Sequential azacitidine and carboplatin induces immune activation in platinum-resistant high-grade serous ovarian cancer cell lines and primes for checkpoint inhibitor immunotherapy.

BACKGROUND: Platinum chemoresistance results in high-grade serous ovarian cancer (HGSOC) disease recurrence. Recent treatment advances using checkpoint inhibitor immunotherapy has not benefited platinum-resistant HGSOC. In ovarian cancer, DNA methyltransferase inhibitors (DNMTi) block methylation and allow expression of silenced genes, primarily affecting immune reactivation pathways. We aimed to determine the epigenome and transcriptome response to sequential treatment with DNMTi and carboplatin in HGSOC. METHODS: In vitro studies with azacitidine or carboplatin alone and in sequential combination. Response was determined by cell growth, death and apoptosis. Genome-wide DNA methylation levels and transcript expression were compared between untreated and azacitidine and carboplatin sequential treatment. RESULTS: Sequential azacitidine and carboplatin significantly slowed cell growth in 50% of cell lines compared to carboplatin alone. The combination resulted in significantly higher cell death in 25% of cell lines, and significantly higher cell apoptosis in 37.5% of cell lines, than carboplatin alone. Pathway analysis of upregulated transcripts showed that the majority of changes were in immune-related pathways, including those regulating response to checkpoint inhibitors. CONCLUSIONS: Sequential azacitidine and carboplatin treatment slows cell growth, and demethylate and upregulate pathways involved in immune response, suggesting that this combination may be used to increase HGSOC response to immune checkpoint inhibitors in platinum-resistant patients who have exhausted all currently-approved avenues of treatment.

SI-CLP inhibits the growth of mouse mammary adenocarcinoma by preventing recruitment of tumor-associated macrophages.

Chitinase-like proteins (CLP) are chitin-binding proteins that lack chitin hydrolyzing activity, but possess cytokine-like and growth factor-like properties, and play crucial role in intercellular crosstalk. Both human and mice express two members of CLP family: YKL-40 and stabilin-1 interacting chitinase-like protein (SI-CLP). Despite numerous reports indicating the role of YKL-40 in the support of angiogenesis, tumor cell proliferation, invasion and metastasis, the role of its structurally related protein SI-CLP in cancer was not reported. Using gain-of-function approach, we demonstrate in the current study that the expression of recombinant SI-CLP in mouse TS/A mammary adenocarcinoma cells results in significant and persistent inhibition of in vivo tumor growth. Using quantitative immunohistochemistry, we show that on the cellular level this phenomenon is associated with reduced infiltration of tumor-associated macrophages (TAMs), CD4+ and FoxP3+ cells in SI-CLP expressing tumors. Gene expression analysis in TAM isolated from SI-CLP-expressing and control tumors demonstrated that SI-CLP does not affect macrophage phenotype. However, SI-CLP significantly inhibited migration of murine bone-marrow derived macrophages and human primary monocytes toward monocyte-recruiting chemokine CCL2 produced in the tumor microenvironment (TME). Mechanistically, SI-CLP did not affect CCL2/CCR2 interaction, but suppressed cytoskeletal rearrangements in response to CCL2. Altogether, our data indicate that SI-CLP functions as a tumor growth inhibitor in mouse breast cancer by altering cellular composition of TME and blocking cytokine-induced TAM recruitment. Taking into consideration weak to absent expression of SI-CLP in human breast cancer, it can be considered as a therapeutic protein to block TAM-mediated support of breast tumor growth.

The immune response in pregnancy and in cancer is active and supportive of placental and tumor cell growth not their destruction.

While many investigators have described the biochemical and physiological similarities between tumor cells and trophoblast cells, in this discourse we will compare primarily their leucocytes, which constitute a large portion of the tumor and its microenvironment as well as the placenta and its microenvironment. There is a remarkable similarity between the cells that support placental growth and development and tumor growth and development. In many cases over half of the cells present in the tumor and the placenta are non-tumor or nontrophoblast cells, immune cells. Most of these immune cells are prevented from attacking the fetal derived placental cells and the self-derived tumor cells. Nevertheless, these leucocytes, in our opinion, are very active and support tumor and placental cell growth through the production of growth factors and angiogenic factors. These cells do this by activating the portion of the immune response which initiates and helps control tissue repair.

Cytokine-induced senescence for cancer surveillance.

The immune response is a first-line systemic defense to curb tumorigenesis and metastasis. Much effort has been invested to design antitumor interventions that would boost the immune system in its fight to defeat or contain cancerous growth. Tumor vaccination protocols, transfer of tumor-associated-antigen-specific T cells, T cell activity-regulating antibodies, and recombinant cytokines are counted among a toolbox filled with immunotherapeutic options. Although the mechanistic underpinnings of tumor immune control remain to be deciphered, these are studied with the goal of cancer cell destruction. In contrast, tumor dormancy is considered as a dangerous equilibrium between cell proliferation and cell death. There is, however, emerging evidence that tumor immune control can be achieved in the absence of overt cancer cell death. Here, we propose cytokine-induced senescence (CIS) by transfer of T helper-1 cells (TH1) or by recombinant cytokines as a novel therapeutic intervention for cancer treatment. Immunity-induced senescence triggers a stable cell cycle arrest of cancer cells. It engages the immune system to construct defensive, isolating barriers around tumors, and prevents tumor growth through the delivery or induction of TH1-cytokines in the tumor microenvironment. Keeping cancer cells in a non-proliferating state is a strategy, which directly copes with the lost homeostasis of aggressive tumors. As most studies show that even after efficient cancer therapies minimal residual disease persists, we suggest that therapies should include immune-mediated senescence for cancer surveillance. CIS has the goal to control the residual tumor and to transform a deadly disease into a state of silent tumor persistence.

Friend or Foe? The Ambiguous Role of Innate Lymphoid Cells in Cancer Development.

The development of immunotherapies represents a major advance towards the effective eradication of malignant tumors. So far, therapeutic approaches have largely focused on T lymphocytes, but the innate arm of the immune system might be similarly important. Innate lymphoid cells (ILCs) are rapidly-responding cells that are functionally analogous to diverse T cell subsets. In recent years these cells have attracted enormous attention owing to their pleiotropic effects in early host defense to infection and organ pathologies. ILCs might also represent promising targets in the context of cancer therapy because they are an innate immune cell population endowed with potent immunomodulatory properties. In this review we discuss the impact of the three ILC subsets and the signature cytokines they release on cancer development and tumor growth.

Inhibition of tumor progression during allergic airway inflammation in a murine model: significant role of TGF-ß.

INTRODUCTION: TGF-ß is an important mediator of pulmonary allergic inflammation, and it has been recently reported to be a potential inhibitor of lung tumor progression. The correlation between cancer and allergic inflammatory diseases remains controversial. Thus, the aim of the present study was to evaluate the effects of pulmonary allergic inflammation and in particular the role of TGF-ß on cancer progression. METHODS: Cancer cells were implanted in a BALB/c mice model of allergic airway inflammation, and tumor growth was measured. Apoptosis was evaluated by TUNEL assay, and TGF-ß was measured by ELISA. Expression of proliferating cell nuclear antigen, TGF-ß, TGF-ß receptors I and II, phospho-Smad2 and phospho-Smad4 was evaluated by immunohistochemistry and quantified using digital pathology. The effect of a TGF-ß activity inhibitor and recombinant TGF-ß on tumor growth was analyzed. The effect of exogenous TGF-ß on cell proliferation and apoptosis was evaluated in vitro. RESULTS: Mice with allergic airway inflammation exhibited decreased tumor volumes due to cell proliferation inhibition and increased apoptosis. TGF-ß was increased in the sera and tumor tissues of allergic mice. TGF-ß activity inhibition increased tumor progression in allergic mice by enhancing proliferation and decreasing apoptosis of tumor cells. The administration of TGF-ß resulted in reduced tumor growth. CONCLUSION: This study is the first to establish an inverse relationship between allergic airway inflammation and tumor progression. This effect appears to be mediated by TGF-ß, which is overexpressed in tumor cells during pulmonary allergic inflammation. This study indicates that TGF-ß is a potential target for antitumor therapy.

Analysis of expression and function of the co-stimulatory receptor SLAMF1 in immune cells from patients with systemic lupus erythematosus (SLE).

The signaling lymphocytic activation molecule SLAMF1 (CD150) is a co-stimulatory molecule that is expressed by most immune cells, including T regulatory (Treg) lymphocytes. Since different abnormalities have been reported regarding the number and function of Foxp3+ Treg cells in patients with systemic lupus erythematosus (SLE), we decided to analyze the expression and function of CD150 in these regulatory lymphocytes in this condition. We isolated peripheral blood mononuclear cells from 20 patients with SLE, and 20 healthy controls. The expression of SLAMF1 was determined by multi-parametric flow cytometry and the suppressive function of CD4+CD25+ lymphocytes, upon engagement or not of CD150 with an agonistic monoclonal antibody, was analyzed by an assay of inhibition of cell proliferation. We observed a significantly increased expression of SLAMF1 by CD3+CD4+ helper T cells and CD19+ B cells in patients with SLE and active disease. However, similar levels of SLAMF1 expression were detected in Foxp3+ Treg cells from patients and controls. In contrast, a higher proportion of SLE patients increased their suppressive function of Treg cells upon CD150 engagement compared to healthy controls. Our data suggest that SLAMF1 is another significant piece in the intricate defective immune-regulatory function of patients with SLE.

Diversified phenotype of antigen specific CD8+ T cells responding to the immunodominant epitopes of IE and pp65 antigens of human cytomegalovirus.

To study the cytomegalovirus (CMV)-specific CD8+ T cells in individuals with HLA A*1101, A*0201 and A*2402, our findings showed that peptide SK-10-2, KI-10 and KV-10 of CMV IE and pp65 antigens were immunodominant in 198 individuals with HLA A*1101, A*0201 and A*2402, the most frequent genotypes in Chinese. Interestingly, SK-10-2 induced the strongest T cell response to produce IFN-γ whereas the others did not induce prominent IFN-γ production despite they all induced remarkable T cell proliferation. The peptides induced different phenotypes including IFN-γ(high)TNF-α(low) and TNF-α(low)Foxp3(low). It suggests that only some of CMV-reactive CD8+ T cells are real protective IFN-γ(high) cytotoxic T cells.

Interleukin-21 sustains inflammatory signals that contribute to sporadic colon tumorigenesis.

Interleukin (IL)-21 triggers inflammatory signals that contribute to the growth of neoplastic cells in mouse models of colitis-associated colorectal cancer (CRC). Because most CRCs are sporadic and arise in the absence of overt inflammation we have investigated the role of IL-21 in these tumors in mouse and man. IL-21 was highly expressed in human sporadic CRC and produced mostly by IFN-γ-expressing T-bet/RORγt double-positive CD3+CD8- cells. Stimulation of human CRC cell lines with IL-21 did not directly activate the oncogenic transcription factors STAT3 and NF-kB and did not affect CRC cell proliferation and survival. In contrast, IL-21 modulated the production of protumorigenic factors by human tumor infiltrating T cells. IL-21 was upregulated in the neoplastic areas, as compared with non-tumor mucosa, of Apc(min/+) mice, and genetic ablation of IL-21 in such mice resulted in a marked decrease of both tumor incidence and size. IL-21 deficiency was associated with reduced STAT3/NF-kB activation in both immune cells and neoplastic cells, diminished synthesis of protumorigenic cytokines (that is, IL-17A, IL-22, TNF-α and IL-6), downregulation of COX-2/PGE2 pathway and decreased angiogenesis in the lesions of Apc(min/+) mice. Altogether, data suggest that IL-21 promotes a protumorigenic inflammatory circuit that ultimately sustains the development of sporadic CRC.

Protein kinase CK2 enables regulatory T cells to suppress excessive TH2 responses in vivo.

The quality of the adaptive immune response depends on the differentiation of distinct CD4(+) helper T cell subsets, and the magnitude of an immune response is controlled by CD4(+)Foxp3(+) regulatory T cells (Treg cells). However, how a tissue- and cell type-specific suppressor program of Treg cells is mechanistically orchestrated has remained largely unexplored. Through the use of Treg cell-specific gene targeting, we found that the suppression of allergic immune responses in the lungs mediated by T helper type 2 (TH2) cells was dependent on the activity of the protein kinase CK2. Genetic ablation of the ß-subunit of CK2 specifically in Treg cells resulted in the proliferation of a hitherto-unexplored ILT3(+) Treg cell subpopulation that was unable to control the maturation of IRF4(+)PD-L2(+) dendritic cells required for the development of TH2 responses in vivo.

Human adipose tissue-derived mesenchymal stem cells abrogate plasmablast formation and induce regulatory B cells independently of T helper cells.

Mesenchymal or stromal stem cells (MSC) interact with cells of the immune system in multiple ways. Modulation of the immune system by MSC is believed to be a therapeutic option for autoimmune disease and transplant rejection. In recent years, B cells have moved into the focus of the attention as targets for the treatment of immune disorders. Current B-cell targeting treatment is based on the indiscriminate depletion of B cells. The aim of this study was to examine whether human adipose tissue-derived MSC (ASC) interact with B cells to affect their proliferation, differentiation, and immune function. ASC supported the survival of quiescent B cells predominantly via contact-dependent mechanisms. Coculture of B cells with activated T helper cells led to proliferation and differentiation of B cells into CD19(+) CD27(high) CD38(high) antibody-producing plasmablasts. ASC inhibited the proliferation of B cells and this effect was dependent on the presence of T cells. In contrast, ASC directly targeted B-cell differentiation, independently of T cells. In the presence of ASC, plasmablast formation was reduced and IL-10-producing CD19(+) CD24(high) CD38(high) B cells, known as regulatory B cells, were induced. These results demonstrate that ASC affect B cell biology in vitro, suggesting that they can be a tool for the modulation of the B-cell response in immune disease.

Four-in-one antibodies have superior cancer inhibitory activity against EGFR, HER2, HER3, and VEGF through disruption of HER/MET crosstalk.

The anti-HER receptor antibodies cetuximab, trastuzumab, and pertuzumab are used widely in clinic to treat metastatic cancer. However, activation of the extensive crosstalk among the HER receptors as well as other RTKs, particularly HER-MET crosstalk, has emerged as a likely source of drug resistance. In this study, we developed two new types of tetra-specific antibodies that recognize EGFR, HER2, HER3, and VEGF. These tetra-specific antibodies, termed FL518 (four-in-one antibody) and CRTB6 (tetra-specific, tetravalent antibody), not only inhibited signaling mediated by these receptors in vitro and in vivo but unexpectedly also disrupted HER-MET crosstalk. When compared with two-in-one antibodies and a series of bispecific antibodies in multiple tumor models, FL518 and CRTB6 were more broadly efficacious. We further showed that tetra-specific antibodies were far more effective than bispecific antibodies in inhibiting the growth of anti-HER-resistant cancer cells, which exhibited elevated levels of MET activation both in vitro and in vivo. Overall, our results establish a new principle to achieve combined HER inhibition and limit drug resistance using a single antibody.

Selective, efficient modulation of activated CD4+ αßT cells by the novel humanized antibody GZ-αßTCR targeting human αßTCR.

Allograft rejection and immunosuppression are two major issues in transplantation medicine. The specific targeting of alloreactive T cells, the initiators and promoters of allograft rejection, would be a promising strategy to reduce unwanted T-cell responses and side effects of lifelong immunosuppression. The novel humanized monoclonal antibody GZ-αßTCR, specific for the human αßT-cell receptor, was tested in vitro and in vivo for its specificity and efficacy to modulate the αßT-cell compartment. GZ-αßTCR moderately induced apoptosis in resting αßT cells in vitro, an effect considerably amplified in activated T cells. A single dose of GZ-αßTCR significantly reduced human CD45(+)CD3(+) T cells in vivo, with a preferential modulation of CD4(+) αßT cells. Importantly, naive T cells, the T-cell subset from which alloreactivity emanates, were significantly reduced. Simultaneously, a significant, compensatory increase of γδ T cells was observed in vitro and in vivo in both humanized mouse models examined. GZ-αßTCR did not induce cytokines and was well tolerated. Thus, specificity and high efficacy make GZ-αßTCR a powerful tool to selectively eliminate putatively detrimental T-cell subsets, a major goal in transplantation medicine. At the same time, GZ-αßTCR spares γδ and natural killer cells, thus leaving the recipient's immune system competent for cell-mediated immunoregulation and cell-mediated immunity.

Regulatory iNKT cells lack expression of the transcription factor PLZF and control the homeostasis of T(reg) cells and macrophages in adipose tissue.

Invariant natural killer T cells (iNKT cells) are lipid-sensing innate T cells that are restricted by the antigen-presenting molecule CD1d and express the transcription factor PLZF. iNKT cells accumulate in adipose tissue, where they are anti-inflammatory, but the factors that contribute to their anti-inflammatory nature, as well as their targets in adipose tissue, are unknown. Here we found that iNKT cells in adipose tissue had a unique transcriptional program and produced interleukin 2 (IL-2) and IL-10. Unlike other iNKT cells, they lacked PLZF but expressed the transcription factor E4BP4, which controlled their IL-10 production. The adipose iNKT cells were a tissue-resident population that induced an anti-inflammatory phenotype in macrophages and, through the production of IL-2, controlled the number, proliferation and suppressor function of regulatory T cells (Treg cells) in adipose tissue. Thus, iNKT cells in adipose tissue are unique regulators of immunological homeostasis in this tissue.

Tumor-induced CD14+HLA-DR (-/low) myeloid-derived suppressor cells correlate with tumor progression and outcome of therapy in multiple myeloma patients.

Myeloid-derived suppressor cells (MDSCs) are heterogeneous, immature, myeloid progenitor cells, which suppress immune responses against tumors. CD14(+)HLA-DR(-/low) monocytic MDSCs (M-MDSC) are increased in patients suffering from multiple myeloma (MM). However, the frequency and function of M-MDSCs with the relationship between the tumor development and outcome of therapy in MM remain unclear. In this study, we analyzed the changes in M-MDSCs in newly diagnosed, relapsed and remission MM patients. In addition, we also assessed the response of M-MDSCs in MM patients treated with a bortezomib-based therapy as well as the impact of bortezomib on the modulation of M-MDSCs in vitro. The levels of M-MDSCs in newly diagnosed and relapsed MM patients were significantly increased compared with those in remission MM patients and healthy donors. Moreover, the levels of M-MDSCs were shown to correlate with tumor progression. The decrease in M-MDSCs after proteasome inhibitory therapy suggested that M-MDSCs could be considered as an indicator for the efficacy of therapy. Finally, we found the plasma from newly diagnosed MM patients, and MM cells were able to induce the accumulation of M-MDSCs in vitro. These results indicated that M-MDSCs could be considered as a prognostic predictor and an important cell type contributing to immune suppressive microenvironment in MM patients. Treatments targeting for M-MDSCs may improve therapeutic outcomes for MM patients.

Antibody blocks acquisition of bacterial colonization through agglutination.

Invasive infection often begins with asymptomatic colonization of mucosal surfaces. A murine model of bacterial colonization with Streptococcus pneumoniae was used to study the mechanism for mucosal protection by immunoglobulin. In previously colonized immune mice, bacteria were rapidly sequestered within large aggregates in the nasal lumen. To further examine the role of bacterial agglutination in protection by specific antibodies, mice were passively immunized with immunoglobulin G (IgG) purified from antipneumococcal sera or pneumococcal type-specific monoclonal human IgA (hIgA1 or hIgA2). Systemically delivered IgG accessed the mucosal surface and blocked acquisition of colonization and transmission between littermates. Optimal protection by IgG was independent of Fc fragment and complement and, therefore, did not involve an opsonophagocytic mechanism. Enzymatic digestion or reduction of IgG before administration showed that protection required divalent binding that maintained its agglutinating effect. Divalent hIgA1 is cleaved by the pneumococcal member of a family of bacterial proteases that generate monovalent Fabα fragments. Thus, passive immunization with hIgA1 blocked colonization by an IgA1-protease-deficient mutant (agglutinated) but not the protease-producing wild-type parent (not agglutinated), whereas protease-resistant hIgA2 agglutinated and blocked colonization by both. Our findings highlight the importance of agglutinating antibodies in mucosal defense and reveal how successful pathogens evade this effect.

Glioma cell-derived placental growth factor induces regulatory B cells.

Tumor specific immune regulatory cells play an important role in the pathogenesis of glioma. The mechanisms have not been fully understood yet. It is suggested that placenta growth factor (PlGF) is involved in the generation of immune regulatory cells. This study aims to investigate the role of glioma cell-derived PlGF in the generation of regulatory B cells (Breg). Glioma cells were isolated from surgically removed glioma tissue. Cytokines were measured by enzyme-linked immunosorbent assay, quantitative real time RT-PCR and Western blotting. Immune suppressor functions of Bregs were assessed by T cell proliferation assay. The results showed that glioma cells expressed PlGF, which was increased after a non-specific activation. Naïve B cells captured the PlGF to differentiate into transforming growth factor-ß positive Bregs. The Bregs were activated upon exposure to protein extracts of glioma tissue to suppress the CD8(+) T cell proliferation and the release of perforin and granzyme B. We conclude that glioma cell-released PlGF can induce Bregs to suppress CD8(+) T cell activities.

Regulatory B cells accelerate hepatocellular carcinoma progression via CD40/CD154 signaling pathway.

Human hepatocellular carcinoma (HCC) is the fifth most common cancer worldwide with a poor prognosis of limited survival. The role of regulatory B cell (Breg), a new important B cell subset, in HCC progression remains unclear. We firstly found that the percentage of B cells at tumor margin was significantly higher than that in tumor and non-tumor regions. Especially, increased intrahepatic B cells at tumor margin were positively associated with tumor invasive features and more tumor recurrence. Besides, HCC patients had a significantly higher percentage of circulating Bregs than healthy people. Increased circulating Bregs were correlated with advanced tumor staging, tumor multiplicity and venous infiltration. Next, we firstly revealed that human Bregs promoted HCC tumor growth independent of Tregs in SCID mice. The migration of Bregs from blood into tumor was also confirmed in mice. Finally, we further explored the molecular mechanism of Bregs promoting proliferation and migration of HCC cells in vitro. Bregs promoted HCC growth and invasiveness by directly interacting with liver cancer cells through the CD40/CD154 signaling pathway.

Enhancement of anti-tumor immune responses by transfection of IFNγ gene into tumor using a novel type synthetic vector.

The existence of Th1 responses in a tumor microenvironment elicits a better prognosis for the patients. Transfection of Th1 polarizing cytokines, such as IFNγ, into tumor cells is an effective way to set up an appropriate microenvironment. Using a novel type synthetic vector composed of polyamidoamine dendrons, we transfected canine IFNγ gene into canine tumor cell lines, and examined direct and indirect effects of dendritic cells (DCs) against tumor growth in vitro. A cloned canine IFNγ gene expressed functional protein that induces maturation of DCs. When the canine IFNγ gene was transfected into canine tumor cell lines using the synthetic vector, most cells secreted canine IFNγ. Secretion of IFNγ reduced with time, but was maintained for 48h. DCs incubated with the IFNγ-transfected tumor cells exhibited greater suppressive activity and induced significantly higher cytotoxic activity against the tumor cells, relative to those incubated with untransfected tumor cells and comparable dose of IFNγ. Successful transfection of IFNγ by the synthetic vector efficiently enhanced the anti-tumor immune function of DCs, and sets up a suitable microenvironment for improvement in tumor therapy.

Interactions between neutrophils and non-small cell lung cancer cells: enhancement of tumor proliferation and inflammatory mediator synthesis.

The inflammatory tumor microenvironment plays a crucial role in tumor progression. In lung cancer, both bacterial infections and neutrophilia are associated with a poor prognosis. In this study, we characterized the effect of isolated human neutrophils on proliferation of the non-small cell lung cancer (NSCLC) cell line A549 and analyzed the impact of A549-neutrophil interactions on inflammatory mediator generation in naive and lipopolysaccharide (LPS)-exposed cell cultures. Co-incubation of A549 cells with neutrophils induced proliferation of resting and LPS-exposed A549 cells in a dose-dependent manner. In transwell-experiments, this effect was demonstrated to depend on direct cell-to-cell contact. This pro-proliferative effect of neutrophils on A549 cells could be attenuated by inhibition of neutrophil elastase activity, but not by oxygen radical neutralization. Correspondingly, neutrophil elastase secretion, but not respiratory burst, was specifically enhanced in co-cultures of A549 cells and neutrophils. Moreover, interference with COX-2 activity by indomethacin or the specific COX-2 inhibitor NS-398 also blunted the increased A549 proliferation in the presence of neutrophils. In parallel, a massive amplification of COX-2-dependent prostaglandin E2 synthesis was detected in A549-neutrophil co-cultures. These findings suggest that direct cell-cell interactions between neutrophils and tumor cells cause release of inflammatory mediators which, in turn, may enhance tumor growth in NSCLC.