cells display an altered phenotype characterized by reduced cytotoxicity or anergy. The goal of this study was to investigate the NK cells' phenotype and activation status in order to get into the pathological process of breast cancer subtypes. In our study, the normal tissues and tumoral breast tissue were fixed in formalin, embedded in paraffin, and the phenotypic marker CD56 and proinflammatory cytokine IL-15 were identified by immunohistology. The distribution and expression of receptors repertoire (NKG2A, NKG2C, NKp46, CD94, CD69, and CD107a) were investigated in peripheral NK cells of mononuclear cells by flow cytometry. mRNA of cytolytic mediators was determined by real-time PCR. The frequency of CD56+ and IL-15+ cells were significantly higher in triple-negative breast cancer tissue. The frequency of NK cell activating receptors was decreased in investigated breast cancer subtypes while the inhibitory NKG2A receptor was increased. Decreased percentage of CD69+/CD107a+ in NK cells could indicate lower cellular activation and cytotoxicity. In luminal B breast cancer, the mRNA of cytolytic mediators was upregulated. In conclusion, modulation of activation status in tumor-infiltration NK cells could be involved in the pathogenesis of molecular breast cancer subtypes. This highlights the importance of NK cells as an appropriate target for potent anti-tumor response in the immunosuppressive tumor microenvironment of breast cancer.

Comparative effects of free doxorubicin, liposome encapsulated doxorubicin and liposome co-encapsulated alendronate and doxorubicin (PLAD) on the tumor immunologic milieu in a mouse fibrosarcoma model.

Background: We have previously shown that alendronate, an amino-bisphosphonate, when reformulated in liposomes, can significantly enhance the efficacy of cytotoxic chemotherapies and help remodel the immunosuppressive tumor microenvironment towards an immune-permissive milieu resulting in increased anticancer efficacy. In addition, we have previously shown that the strong metal-chelating properties of alendronate can be exploited for nuclear imaging of liposomal biodistribution. To further improve anticancer efficacy, a pegylated liposome formulation co-encapsulating alendronate and doxorubicin (PLAD) has been developed. In this study, we examined the effects of PLAD on the tumor immunologic milieu in a mouse fibrosarcoma model in which the tumor microenvironment is heavily infiltrated with tumor-associated macrophages (TAM) that are associated with poor prognosis and treatment resistance.Methods: Doxorubicin biodistribution, characterization of the tumor immunologic milieu, cellular doxorubicin uptake, and tumor growth studies were performed in Balb/c mice bearing subcutaneously implanted WEHI-164 fibrosarcoma cells treated intravenously with PLAD, pegylated liposomal doxorubicin (PLD), free doxorubicin, or vehicle.Results: PLAD delivery resulted in a high level of tumor doxorubicin that was 20 to 30-fold greater than in free doxorubicin treated mice, and non-significantly higher than in PLD treated mice. PLAD also resulted in increased uptake in spleen and slightly lower plasma levels as compared to PLD. Importantly, our results showed that PLAD, and to a lesser extent PLD, shifted cellular drug uptake to TAM and to monocytic myeloid-derived suppressor cells (MDSC), while there was no drug uptake in neutrophilic MDSC or lymphoid cells. Free doxorubicin cellular drug uptake was below detectable levels. PLAD, and to a lesser extent PLD, also induced significant changes in number and functionality of tumor-infiltrating TAM, MDSC, Treg, NKT, and NK cells that are consistent with enhanced antitumor immune responses in the tumor microenvironment. In contrast, free doxorubicin induced moderate changes in the tumor microenvironment that could promote (decreased Treg) or be detrimental to antitumor immune responses (decreased M1 TAM and NK cells). These immune modulatory effects are reflected in the therapeutic study which showed that PLAD and PLD inhibited tumor growth and significantly prolonged survival, while free doxorubicin showed little or no anticancer activity.Conclusion: We show that liposomal delivery of doxorubicin not only alters pharmacokinetics, but also dramatically changes the immune modulatory activity of the drug cargo. In addition, our data support that the PLAD nanotheranostic platform further enhances some immune changes that may act in synergy with its cytotoxic chemotherapy effects.

608 Synergistic effect of the combination of ATG-017, an ERK1/2 inhibitor, and immune checkpoint inhibitor in preclinical cancer models

BackgroundThe RAS/MAPK pathway has emerged as a critical pathway for therapeutic targeting in a spectrum of solid tumor and hematological malignances. Inhibitors targeting MAPK pathway targets, such as RAS, BRAF, or MEK have been approved for the treatment of cancer either as monotherapy or in combination. However, there has been no approved drug targeting ERK1/2, the terminal kinases in the Ras-Raf-MEK-ERK signal transduction cascade. The combination of ERK1/2 inhibitor and checkpoint inhibitors as therapeutic strategy has not been explored in clinic. ATG-017 is an oral, potent, and highly selective inhibitor of ERK1/2, which is under Phase 1 clinical investigation. ATG-012 is a novel inhibitor of KRAS G12C. In this study, we tested the anti-tumor effect induced by the combination of ATG-017, or ATG-012, and an anti-PDL1 antibody in preclinical cancer models.MethodsWe assessed the anti-cancer effects of ERK1/2, KRAS G12C and PD1/PDL1 inhibition as monotherapy, and as combinations. Anti-PDL1 antibody (atezolizumab), the combination of ATG-012 or ATG-017 and atezolizumab, and the triple combination of atezolizumab, ATG-012 and ATG-017 were tested in a PD(L)1 blockade insensitive syngeneic lung cancer model,LL/2. The ATG-017-atezolizumab combination was also evaluated in a KRAS G13C-mutant,PD(L)1 blockade insensitive lymphoma model, EL4. To further investigate if ERK1/2 inhibition could enhance the efficacy of atezolizumab in a MAPK aberration-independent manner, the ATG-017-atezolizumab combination was tested in a MAPK wild type lymphoma model, A20. As well as assessment of impact on tumor growth, the impact of the drugs on tumor infiltrating leukocytes were analyzed by flow cytometry.ResultsThe combination of ERK1/2 inhibition and an anti-PDL1 antibody showed enhanced efficacy in mouse syngeneic tumor models. In the EL4 model, neither ATG-017 nor atezolizumab showed single agent activity, while the combination showed significant tumor growth inhibition (TGI=22%) on day 9. No body weight loss was observed. The percentage of infiltrating CD8+ T cells, NK cells and CD8:CD4 ratio were found increased in the combination group. The mean percentage of CD8+T cells among CD45+ cells increased from 4.17% (IgG +Vehicle control), 3.81% (ATG-017), 3.23% (atezolizumab), to 12.92% in the combination group. The CD8:CD4 ratio were 0.25 (IgG+Vehicle), 0.32(ATG-017), 0.17 (atezolizumab) and 0.67 (combination), respectively (Figure 1). More data from LL/2 and A20 model are being generated.ConclusionsSynergism has been observed for the combination of checkpoint inhibition and ERK1/2 inhibition in vivo, suggesting promising therapeutic strategies for cancer patients that warrants further clinical investigation.Ethics ApprovalThe protocol and any amendment(s) or procedures involving the care and use of animals in this study were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) of CrownBio prior to execution. During the study, the care and use of animals were conducted in accordance with the regulations of the Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC).All studies were conducted following an approved IACUC protocol. AUP NO.: AN-2004-09-309Abstract 608 Figure 1ATG-017-Atezolizumab combination showed in vivo synergism(A) Tumor growth curve and (B) mean body weight of EL4 syngeneic model; N=8 for each treatment group. (C) ATG-017-Atezolizumab combination increased tumor infiltrating CD8+T cells (left), CD8+/CD4+ ratio (middle), and tumor infiltrating NK cells (right). The TILs were isolated from tumor samples shown in (A). N=3 for each group.

Reprogramming NK cells and macrophages via combined antibody and cytokine therapy primes tumors for elimination by checkpoint blockade.

SUMMARYTreatments aiming to augment immune checkpoint blockade (ICB) in cancer often focus on T cell immunity, but innate immune cells may have important roles to play. Here, we demonstrate a single-dose combination treatment (termed AIP) using a pan-tumor-targeting antibody surrogate, half-life-extended interleukin-2 (IL-2), and anti-programmed cell death 1 (PD-1), which primes tumors to respond to subsequent ICB and promotes rejection of large established tumors in mice. Natural killer (NK) cells and macrophages activated by AIP treatment underwent transcriptional reprogramming; rapidly killed cancer cells; governed the recruitment of cross-presenting dendritic cells (DCs) and other leukocytes; and induced normalization of the tumor vasculature, facilitating further immune infiltration. Thus, innate cell-activating therapies can initiate critical steps leading to a self-sustaining cycle of T cell priming driven by ICB.

933 Immune infiltrates differences between central and periphery regions of colorectal and pancreas adenocarcinomas

BackgroundThe tumor microenvironment (TME) is a complex system, where malignant cells co-exist and communicate with immune and non-immune cells.1 This interaction orchestrates an immune response that regulates and recruits other immune cells that may either promote cancer growth or mediate tumor regression.2 The number and spatiotemporal distribution of tumor-infiltrating lymphocytes (TILs) in the TME, as well as NK cells, have been correlated with favorable prognosis in patients with colorectal cancer or pancreatic adenocarcinomas (CRC and PDAC).3–5 A more detailed analysis of the TME landscape composition will help to understand which specific areas will give rise to immune cells with clinically relevant anti–cancer-directed responses and could be preferentially expanded for immunotherapy using tumor-infiltrating NK cells.MethodsFresh CRC (n=6) and PDAC (n=6) tumor specimens were obtained within 20 minutes after surgery. A certified GI pathologist collected central and periphery tumor regions. From each region, half of the specimen was used for immunophenotypic analysis, the other half was cultured with IL-2 (1000 IU/mL) for 12 days. Immunophenotyping was performed using CD45, CD19, CD3, CD4, CD8, CD56, CD16, and LiveDead marker by flow cytometry at days 0, 6, and 12. Parallel formalin-fixed paraffin-embedded blocks were generated and analyzed by immunohistochemistry (IHC) for CD8 and CD56.ResultsThe immuneprofile of the tumor center and periphery is different both in CRC and PDAC. The mean percentages of B-cells, T-cells (CD4+ and CD8+ T-cells) and NK cells (CD56bright CD16- and CD56+ CD16+) of tumor regions are presented in figure 1) are more abundant in the TME and formed larger clusters than NK cells (figure 2). The majority of NK cells were found in the stroma however, in some cases, NK cells were located within the malignant epithelium.Abstract 933 Table 1Representation of percentage of immune cells in the central and periphery tumor regions of both CRC and PDAC tissues. The mean percentage of B-cells, T-cells (including CD4+ and CD8+-T-cells) and NK cells (including CD56bright CD16- and CD56+ CD16+ subpopulation) during ex vivo expansion (day 0, day 6 and day 12) are represented from the different regions of both CRC (n=6) and PDA (n=6).Abstract 933 Figure 1Representative IHC images of CD8+- T-cells in different regions: stroma (periphery and intratumoral) and intraepithelial of both CRC (n=3) and PDAC (n=3). Semi-quantitative analysis of CD8+- T-cells (from negative (-) to high numbers (+++)). Yellow arrows point to intraepithelial CD8+- T-cells. Scale bar corresponds to 50 µm.Abstract 933 Figure 2Representative IHC images of CD56+ NK cells in different regions: stroma (periphery and intratumoral) and intraepithelial of both CRC (n=3) and PDAC (n=3). Semi-quantitative analysis of CD56+ NK cells (from negative (-) to high numbers (+++)). Yellow arrows point to intraepithelial CD56+ NK cells. Scale bar corresponds to 50 µm.ConclusionsOur data in this small set of specimens show that TME immune cells in central and periphery tumor regions present different phenotypes in both tumor types. The tumor periphery exhibits a stronger infiltration with TIL and NK cells as compared to the center. We also observed that immune cells are in close physical contact with tumor cells since they were identified inside the malignant epithelium in some specimens. NK-cells and particularly TIL close to cancer cells may be preferentially harvested to obtain improved anti-cancer-directed NK or TCR alpha-beta-directed immune cell products for active immunotherapy.AcknowledgementsThe authors would like to thank the Champalimaud Foundation Biobank of the Champalimaud Centre for the Unknown for anonymously releasing tissue specimens.Trial RegistrationN/AReferencesGiraldo NA, Sanchez-Salas R, Peske JD, Vano Y, Becht E, Petitprez F, Validire P, Ingels A, Cathelineau X, Fridman WH, et al. The clinical role of the TME in solid cancer. Br J Cancer (2019);120:45–53.Li L, Yu R, Cai T, Chen Z, Lan M, Zou T, Wang B, Wang Q, Zhao Y, Cai Y. Effects of immune cells and cytokines on inflammation and immunosuppression in the tumor microenvironment. Int Immunopharmacol (2020);88:106939.Lianyuan T, Dianrong X, Chunhui Y, Zhaolai M, Bin J. The predictive value and role of stromal tumor-infiltrating lymphocytes in pancreatic ductal adenocarcinoma (PDAC). Cancer Biol Ther (2018);19:296–305.Idos GE, Kwok J, Bonthala N, Kysh L, Gruber SB, Qu C. The prognostic implications of tumor-infiltrating lymphocytes in colorectal cancer: a systematic review and meta-analysis. Sci Rep (2020);10:3360.Zhang S, Liu W, Hu B, Wang P, Lv X, Chen S, Shao Z. Prognostic significance of tumor-infiltrating natural killer cells in solid tumors: a systematic review and meta-analysis. Front Immunol (2020);11:1–11.Ethics ApprovalThis study was approved by the Champalimaud Foundation Ethics Committee and by the Ethics Research Committee of NOVA Medical School of NOVA University of Lisbon; approval number 56.ConsentWritten informed consent was obtained from the patient for publication of this abstract and any accompanying images. A copy of the written consent is available for review by the Editor of this journal.

SIRT2 Promotes Murine Melanoma Progression Through Natural Killer Cell Inhibition

SIRT2, an NAD+-dependent histone deacetylase, has been shown to play a pivotal role in various physiological processes, however, its role in cancer is currently controversial. In recent years, SIRT2 has been described as both a tumor suppressor and oncogene with divergent expression and function in various malignancies. With established roles in inflammation and glucose and iron metabolism, systemic and microenvironment SIRT2, rather than just intrinsic tumor cell expression, might have implications in tumor progression. Moreover, recent interest in the tumor immune microenvironment has further characterized immune cells involved in tumor suppression, including natural killer (NK) cells. Here, we sought to investigate the biological role of systemic SIRT2 in melanoma tumor progression and to evaluate its interaction with key tumor suppressive immune cells.Murine models were employed to investigate the effect of systemic SIRT2 on melanoma tumor progression. Tumor progression was measured in terms of weight and volume for 20 days following subcutaneous inoculation of Sirt2-KI and WT mice with B16-F10 melanoma cells. Differences in tumor-infiltrating immune cell composition were determined by flow cytometry. Granzyme B and Ki-67 immunohistochemical staining was used to measure NK cell functional activity and proliferation in in vivo melanoma samples. NK cell function was further evaluated using in vitro and ex vivo mouse models as well as an in vitro human model. Antibody-mediated NK cell depletion established NK cells as an integral player in SIRT2-mediated tumor promotion. Finally, SIRT2 was pharmacologically inhibited by SirReal2, and the effect on melanoma progression and NK cell tumor infiltration was established.Increased systemic expression of SIRT2 promoted melanoma progression in mice, resulting in larger and heavier tumors. Mechanistically, systemic SIRT2 overexpression reduced the number of tumor-infiltrating NK cells and suppressed NK cell activation and proliferation within the tumor microenvironment. Furthermore, despite the effect of increased tumor growth rate and tumor volume in wild-type littermate mice, NK cell depletion did not affect that in SIRT2-overexpressing mice. Lastly, pharmacological inhibition of SIRT2 increased NK cell tumor infiltration and suppressed melanoma tumor growth.Systemic SIRT2 and NK cell activity exhibit a dynamic functional interaction which promotes melanoma tumor progression. Given the recent renewed interest in NK-cell-mediated immunotherapy response, SIRT2 could represent a new opportunity to mediate immunotherapy response and resistance.

METTL3-mediated m6A RNA methylation promotes the anti-tumour immunity of natural killer cells

Natural killer (NK) cells exert critical roles in anti-tumor immunity but how their functions are regulated by epitranscriptional modification (e.g., N6-methyladenosine (m6A) methylation) is unclear. Here we report decreased expression of the m6A “writer” METTL3 in tumor-infiltrating NK cells, and a positive correlation between protein expression levels of METTL3 and effector molecules in NK cells. Deletion of Mettl3 in NK cells alters the homeostasis of NK cells and inhibits NK cell infiltration and function in the tumor microenvironment, leading to accelerated tumor development and shortened survival in mice. The gene encoding SHP-2 is m6A modified, and its protein expression is decreased in METTL3-deficient NK cells. Reduced SHP-2 activity renders NK cells hyporesponsive to IL-15, which is associated with suppressed activation of the AKT and MAPK signaling pathway in METTL3-deficient NK cells. These findings show that m6A methylation safeguards the homeostasis and tumor immunosurveillance function of NK cells.

Adaptive NK Cell Therapy Modulated by Anti-PD-1 Antibody in Gastric Cancer Model.

Recently, adaptive NK cell therapy has become a promising treatment but has limited efficacy as a monotherapy. The identification of immune checkpoint inhibitor (ICI) molecules has opened a new horizon of immunotherapy. Herein, we aimed to demonstrate the cytotoxic effects of a polytherapy consisting of ex vivo expanded IL-2-activated NK cells combined with human anti-PD-1 antibody as an important checkpoint molecule in a xenograft gastric cancer mouse model. EBV-LCL cell is used as a feeder to promote NK cell proliferation with a purity of 93.4%. Mice (NOG, female, 6–8 weeks old) with xenograft gastric tumors were treated with PBS, ex vivo IL-2-activated NK cells, IL-2-activated NK cell along with human anti-PD-1 (Nivolumab), and IL-2-activated pretreated NK cells with anti-PD-1 antibody. The cytotoxicity of ex vivo expanded NK cells against MKN-45 cells was assessed by a lactate dehydrogenase (LDH) assay. Tumor volume was evaluated for morphometric properties, and tumor-infiltrating NK cells were assessed by immunohistochemistry (IHC) and quantified by flow cytometry. Pathologic responses were considered by H and E staining. Ex vivo LDH evaluation showed the cytotoxic potential of treated NK cells against gastric cancer cell line. We indicated that the adoptive transfer of ex vivo IL-2-activated NK cells combined with anti-PD-1 resulted in tumor growth inhibition in a xenograft gastric cancer model. Mitotic count was significantly decreased (*p < 0.05), and the tumor was associated with improved infiltration of NK cells in the NK-anti-PD-1 pretreated group (*p < 0.05). In conclusion, the combination approach of activated NK cells and anti-PD-1 therapy results in tumor growth inhibition, accompanied by tumor immune cell infiltration in the gastric tumor model.

MP66-20 BLADDER CANCER EXTRACELLULAR VESICLES: A NOVEL ANTI-TUMOR VACCINE

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology III (MP66)1 Sep 2021MP66-20 BLADDER CANCER EXTRACELLULAR VESICLES: A NOVEL ANTI-TUMOR VACCINE Carlos Ortiz-Bonilla, Taylor Ucello, Scott Gerber, Hiroshi Miyamoto, Edith Lord, Edward Messing, and Yi-Fen Lee Carlos Ortiz-BonillaCarlos Ortiz-Bonilla More articles by this author , Taylor UcelloTaylor Ucello More articles by this author , Scott GerberScott Gerber More articles by this author , Hiroshi MiyamotoHiroshi Miyamoto More articles by this author , Edith LordEdith Lord More articles by this author , Edward MessingEdward Messing More articles by this author , and Yi-Fen LeeYi-Fen Lee More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000002106.20AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Extracellular vesicles (EV) comprise a diverse group of small vesicles secreted by almost all types of cells into the extracellular space. EV have been shown to play crucial roles in carrying and presenting functional MHC-peptide complexes to modulate antigen-specific CD8 T cell responses. However, the therapeutic potential and immunogenicity of EV-based vaccines has not yet tested. In this project, we aim to investigate the potential of using bladder cancer (BC) EV as a cancer vaccine in a pre-clinical model. METHODS: EV derived from murine MB49 BC cells were characterized by Nanoparticle Tracking Analysis, and EV cargo proteins and encapsulated cytokines were analyzed by Mass Spectrometry and pro-inflammatory cytokine array. The anti-tumor immunity of BCEV was evaluated in the MB49 syngeneic mouse model where mice were primed with MB49 EV prior MB49 tumors inoculation. Tumor growth was monitored and tumor-infiltrated immune cell populations were determined by flow cytometry. Anti-CD8 neutralizing antibody as well as IgG isotype control antibody were administrated to mice to test whether BCEV-mediated anti-tumor immunity is dependent on CD8 T cells. Moreover, we determined whether priming mice with MB49EV can prevent colonization of un-related colon MC-38 tumors in vivo. RESULTS: Proteomic and cytokine array analysis have revealed that MB49 EV encapsulated enriched immuno-active cargo proteins and cytokines with function linked to both innate and adaptive immune responses. To our surprise, priming mice with MB49 EV completely prevented MB49 tumor growth in vivo and neutralizing CD8 T cells abolished the MB49 EV-mediated anti-tumor effect. Interestingly, MB49 EV priming was unable to prevent MC-38 tumor growth but hindered significantly their growth in vivo. Flow cytometry analyses revealed increased levels of tumor-infiltrated granulocytes, inflammatory monocytes, and natural killer cells in EV-primed MC-38 tumors. In addition, those tumors also showed increased numbers of infiltrated antigen-presenting dendritic cells and antigen-specific CD4 and CD8 T cells. CONCLUSIONS: For the first time, we provided direct in vivo evidence that BCEV can prevent isogenic tumor growth, and such action is mainly mediated by CD8 T cells. They encapsulated enriched immunogenic cargo proteins and cytokines that could even promote anti-tumor host response against unrelated tumor growth. In summary, our study support the potential application of tumor-derived EV in personalized medicine, and open new avenue for developing therapeutic cancer vaccine based on patient-derived EV to prevent BC recurrence. Source of Funding: NCI RO1 CA173986 © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e1137-e1137 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Carlos Ortiz-Bonilla More articles by this author Taylor Ucello More articles by this author Scott Gerber More articles by this author Hiroshi Miyamoto More articles by this author Edith Lord More articles by this author Edward Messing More articles by this author Yi-Fen Lee More articles by this author Expand All Advertisement Loading ...

The Ratio of Exhausted to Resident Infiltrating Lymphocytes Is Prognostic for Colorectal Cancer Patient Outcome.

Immunotherapy success in colorectal cancer is mainly limited to patients whose tumors exhibit high microsatellite instability (MSI). However, there is variability in treatment outcomes within this group, which is in part driven by the frequency and characteristics of tumor-infiltrating immune cells. Indeed, the presence of specific infiltrating immune-cell subsets has been shown to correlate with immunotherapy response and is in many cases prognostic of treatment outcome. Tumor-infiltrating lymphocytes (TIL) can undergo distinct differentiation programs, acquiring features of tissue-residency or exhaustion, a process during which T cells upregulate inhibitory receptors, such as PD-1, and lose functionality. Although residency and exhaustion programs of CD8+ T cells are relatively well studied, these programs have only recently been appreciated in CD4+ T cells and remain largely unknown in tumor-infiltrating natural killer (NK) cells. In this study, we used single-cell RNA sequencing (RNA-seq) data to identify signatures of residency and exhaustion in colorectal cancer-infiltrating lymphocytes, including CD8+, CD4+, and NK cells. We then tested these signatures in independent single-cell data from tumor and normal tissue-infiltrating immune cells. Furthermore, we used versions of these signatures designed for bulk RNA-seq data to explore tumor-intrinsic mutations associated with residency and exhaustion from TCGA data. Finally, using two independent transcriptomic datasets from patients with colon adenocarcinoma, we showed that combinations of these signatures, in particular combinations of NK-cell activity signatures, together with tumor-associated signatures, such as TGFβ signaling, were associated with distinct survival outcomes in patients with colon adenocarcinoma.

Targeting the αv integrin/TGF-β axis improves natural killer cell function against glioblastoma stem cells.

Glioblastoma multiforme (GBM), the most aggressive brain cancer, recurs because glioblastoma stem cells (GSCs) are resistant to all standard therapies. We showed that GSCs, but not normal astrocytes, are sensitive to lysis by healthy allogeneic natural killer (NK) cells in vitro. Mass cytometry and single-cell RNA sequencing of primary tumor samples revealed that GBM tumor-infiltrating NK cells acquired an altered phenotype associated with impaired lytic function relative to matched peripheral blood NK cells from patients with GBM or healthy donors. We attributed this immune evasion tactic to direct cell-to-cell contact between GSCs and NK cells via αv integrin-mediated TGF-β activation. Treatment of GSC-engrafted mice with allogeneic NK cells in combination with inhibitors of integrin or TGF-β signaling or with TGFBR2 gene-edited allogeneic NK cells prevented GSC-induced NK cell dysfunction and tumor growth. These findings reveal an important mechanism of NK cell immune evasion by GSCs and suggest the αv integrin/TGF-β axis as a potentially useful therapeutic target in GBM.

Blockade of checkpoint receptor PVRIG unleashes anti-tumor immunity of NK cells in murine and human solid tumors

BackgroundAlthough checkpoint-based immunotherapy has shown exciting results in the treatment of tumors, around 70% of patients have experienced unresponsiveness. PVRIG is a recently identified immune checkpoint receptor and blockade of which could reverse T cell exhaustion to treat murine tumor; however, its therapeutic potential via NK cells in mice and human remains seldom reported.MethodsIn this study, we used patient paraffin-embedded colon adenocarcinoma sections, various murine tumor models (MC38 colon cancer, MCA205 fibrosarcoma and LLC lung cancer), and human NK cell- or PBMC-reconstituted xenograft models (SW620 colon cancer) to investigate the effect of PVRIG on tumor progression.ResultsWe found that PVRIG was highly expressed on tumor-infiltrating NK cells with exhausted phenotype. Furthermore, either PVRIG deficiency, early blockade or late blockade of PVRIG slowed tumor growth and prolonged survival of tumor-bearing mice by inhibiting exhaustion of NK cells as well as CD8+ T cells. Combined blockade of PVRIG and PD-L1 showed better effect in controlling tumor growth than using either one alone. Depletion of NK or/and CD8+ T cells in vivo showed that both cell types contributed to the anti-tumor efficacy of PVRIG blockade. By using Rag1−/− mice, we demonstrated that PVRIG blockade could provide therapeutic effect in the absence of adaptive immunity. Further, blockade of human PVRIG with monoclonal antibody enhanced human NK cell function and inhibited human tumor growth in NK cell- or PBMC-reconstituted xenograft mice.ConclusionsOur results reveal the importance of NK cells and provide novel knowledge for clinical application of PVRIG-targeted drugs in future.

Imbalance between Tolerant and Cytotoxic Functional Subsets of NK Cells in a JAK2V617F Myeloproliferative Neoplasm Model

Natural killer (NK) cells are lymphocytes that play a critical role in both innate and adaptive immune responses. It has been shown that CD27 and CD11b are antigens expressed in the last stage of NK cells development and allow the classification of NK cells into three functional subsets. Tolerant NK cells are double negative (DN) for CD27 and CD11b, have an immature phenotype and present low secretory and cytotoxic potential. The secretory NK cells are CD27 positive, have a great ability to release cytokines, but do not exhibit cytotoxicity properties. The cytotoxic NK cells express CD11b, release granzymes and perforins, but are less capable of secreting cytokines. While cytotoxic NK cells are essential for the natural defenses against infected or malignant cells, secretory NK cells, when activated, produce different cytokines, including IFNγ, TNFα, TGFβ, GM-CSF, MIP1α, MIP1β, IL-10 and others, which may modify the functions or other hematopoietic cells including the hematopoietic stem cells (HSC). We hypothesized that deregulation of the balance between secretory or cytotoxic subtypes of NK cells may favor HSC malignant transformation and contribute to stem cell-derived diseases. In this regard, few studies have shown that NK cells are defective in chronic myeloid leukemia (CML). In contrast to CML, the role of NK cells in BCR-ABL1 negative-myeloproliferative neoplasms (MPN) is currently unexplored. We aimed to study the activity of NK cells and their subsets in murine primary cells from MPN. NK cells subsets were quantified by flow cytometry by the use of spleen cells from a pre-established conditional vavCre knockin Jak2V617F (Jak2VF) murine model. Besides genotyping to identify controls (Jak2 wt/wt vavCre+, or Jak2WT) and Jak2-mutated mice (Jak2 wt/V617F vavCre+, Jak2VF), the presence of polycythemia and splenomegaly was confirmed in the animals with MPN. Mice (n=5 per group) were euthanized between 8-12 weeks of age. For immunophenotyping, spleen cells were isolated, submitted to red cell lyse, and stained with fluorescence-conjugated antibodies against Ter119, CD19, CD4, CD8, CD3, NK1.1 and CD27 and CD11b. All the stainings were performed at 4°C for 20 minutes and acquisition was performed in a FACSCanto™II flow cytometer (BD Biosciences). Data was analyzed by the use of the Flowjo software version X. When compared to Jak2WT controls, the Jak2VF mice had lower numbers of NK cells (1,447 x 106 versus 0,381 x 106 p<0.05) and T cells (0,638 x 106 versus 0,158 x 106 of the spleen; p<0.05) among the total splenocytes. The analysis of NK subsets showed an increase in the percentages of DN NK cells (CD27-CD11b-) in the spleen of Jak2VF mice as compared to controls (14.84% versus 4.97%; p<0.05). CD27+ immature and mature secretory NK (CD27+CD11b-/+) cells also seemed to be increased in Jak2-mutated mice but this data did not reach statistical significance (37.43% versus 17.65%; p=0.1). On the other hand, when compared to Jak2WT controls, the Jak2VF mice presented lower percentages of CD11b+ cytotoxic NK cells (77.07% versus 47.80% of the spleen cells; p<0.05). In agreement, when the expression of NK receptors was analyzed by flow cytometry, the inhibitory NKG2A receptor was found to be higher expressed in Jak2VF NK cells as compared to non-mutated cells, while the expression of the activatory receptors Ly49H and Ly49D was reduced in Jak2-mutated NK cells. Of note, low levels of the Jak2V617F mutation were detected in sorted NK cells isolated from Jak2VF mice as compared to controls by Real Time quantitative RT-PCR, thus suggesting that the distribution between NK cell subtypes observed here is associated with the JAK2 mutational status. In summary, when compared to controls, Jak2-mutated mice presented an increased proportion of tolerant NK cells, previously described as immature cells with low secretory and cytotolytic potential. To date, CD11b-/CD27- tolerant cells have been observed among solid tumor-infiltrating NK cells, but have not been previously associated with MPN. Moreover, an inverted ratio between the secretory and cytotoxic NK subsets was also detected in Jak2VF animals, suggesting that a cytotoxic deficiency occurs and may contribute to the leukemic stem cell expansion. The expansion of the NK secretory potential deserves further elucidation. Our study provided new insights into the distribution of JAK2V617F NK functional subsets that may be used as future therapeutic targets for MPN. DisclosuresKobayashi:Pfizer: Consultancy.

SIRT2 promotes murine melanoma progression through natural killer cell inhibition

SIRT2, an NAD+-dependent histone deacetylase, has been shown to play a pivotal role in various physiological processes, however, its role in cancer is currently controversial. In recent years, SIRT2 has been described as both a tumor suppressor and oncogene with divergent expression and function in various malignancies. Using murine allograft melanoma models, our results suggest increased systemic expression of SIRT2 promotes tumor progression. In this study, SIRT2-overexpressing mice exhibited enhanced tumor growth and larger tumor volumes compared to their wild-type littermates. Mechanistically, systemic overexpression of SIRT2 reduces the number of tumor-infiltrating natural killer (NK) cells and suppresses NK cell function and proliferation within the tumor microenvironment (TME). Furthermore, despite the enhancing effect of NK cell depletion on tumor volume and growth rate in wild-type littermate mice, this effect was diminished in SIRT2-overexpressing mice. Lastly, pharmacological inhibition of SIRT2 increases NK cell tumor infiltration and suppresses allograft melanoma tumor growth. The findings of this study identify a dynamic functional interaction between systemic SIRT2 and NK cell activity, which controls melanoma tumor progression. Given the recent renewed interest in NK-cell-mediated immunotherapy response, SIRT2 could present a new opportunity to mediate immunotherapy response and resistance.

Circulating and Tumor-Infiltrating NK Cells From Clear Cell Renal Cell Carcinoma Patients Exhibit a Predominantly Inhibitory Phenotype Characterized by Overexpression of CD85j, CD45, CD48 and PD-1.

Although natural killer (NK) cells infiltrate clear cell renal cell carcinomas (ccRCC), the most frequent malignancy of the kidney, tumor progression suggests that they become dysfunctional. As ccRCC-driven subversion of NK cell effector functions is usually accompanied by phenotypic changes, analysis of such alterations might lead to the identification of novel biomarkers and/or targets in immuno-oncology. Consequently, we performed a phenotypic analysis of peripheral blood NK cells (PBNK) and tumor-infiltrating NK cells (TINK) from ccRCC patients. Compared to HD, PBNK from ccRCC patients exhibited features of activated cells as shown by CD25, CD69 and CD62L expression. They also displayed increased expression of DNAM-1, CD48, CD45, MHC-I, reduced expression of NKG2D, and higher frequencies of CD85j+ and PD-1+ cells. In addition, compared to PBNK from ccRCC patients, TINK exhibited higher expression of activation markers, tissue residency features and decreased expression of the activating receptors DNAM-1, NKp30, NKp46, NKp80 and CD16, suggesting a more inhibitory phenotype. Analysis of The Cancer Genome Atlas (TCGA) revealed that CD48, CD45, CD85j and PD-1 are significantly overexpressed in ccRCC and that their expression is associated with an NK cell infiltration signature. Calculation of z-scores revealed that their expression on PBNK, alone or combined, distinguished ccRCC patients from HD. Therefore, these molecules emerge as novel potential biomarkers and our results suggest that they might constitute possible targets for immunotherapy in ccRCC patients.

Natural Killer Cells and T Cells in Hepatocellular Carcinoma and Viral Hepatitis: Current Status and Perspectives for Future Immunotherapeutic Approaches.

Natural killer (NK) cells account for 25–50% of the total number of hepatic lymphocytes, which implicates that NK cells play an important role in liver immunity. The frequencies of both circulating and tumor infiltrating NK cells are positively correlated with survival benefit in hepatocellular cancer (HCC) and have prognostic implications, which suggests that functional impairment in NK cells and HCC progression are strongly associated. In HCC, T cell exhaustion is accompanied by the interaction between immune checkpoint ligands and their receptors on tumor cells and antigen presenting cells (APC). Immune checkpoint inhibitors (ICIs) have been shown to interfere with this interaction and have altered the therapeutic landscape of multiple cancer types including HCC. Immunotherapy with check-point inhibitors, aimed at rescuing T-cells from exhaustion, has been applied as first-line therapy for HCC. NK cells are the first line effectors in viral hepatitis and play an important role by directly eliminating virus infected cells or by activating antigen specific T cells through IFN-γ production. Furthermore, chimeric antigen receptor (CAR)-engineered NK cells and T cells offer unique opportunities to create CAR-NK with multiple specificities learning from the experience gained with CAR-T cells with potentially less adverse effects. This review focus on the abnormalities of NK cells, T cells, and their functional impairment in patients with chronic viral hepatitis, which contributes to progression to hepatic malignancy. Furthermore, we discuss and summarize recent advances in the NK cell and T cell based immunotherapeutic approaches in HCC.

NK cell and ILC heterogeneity in colorectal cancer. New perspectives from high dimensional data

Innate lymphoid cells (ILCs) and tissue-resident natural killer (NK) cells ensure immunity at environmental interfaces and help maintain barrier integrity of the intestinal tract. This wide range of innate lymphocytes is able to provide fast and potent inflammatory responses that, when deregulated, have been associated with pathogenesis of inflammatory bowel disease (IBD) and colorectal cancer (CRC). While the presence of tumor-infiltrating NK cells is generally associated with a favorable outcome in CRC patients, emerging evidence reveals distinct roles for ILCs in regulating CRC pathogenesis and progression. Advances in next generation sequencing technology, and in particular of single-cell RNA-seq approaches, along with multidimensional flow cytometry analysis, have helped to deconvolute the complexity and heterogeneity of the ILC system both in homeostatic and pathological contexts. In this review, we discuss the protective and detrimental roles of NK cells and ILCs in the pathogenesis of CRC, focusing on the phenotypic and transcriptional modifications these cells undergo during CRC development and progression.

Developing natural killer cells producing hypoxia-inducible cytokines for effective immunotherapy against solid tumors.

Abstract Natural Killer (NK) cells are a promising means for adoptive immunotherapy for cancers. NK cell therapies against hematological cancers such as acute myeloid leukemia have demonstrated good efficacy. However, the poor effector function of tumor-infiltrating NK cells limits the potential of NK cells against solid tumors. Accumulating data suggests that hypoxia in the tumor microenvironment (TME) suppresses the effector function of NK cells. One of the immunosuppressive features of hypoxia is the generation of adenosine, a metabolite that highly suppresses NK cell cytotoxicity and proliferation. Although engineering NK cells to overexpress stimulatory cytokines before the adoptive immunotherapy has demonstrated promising potential for cancer therapy, it raises a concern that high systemic levels of cytokines may lead to cytokine-mediated pathology. Therefore, we hypothesize that driving overexpression of the potent stimulatory cytokines exclusively in the TME would circumvent systemic toxicity. An attractive strategy to achieve this goal is to take advantage of the hypoxic condition, a key characteristic of the TME. Hypoxia-inducible factor 1 alpha (HIF1-α) plays a pivotal role in expressing hypoxia-responsive genes by associating with promoters containing hypoxia response element (HRE). By mimicking the biological system, HRE-induced cytokine expression vectors were generated and tested in NK cells. The expression was inducible under hypoxic conditions and was reversible upon re-oxygenated conditions. Our results suggest that the hypoxia-inducible vector system can be beneficial to trigger TME specific cytokine expressions in NK cells and provide insight into efficient immunotherapy against solid tumors.

Abstract IA-024: Sirt2: A tumor suppressor or promotor?

Abstract SIRT2, an NAD+-dependent histone deacetylase, has been shown to play a pivotal role in various physiological processes, however, its role in cancer is currently controversial. In recent years, SIRT2 has been described as both a tumor suppressor and oncogene with divergent expression and function in various malignancies. Using murine xenograft melanoma models, our results suggest increased systemic expression of SIRT2 promotes tumor progression. In this study, SIRT2-overexpressing mice exhibited enhanced tumor growth and larger tumor volumes compared to their wild-type littermates. Mechanistically, systemic overexpression of SIRT2 reduces the number of tumor-infiltrating natural killer (NK) cells and suppresses NK cell activation and proliferation within tumor microenvironment (TME). Furthermore, despite the effect of increased tumor growth rate and tumor volume in wild-type littermate mice, NK cell depletion does not affect that in SIRT2-overexpressing mice. Lastly, pharmacological inhibition of SIRT2 increases NK cell tumor infiltration and suppresses melanoma tumor growth in mice. The findings of this study identify a dynamic functional interaction between systemic SIRT2 and NK cell activity, which promotes melanoma tumor progression. Given the recent renewed interest in NK-cell-mediated immunotherapy response, SIRT2 could present a new opportunity to mediate immunotherapy response and resistance. Citation Format: Fen Xia. Sirt2: A tumor suppressor or promotor? [abstract]. In: Proceedings of the AACR Virtual Special Conference on Radiation Science and Medicine; 2021 Mar 2-3. Philadelphia (PA): AACR; Clin Cancer Res 2021;27(8_Suppl):Abstract nr IA-024.

Abstract LT006: NK cells from prostate cancer patients acquire a pro-angiogenic phenotype and polarize macrophages towards a M2-like/TAM subset

Abstract 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.