Early detection of tumor cells by innate immune cells leads to T(reg) recruitment through CCL22 production by tumor cells

In breast carcinomas, patient survival seems to be negatively affected by the recruitment of regulatory T cells (T(reg)) within lymphoid aggregates by CCL22. However, the mechanisms underpinning this process, which may be of broader significance in solid tumors, have yet to be described. In this study, we determined how CCL22 production is controlled in tumor cells. In human breast carcinoma cell lines, CCL22 was secreted at low basal levels that were strongly increased in response to inflammatory signals [TNF-α, IFN-γ, and interleukin (IL)-1β], contrasting with CCL17. Primary breast tumors and CD45(+) infiltrating immune cells appeared to cooperate in driving CCL22 secretion, as shown clearly in cocultures of breast tumor cell lines and peripheral blood mononuclear cells (PBMC) or their supernatants. We determined that monocyte-derived IL-1β and TNF-α are key players as monocyte depletion or neutralization of these cytokines attenuated secretion of CCL22. However, when purified monocytes were used, exogenous human IFN-γ was also required to generate this response suggesting a role for IFN-γ-producing cells within PBMCs. In this setting, we found that human IFN-γ could be replaced by the addition of (i) IL-2 or K562-activated natural killer (NK) cells or (ii) resting NK cells in the presence of anti-MHC class I antibody. Taken together, our results show a dialogue between NK and tumor cells leading to IFN-γ secretion, which in turn associates with monocyte-derived IL-1β and TNF-α to drive production of CCL22 by tumor cells and subsequent recruitment of T(reg). As one validation of this conclusion in primary breast tumors, we showed that NK cells and macrophages tend to colocalize within tumors. In summary, our findings suggest that at early times during tumorigenesis, the detection of tumor cells by innate effectors (monocytes and NK cells) imposes a selection for CCL22 secretion that recruits T(reg) to evade this early antitumor immune response.     

趋化因子CXCL9和CXCL10介导的胃癌免疫细胞浸润的探讨

目的:探讨不同刺激因子对胃癌中CXCL9和CXCL10表达的影响,及不同血样中免疫细胞经不同方式活化后,其膜表面CXCR3的表达情况,建立并评价CXCR3和CXCL9、CXCL10介导的靶向抗胃癌免疫治疗新技术。方法:采用IFN-γ、TNF-α、Poly（I∶C）、LPS和PHA刺激胃癌细胞（BGC-823、SGC-7901及MKN-28）,ELISA法测定肿瘤细胞分泌CXCL9和CXCL10的情况。将免疫细胞分别活化为CIK和CAPRI,流式细胞术检测活化前后免疫细胞表面CXCR3的表达情况。四甲基偶氮唑蓝（MTT）法评价刺激因子［IFN-γ、TNF-α、Poly（I∶C）、LPS和PHA］本身对肿瘤细胞增殖的影响。Transwell趋化实验比较趋化因子CXCL9和CXCL10及其受体CXCR3的表达差异对免疫细胞靶向归巢至肿瘤的数量的影响。结果:胃癌细胞株基础状态无明显CXCL9和CXCL10的表达,IFN-γ可以显著升高CXCL9和CXCL10的表达水平,TNF-α、Poly（I∶C）、LPS和PHA均可升高CXCL9和/或CXCL10的表达水平,与IFN-γ有一定的协同作用,其中Poly（I∶C）效果最强。IFN-γ、TNF-α、Poly（I∶C）、LPS和PHA等刺激因子本身在单独或联合应用时对3种肿瘤细胞均无明显增殖抑制作用。相同刺激条件下,胃癌高中分化细胞株的CXCL9和CXCL10表达显著高于低分化细胞株。CIK、CAPRI两种方式活化后细胞膜表面CXCR3显著升高。CIK、CAPRI两种活化方式刺激CXCR3的升高效能相当,无统计学差异。3种血样［健康足月新生儿脐带血、健康成人外周血单个核细胞（PBMC）、肿瘤患者PBMC］在相同活化方式刺激条件下CXCR3的表达无统计学差异。免疫活性细胞经由CXCL9、CXCL10与CXCR3介导,可以更多数量归巢至肿瘤。结论:趋化因子CXCL9和CXCL10及其受体CXCR3的高表达可以有效促进免疫细胞靶向归巢至肿瘤。     

Modulation of CXCR3 ligand secretion by prostaglandin E2 and cyclooxygenase inhibitors in human breast cancer

Introduction

In murine breast cancer models, the two interferon-gamma (IFN-γ) inducible chemokines and CXC-chemokine receptor 3 (CXCR3) receptor ligands, monokine induced by γ-interferon (CXCL9) and interferon-γ-inducible protein-10 (CXCL10) impair tumor growth and metastasis formation through recruitment of natural killer (NK) cells and tumor-suppressive T lymphocytes. In human breast cancer, CXCL9 mRNA overexpression correlates with the number of tumor infiltrating lymphocytes and predicts response to different chemotherapeutic regimens. Raising the intratumoral CXCR3 ligand concentration is therefore a possible way to enhance immune intervention in breast cancer. Little is known, however, about expression levels and regulation of these chemokines in human breast cancer. Since the inhibition of cyclooxygenases (COX) has been shown to reduce tumor growth and incidence of metastases in a lymphocytic and IFN-γ dependent manner, we argued that COX isoenzymes are a pharmacologic target to increase intratumoral CXCR3 ligand concentration in human breast cancer.

Methods

CXCL9 was visualized in breast cancer specimens by immunohistochemistry, expression levels of CXCL9 and cyclooxygenases were determined by ELISA and western blotting, respectively. For regulation studies, Michigan Cancer Foundation-7 (MCF-7) and M.D. Anderson - Metastatic Breast 231 (MDA-MB 231) breast cancer cells were stimulated with IFN-γ with or without prostaglandin E₂ (PGE₂) or COX inhibitors (indomethacin, acetylsalicylic acid (ASA), celecoxib). CXCR3 ligand release from cells was measured by ELISA.

Results

Within the tumor microenvironment, cancer cells are the major source of CXCL9. PGE₂ impairs IFN-γ mediated CXCL9 and CXCL10 release from MCF-7 and MDA-MB 231 cells, and inhibition of endogenous cyclooxygenases by indomethacin or ASA correspondingly increases this secretion. Otherwise, high concentrations of the Cyclooxygenase-2 (COX-2) specific antagonist celecoxib have opposite effects and impair CXCL9 and CXCL10 release. In human breast cancer tissue specimens there is an inverse correlation between COX-2 overexpression and CXCL9 concentration, suggesting that the observed in vitro effects are of importance in vivo as well.

Conclusions

Suppressing endogenous PGE₂ synthesis by cyclooxygenase inhibition increases CXCL9 and CXCL10 release from breast cancer cells and is therefore a pharmacologic candidate to enhance intratumoral immune infiltration. Yet, to this end the unselective COX inhibitors ASA and indomethacin seem preferable to celecoxib that at higher concentrations reduces CXCR3 ligand release most probably due to COX independent mechanisms.     

Interleukin-7 Enhances the in Vivo Anti-tumor Activity of Tumor-reactive CD8+ T cells with Induction of IFN-gamma in a Murine Breast Cancer Model

Interleukin-7 (IL-7) is a potent anti-apoptotic cytokine that enhances immune effector cell functionsand is essential for lymphocyte survival. While it known to induce differentiation and proliferation in somehaematological malignancies, including certain types of leukaemias and lymphomas, little is known about itsrole in solid tumours, including breast cancer. In the current study, we investigated whether IL-7 could enhancethe in vivo antitumor activity of tumor-reactive CD8+ T cells with induction of IFN-γ in a murine breast cancermodel. Human IL-7 cDNA was constructed into the eukaryotic expression plasmid pcDNA3.1, and then therecombinational pcDNA3.1-IL-7 was intratumorally injected in the TM40D BALB/C mouse graft model. Serumand intracellular IFN-γ levels were measured by ELISA and flow cytometry, respectively. CD8+ T cell-mediatedcytotoxicity was analyzed using the MTT method. Our results showed that IL-7 administration significantlyinhibited tumor growth from day 15 after direct intratumoral injection of pcDNA3.1-IL-7. The anti-tumoreffect correlated with a marked increase in the level of IFN-γ and breast cancer cells-specific CTL cytotoxicity.In vitro cytotoxicity assays showed that IL-7-treatment could augment cytolytic activity of CD8+ T cells fromtumor bearing mice, while anti-IFN-γ blocked the function of CD8+ T cells, suggesting that IFN-γ mediated thecytolytic activity of CD8+ T cells. Furthermore, in vivo neutralization of CD8+ T lymphocytes by CD8 antibodiesreversed the antitumor benefit of IL-7. Thus, we demonstrated that IL-7 exerts anti-tumor activity mainly throughactivating CD8+ T cells and stimulating them to secrete IFN-γ in a murine breast tumor model. Based on theseresults, our study points to a potential novel way to treat breast cancer and may have important implicationsfor clinical immunotherapy.     

DcR2 (TRAIL-R4) siRNA and adenovirus delivery of TRAIL (Ad5hTRAIL) break down in vitro tumorigenic potential of prostate carcinoma cells

High levels of decoy receptor 2 (DcR2; TRAIL-R4) expression are correlated with TRAIL resistance in prostate cancer cells. In addition, upregulation of TRAIL death receptor (DR4 and DR5) expression, either by ionizing radiation or chemotherapy, can sensitize cancer cells to TRAIL. Considering more than half of human cancers are TRAIL resistant, modulation of surface TRAIL receptor expression appears to be an attractive treatment modality to counteract TRAIL resistance. In this study, three siRNA duplexes targeting DcR2 receptor were tested. Ad5hTRAIL infections were performed to overexpress human full-length TRAIL to induce cell death, and the in vitro tumorigenic potential of prostate cancer cells was assessed using colony-forming assays on soft agar. The DU145 and LNCaP prostate cancer cell lines, which express high levels of DcR2, were resistant to Ad5hTRAIL-induced death. Downregulation of surface DcR2 expression by siRNA sensitized these prostate cancer cell lines to Ad5hTRAIL. In addition, DcR2 siRNA-mediated knockdown of DcR2, followed by Ad5hTRAIL infection, dramatically reduced the in vitro tumorigenic potential of prostate cancer cells. Collectively, our results suggest the potential for combining receptor-specific siRNA with TRAIL in the treatment of certain cancers.     

IL-2 costimulation enables statin-mediated activation of human NK cells, preferentially through a mechanism involving CD56+ dendritic cells

Statins are inhibitors of cholesterol biosynthesis and protein prenylation that also have been studied in cancer therapy and chemoprevention. With regard to natural killer (NK) cells, only inhibitory effects of statins such as suppression of granule exocytosis have been reported so far. In this study, we show that statins can cooperate with IL-2 to potently induce the activation of CD56(dim) NK cells in a synergistic, time- and dose-dependent fashion. Supplementation experiments revealed that the statin effect was specific to inhibition of their target hydroxymethylglutaryl coenzyme A reductase and that downstream depletion of geranylgeranyl pyrophosphate was responsible for cooperating with IL-2 in NK cell activation. Mechanistic studies revealed that CD56(+)HLA-DR(+)CD14(+) dendritic cell (DC)-like accessory cells mediated the ability of statin to activate NK cells. In contrast, BDCA-1(+) (CD1c(+)) myeloid DCs, which partially expressed CD56, were somewhat less potent. Conventional blood monocytes, which lack CD56, exhibited the lowest accessory cell capacity. NK cell IFN-γ production was IL-12 independent but required endogenous IL-18, IL-1β, and caspase-1 activity. Statins directly induced apoptosis in human cancer cell lines and cooperated with NK cell-derived IFN-γ to generate potent cytotoxic antitumor effects in vitro even in the presence of statin-mediated inhibitory effects on granule exocytosis. Our work reveals novel and unexpected immunomodulatory properties of statins, which might be harnessed for the treatment of cancer.     

Aberrant methylation of trail decoy receptor genes is frequent in multiple tumor types

TNF-related apoptosis-inducing ligand (TRAIL) selectively induces programmed cell death (apoptosis) in various cancer cells but not in normal cells. TRAIL is known to bind to 4 different receptors, 2 proapoptotic (DR4 and DR5), and 2 potentially antiapoptotic receptors lacking death domains (DcR1 and DcR2). Aberrant promoter methylation and resultant silencing of tumor suppressor genes play an important role in the pathogenesis of many tumor types. Recently aberrant methylation of TRAIL decoy receptors was reported in pediatric tumor cell lines and neuroblastomas. We examined the methylation and expression status of TRAIL receptor genes in cancers of breast, lung, mesothelioma, prostate, bladder, cervix, ovary, brain and in hematopoietic malignancies. Aberrant methylation of DcR1 or DcR2 was present in 70% of primary breast cancers, 31% of primary lung cancers, in 63% of primary malignant mesothelioma (MM), in 60% of prostate cancer, in 42% of bladder cancer, in 100% of cervical cancer, in 43% of ovarian cancer, in 41% of lymphoma, in 26% of leukemia and in 56% of multiple myeloma. Methylation of DR4 and DR5 was rare in all the tumor types examined. Methylation of all the 4 receptors was rare in non malignant tissues. In cell lines, aberrant methylation of DcR1 was present in 11 of 23 (48%) breast, 10 of 27 (37%) lung and 3 of 7 (43%) MM, whereas aberrant methylation of DcR2 was present in 17 of 23 (74%) breast, 13 of 27 (48%) lung and 5 of 7 (71%) MM. The concordance between loss of gene expression and aberrant methylation ranged from 70-100%. Treatment with 5-aza-2'-deoxycytidine restored DcR1 and DcR2 expression in 9 methylated cell lines confirming that aberrant methylation was the cause for silencing of DcR1 and DcR2 expression. Our results demonstrate that DcR1 and DcR2 genes are frequently methylated in various tumor types, and that the role of decoy receptors in tumor pathogenesis needs to be re-evaluated.     

IFN-γ–STAT1-mediated NK2R expression is involved in the induction of antitumor effector CD8+ T cells in vivo

The induction of antitumor effector T cells in the tumor microenvironment is a crucial event for cancer immunotherapy. Neurokinin receptor 2 (NK2R), a G protein-coupled receptor for neurokinin A (NKA), regulates diverse physiological functions. However, the precise role of NKA–NK2R signaling in antitumor immunity is unclear. Here, we found that an IFN-γ–STAT1 cascade augmented NK2R expression in CD8⁺ T cells, and NK2R-mediated NKA signaling was involved in inducing antitumor effector T cells in vivo. The administration of a synthetic analog of double-stranded RNA, polyinosinic–polycytidylic acid (poly I:C), into a liver cancer mouse model induced type I and type II IFNs and significantly suppressed the tumorigenesis of Hepa1-6 liver cancer cells in a STAT1-dependent manner. The reduction in tumor growth was diminished by the depletion of CD8⁺ T cells. IFN-γ stimulation significantly induced NK2R and tachykinin precursor 1 (encodes NKA) gene expression in CD8⁺ T cells. NKA stimulation combined with anti-CD3 monoclonal antibody (mAb) treatment significantly augmented IFN-γ and granzyme B production by CD8⁺ T cells compared with the anti-CD3 mAb alone in vitro. ERK1/2 phosphorylation and IκBα degradation in activated CD8⁺ T cells were suppressed under NK2R deficiency. Finally, we confirmed that tumor growth was significantly increased in NK2R-deficient mice compared with that in wild-type mice, and the antitumor effects of poly I:C were abolished by NK2R absence. These findings suggest that IFN-γ–STAT1-mediated NK2R expression is involved in the induction of antitumor effector T cells in the tumor microenvironment, which contributes to the suppression of cancer cell tumorigenesis in vivo. In this study, we revealed that IFN-γ–STAT1-mediated NK2R expression is involved in the induction of antitumor effector CD8⁺ T cells in the tumor microenvironment, which contributes to suppressing the tumorigenesis of liver cancer cells in vivo.     

Interleukin-21 and rituximab enhance NK cell functionality in patients with B-cell chronic lymphocytic leukaemia

We have examined natural killer (NK) cell functionality of 54 B-CLL patients upon in vitro stimulation with interleukin-21 (IL-21), together with the anti-CD20 antibody, rituximab. Upon stimulation with rituximab-coated target cells IFN-γ production was reduced in patients’ NK cells compared to healthy donors’, while both natural- and antibody-dependent cytotoxicity (ADCC) was normal. Following additional stimulation with IL-21, IFN-γ production, natural cytotoxicity and ADCC were significantly augmented in patients. A complete restoration of IFN-γ production, however, required the depletion of malignant cells prior to stimulation. Collectively, our data show that NK cells of B-CLL patients are reversibly inhibited, but that their functionality can be normalized by stimulation with IL-21 and when inhibitory effects of the malignant B-CLL cells are eliminated by depletion.     

乳腺癌患者可溶性MICA对自然杀伤细胞受体的影响

目的观察乳腺癌患者外周血自然杀伤（NK）细胞杀伤活性及受体的变化,探讨可溶性MICA（sMICA）对NK细胞受体及杀伤活性的影响。方法ELISA法检测外周血血清sMICA的含量。流式细胞术（FCM）检测NK细胞百分比、NK细胞活化性受体NKG2D、抑制性受体KIR（CD158b）表达。MTT法检测NK细胞对乳腺癌细胞株MCF-7的杀伤活性。结果与健康人比较,乳腺癌患者中81．6％表达sMICA,含量为（205．36±71．27）ng／L,且sMICA含量与TNM分期呈正相关。乳腺癌患者外周血NK细胞所占百分比无明显差异,但血清sMICA阳性的乳腺癌患者中NK细胞杀伤活性明显降低,NKG2D表达下降,CD158b表达增高。当NK细胞培养体系中加入sMICA阳性的乳腺癌血清时,其杀瘤活性明显降低【（76．2±6．7）％与（48．4±4．1）％】,NKG2D的表达明显下调[（92．5±7．1）％与（62．5±6．4）％],而CD158b的表达明显上升【（10．6±3．2）％与（43．6±3．4）％】。sMICA阳性的乳腺癌患者NK细胞与细胞因子IL-15共培养,NK细胞的杀瘤活性、NKG2D的表达明显升高,KIR（CD158b）的表达明显下降。结论乳腺癌外周血血清中sMICA可通过下调NK细胞NKG2D表达以及上调KIR表达,降低NK细胞杀瘤活性。IL-15可逆转sMICA对NK细胞的免疫下调作用。     

Predominant Bcl-XL knockdown disables antiapoptotic mechanisms: tumor necrosis factor-related apoptosis-inducing ligand-based triple chemotherapy overcomes chemoresistance in pancreatic cancer cells in vitro

Pancreatic cancer is lethal because of its invasiveness, rapid progression, and profound resistance to chemotherapy and radiation therapy. To identify the molecular mechanisms underlying this, we have examined the expression and potency of three major death receptors: tumor necrosis factor receptor (TNF-R), TNF-related apoptosis-inducing ligand receptor (TRAIL-R), and Fas in mediating cytotoxicity in four invasive pancreatic cancer cell lines. We have analyzed the expression of major antiapoptotic factors, cell cycle regulators and death receptor decoys (DcR) in comparison with normal pancreas tissues and five other human malignant tumor cell lines. We have found that different pancreatic cancer cell lines coexpress high-level TRAIL-R, Fas, and TNF-R1 but are strongly resistant to apoptosis triggered by the death receptors. DcR2 and DcR3 overexpression may partly contribute to the resistance of pancreatic cancer cells to TRAIL-R- and Fas-mediated cytotoxicity. Bcl-XL and Bcl-2 are predominantly overexpressed in pancreatic cancer cell lines, respectively. Bcl-XL is also predominantly overexpressed in prostate, colorectal, and intestinal cancer cells. The knockdown of the predominant Bcl-XL overexpression significantly reduces the viability of pancreatic cancer cells to TNFalpha- and TRAIL-mediated apoptosis by sublethal-dose single and combined antitumor drugs, including geldanamycin, PS-341, Trichostatin A, and doxorubicine. Geldanamyin and PS-341 synergistically block NFkappaB activation, suppress Akt/PKB pathway, and down-regulate Bcl-XL, Bcl-2, cIAP-1, and cyclin D1 expression. This combined regimen dramatically enhances TRAIL cytotoxic effects and breaks through chemoresistance. Bcl-XL plays a vital role in pancreatic cancer chemoresistance. Geldanamycin, PS-341, and TRAIL triple combination may be a novel therapeutic strategy for pancreatic cancer.     

TRAIL及其受体在乳腺癌组织中的表达及意义

目的探讨肿瘤坏死因子相关凋亡诱导配体（TRAIL）及其受体（DR4、DR5、DcR1、DcR2）在乳腺癌组织的表达及意义。方法用反转录聚合酶链反应（RT-PCR）法检测60例乳腺癌及对应的正常乳腺组织中TRAIL及其受体的表达。结果乳腺癌组织中TRAIL、DcR1、DcR2表达均低于正常乳腺组织(P＜0.05),且TRAIL及其受体的表达与乳腺癌的分期、分级等无关(P＞0.05)。结论TRAIL及其受体在乳腺癌凋亡过程中起着重要的作用,TRAIL及其诱骗受体低表达同乳腺癌的发生关系密切。     

Mesenchymal stem cells promote mammary cancer cell migration in vitro via the CXCR2 receptor

Bone metastasis is a common event during breast cancer progression. Recently, mesenchymal stem cells (MSCs) have been implicated in the metastasis of primary mammary cancer. Given that bone is the native environment for MSCs, we hypothesized MSCs facilitate the homing of circulating mammary cancer cells to the bone. To test this hypothesis, we examined in vitro whether bone derived MSCs from FVB mice could influence the migration of syngeneic murine mammary cancer cell lines derived from the polyoma virus middle-T (PyMT) model of mammary gland tumorigenesis. Our data show that conditioned media derived from MSCs significantly enhanced the migration of PyMT mammary cancer cell lines. Analysis of conditioned media using a cytokine array revealed the presence of numerous cytokines in the MSC conditioned media, most notably, the murine orthologs of CXCL1 and CXCL5 that are cognate ligands of the CXCR2 receptor. Further investigation identified that: (1) CXCL1, CXCL5 and CXCR2 mRNA and protein were expressed by the MSCs and PyMT cell lines and; (2) neutralizing antibodies to CXCL1, CXCL5 and CXCR2 or a CXCR2 small molecule inhibitor (SB265610) significantly abrogated the migratory effect of the MSC conditioned media on the PyMT cells. Therefore, in vitro evidence demonstrates that bone derived MSCs play a role in the migration of mammary cancer cells, a conclusion that has potential implications for breast to bone metastasis in vivo.     

重组可溶性MICA蛋白负调控NK细胞对白血病细胞的杀伤活性

目的:体外研究重组可溶性MHCⅠ类分子相关A(soluble MHC classⅠchain-related gene A,sMICA)蛋白对自然杀伤细胞(natural killer cell,NK cell)表面活化性受体NKG2D(natural killer group 2 member D)的表达、杀伤白血病细胞的活性和分泌IFN-γ的影响。方法:免疫磁珠分选健康人外周血NK细胞,分为空白对照组、重组sMICA组(200、500、800μg/L三亚组),相互作用24 h后,流式细胞术检测NK细胞NKG2D表达水平,LDH释放法检测NK细胞对白血病细胞K562的杀伤活性,ELISA法检测培养上清中IFN-γ的水平。结果:200、500、800μg/L sMICA作用组与对照组相比,NK细胞表面NKG2D的表达均下调[(68.79±6.87)%,(55.75±9.31)%、(45.14±5.70)%vs(92.75±6.91)%,P<0.05或P<0.01],均抑制NK细胞杀伤白血病细胞K562的活性[(18.67±2.35)%、(15.01±2.25)%、(7.33±2.52)%vs(36.33±2.51)%,P<0.05或P<0.01],均抑制IFN-γ的分泌[(164.48±22.48)、(112.71±10.89)、(70.23±9.64)vs(313.72±16.06)pg/ml,P<0.05,P<0.01]。结论:急性白血病细胞表面脱落的sMICA可下调NK细胞NKG2D的表达和IFN-γ的分泌,抑制了NK细胞对白血病细胞的杀伤活性。     

TRAIL death receptor-4 expression positively correlates with the tumor grade in breast cancer patients with invasive ductal carcinoma

PURPOSE: Tumor necrosis factor-related apoptosis inducing ligand (TRAIL) selectively induces apoptosis in cancer cells but not in normal cells, and a number of clinical trials have recently been initiated to test the safety and antitumoral potential of TRAIL in cancer patients. Four different receptors have been identified to interact with TRAIL: two are death-inducing receptors (TRAIL-R1 [DR4] and TRAIL-R2 [DR5]), whereas the other two (TRAIL-R3 [DcR1] and TRAIL-R4 [DcR2]) do not induce death upon ligation and are believed to counteract TRAIL-induced cytotoxicity. Because high levels of DcR2 expression have recently been correlated with carcinogenesis in the prostate and lung, this study investigated the importance of TRAIL and TRAIL receptor expression in breast cancer patients with invasive ductal carcinoma, taking various prognostic markers into consideration. METHODS AND MATERIALS: Immunohistochemical analyses were performed on 90 breast cancer patients with invasive ductal carcinoma using TRAIL and TRAIL receptor-specific antibodies. Age, menopausal status, tumor size, lymph node status, tumor grade, lymphovascular invasion, perineural invasion, extracapsular tumor extension, presence of an extensive intraductal component, multicentricity, estrogen and progesterone receptor status, and CerbB2 expression levels were analyzed with respect to TRAIL/TRAIL receptor expression patterns. RESULTS: The highest TRAIL receptor expressed in patients with invasive ductal carcinoma was DR4. Although progesterone receptor-positive patients exhibited lower DR5 expression, CerbB2-positive tissues displayed higher levels of both DR5 and TRAIL expressions. CONCLUSIONS: DR4 expression positively correlates with the tumor grade in breast cancer patients with invasive ductal carcinoma.     

Differential susceptibility of metastatic and primary oral cancer cells to TRAIL-induced apoptosis

TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) preferentially induces apoptosis of cancer cells without toxicity in normal cells. TRAIL plays an important role in host immune surveillance against tumor metastasis. Cathepsin B (CB) is a mediator of apoptosis whose activity is regulated by its inhibitors, known as cystatins. We examined the TRAIL-mediated cytotoxicity rates of clonally-related primary and metastatic oral cancer (OC) cells and correlated them with the expression levels of TRAIL receptors, cathepsin B and cystatins A, B, C and M. Two pairs of primary (686Tu and 101A) and metastatic (686Ln and 101B) OC cell lines were treated with various concentrations (5 to 1000 ng/ml) of recombinant human TRAIL protein for 14 h, and cell viability and apoptotic rate were determined. In both pairs of cell lines, primary OC cells revealed greater susceptibility to TRAIL than their metastatic counterparts. The protein synthesis inhibitor cycloheximide markedly increased the TRAIL sensitivity of these cell lines, whereas the CB-specific chemical inhibitor CA-074 markedly reduced the sensitivity of primary OC cells to TRAIL. DNA laddering and M30 CytoDEATH immunodetection assays confirmed that TRAIL-induced OC cell death is an apoptotic process. Expression levels of TRAIL death (DR4 and DR5) and decoy (DcR1 and DcR2) receptors were not different between primary and metastatic OC cells. However, expression levels of cystatins were higher in metastatic OC cells than in their respective primary cells, whereas CB levels remain unchanged. Cathepsin B is a mediator of TRAIL-induced apoptosis in OC cells. Elevated levels of cystatins in metastatic OC cells may cause their greater resistance to TRAIL-induced apoptosis. Our data suggest that high expression of cystatins in OC cells may confer a metastatic phenotype by enhancing their resistance to TRAIL.     

Doxorubicin potentiates TRAIL cytotoxicity and apoptosis and can overcome TRAIL-resistance in rhabdomyosarcoma cells

Doxorubicin (DOX) and ifosfamide (IFO) are the most active single agents in soft tissue sarcomas (STS). Tumour necrosis factor-alpha (TNF-alpha) is used for STS in the setting of isolated limb perfusions. Like TNF-alpha, TNF-related apoptosis-inducing ligand (TRAIL) induces apoptosis. In contrast to TNF-alpha preliminary studies suggest that TRAIL lacks systemic side effects. The effects of TRAIL alone and in combination with DOX or 4-hydroxy-IFO were evaluated in the TNF-alpha sensitive rhabdomyosarcoma cell line KYM-1, its 5-fold TNF-alpha sensitive subline KD4 and its >150-fold TNF-alpha resistant subline 37B8R. Membrane expression of TRAIL-receptors DR4 (death receptor 4), DR5 (pro-apoptotic), DcR1 (decoy receptor 1), DcR2 (anti-apoptotic) was assessed by flow cytometry. Cytotoxicity was determined by microculture tetrazolium assays. Apoptosis assays were performed with acridine orange. DOX (doxorubicin) and 4-OH-IFO decreased survival in all cell lines; a 2-fold resistance was observed for both drugs in 37B8R. All cell lines expressed DR4 and DR5, but hardly any DcR1 or DcR2. TRAIL was cytotoxic in KYM-1, even more in KD4 and induced massive apoptosis; 37B8R was >500-fold resistant to TRAIL and little apoptosis could be observed. TRAIL plus DOX showed synergistic cytotoxicity in KYM-1 and 37B8R. TRAIL plus 4-OH-IFO showed addition in all three cell lines. DOX plus TRAIL-induced more cytotoxicity and apoptosis in all cell lines compared to TRAIL alone. In 37B8R, DOX overcame resistance to TRAIL. In KYM-1, KD4 and 37B8R, sensitivity and resistance to TNF-alpha and TRAIL parallels. TRAIL-resistance was independent from expression of TRAIL-receptors. DOX with TRAIL could overcome TRAIL-resistance in 37B8R cells.     

T helper responses are maintained by basal-like breast cancer cells and confer to immune modulation via upregulation of PD-1 ligands

A conspicuous T cell infiltration is frequently observed in triple-negative and/or basal-like breast cancers. Since the immunological course of breast cancer is explicitly directed by helper T cells, this study aims to determine the influence of basal-like breast cancer (BLBC) cells on CD4⁺ T cell responses. Co-cultures were established with breast cancer cell lines and CD4⁺ T cells under stimulatory conditions. Helper T cell activation, proliferation, cytokine secretion, and differentiation were assessed. Protein and mRNA expression of PD-1 ligands were determined on breast cancer cell lines. Blockade assays were performed in order to determine the functional assets of PD-1 ligation. In contrast to luminal breast cancer cells, BLBC cells allowed CD4⁺ T cell activation, proliferation, and IFN-γ secretion, but only to a certain extent. A substantial population of CD25⁺CD127^low/? regulatory T (Treg) cells was also induced in BLBC co-cultures. In return, IFN-γ stimulated the upregulation of PD-L1 (B7-H1) and/or PD-L2 (B7-DC) inhibitory molecules on the basal-like cells. In prolonged periods of co-culturing, blockade of PD-1 ligands on BLBC cell lines impaired Treg differentiation, restored IL-2 secretion, and increased CD8⁺ T cell activation. In conclusion, T helper responses were maintained by BLBC cells. On the other hand, IFN-γ secreted from Th1 and other immune cells upregulated the expression of PD-1 ligands on BLBC cells and modulated the immune reactions. Our results indicate the capacity of BLBCs to adapt to IFN-γ-mediated anti-tumor immune responses and to evade immunity via upregulation of PD-1 ligands.     

Natural killer cells in children with acute leukemia. The effect of interleukin-2

The purpose of this study was to determine (1) the number and activity of natural killer (NK) cells in children with acute leukemia at different stages of their disease; and (2) the effect of interleukin-2 (IL-2) in enhancing NK activity of these patients' cells. The mean percentage of Leu 11+ NK cells in patients at diagnosis (5% of peripheral blood (PB) mononuclear cells) was significantly lower than for patients on maintenance (23%), post-treatment (21%) and for normal children (20%). The mean PB NK cell cytotoxicity for patients at diagnosis (16% lysis versus K562) and during maintenance (20%) was significantly lower than for post-treatment (41%) and normal controls (40%). After NK cells were incubated for 5 days with IL-2, NK cells from 82% (36/44) of patients showed enhanced cytotoxicity toward K562 and several acute leukemia cell lines as well as toward autologous leukemic cells. Cytotoxicity toward autologous cells was very low (0% to 5%, 16 hour assay) before IL-2 stimulation, and significantly increased (23% to 69%) after stimulation, suggesting that IL-2 may be a useful agent for enhancing the antileukemic immune response.