NFκB-Activated COX2/PGE2/EP4 Axis Controls the Magnitude and Selectivity of BCG-Induced Inflammation in Human Bladder Cancer Tissues.

Bacillus Calmette-Guérin (BCG) is commonly used in the immunotherapy of bladder cancer (BlCa) but its effectiveness is limited to only a fraction of patients. To identify the factors that regulate the response of human BlCa tumor microenvironment (TME) to BCG, we used the ex vivo whole-tissue explant model. The levels of COX2 in the BCG-activated explants closely correlated with the local production of Treg- and MDSCS attractants and suppressive factors, while the baseline COX2 levels did not have predictive value. Accordingly, we observed that BCG induced high levels of MDSC- and Treg-attracting chemokines (CCL22, CXCL8, CXCL12) and suppressive factors (IDO1, IL-10, NOS2). These undesirable effects were associated with the nuclear translocation of phosphorylated NFκB, induction of COX2, the key enzyme controlling PGE2 synthesis, and elevation of a PGE2 receptor, EP4. While NFκB blockade suppressed both the desirable and undesirable components of BCG-driven inflammation, the inhibitors of PGE2 synthesis (Celecoxib or Indomethacin) or signaling (EP4-selective blocker, ARY-007), selectively eliminated the induction of MDSC/Treg attractants and immunosuppressive factors but enhanced the production of CTL attractants, CCL5, CXCL9 and CXCL10. PGE2 blockade allowed for the selectively enhanced migration of CTLs to the BCG-treated BlCa samples and eliminated the enhanced migration of Tregs. Since the balance between the CTLs and suppressive cells in the TME predicts the outcomes in patients with BlCa and other diseases, our data help to elucidate the mechanisms which limit the effectiveness of BCG therapies and identify new targets to enhance their therapeutic effects.

Mitochondrial hypoxic stress induces widespread RNA editing by APOBEC3G in natural killer cells.

BACKGROUND: Protein recoding by RNA editing is required for normal health and evolutionary adaptation. However, de novo induction of RNA editing in response to environmental factors is an uncommon phenomenon. While APOBEC3A edits many mRNAs in monocytes and macrophages in response to hypoxia and interferons, the physiological significance of such editing is unclear. RESULTS: Here, we show that the related cytidine deaminase, APOBEC3G, induces site-specific C-to-U RNA editing in natural killer cells, lymphoma cell lines, and, to a lesser extent, CD8-positive T cells upon cellular crowding and hypoxia. In contrast to expectations from its anti-HIV-1 function, the highest expression of APOBEC3G is shown to be in cytotoxic lymphocytes. RNA-seq analysis of natural killer cells subjected to cellular crowding and hypoxia reveals widespread C-to-U mRNA editing that is enriched for genes involved in mRNA translation and ribosome function. APOBEC3G promotes Warburg-like metabolic remodeling in HuT78 T cells under similar conditions. Hypoxia-induced RNA editing by APOBEC3G can be mimicked by the inhibition of mitochondrial respiration and occurs independently of HIF-1α. CONCLUSIONS: APOBEC3G is an endogenous RNA editing enzyme in primary natural killer cells and lymphoma cell lines. This RNA editing is induced by cellular crowding and mitochondrial respiratory inhibition to promote adaptation to hypoxic stress.

Helicase-Driven Activation of NFκB-COX2 Pathway Mediates the Immunosuppressive Component of dsRNA-Driven Inflammation in the Human Tumor Microenvironment.

Presence of cytotoxic CD8+ T cells (CTL) in tumor microenvironments (TME) is critical for the effectiveness of immune therapies and patients' outcome, whereas regulatory T(reg) cells promote cancer progression. Immune adjuvants, including double-stranded (ds)RNAs, which signal via Toll-like receptor-3 (TLR3) and helicase (RIG-I/MDA5) pathways, all induce intratumoral production of CTL-attractants, but also Treg attractants and suppressive factors, raising the question of whether induction of these opposing groups of immune mediators can be separated. Here, we use human tumor explant cultures and cell culture models to show that the (ds) RNA Sendai Virus (SeV), poly-I:C, and rintatolimod (poly-I:C12U) all activate the TLR3 pathway involving TRAF3 and IRF3, and induce IFNα, ISG-60, and CXCL10 to promote CTL chemotaxis to ex vivo-treated tumors. However, in contrast with SeV and poly I:C, rintatolimod did not activate the MAVS/helicase pathway, thus avoiding NFκB- and TNFα-dependent induction of COX2, COX2/PGE2-dependent induction of IDO, IL10, CCL22, and CXCL12, and eliminating Treg attraction. Induction of CTL-attractants by either poly I:C or rintatolimod was further enhanced by exogenous IFNα (enhancer of TLR3 expression), whereas COX2 inhibition enhanced the response to poly-I:C only. Our data identify the helicase/NFκB/TNFα/COX2 axis as the key suppressive pathway of dsRNA signaling in human TME and suggest that selective targeting of TLR3 or elimination of NFκB/TNFα/COX2-driven suppression may allow for selective enhancement of type-1 immunity.Significance: This study characterizes two different poly-I:C-induced signaling pathways in their induction of immunostimulatory and suppressive factors and suggests improved ways to reprogram the TME to enhance the antitumor efficacy of immunotherapies. Cancer Res; 78(15); 4292-302. ©2018 AACR.

Adoptive transfer of natural killer cells promotes the anti-tumor efficacy of T cells.

The density of NK cells in tumors correlates positively with prognosis in many types of cancers. The average number of infiltrating NK cells is, however, quite modest (approximately 30 NK cells/sq.mm), even in tumors deemed to have a "high" density of infiltrating NK cells. It is unclear how such low numbers of tumor-infiltrating NK cells can influence outcome. Here, we used ovalbumin-expressing tumor cell lines and TCR transgenic, OVA-specific cytotoxic T lymphocytes (OT-I-CTLs) to determine whether the simultaneous attack by anti-tumor CTLs and IL-2-activated NK (A-NK) cells synergistically increases the overall tumor cell kill and whether upregulation of tumor MHC class-I by NK cell-derived interferon-gamma (IFNγ) improves tumor-recognition and kill by anti-tumor CTLs. At equal E:T ratios, A-NK cells killed OVA-expressing tumor cells better than OT-I-CTLs. The cytotoxicity against OVA-expressing tumor cells increased by combining OT-I-CTLs and A-NK cells, but the increase was additive rather than synergistic. A-NK cells adenovirally-transduced to produce IL-12 (A-NKIL-12) produced high amounts of IFNγ. The addition of a low number of A-NKIL-12 cells to OT-I-CTLs resulted in a synergistic, albeit modest, increase in overall cytotoxicity. Pre-treatment of tumor cells with NK cell-conditioned medium increased tumor MHC expression and sensitivity to CTL-mediated killing. Pre-treatment of CTLs with NK cell-conditioned medium had no effect on CTL cytotoxicity. In vivo, MHC class-I expression by OVA-expressing B16 melanoma lung metastases increased significantly within 24-48h after adoptive transfer of A-NKIL-12 cells. OT-I-CTLs and A-NKIL-12 cells localized selectively and equally well into OVA-expressing B16 lung metastases and treatment of mice bearing 7-days-old OVA-B16 lung metastases with both A-NKIL-12 cells and OT-I-CTLs lead to a significant prolongation of survival. Thus, an important function of tumor-infiltrating NK cells may be to increase tumor cell expression of MHC class-I through secretion of IFNγ, to prepare them for recognition by tumor-specific CTLs.

Cancer Stem Cells Protect Non-Stem Cells From Anoikis: Bystander Effects.

Cancer stem cells (CSCs) are capable of initiation and metastasis of tumors. Therefore, understanding the biology of CSCs and the interaction between CSCs and their counterpart non-stem cells is crucial for developing a novel cancer therapy. We used CSC-like and non-stem breast cancer MDA-MB-231 and MDA-MB-453 cells to investigate mammosphere formation. We investigated the role of the epithelial cadherin (E-cadherin)-extracellular signal-regulated kinase (Erk) axis in anoikis. Data from E-cadherin small hairpin RNA assay and mitogen-activated protein kinase kinase (MEK) inhibitor study show that activation of Erk, but not modulation of E-cadherin level, may play an important role in anoikis resistance. Next, the two cell subtypes were mixed and the interaction between them during mammosphere culture and xenograft tumor formation was investigated. Unlike CSC-like cells, increased secretion of interleukin-6 (IL-6) and growth-related oncogene (Gro) chemokines was detected during mammosphere culture in non-stem cells. Similar results were observed in mixed cells. Interestingly, CSC-like cells protected non-stem cells from anoikis and promoted tumor growth. Our results suggest bystander effects between CSC-like cells and non-stem cells. J. Cell. Biochem. 117: 2289-2301, 2016. © 2016 Wiley Periodicals, Inc.

Exogenous activated NK cells enhance trafficking of endogenous NK cells to endometriotic lesions.

BACKGROUND: Endometriosis is defined as the presence of endometrial glands and stroma at ectopic locations. Although the prevalence of endometriosis is as high as 35%-50%, its pathogenesis remains controversial. An increasing number of studies suggest that changes in immune reactivity may be primarily involved in the development of endometriosis development. In this sense, it has been strongly suggested that a fundamental part of immunologic system, the natural killer cells (NK cells), are an important part of this process. NK cells, a component of the innate immune system, have been extensively studied for their ability to defend the organism against infections and malignancy. Recent studies have shown that IL-2-activated NK (A-NK) cells are able to attack and destroy tumors in lungs and livers of mice, demonstrating the therapeutic potential of these cells. Similarly to metastatic tumor cells, endometrial cells are able to adhere, infiltrate and proliferate at ectopic locations. Therefore, in this study, we evaluated the ability of adoptively transferred and endogenous NK cells to infiltrate endometriosis lesions. METHODS: As NK cells donors were used C57BL/6 B6. PL- Thy 1.1 female mice. As uterine horns donors were used C57/BL6+GFP female mice and as endometriosis recipients C57BL/6 Thy1.2 female mice. Endometriosis induction was made by injection of endometrial tissue fragments. After 4 weeks, necessary for endometriosis lesions establishment the animals were divided in 3 experimental groups with 10 animals each. Group 1 received i.v doses of 5x106 A-NK in 200µl RPMI; Group 2 received i.p dose of 5x106 A-NK in 200µl RPMI and Group 3 received i.p dose of IL2 (0.5 mL RPMI containing 5.000U of IL2). RESULTS: Our data show that exogenous A-NK cells injected via ip combined with endogenous A-NK cells seems to be the most efficient way for activated NK cells track and infiltrate endometriosis. CONCLUSION: For the first time, it was shown that both endogenous as exogenous A-NK cells are able to track, migrate and infiltrate endometriosis lesion. This seems to be a promising result, and if confirmed the efficiency of A-NK cells in killing endometriosis lesions, maybe in the future we could use this approach as an alternative treatment for women with endometriosis.

The HSP90 Inhibitor Ganetespib Radiosensitizes Human Lung Adenocarcinoma Cells.

The molecular chaperone HSP90 is involved in stabilization and function of multiple client proteins, many of which represent important oncogenic drivers in NSCLC. Utilization of HSP90 inhibitors as radiosensitizing agents is a promising approach. The antitumor activity of ganetespib, HSP90 inhibitor, was evaluated in human lung adenocarcinoma (AC) cells for its ability to potentiate the effects of IR treatment in both in vitro and in vivo. The cytotoxic effects of ganetespib included; G2/M cell cycle arrest, inhibition of DNA repair, apoptosis induction, and promotion of senescence. All of these antitumor effects were both concentration- and time-dependent. Both pretreatment and post-radiation treatment with ganetespib at low nanomolar concentrations induced radiosensitization in lung AC cells in vitro. Ganetespib may impart radiosensitization through multiple mechanisms: such as down regulation of the PI3K/Akt pathway; diminished DNA repair capacity and promotion of cellular senescence. In vivo, ganetespib reduced growth of T2821 tumor xenografts in mice and sensitized tumors to IR. Tumor irradiation led to dramatic upregulation of ß-catenin expression in tumor tissues, an effect that was mitigated in T2821 xenografts when ganetespib was combined with IR treatments. These data highlight the promise of combining ganetespib with IR therapies in the treatment of AC lung tumors.

Viral infection of implanted meningeal tumors induces antitumor memory T-cells to travel to the brain and eliminate established tumors.

BACKGROUND: Leptomeningeal metastases occur in 2%-5% of patients with breast cancer and have an exceptionally poor prognosis. The blood-brain and blood-meningeal barriers severely inhibit successful chemotherapy. We have developed a straightforward method to induce antitumor memory T-cells using a Her2/neu targeted vesicular stomatitis virus. We sought to determine whether viral infection of meningeal tumor could attract antitumor memory T-cells to eradicate the tumors. METHODS: Meningeal implants in mice were studied using treatment trials and analyses of immune cells in the tumors. RESULTS: This paper demonstrates that there is a blood-meningeal barrier to bringing therapeutic memory T-cells to meningeal tumors. The barrier can be overcome by viral infection of the tumor. Viral infection of the meningeal tumors followed by memory T-cell transfer resulted in 89% cure of meningeal tumor in 2 different mouse strains. Viral infection produced increased infiltration and proliferation of transferred memory T-cells in the meningeal tumors. Following viral infection, the leukocyte infiltration in meninges and tumor shifted from predominantly macrophages to predominantly T-cells. Finally, this paper shows that successful viral therapy of peritoneal tumors generates memory CD8 T-cells that prevent establishment of tumor in the meninges of these same animals. CONCLUSIONS: These results support the hypothesis that a virally based immunization strategy can be used to both prevent and treat meningeal metastases. The meningeal barriers to cancer therapy may be much more permeable to treatment based on cells than treatment based on drugs or molecules.

Relocation of CLIC1 promotes tumor cell invasion and colonization of fibrin.

UNLABELLED: Chloride intracellular channel 1 (CLIC1) has been shown to be upregulated in various malignancies but its exact function remains unclear. Here, it is revealed that CLIC1 is critical for the stability of invadopodia in endothelial and tumor cells embedded in a 3-dimensional (3D) matrix of fibrin. Invadopodia stability was associated with the capacity of CLIC1 to induce stress fiber and fibronectin matrix formation following its ß3 integrin (ITGB3)-mediated recruitment into invadopodia. This pathway, in turn, was relevant for fibrin colonization as well as slug (SNAI2) expression and correlated with a significant role of CLIC1 in metastasis in vivo. Mechanistically, a reduction of myosin light chain kinase (MYLK) in CLIC1-depleted as well as ß3 integrin-depleted cells suggests an important role of CLIC1 for integrin-mediated actomyosin dynamics in cells embedded in fibrin. Overall, these results indicate that CLIC1 is an important contributor to tumor invasion, metastasis, and angiogenesis. IMPLICATIONS: This study uncovers an important new function of CLIC1 in the regulation of cell-extracellular matrix interactions and ability of tumor cells to metastasize to distant organs.

Novel nuclear localization of fatty acid synthase correlates with prostate cancer aggressiveness.

Fatty acid synthase is up-regulated in a variety of cancers, including prostate cancer. Up-regulation of fatty acid synthase not only increases production of fatty acids in tumors but also contributes to the transformed phenotype by conferring growth and survival advantages. In addition, increased fatty acid synthase expression in prostate cancer correlates with poor prognosis, although the mechanism(s) by which this occurs are not completely understood. Because fatty acid synthase is expressed at low levels in normal cells, it is currently a major target for anticancer drug design. Fatty acid synthase is normally found in the cytosol; however, we have discovered that it also localizes to the nucleus in a subset of prostate cancer cells. Analysis of the fatty acid synthase protein sequence indicated the presence of a nuclear localization signal, and subcellular fractionation of LNCaP prostate cancer cells, as well as immunofluorescent confocal microscopy of patient prostate tumor tissue and LNCaPs confirmed nuclear localization of this protein. Finally, immunohistochemical analysis of prostate cancer tissue indicated that nuclear localization of fatty acid synthase correlates with Gleason grade, implicating a potentially novel role in prostate cancer progression. Possible clinical implications include improving the accuracy of prostate biopsies in the diagnosis of low- versus intermediate-risk prostate cancer and the uncovering of novel metabolic pathways for the therapeutic targeting of androgen-independent prostate cancer.

NK cells in the tumor microenvironment.

The presence of natural killer (NK) cells in the tumor microenvironment correlates with outcome in a variety of cancers. However, the role of intratumoral NK cells is unclear. Preclinical studies have shown that, while NK cells efficiently kill circulating tumor cells of almost any origin, they seem to have very little effect against the same type of tumor cells when these have extravasated. The ability to kill extravasated tumor cells is, however, is dependent of the level of activation of the NK cells, as more recent published and unpublished studies, discussed below, have demonstrated that interleukin-2-activated NK cells are able to attack well-established solid tumors.

Matrix metalloproteinases in cytotoxic lymphocytes impact on tumour infiltration and immunomodulation.

To efficiently combat solid tumours, endogenously or adoptively transferred cytotoxic T cells and natural killer (NK) cells, need to leave the vasculature, traverse the interstitium and ultimately infiltrate the tumour mass. During this locomotion and migration in the three dimensional environment many obstacles need to be overcome, one of which is the possible impediment of the extracellular matrix. The first and obvious one is the sub-endothelial basement membrane but the infiltrating cells will also meet other, both loose and tight, matrix structures that need to be overridden. Matrix metalloproteinases (MMPs) are believed to be one of the most important endoprotease families, with more than 25 members, which together have function on all known matrix components. This review summarizes what is known on synthesis, expression patterns and regulation of MMPs in cytotoxic lymphocytes and their possible role in the process of tumour infiltration. We also discuss different functions of MMPs as well as the possible use of other lymphocyte proteases for matrix degradation.

Regenerated luminal epithelial cells are derived from preexisting luminal epithelial cells in adult mouse prostate.

Determining the source of regenerated luminal epithelial cells in the adult prostate during androgen deprivation and replacement will provide insights into the origin of prostate cancer cells and their fate during androgen deprivation therapy. Prostate stem cells in the epithelial layer have been suggested to give rise to luminal epithelium. However, the extent of stem cell participation to prostate regrowth is not clear. In this report, using prostate-specific antigen-CreER(T2)-based genetic lineage marking/tracing in mice, preexisting luminal epithelial cells were shown to be a source of regenerated luminal epithelial cells in the adult prostate. Prostatic luminal epithelial cells could survive androgen deprivation and were capable of proliferating upon androgen replacement. Prostate cancer cells, typically exhibiting a luminal epithelial phenotype, may retain this intrinsic capability to survive and regenerate in response to changes in androgen signaling, providing part of the mechanism for the ultimate failure of androgen deprivation therapy in prostate cancer.

Merkel cell polyomavirus large T antigen disrupts lysosome clustering by translocating human Vam6p from the cytoplasm to the nucleus.

Merkel cell polyomavirus (MCV) has been recently described as the cause for most human Merkel cell carcinomas. MCV is similar to simian virus 40 (SV40) and encodes a nuclear large T (LT) oncoprotein that is usually mutated to eliminate viral replication among tumor-derived MCV. We identified the hVam6p cytoplasmic protein involved in lysosomal processing as a novel interactor with MCV LT but not SV40 LT. hVam6p binds through its clathrin heavy chain homology domain to a unique region of MCV LT adjacent to the retinoblastoma binding site. MCV LT translocates hVam6p to the nucleus, sequestering it from involvement in lysosomal trafficking. A naturally occurring, tumor-derived mutant LT (MCV350) lacking a nuclear localization signal binds hVam6p but fails to inhibit hVam6p-induced lysosomal clustering. MCV has evolved a novel mechanism to target hVam6p that may contribute to viral uncoating or egress through lysosomal processing during virus replication.

Tumor disposition of pegylated liposomal CKD-602 and the reticuloendothelial system in preclinical tumor models.

Liposomes, such as pegylated-liposomal CKD-602 (S-CKD602), undergo catabolism by macrophages and dendritic cells (DCs) of the reticuloendothelial system (RES). The relationship between plasma and tumor disposition of S-CKD602 and RES was evaluated in mice bearing A375 melanoma or SKOV-3 ovarian xenografts. Area under the concentration-time curves (AUCs) of liposomal encapsulated, released, and sum total (encapsulated + released) CKD-602 in plasma, tumor, and tumor extracellular fluid (ECF) were estimated. A375 and SKOV-3 tumors were stained with cd11b and cd11c antibodies as measures of macrophages and DC. The plasma disposition of S-CKD602 was similar in both xenograft models. The ratio of tumor sum total AUC to plasma sum total AUC was 1.7-fold higher in mice bearing human SKOV-3 xenografts, compared with A375. The ratio of tumor ECF AUC to tumor sum total AUC was 2-fold higher in mice bearing human SKOV-3 xenografts, compared with A375. The staining of cd11c was 4.5-fold higher in SKOV-3, compared with A375 (P < 0.0001). The increased tumor delivery and release of CKD-602 from S-CKD602 in the ovarian xenografts, compared with the melanoma xenografts, was consistent with increased cd11c staining, suggesting that variability in the RES may affect the tumor disposition of liposomal agents.

Zinc in innate and adaptive tumor immunity.

Zinc is important. It is the second most abundant trace metal with 2-4 grams in humans. It is an essential trace element, critical for cell growth, development and differentiation, DNA synthesis, RNA transcription, cell division, and cell activation. Zinc deficiency has adverse consequences during embryogenesis and early childhood development, particularly on immune functioning. It is essential in members of all enzyme classes, including over 300 signaling molecules and transcription factors. Free zinc in immune and tumor cells is regulated by 14 distinct zinc importers (ZIP) and transporters (ZNT1-8). Zinc depletion induces cell death via apoptosis (or necrosis if apoptotic pathways are blocked) while sufficient zinc levels allows maintenance of autophagy. Cancer cells have upregulated zinc importers, and frequently increased zinc levels, which allow them to survive. Based on this novel synthesis, approaches which locally regulate zinc levels to promote survival of immune cells and/or induce tumor apoptosis are in order.

Effects of IL-2 on MMP expression in freshly isolated human NK cells and the IL-2-independent NK cell line YT.

Interleukin-2 is an important activation factor for natural killer (NK) cells but its effect on NK cell matrix metalloproteinases (MMP) production and matrix degradation is less well investigated. We have used freshly isolated human NK cells and the IL-2-independent NK cell line, YT, to investigate the effects of IL-2 stimulation on NK cell invasion of Matrigel and on MMP expression and production. In YT cells, we found opposing early and late effects of IL-2 stimulation with an early (2 h) increase in MMP-9 protein level and enhanced migration in the Matrigel invasion assay and by 30 hours a decreased mRNA expression of MMP-2, MMP-9, MMP-13, MT3-MMP, and MT6-MMP. We also found a preculture period of 48 hours with IL-2 to negatively affect YT cell migration. We furthermore found that freshly isolated human NK cells Matrigel invasion was MMP-dependent and it increased in response to IL-2. Importantly, in freshly isolated human NK cells we did not see a downregulation of MMPs after 24 hours IL-2 stimulation, but instead a significant upregulation of MT6-MMP mRNA. Because of the cellular localisation of MT6-MMP, which ensures a focalized proteolytic activity, and its high expression compared with the other MMPs in freshly isolated human NK cells makes it of interest to study further.

IL-4 suppresses very late antigen-4 expression which is required for therapeutic Th1 T-cell trafficking into tumors.

Murine CD4 T cells cultured under type 1 polarizing conditions selectively express significantly higher levels of the very late antigen (VLA)-4 and VLA-6 integrins when compared with T cells cultured under type 2 or nonpolarizing (type 0) conditions. This difference appears due to the action of interleukin (IL)-4, as loss of VLA-4/-6 expression on Th cells was prevented by inclusion of neutralizing anti-IL-4 mAb during the initial culture period. We also observed that CD4 T cells deficient in Stat6, a critical component of the IL-4R signaling cascade, retained high levels of VLA-4 and VLA-6 expression, regardless of IL-4 status in the culture conditions. When applied to committed Th1 cells, rIL-4 readily inhibited VLA-4 and VLA-6 expression to levels observed for Th2 cells, without altering the type 1 functional status of these cells. Conversely, low levels of VLA-4/VLA-6 expressed by committed Th2 cells could not be resurrected by culture in the presence of the Th1-kines IL-12p70 and interferon-gamma. Predictably, among the Th populations evaluated, Th1 cells alone adhered efficiently to, and were costimulated by, plate-bound VCAM-1 and laminin in a VLA-4-dependent or VLA-6-dependent manner, respectively. Finally, adoptive-transferred Th1 (but not Th2) cells developed from OT-II mice were uniquely competent to traffick into OVA M05 melanoma lesions in vivo, thereby enhancing the therapeutic benefits associated with cotransferred OVA-specific type 1 CD8 (OT-I) cells. These data suggest that treatment strategies capable of sustaining/enhancing VLA-4/VLA-6 expression on Th1 effector cells may yield improved clinical efficacy in the cancer setting.

Human NK cell lines migrate differentially in vitro related to matrix interaction and MMP expression.

Matrix metalloproteinases (MMPs) are thought to be of importance for the migratory ability of natural killer (NK) cells. Their expression and production may influence the amount of tumour-infiltrating NK cells and thereby any therapeutic capability. In this study, we sought to investigate the importance of MMPs for human NK cells' ability to degrade and migrate through the extracellular matrix (ECM). The two human NK cell lines, NK-92 and YT, migratory ability, MMP expression and production as well as their morphological appearance when cultured in the ECM equivalent Matrigel were analysed and compared. The quantitatively more migratory NK-92 cells were found to express invadopodia/podosomes at a significantly higher degree when cultured in Matrigel and gave rise to a general disintegration of the Matrigel. The NK-92 cells had a higher mRNA expression of MMP-2, -9, -13, MT1-, MT3- and MT6-MMP and a significantly higher production of MMP-9 compared to YT cells. These differences could explain the substantial functional difference observed between the two cell lines with respect to migratory capacity. In addition, the number of Matrigel invading NK-92 cells decreased significantly in the presence of the MMP inhibitor GM6001, demonstrating that MMPs have a critical function in their migration.

Pharmacologic administration of interleukin-2.

The development of biologic therapies for patients with cancer has in part been impeded by the extraordinary complexity and intrinsic feedback mechanisms promoting homeostasis in tissue injury, repair, inflammation, and immunity. Recombinant interleukin 2 (IL-2) therapy was initiated in 1984 based on its role as the prototypic T-cell growth factor, with novel roles deduced late after its FDA approval in regulating not only effector T cells but also regulatory T cells. Complicating its application, even in the most sophisticated centers, has been the manageable but difficult toxicities attendant on its use in spite of clear evidence of complete responses in 5-10% of treated patients with melanoma and renal cell carcinoma with extraordinary durability lasting now for almost 25 years, thus tantamount to "cures." Although efforts have been made to diminish toxicity or enhance efficacy the only substantive advance in combination therapy has been the application of tumor-infiltrating lymphocytes and the antibody to CTLA4. A deeper understanding of the "limiting" toxicity associated with mild flu-like symptoms and more debilitating cytokine "storm" not forthcoming. Here we propose the notion that the systemic syndrome associated with IL-2 administration is due to global cytokine-induced autophagy and temporally limited tissue dysfunction. The possible role of autophagy inhibitors to enhance efficacy and limit toxicity as well as possible problems with this approach are considered.