The C-terminal peptide of CCL21 drastically augments CCL21 activity through the dendritic cell lymph node homing receptor CCR7 by interaction with the receptor N-terminus.

The endogenous chemokines CCL19 and CCL21 signal via their common receptor CCR7. CCL21 is the main lymph node homing chemokine, but a weak chemo-attractant compared to CCL19. Here we show that the 41-amino acid positively charged peptide, released through C-terminal cleavage of CCL21, C21TP, boosts the immune cell recruiting activity of CCL21 by up to 25-fold and the signaling activity via CCR7 by ~ 100-fold. Such boosting is unprecedented. Despite the presence of multiple basic glycosaminoglycan (GAG) binding motifs, C21TP boosting of CCL21 signaling does not involve interference with GAG mediated cell-surface retention. Instead, boosting is directly dependent on O-glycosylations in the CCR7 N-terminus. As dictated by the two-step binding model, the initial chemokine binding involves interaction of the chemokine fold with the receptor N-terminus, followed by insertion of the chemokine N-terminus deep into the receptor binding pocket. Our data suggest that apart from a role in initial chemokine binding, the receptor N-terminus also partakes in a gating mechanism, which could give rise to a reduced ligand activity, presumably through affecting the ligand positioning. Based on experiments that support a direct interaction of C21TP with the glycosylated CCR7 N-terminus, we propose that electrostatic interactions between the positively charged peptide and sialylated O-glycans in CCR7 N-terminus may create a more accessible version of the receptor and thus guide chemokine docking to generate a more favorable chemokine-receptor interaction, giving rise to the peptide boosting effect.

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.

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.

Non-small cell lung cancer cells survived ionizing radiation treatment display cancer stem cell and epithelial-mesenchymal transition phenotypes.

Ionizing radiation (IR) is used for patients diagnosed with unresectable non small cell lung cancer (NSCLC), however radiotherapy remains largely palliative due to radioresistance. Cancer stem cells (CSCs), as well as epithelial-mesenchymal transition (EMT), may contribute to drug and radiation resistance mechanisms in solid tumors. Here we investigated the molecular phenotype of A549 and H460 NSCLC cells that survived treatment with IR (5Gy) and are growing as floating tumor spheres and cells that are maintained in a monolayer after irradiation.Non-irradiated and irradiated cells were collected after one week, seeded onto ultra low attachment plates and propagated as tumor spheres. Bulk NSCLC cells which survived radiation and grew in spheres express cancer stem cell surface and embryonic stem cell markers and are able to self-renew, and generate differentiated progeny. These cells also have a mesenchymal phenotype. Particularly, the radiation survived sphere cells express significantly higher levels of CSC markers (CD24 and CD44), nuclear ß-catenin and EMT markers (Snail1, Vimentin, and N-cadherin) than non-irradiated lung tumor sphere cells. Upregulated levels of Oct-4, Sox2 and beta-catenin were detected in H460 cells maintained in a monolayer after irradiation, but not in radiation survived adherent A459 cells.PDGFR-beta was upregulated in radiation survived sphere cells and in radiation survived adherent cells in both A549 and H460 cell lines. Combining IR treatment with axitinib or dasatinib, inhibitors with anti-PDFGR activity, potentiates the efficacy of NSCLC radiotherapy in vitro.Our findings suggest that radiation survived cells have a complex phenotype combining the properties of CSCs and EMT. CD44, SNAIL and PDGFR-beta are dramatically upregulated in radiation survived cells and might be considered as markers of radiotherapy response in NSCLC.

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.

Hyperthermia-enhanced TRAIL- and mapatumumab-induced apoptotic death is mediated through mitochondria in human colon cancer cells.

Colorectal cancer is the third leading cause of cancer-related mortality in the world; death usually results from uncontrolled metastatic disease. Previously, we developed a novel strategy of TNF-related apoptosis-inducing ligand (Apo2L/TRAIL) in combination with hyperthermia to treat hepatic colorectal metastases. However, previous studies suggest a potential hepatocyte cytotoxicity with TRAIL. Unlike TRAIL, anti-human TRAIL receptor antibody induces apoptosis without hepatocyte toxicity. In this study, we evaluated the anti-tumor efficacy of humanized anti-death receptor 4 (DR4) antibody mapatumumab (Mapa) by comparing it with TRAIL in combination with hyperthermia. TRAIL, which binds to both DR4 and death receptor 5 (DR5), was approximately tenfold more effective than Mapa in inducing apoptosis. However, hyperthermia enhances apoptosis induced by either agent. We observed that the synergistic effect was mediated through elevation of reactive oxygen species, c-Jun N-terminal kinase activation, Bax oligomerization, and translocalization to the mitochondria, loss of mitochondrial membrane potential, release of cytochrome c to cytosol, activation of caspases, and increase in poly(ADP-ribose) polymerase cleavage. We believe that the successful outcome of this study will support the application of Mapa in combination with hyperthermia to colorectal hepatic metastases.

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.

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.

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.

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.

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.

Cytokine gene therapy using adenovirally transduced, tumor-seeking activated natural killer cells.

We previously demonstrated that adoptively transferred, interleukin (IL)-2-activated natural killer (A-NK) cells are effective in reducing B16 lung tumors in tumor-bearing animals. This effect depends on high and often toxic doses of IL-2 to support the survival and antitumor functions of the transferred A-NK cells. We hypothesized that A-NK cells transduced to express pro-NK cell cytokines would become less dependent on high and potentially toxic amounts of IL-2. Here, we demonstrate that A-NK cells adenovirally transduced to express mIL-12 survive well and function efficiently in mice bearing B16 lung tumors when supported with low, nontoxic doses of IL-2. The intratumoral survival of nontransduced "bystander'' A-NK cells also increased when they were coinjected with IL-12 gene-transduced A-NK cells. The enhanced survival of exogenously delivered, IL-12 gene-transduced A-NK cells resulted in greater antitumor responsiveness. This led to a 7- to 10-day increase in median survival time compared with tumor-bearing mice receiving mock-transduced A-NK cells. These data show that the presence of IL-12 around tumor-infiltrating A-NK cells enhances their antitumor activity while reducing their requirement for systemically administered IL-2.

Helper role of NK cells during the induction of anticancer responses by dendritic cells.

Recent reports demonstrate that natural killer (NK) cells and dendritic cells (DC) support each other's activity in a positive feedback. We observed that activated NK cells induce the maturation of DCs into stable type-1 polarized DCs (DC1), characterized by up to 100-fold enhanced ability to produce IL-12p70 in response to subsequent interaction with Th cells. DC1 induction depends on NK cell-produced IFN-gamma and TNF-alpha, with a possible involvement of additional factors. DC1, induced by NK cells or by NK cell-related soluble factors, are stable, resistant to tumor-related suppressive factors, and show strongly enhanced ability to induce Th1 and CTL responses. In analogy to resting T cells, the induction of "helper" function of NK cells relies on a two-signal activation paradigm. While NKG2D-dependent tumor cell recognition is sufficient to induce the cytotoxic "effector" function of NK cells, the induction of "NK cell help" requires additional signals from type-1 IFNs, products of virally-infected cells, or from IL-2. Compared to non-polarized DCs currently-used in clinical trials, DC1s act as superior inducers of anti-cancer CTL responses during in vitro sensitization. The current data provides rationale for the clinical use of DC1s in cancer and chronic infections (such as HIV), as a new generation DC-based vaccines, uniquely combining fully mature DC status with an elevated, rather than "exhausted" ability to produce bioactive IL-12p70. We are currently implementing stage I/II clinical trials, testing the effectiveness of DC1s induced by NK cells or by NK cell-related factors, as therapeutic vaccines against melanoma.

Neither epithelial nor mesenchymal circulating tumor cells isolated from breast cancer patients are tumorigenic in NOD-scid Il2rgnull mice.

The quantitative evaluation of circulating EpCAM+ tumor cells (CTCs) in the peripheral blood of breast cancer patients provides an independent predictor of risk of progression in patients with metastatic disease. The present study investigated the tumorigenic potential of CTCs from cryopreserved mobilized leukapheresis products obtained from three metastatic breast cancer patients in remission. Cells were immunomagnetically separated if they expressed either the epithelial cell surface marker EpCAM, or CD90, a mesenchymal stromal cell marker associated with tumorigenic stem-like cancer cells. Cells were injected into the mammary fat pads of NOD-scid Il2rgnull mice. The injection of very large numbers of CTCs (0.3-1.5×106 CTCs per site, 20 sites per sample) in an optimized xenograft model did not result in the establishment of human-derived tumor xenografts. Four orders of magnitude fewer cells of the same CD90+ phenotype, but obtained from metastatic breast cancer pleural effusions, were highly tumorigenic in the same model system. These results favor the interpretation that circulating tumor cell load does not directly bear on metastatic potential, and that tumorigenic circulating breast cancer cells in patients with metastatic breast cancer are exceedingly rare. Furthermore, the CD44+/CD90+ phenotypic signature indicative of tumorigenicity in cells separated from metastatic or primary breast tumors does not have the same significance in circulating tumor cells.

Secretory TRAIL-Armed Natural Killer Cell-Based Therapy: In Vitro and In Vivo Colorectal Peritoneal Carcinomatosis Xenograft.

Since its discovery in 1995, TNF-related apoptosis-inducing ligand (TRAIL) has sparked growing interest among oncologists due to its remarkable ability to induce apoptosis in malignant human cells, but not in most normal cells. However, one major drawback is its fast clearance rate in vivo Thus, the development of an alternative means of delivery may increase the effectiveness of TRAIL-based therapy. In this study, we developed a secretory TRAIL-armed natural killer (NK) cell-based therapy and assessed its cytotoxic effects on colorectal cancer cells and its tumoricidal efficacy on colorectal peritoneal carcinomatosis xenograft. We generated genetically modified NK cells by transduction with a lentiviral vector consisting of a secretion signal domain, a trimerization domain, and an extracellular domain of the TRAIL gene. These NK cells secreted a glycosylated form of TRAIL fusion protein that induced apoptotic death. Intraperitoneally, but not intravenously, injected NK cells effectively accumulated at tumor sites, infiltrated tumor tissue, induced apoptosis, and delayed tumor growth. These results shed light on the therapeutic potential of genetically engineered NK cells to treat peritoneal carcinomatosis. Mol Cancer Ther; 15(7); 1591-601. ©2016 AACR.

Evaluation of Silicon Phthalocyanine 4 Photodynamic Therapy Against Human Cervical Cancer Cells In Vitro and in Mice.

BACKGROUND: Cervical cancer is the second most common cancer in women worldwide [1]. Photodynamic therapy has been used for cervical intraepithelial neoplasia with good responses, but few studies have used newer phototherapeutics. We evaluated the effectiveness of photodynamic therapy using Pc 4 in vitro and in vivo against human cervical cancer cells. METHODS: CaSki and ME-180 cancer cells were grown as monolayers and spheroids. Cell growth and cytotoxicity were measured using a methylthiazol tetrazolium assay. Pc 4 cellular uptake and intracellular distrubtion were determined. For in vitro Pc 4 photodynamic therapy cells were irradiated at 667nm at a fluence of 2.5 J/cm2 at 48 h. SCID mice were implanted with CaSki and ME-180 cells both subcutaneously and intracervically. Forty-eight h after Pc 4 photodynamic therapy was administered at 75 and 150 J/cm2. RESULTS: The IC50s for Pc 4 and Pc 4 photodynamic therapy for CaSki and ME-180 cells as monolayers were, 7.6µM and 0.016µM and >10µM and 0.026µM; as spheroids, IC50s of Pc 4 photodynamic therapy were, 0.26µM and 0.01µM. Pc 4 was taken up within cells and widely distributed in tumors and tissues. Intracervical photodynamic therapy resulted in tumor death, however mice died due to gastrointestinal toxicity. Photodynamic therapy resulted in subcutaneous tumor death and growth delay. CONCLUSIONS: Pc 4 photodynamic therapy caused death within cervical cancer cells and xenografts, supporting development of Pc 4 photodynamic therapy for treatment of cervical cancer. Support: P30-CA47904, CTSI BaCCoR Pilot Program.

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.