3D Microfluidic model for evaluating immunotherapy efficacy by tracking dendritic cell behaviour toward tumor cells

Immunotherapy efficacy relies on the crosstalk within the tumor microenvironment between cancer and dendritic cells (DCs) resulting in the induction of a potent and effective antitumor response. DCs have the specific role of recognizing cancer cells, taking up tumor antigens (Ags) and then migrating to lymph nodes for Ag (cross)-presentation to naïve T cells. Interferon-α-conditioned DCs (IFN-DCs) exhibit marked phagocytic activity and the special ability of inducing Ag-specific T-cell response. Here, we have developed a novel microfluidic platform recreating tightly interconnected cancer and immune systems with specific 3D environmental properties, for tracking human DC behaviour toward tumor cells. By combining our microfluidic platform with advanced microscopy and a revised cell tracking analysis algorithm, it was possible to evaluate the guided efficient motion of IFN-DCs toward drug-treated cancer cells and the succeeding phagocytosis events. Overall, this platform allowed the dissection of IFN-DC-cancer cell interactions within 3D tumor spaces, with the discovery of major underlying factors such as CXCR4 involvement and underscored its potential as an innovative tool to assess the efficacy of immunotherapeutic approaches

A multidisciplinary study using in vivo tumor models and microfluidic cell-on-chip approach to explore the cross-talk between cancer and immune cells

A full elucidation of events occurring inside the cancer microenvironment is fundamental for the optimization of more effective therapies. In the present study, the cross-talk between cancer and immune cells was examined by employing mice deficient (KO) in interferon regulatory factor (IRF)-8, a transcription factor essential for induction of competent immune responses. The in vivo results showed that IRF-8 KO mice were highly permissive to B16.F10 melanoma growth and metastasis due to failure of their immune cells to exert proper immunosurveillance. These events were found to be dependent on soluble factors released by cells of the immune system capable of shaping the malignant phenotype of melanoma cells. An on-chip model was then generated to further explore the reciprocal interactions between the B16.F10 and immune cells. B16.F10 and immune cells were co-cultured in a microfluidic device composed of three culturing chambers suitably inter-connected by an array of microchannels; mutual interactions were then followed using time-lapse microscopy. It was observed that WT immune cells migrated through the microchannels towards the B16.F10 cells, establishing tight interactions that in turn limited tumor spread. In contrast, IRF-8 KO immune cells poorly interacted with the melanoma cells, resulting in a more invasive behavior of the B16.F10 cells. These results suggest that IRF-8 expression plays a key role in the cross-talk between melanoma and immune cells, and under-score the value of cell-on-chip approaches as useful in vitro tools to reconstruct complex in vivo microenvironments on a microscale level to explore cell interactions such as those occurring within a cancer immunoenvironment

Targeting CXCR4 reverts the suppressive activity of T-regulatory cells in renal cancer

With the intent to identify biomarkers in renal cell carcinoma (RCC) the functional status of T-regulatory cells (Tregs) was investigated in primary RCC. Tregs were isolated from tumoral-(TT), peritumoral tissue-(PT) and peripheral blood-(PB) of 42 primary RCC patients and function evaluated through effector T cells (Teff) proliferation, cytokines release and demethylation of Treg Specific Region (TSDR). The highest value of Tregs was detected in TT with the uppermost amount of effector-Tregs-(CD4+CD25hiFOXP3hiCD45RA-). PB-RCC Tregs efficiently suppress Teff proliferation compared to healthy donor (HD)-Tregs and, at the intrapatient evaluation, TT-derived Tregs were the most suppressive. Higher demethylation TSDR was detected in TT- and PB-RCC Tregs vs HD-Tregs (P <0,001). CXCR4 is highly expressed on Tregs, thus we wished to modulate Tregs function through CXCR4 inhibition. CXCR4 antagonism, elicited by a new peptidic antagonist, Peptide-R29, efficiently reversed Tregs suppression of Teff proliferation. Thus Tregs functional evaluation precisely reflects Tregs status and may be a reliable biomarker of tumoral immune response. In addition, treatment with CXCR4 antagonist, impairing Tregs function, could improve the anticancer immune response, in combination with conventional therapy and/or immunotherapy such as checkpoints inhibitors

Role of interferon regulatory factor 1 in governing Treg depletion, Th1 polarization, inflammasome activation and antitumor efficacy of cyclophosphamide

The antitumor effectiveness of cyclophosphamide (CTX) and other chemotherapeutics was shown to rely not only on direct cytotoxicity but also on immunogenic tumor cell death and systemic immunomodulatory mechanisms, including regulatory T cell (Treg) depletion, Th1 cell polarization, type I interferon (IFN) and proinflammatory cytokine production. IFN regulatory factor (IRF)-1 is a transcriptional regulator of IFNs and IFN-inducible genes, involved in the control of Th1 and Treg differentiation and in sterile inflammation. Aim of this study was to explore the role of IRF-1 in CTX-induced antitumor effects and related immune activities. This study shows for the first time that IRF-1 is important for the antitumor efficacy of CTX in mice. Moreover, experiments in tumor-bearing C57BL/6 mice showed that Irf1 gene expression in the spleen was transiently increased following CTX administration and correlated with the induction of Th1 cell expansion and of Il12p40 gene expression, which is the main Th1-driving cytokine. At the same time, CTX administration reduced both Foxp3 expression and Treg cell percentages. These effects were abrogated in Irf1(-/-) mice. Further experiments showed that the gene and/or protein expression of caspase-1, iNOS, IL-1β, IL-6 and CXCL10 and the levels of nitric oxide were modulated following CTX in an IRF-1-direct- or -indirect-dependent manner, and highlighted the importance of caspase-1 in driving the sterile inflammatory response to CTX. Our data identify IRF-1 as important for the antitumor efficacy of CTX and for the regulation of many immunomodulatory activities of CTX, such as Th1 polarization, Treg depletion and inflammation

IRF-8 Controls Melanoma Progression by Regulating the Cross Talk between Cancer and Immune Cells within the Tumor Microenvironment

The transcription factor interferon regulatory factor-8 (IRF-8) is crucial for myeloid cell development and immune response and also acts as a tumor suppressor gene. Here, we analyzed the role of IRF-8 in the cross talk between melanoma cells and tumor-infiltrating leukocytes. B16-F10 melanoma cells transplanted into IRF-8-deficient (IRF-8-/-) mice grow more rapidly, leading to higher numbers of lung metastasis, with respect to control animals. These events correlated with reduced dendritic cell and T cell infiltration, accumulation of myeloid-derived suppressor cells and a chemokine/chemokine receptor expression profile within the tumor microenvironment supporting tumor growth, angiogenesis, and metastasis. Noticeably, primary tumors developing in IRF-8-/- mice displayed a clear-cut inhibition of IRF-8 expression in melanoma cells. Injection of the demethylating agent 5-aza-2′-deoxycytidine into melanoma-bearing IRF-8-/- animals induced intratumoral IRF-8 expression and resulted in the re-establishment of a chemokine/ chemokine receptor pattern favoring leukocyte infiltration and melanoma growth arrest. Importantly, intrinsic IRF-8 expression was progressively down-modulated during melanoma growth in mice and in human metastatic melanoma cells with respect to primary tumors. Lastly, IRF-8 expression in melanoma cells was directly modulated by soluble factors, among which interleukin-27 (IL-27), released by immune cells from tumor-bearing mice. Collectively, these results underscore a key role of IRF-8 in the cross talk between melanoma and immune cells, thus revealing its critical function within the tumor microenvironment in regulating melanoma progression and invasiveness

Critical role of IRF-8 in negative regulation of TLR3 expression by Src homology 2 domain-containing protein tyrosine phosphatase-2 activity in human myeloid dendritic cells

Despite extensive studies that unraveled ligands and signal transduction pathways triggered by TLRs, little is known about the regulation of TLR gene expression. TLR3 plays a crucial role in the recognition of viral pathogens and induction of immune responses by myeloid DCs. IFN regulatory factor (IRF)-8, a member of the IRF family, is a transcriptional regulator that plays essential roles in the development and function of myeloid lineage, affecting different subsets of myeloid DCs. In this study, we show that IRF-8 negatively controls TLR3 gene expression by suppressing IRF-1– and/or polyinosinic-polycytidylic acid-stimulated TLR3 expression in primary human monocyte-derived DCs (MDDCs). MDDCs expressed TLR3 increasingly during their differentiation from monocytes to DCs with a peak at day 5, when TLR3 expression was further enhanced upon stimulation with polyinosinic-polycytidylic acid and then was promptly downregulated. We found that both IRF-1 and IRF-8 bind the human TLR3 promoter during MDDC differentiation in vitro and in vivo but with different kinetic and functional effects. We demonstrate that IRF-8–induced repression of TLR3 is specifically mediated by ligand-activated Src homology 2 domain-containing protein tyrosine phosphatase association. Indeed, Src homology 2 domain-containing protein tyrosine phosphatase–dephosphorylated IRF-8 bound to the human TLR3 promoter competing with IRF-1 and quashing its activity by recruitment of histone deacetylase 3. Our findings identify IRF-8 as a key player in the control of intracellular viral dsRNA-induced responses and highlight a new mechanism for negative regulation of TLR3 expression that can be exploited to block excessive TLR activation

The natural agonist of estrogen receptor β silibinin plays an immunosuppressive role representing a potential therapeutic tool in rheumatoid arthritis

Estrogens, in particular 17β-estradiol (E2), have a strong influence on the immune system and also affect pathological conditions such as autoimmune diseases. The biological effects of E2 are mediated by two intracellular receptors, i.e., estrogen receptor (ER)α and ERβ, which function as ligand-activated nuclear transcription factors producing genomic effects. Immune cells express both ERα and ERβ that play a complex role in modulating inflammation. Phytoestrogens display estrogen-like effects. Among them, silibinin, the major active constituent of silymarin extracted by the milk thistle (Silybum marianum), has been suggested to have an ERβ selective binding. Silibinin is known to have anti-inflammatory, hepatoprotective, and anticarcinogenic effects; however, the role of silibinin in modulating human immune responses and its impact on autoimmunity remains unclear. Aim of this study was to dissect the ability of the ERβ natural ligand silibinin to modulate T cell immunity, taking into account possible differences between females and males, and to define its possible role as therapeutic tool in immune-mediated diseases. To this purpose, female and age-matched male healthy subjects and patients with active rheumatoid arthritis (RA) were recruited. We evaluated the ability of silibinin to modulate ERβ expression in T lymphocytes and its effects on T cell functions (i.e., apoptosis, proliferation, and cytokine production). We also analyzed whether silibinin was able to modulate the expression of microRNA-155 (miR-155), which strongly contributes to the pathogenesis of RA driving aberrant activation of the immune system. We demonstrated that silibinin upregulated ERβ expression, induced apoptosis, inhibited proliferation, and reduced expression of the pro-inflammatory cytokines IL-17 and TNF-α, through ERβ binding, in T lymphocytes from female and male healthy donors. We obtained similar results in T lymphocytes from patients with active RA in term of apoptosis, proliferation, and cytokine production. In addition, we found that silibinin acted as an epigenetic modifier, down-modulating the expression of miR-155. In conclusion, our data demonstrated an immunosuppressive role of silibinin, supporting its application in the treatment of autoimmune diseases as drug, but also as dietary nutritional supplement, opening new perspective in the field of autoimmune disease management