Changes in circulating exosome molecular profiles following surgery/(chemo)radiotherapy: early detection of response in head and neck cancer patients.

BACKGROUND: Head and neck cancers (HNSCC) are highly immunosuppressive. Plasma-derived exosomes of HNSCC patients carry immunomodulatory molecules, and their cargo correlates with clinical parameters. Here, we evaluated the exosomal molecular profile for early detection of treatment failure in locally advanced HNSCC patients treated with conventional therapy. METHODS: Plasma from 17 HNSCC patients was collected before, during, and after treatment by surgery with adjuvant (chemo)radiation and at recurrence. Exosomes were isolated by size-exclusion chromatography. Total exosomal protein (TEP) was used to estimate exosome load and on-bead flow cytometry to evaluate relative fluorescence intensity (RFI) of tumour-associated and immunoregulatory proteins on exosomes. Exosomal effects on the activity of and adenosine production by T cells was assessed by flow cytometry and mass spectrometry. RESULTS: TEP and the ratio of tumour-/immune-cell-derived exosomes varied during and after therapy with an overall decrease in the tumour-free follow-up but an increase at recurrence. RFI values of immunoregulatory proteins on exosomes, their ability for T cell inhibition and adenosine production changed during and after therapy. PD-L1 was the earliest discriminator for treatment failure and disease-free survival. CONCLUSIONS: Monitoring of plasma exosomes during therapy represents a promising opportunity for early detection of treatment failure and risk stratification to delay/avoid recurrence.

Targeting CSPG4 for isolation of melanoma cell-derived exosomes from body fluids.

This manuscript describes the functional properties of the exosomes released from melanoma cells. It details the characteristics of the tumor antigen chondroitin sulfate proteoglycan 4 (CSPG4), which is used as a marker to separate exosomes released by melanoma cells from exosomes released by nonmalignant cells. The results are discussed in view of the potential role of melanoma cell-derived exosomes in the escape of malignant cells from the host's immune system.

Exosomes in HNSCC plasma as surrogate markers of tumour progression and immune competence.

Exosomes in plasma of head and neck squamous cell carcinoma (HNSCC) patients comprise subsets of vesicles derived from various cells. Recently, we separated CD3(+) from CD3(-) exosomes by immune capture. CD3(-) exosomes were largely tumour-derived (CD44v3+ ). Both subsets carried immunosuppressive proteins and inhibited functions of human immune cells. The role of these subsets in immune cell reprogramming by the tumour was investigated by focusing on the adenosine pathway components. Spontaneous adenosine production by CD3(+) or CD3(-) exosomes was measured by mass spectrometry, as was the production of adenosine by CD4+ CD39+ regulatory T cells (Treg ) co-incubated with these exosomes. The highest level of CD39/CD73 ectoenzymes and of adenosine production was found in CD3(-) exosomes in patients with the stages III/IV HNSCCs). Also, the production of 5'-AMP and purines was significantly higher in Treg co-incubated with CD3(-) than CD3(+) exosomes. Consistently, CD26 and adenosine deaminase (ADA) levels were higher in CD3(+) than CD3(-) exosomes. ADA and CD26 levels in CD3(+) exosomes were significantly higher in patients with early (stages I/II) than advanced (stages III/IV) disease. HNSCC patients receiving and responding to photodynamic therapy had increased ADA levels in CD3(+) exosomes with no increase in CD3(-) exosomes. The opposite roles of CD3(+) ADA+ CD26+ and CD3(-) CD44v3+ adenosine-producing exosomes in early versus advanced HNSCC suggest that, like their parent cells, these exosomes serve as surrogates of immune suppression in cancer.

Polyfunctionality of CD4+ T lymphocytes is increased after chemoradiotherapy of head and neck squamous cell carcinoma.

BACKGROUND: For head and neck squamous cell cancer (HNSCC), standard therapy consists of surgery, radiation, and/or chemotherapy. Antineoplastic immunotherapy could be an option in an adjuvant setting and is already in palliation. A functional immune system is a prerequisite for successful immunotherapy. However, effects of the standard-of-care therapy on the patients' immune system are not fully understood. METHODS: Peripheral blood mononuclear cells (PBMC) were collected from patients with HNSCC (n = 37) and healthy controls (n = 10). PBMC were stimulated with staphylococcal enterotoxin B (SEB). Simultaneous expression of various cytokines was measured in CD4+ and CD8+ T cells by multicolor flow cytometry, and polyfunctional cytokine expression profiles were determined on a single-cell basis. RESULTS: Expression levels of all measured cytokines in CD4+ T cells were higher in patients after chemoradiotherapy (CRT) as compared to untreated HNSCC patients or normal controls. After CRT, the frequency of polyfunctional CD4+ T cells, which simultaneously expressed multiple cytokines, was significantly increased as compared to untreated patients (p < 0.01). CONCLUSION: CRT increases polyfunctionality of CD4+ T cells in HNSCC patients, suggesting that standard-of-care therapy can promote immune activity in immune cells. These polyfunctional CD4+ T cells in the blood of treated HNSCC patients are expected to be responsive to subsequent immunotherapeutic approaches.

Separation of plasma-derived exosomes into CD3(+) and CD3(-) fractions allows for association of immune cell and tumour cell markers with disease activity in HNSCC patients.

Head and neck squamous cell carcinoma (HNSCC) is a highly immunosuppressive malignancy. Exosomes in HNSCC patients' plasma are enriched in inhibitory cargo and mediate immunosuppression. As these exosomes are products of various cells, the cellular origin of immunoregulatory proteins they carry is unknown. To test whether tumour- or T cell-derived exosomes in patients' plasma are immunosuppressive and impact upon disease activity, we separated CD3(-) from CD3(+) exosomes by immunocapture using anti-CD3 antibodies. The exosome protein cargo was evaluated for immunoregulatory proteins using on-bead flow cytometry. Tumour protein-enriched CD3(-) exosomes were CD44v3(+) . Surprisingly, mean levels of programmed death ligand 1 (PD-L1), cytotoxic T lymphocyte antigen 4 (CTLA-4) and cyclooxygenase-2 (COX-2) were similar in CD3(+) and CD3(-) exosomes, although the latter induced higher (P < 0·0025) ex-vivo apoptosis of CD8(+) T cells and greater (P < 0·005) conversion of CD4+ T cells to CD4(+) CD39(+) regulatory T cells (Treg ). CD3(+) and CD3(-) exosomes carrying high levels of immunosuppressive proteins were highly effective in mediating these functions. Exosomes of patients with Union for International Cancer Control (UICC) stages III/IV disease had higher levels of PD-L1 and COX-2 than stages I/II patients (P < 0·005). Patients with nodal involvement had exosomes with the higher inhibitory protein content than N0 patients (P < 0·03). CD3(+) and CD3(-) exosomes of HNSCC patients had higher PD-L1, COX-2 and CD15s levels than healthy donors' exosomes (P < 0·009), although levels of immunostimulatory OX40 or OX40L were not different. By isolating CD3(-) /CD44v3-enriched and CD3(+) exosomes from plasma, the cellular origins of immunoregulatory proteins they carry were identified. Association of exosome molecular profiles with disease progression supports the exosome potential as future cancer biomarkers.

Exosomes carrying immunoinhibitory proteins and their role in cancer.

Recent emergence of exosomes as information carriers between cells has introduced us to a new previously unknown biological communication system. Multi-directional cross-talk mediated by exosomes carrying proteins, lipids and nucleic acids between normal cells, cells harbouring a pathogen or cancer and immune cells has been instrumental in determining outcomes of physiological as well as pathological conditions. Exosomes play a key role in the broad spectrum of human diseases. In cancer, tumour-derived exosomes carry multiple immunoinhibitory signals, disable anti-tumour immune effector cells and promote tumour escape from immune control. Exosomes delivering negative signals to immune cells in cancer, viral infections, autoimmune or other diseases may interfere with therapy and influence outcome. Exosomes can activate tissue cells to produce inhibitory factors and thus can suppress the host immune responses indirectly. Exosomes also promise to be non-invasive disease biomarkers with a dual capability to provide insights into immune dysfunction as well as disease progression and outcome.

The emerging roles of tumor-derived exosomes in hematological malignancies.

Exosomes are small (30-150 nm) membranous vesicles of endocytic origin produced by all cells under physiological and pathological conditions. They have recently emerged as vehicles for intercellular transfer of molecular and genetic contents from parent to recipient cells. Exosome-mediated transfer of proteins or genes (RNA, miRNA, DNA) results in reprogramming of recipient cell functions. Exosomes carry and deliver information that is essential for health, and they participate in pathological events, including malignant transformation. Within the hematopoietic system, exosomes maintain crosstalk between cells located in the bone marrow compartment and at distant tissue sites. In hematological malignancies, tumor-derived exosomes (TEX) reprogram the bone marrow environment, suppress anti-leukemia immunity, mediate drug resistance and interfere with immunotherapies. TEX are also viewed as promising biomarkers of malignant progression and as potential therapeutic targets. The involvement of TEX in nearly all aspects of malignant transformation has generated much interest in their biology, mechanisms responsible for information transfer and the role they play in cancer escape from the host immune system.

Phenotypic and functional characteristics of CD39high human regulatory B cells (Breg).

CD39 and CD73 are key enzymes in the adenosine (ADO) pathway. ADO modulates pathophysiological responses of immune cells, including B cells. It has recently emerged that a subpopulation of ADO-producing CD39+CD73+ B cells has regulatory properties. Here, we define the CD39high subset of these cells as the major contributor to the regulatory network operated by human B lymphocytes. Peripheral blood B cells were sorted into CD39neg, CD39inter and CD39high subsets. The phenotype, proliferation and IL-10 secretion by these B cells were studied by flow cytometry. 5'-AMP and ADO levels were measured by mass spectrometry. Agonists or antagonists of A1R, A2AR and A3R were used to study ADO-receptor signaling in B cells. Inhibition of effector T-cell (Teff) activation/proliferation by B cells was assessed in co-cultures. Cytokine production was measured by Luminex. Upon in vitro activation and culture of B cells, the subset of CD39high B cells increased in frequency (p < 0.001). CD39high B cells upregulated CD73 expression, proliferated (approximately 40% of CD39high B cells were Ki-67+ and secreted fold-2 higher IL-10 and ADO levels than CD39neg or CD39inter B cells. CD39high B cells co-cultured with autologous Teff suppressed T-cell activation/proliferation and secreted elevated levels of IL-6 and IL-10. The A1R and A2AR agonists promoted expansion and functions of CD39high B cells. CD39 ectonucleotidase is upregulated in a subset of in vitro-activated B cells which utilize ADO and IL-10 to suppress Teff functions. Proliferation and functions of these CD39high B cells are regulated by A1R- and A2AR-mediated autocrine signaling.

Human CD4+ CD39+ regulatory T cells produce adenosine upon co-expression of surface CD73 or contact with CD73+ exosomes or CD73+ cells.

While murine CD4(+) CD39(+) regulatory T cells (T(reg)) co-express CD73 and hydrolyze exogenous (e) adenosine triphosphate (ATP) to immunosuppressive adenosine (ADO), surface co-expression of CD73 on human circulating CD4(+) CD39(+) T(reg) is rare. Therefore, the ability of human T(reg) to produce and utilize ADO for suppression remains unclear. Using mass spectrometry, we measured nucleoside production by subsets of human CD4(+) CD39(+) and CD4(+) CD39(-)CD73(+) T cells or CD19(+) B cells isolated from blood of 30 volunteers and 14 cancer patients. CD39 and CD73 expression was evaluated by flow cytometry, Western blots, confocal microscopy or reverse transcription-polymerase chain reaction (RT-PCR). Circulating CD4(+) CD39(+) T(reg) which hydrolyzed eATP to 5'-AMP contained few intracytoplasmic granules and had low CD73 mRNA levels. Only ∼1% of these T(reg) were CD39(+) CD73(+) . In contrast, CD4(+) CD39(neg) CD73(+) T cells contained numerous CD73(+) granules in the cytoplasm and strongly expressed surface CD73. In vitro-generated T(reg) (Tr1) and most B cells were CD39(+) CD73(+) . All these CD73(+) T cell subsets and B cells hydrolyzed 5'-AMP to ADO. Exosomes isolated from plasma of normal control (NC) or cancer patients carried enzymatically active CD39 and CD73(+) and, when supplied with eATP, hydrolyzed it to ADO. Only CD4(+) CD39(+) T(reg) co-incubated with CD4(+) CD73(+) T cells, B cells or CD39(+) CD73(+) exosomes produced ADO. Thus, contact with membrane-tethered CD73 was sufficient for ADO production by CD4(+) CD39(+) T(reg). In microenvironments containing CD4(+) CD73(+) T cells, B cells or CD39(+) CD73(+) exosomes, CD73 is readily available to CD4(+) CD39(+) CD73(neg) T(reg) for the production of immunosuppressive ADO.

Intratumoral regulatory T cells upregulate immunosuppressive molecules in head and neck cancer patients.

BACKGROUND: Although regulatory T cells (Treg) are highly enriched in human tumours compared with peripheral blood, expression of the immune-checkpoint receptors, immunosuppressive molecules and function of Treg in these two sites remains undefined. METHODS: Tumour-infiltrating lymphocytes and peripheral blood lymphocytes were isolated from a cohort of head and neck squamous cell carcinoma (HNSCC) patients. The immunosuppressive phenotypes and function of intratumoral Treg were compared with those of peripheral blood Treg. RESULTS: The frequency of immune-checkpoint receptor-positive cells was higher on intratumoral FOXP3(+)CD25(hi) Treg compared with circulating Treg (CTLA-4, P=0.002; TIM-3, P=0.002 and PD-1, P=0.002). Immunosuppressive effector molecules, LAP and ectonucleotidase CD39 were also upregulated on intratumoral FOXP3(+) Treg (P=0.002 and P=0.004, respectively). CTLA-4 and CD39 were co-expressed on the majority of intratumoral FOXP3(+)CD4(+) Treg, suggesting that these molecules have a key role in regulatory functions of these cells in situ. Notably, intratumoral Treg exhibited more potently immunosuppressive activity than circulating Treg. CONCLUSION: These results indicate that intratumoral Treg are more immunosuppressive than circulating Treg and CTLA-4 and CD39 expressed can be potential target molecules to inhibit suppressive activities of intratumoral Treg in situ.

IRX-2, a novel immunotherapeutic, enhances and protects NK-cell functions in cancer patients.

BACKGROUND: IRX-2 is a primary biologic which has been used for the therapy of head and neck squamous cell cancer (HNSCC) with promising clinical results. Since NK-cell function is compromised in HNSCC patients, we tested the effects of IRX-2 on the restoration of human NK-cell functions in vitro. METHODS: Peripheral blood mononuclear cells (PBMC) were isolated from 23 HNSCC patients and 10 normal controls (NC). The NK-cell phenotype and functions were compared before and after culture ± IRX-2 or ± 50 IU/ml rhIL-2. Flow cytometry was used to study the NK-cell phenotype, cytotoxic activity and cytokine expression. RESULTS: Impaired NK-cell cytotoxicity in HNSCC patients was related to lower expression of NKG2D, NKp30 and NKp46 receptors (P < 0.05) and not to a decreased frequency of NK cells. Incubation of patients' NK cells with IRX-2 up-regulated the percentage of receptor-positive NK cells (P < 0.05). It also up-regulated cytotoxicity of patients' NK cells (P < 0.01) more effectively than rhIL-2 (P < 0.01). IRX-2, but not rhIL-2, protected NK cells from suppression mediated by TGF-ß, and it restored (P < 0.05) expression of activating NK-cell receptors and NK-cell cytotoxicity suppressed by TGF-ß. Expression of pSMAD was decreased in NK cells treated with IRX-2 but not in those treated with rhIL-2. CONCLUSIONS: IRX-2 was more effective than IL-2 in enhancing NK-cell cytotoxicity and protecting NK-cell function of HNSCC patients in vitro, emphasizing the potential advantage of IRX-2 as a component of future therapies for HNSCC.

The role of the adenosinergic pathway in immunosuppression mediated by human regulatory T cells (Treg).

Tumor-induced dysfunction of immune cells is a common problem in cancer. Tumors induce immune suppression by many different mechanisms, including accumulation of regulatory T cells (Treg). Adaptive Treg (Tr1) generated in the tumor microenvironment express CD39 and CD73 ectonucleotidases, produce adenosine and are COX2+PGE2+. Adenosine and PGE2 produced by Tr1 or tumor cells bind to their respective receptors on the surface of T effector cells (Teff) and cooperate in up-regulating cytosolic 3'5'-cAMP levels utilizing adenylyl cyclase isoform 7 (AC-7). In Teff, increased cAMP mediates suppression of anti-tumor functions. Treg, in contrast to Teff, seem to require high cAMP levels for mediating suppression. This differential requirement of Treg and Teff for cAMP offers an opportunity for pharmacologic interventions using selected inhibitors of the adenosine/PGE2 pathways. Blocking of adenosine/PGE2 production by Tr1 or blocking binding of these factors to their receptors on T cells or inhibition of cAMP synthesis in Teff all represent novel therapeutic strategies that used in combination with conventional therapies could restore anti-tumor functions of Teff . At the same time, these inhibitors could disarm Tr1 cells by depriving them of the factors promoting their generation and activity or by down-regulating 3'5'-cAMP levels. Thus, the pharmacologic control of Treg-Teff interactions offers a novel strategy for restoration of anti-tumor Teff functions and silencing of Treg. Used in conjunction with anti-cancer drugs or with immune therapies, this strategy has a potential to improve therapeutic effects by preventing or reversing tumor-induced immune suppression.

Dendritic cell generation and CD4+ CD25high FOXP3+ regulatory t cells in human head and neck carcinoma during radio-chemotherapy.

BACKGROUND: Regulatory T cells (Treg) and dendritic cells (DC) play an important role in tumor immunity and immune escape. However, their interplay and the effects of anti-cancer therapy on the human immune system are largely unknown. METHODS: For DC generation, CD14⊃+ monocytes were enriched by immunomagnetic selection from peripheral blood of advanced head and neck squamous cell carcinoma (HNSCC) patients and differentiated into immature DC using GM-SCF and IL-4. DC maturation was induced by addition of TNFα. The frequency of CD4⊃+CD25⊃highFOXP3⊃+ Treg in HNSCC patients was analyzed before and after radio-chemotherapy (RCT) by four-color flow cytometry. RESULTS: In HNSCC patients, the frequency of Treg (0.33 ± 0.06%) was significantly (p = 0.001) increased compared to healthy controls (0.11 ± 0.02%), whereas RCT had variable effects on the Treg frequency inducing its increase in some patients and decrease in others. After six days in culture, monocytes of all patients had differentiated into immature DC. However, DC maturation indicated by CD83 up-regulation (70.7 ± 5.5%) was successful only in a subgroup of patients and correlated well with lower frequencies of peripheral blood Treg in those patients. CONCLUSION: The frequency of regulatory T cells is elevated in HNSCC patients and may be modulated by RCT. Monocyte-derived DC in HNSCC patients show a maturation deficiency ex vivo. Those preliminary data may have an impact on multimodality clinical trials integrating cellular immune modulation in patients with advanced HNSCC.

Prolactin receptor is a negative prognostic factor in patients with squamous cell carcinoma of the head and neck.

BACKGROUND: The influence of human prolactin (hPRL) on the development of breast and other types of cancer is well established. Little information, however, exists on the effects of hPRL on squamous cell carcinomas of the head and neck (SCCHNs). METHODS: In this study, we evaluated prolactin receptor (PRLR) expression in SCCHN cell lines and assessed by immunohistochemistry the expression in 89 patients with SCCHNs. The PRLR expression was correlated with clinicopathological characteristics as well as clinical outcome. The effect of hPRL treatment on tumour cell growth was evaluated in vitro. RESULTS: Immunoreactivity for PRLR was observed in 85 out of 89 (95%) tumours. Multivariate COX regression analysis confirmed high levels of PRLR expression (>25% of tumour cells) to be an independent prognostic factor with respect to overall survival (HR=3.70, 95% CI: 1.14-12.01; P=0.029) and disease-free survival (P=0.017). Growth of PRLR-positive cancer cells increased in response to hPRL treatment. CONCLUSION: Our data indicate that hPRL is an important growth factor for SCCHN. Because of PRLR expression in a vast majority of tumour specimens and its negative impact on overall survival, the receptor represents a novel prognosticator and a promising drug target for patients with SCCHNs.

Impact of HIV on liver fibrosis in men with hepatitis C infection and haemophilia.

Hepatitis C virus (HCV) is the major cause of liver disease in haemophilia. Few data exist on the proportion with liver fibrosis in this group after long-term HCV and HIV co-infection. We conducted a cross-sectional multi-centre study to determine the impact of HIV on the prevalence and risk factors for fibrosis in haemophilic men with chronic hepatitis C. Biopsies were independently scored by Ishak, Metavir and Knodell systems. Variables were tested for associations with fibrosis using logistic regression and receiver operating curves (ROC). Of 220 biopsied HCV(+) men, 23.6% had Metavir ≥ F3 fibrosis, with higher mean Metavir fibrosis scores among HIV/HCV co-infected than HCV mono-infected, 1.6 vs. 1.3 (P = 0.044). Variables significantly associated with fibrosis included AST, ALT, APRI score (AST/ULN × 100/platelet × 10(9) /L), alpha-fetoprotein (all P < 0.0001), platelets (P = 0.0003) and ferritin (P = 0.0008). In multiple logistic regression of serum markers, alpha-fetoprotein, APRI and ALT were significantly associated with ≥ F3 fibrosis [AUROC = 0.77 (95% CI 0.69, 0.86)]. Alpha-fetoprotein, APRI and ferritin were significant in HIV(-) [AUROC = 0.82 (95% CI 0.72, 0.92)], and alpha-fetoprotein and platelets in HIV(+) [AUROC = 0.77 (95% CI 0.65, 0.88]. In a multivariable model of demographic and clinical variables, transformed (natural logarithm) of alpha-fetoprotein (P = 0.0003), age (P = 0.006) and HCV treatment (P = 0.027) were significantly associated with fibrosis. Nearly one-fourth of haemophilic men have Metavir ≥ 3 fibrosis. The odds for developing fibrosis are increased in those with elevated alpha-fetoprotein, increasing age and past HCV treatment.

In vitro chemosensitivity of head and neck cancer cell lines.

BACKGROUND: Systemic treatment of head and neck squamous cell carcinoma (HNSCC) includes a variety of antineoplastic drugs. However, drug-resistance interferes with the effectiveness of chemotherapy. Preclinical testing models are needed in order to develop approaches to overcome chemoresistance. - METHODS: Ten human cell lines were obtained from HNSCC, including one with experimentally-induced cisplatin resistance. Inhibition of cell growth by seven chemotherapeutic agents (cisplatin, carboplatin, 5- fluorouracil, methotrexate, bleomycin, vincristin, and paclitaxel) was measured using metabolic MTT-uptake assay and correlated to clinically-achievable plasma concentrations. - RESULTS: All drugs inhibited cell growth in a concentration-dependent manner with an IC50 comparable to that achievable in vivo. However, response curves for methotrexate were unsatisfactory and for paclitaxel, the solubilizer cremophor EL was toxic. Cross-resistance was observed between cisplatin and carboplatin. - CONCLUSION: Chemosensitivity of HNSCC cell lines can be determined using the MTT-uptake assay. For DNA-interfering cytostatics and vinca alkaloids this is a simple and reproducible procedure. Determined in vitro chemosensitivity serves as a baseline for further experimental approaches aiming to modulate chemoresistance in HNSCC with potential clinical significance.

TLR4 signaling induced by lipopolysaccharide or paclitaxel regulates tumor survival and chemoresistance in ovarian cancer.

Toll-like receptors (TLRs) expressed on immune cells trigger inflammatory responses. TLRs are also expressed on ovarian cancer (OvCa) cells, but the consequences of signaling by the TLR4/MyD88 pathway in these cells are unclear. Here, TLR4 and MyD88 expression in OvCa tissues (n=20) and cell lines (OVCAR3, SKOV3, AD10, A2780 and CP70) was evaluated by reverse transcriptase-PCR, western blots and immunohistochemistry. Cell growth, apoptosis, nuclear factor-kappaB (NF-kappaB) translocation, IRAK4 and TRIF expression and cJun phosphorylation were measured following tumor cell exposure to the TLR4 ligands, lipopolysaccharide (LPS) or paclitaxel (PTX). Culture supernatants were tested for cytokine levels. TLR4 was expressed in all tumors, tumor cell lines and normal epithelium. MyD88 was detectable in tumor tissues and in 3/5 OvCa lines but not in normal cells. In MyD88(+) SCOV3 cells, LPS or PTX binding to TLR4 induced IRAK4 activation and cJun phosphorylation, activated the NF-kappaB pathway and promoted interleukin (IL)-8, IL-6, vascular endothelial growth factor and monocyte chemotactic protein-1 production and resistance to drug-induced apoptosis. Silencing of TLR4 in SCOV3 cells with small interference RNA resulted in phosphorylated-cJun (p-cJun) downregulation and a loss of PTX resistance. In PTX-sensitive, MyD88(neg) A2780 cells, TLR4 stimulation upregulated TRIF, and TLR4 silencing eliminated this effect. Thus, TLR4/MyD88 signaling supports OvCa progression and chemoresistance, promoting immune escape.

IRX-2, a novel immunotherapeutic, protects human T cells from tumor-induced cell death.

IRX-2 is a cytokine-based biologic agent that has the potential to enhance antitumor immune responses. We investigated whether IRX-2 can protect T cells from tumor-induced apoptosis. Tumor-derived microvesicles (MV) expressing FasL were purified from supernatants of tumor cells and incubated with activated CD8(+) T cells. MV induced significant CD8(+) T-cell apoptosis, as evidenced by Annexin binding (64.4+/-6.4%), caspase activation (58.1+/-7.6%), a loss of mitochondrial membrane potential (82.9+/-3.9%) and DNA fragmentation. T-cell pretreatment with IRX-2 prevented apoptosis. IRX-2-mediated cytoprotection was dose and time dependent and was comparable to effects of IL-2, IL-7 or IL-15. IRX-2 prevented MV-induced downregulation of JAK3 and TCRzeta chain and induced STAT5 activation in T cells. IRX-2 prevented MV-induced Bax and Bim upregulation (P<0.005-0.05), prevented cytochrome c release and Bid cleavage, and concurrently restored the expression of Bcl-2, Bcl-xL, FLIP and Mcl-1 (P<0.005-0.01) in T cells. In addition, IRX-2 reversed MV-induced inhibition of the PI3K/Akt pathway. An Akt inhibitor (Akti-1/2) abrogated protective effects of IRX-2, suggesting that Akt is a downstream target of IRX-2 signaling. Thus, ex vivo pretreatment of CD8(+) T cells with IRX-2 provided potent protection from tumor-induced apoptosis. IRX-2 application to future cancer biotherapies could improve their effectiveness by bolstering T-cell resistance to tumor-induced immunosuppression.

The tumor microenvironment and its role in promoting tumor growth.

The tumor microenvironment is created by the tumor and dominated by tumor-induced interactions. Although various immune effector cells are recruited to the tumor site, their anti-tumor functions are downregulated, largely in response to tumor-derived signals. Infiltrates of inflammatory cells present in human tumors are chronic in nature and are enriched in regulatory T cells (T(reg)) as well as myeloid suppressor cells (MSC). Immune cells in the tumor microenvironment not only fail to exercise antitumor effector functions, but they are co-opted to promote tumor growth. Sustained activation of the NF-kappaB pathway in the tumor milieu represents one mechanism that appears to favor tumor survival and drive abortive activation of immune cells. The result is tumor escape from the host immune system. Tumor escape is accomplished through the activation of one or several molecular mechanisms that lead to inhibition of immune cell functions or to apoptosis of anti-tumor effector cells. The ability to block tumor escape depends on a better understanding of cellular and molecular pathways operating in the tumor microenvironment. Novel therapeutic strategies that emerge are designed to change the pro-tumor microenvironment to one favoring acute responses and potent anti-tumor activity.