Tumor-derived Soluble Factors Research Articles

Metformin Suppresses Both PD-L1 Expression in Cancer Cells and Cancer-Induced PD-1 Expression in Immune Cells to Promote Antitumor Immunity.

Metformin, a drug prescribed for patients with type 2 diabetes, has potential efficacy in enhancing antitumor immunity; however, the detailed underlying mechanisms remain to be elucidated. Therefore, we aimed to identify the inhibitory molecular mechanisms of metformin on programmed death ligand 1 (PD-L1) expression in cancer cells and programmed death 1 (PD-1) expression in immune cells. We employed a luciferase reporter assay, quantitative real-time PCR, immunoblotting analysis, immunoprecipitation and ubiquitylation assays, and a natural killer (NK) cell-mediated tumor cell cytotoxicity assay. A mouse xenograft tumor model was used to evaluate the effect of metformin on tumor growth, followed by flow-cytometric analysis using tumor-derived single-cell suspensions. Metformin decreased AKT-mediated β-catenin S552 phosphorylation and subsequent β-catenin transactivation in an adenosine monophosphate-activated protein kinase (AMPK) activation-dependent manner, resulting in reduced CD274 (encoding PD-L1) transcription in cancer cells. Tumor-derived soluble factors enhanced PD-1 protein stability in NK and T cells via dissociation of PD-1 from ubiquitin E3 ligases and reducing PD-1 polyubiquitylation. Metformin inhibited the tumor-derived soluble factor-reduced binding of PD-1 to E3 ligases and PD-1 polyubiquitylation, resulting in PD-1 protein downregulation in an AMPK activation-dependent manner. These inhibitory effects of metformin on both PD-L1 and PD-1 expression ameliorated cancer-reduced cytotoxic activity of immune cells in vitro and decreased tumor immune evasion and growth in vivo. Metformin blocks both PD-L1 and PD-1 within the tumor microenvironment. This study provided a mechanistic insight into the efficacy of metformin in improving immunotherapy in human cancer.

Assessment of pDCs functional capacity upon exposure to tumor-derived soluble factors.

Plasmacytoid dendritic cells (pDCs) are a minority subset of dendritic cells that despite their tiny quantity play an important role in the immune system, especially in antiviral immunity. They are known mostly as the major producers of type I IFN, which they secrete upon stimulation of endosomal Toll-like receptors 7 and 9 with viral RNA and DNA. However, the functionality of pDCs is more complex, as they were shown to be also involved in autoimmunity, inflammation, and cancer. In the context of the tumor microenvironment, pDCs mostly show substantial functional defects and thus contribute to establishing immunosuppressive micromilieu. Indeed, tumor-infiltrating pDCs were shown to be predominantly pro-tumorigenic, with reduced ability to produce IFNα and capacity to prime regulatory T cells via the ICOS/ICOS-L pathway. Here we describe in detail a method to assess the functional capacity of pDCs upon exposure to tumor-derived cell culture supernatants. The same technique can be implemented with minimal variations to test any soluble factor's impact on pDC phenotype and function.

Role of myokines and osteokines in cancer cachexia.

Cancer-induced muscle wasting, i.e. cachexia, is associated with different types of cancer such as pancreatic, colorectal, lung, liver, gastric and esophageal. Cachexia affects prognosis and survival in cancer, and it is estimated that it will be the ultimate cause of death for up to 30% of cancer patients. Musculoskeletal alterations are known hallmarks of cancer cachexia, with skeletal muscle atrophy and weakness as the most studied. Recent evidence has shed light on the presence of bone loss in cachectic patients, even in the absence of bone-metastatic disease. In particular, we and others have shown that muscle and bone communicate by exchanging paracrine and endocrine factors, known as myokines and osteokines. This review will focus on describing the role of the most studied myokines, such as myostatin, irisin, the muscle metabolite β-aminoisobutyric acid, BAIBA, and IL-6, and osteokines, including TGF-β, osteocalcin, sclerostin, RANKL, PTHrP, FGF23, and the lipid mediator, PGE2 during cancer-induced cachexia. The interplay of muscle and bone factors, together with tumor-derived soluble factors, characterizes a complex clinical scenario in which musculoskeletal alterations are amongst the most debilitating features. Understanding and targeting the "secretome" of cachectic patients will likely represent a promising strategy to preserve bone and muscle during cancer cachexia thereby enhancing recovery.

Platelet-expressed immune checkpoint regulator GITRL in breast cancer

Owing to their key role in several diseases including cancer, activating and inhibitory immune checkpoint molecules are increasingly exploited as targets for immunotherapy. Recently, we demonstrated that platelets, which largely influence tumor progression and immune evasion, functionally express the ligand of the checkpoint molecule GITR. This immunoreceptor modulates effector functions of T cells and NK cells with its function varying dependent on cellular context and activation state. Here, we provide a comparative analysis of platelet-derived GITRL (pGITRL) in breast cancer patients and healthy volunteers. The levels of pGITRL were found to be higher on platelets derived from cancer patients and appeared to be specifically regulated during tumor progression as exemplified by several clinical parameters including tumor stage/grade, the occurrence of metastases and tumor proliferation (Ki67) index. In addition, we report that pGITRL is upregulated during platelet maturation and particularly induced upon exposure to tumor-derived soluble factors. Our data indicate that platelets modulate the GITR/GITRL immune checkpoint in the context of malignant disease and provide a rationale to further study the GITR/GITRL axis for exploitation for immunotherapeutic intervention in cancer patients.

Glycolytic activation of monocytes regulates the accumulation and function of neutrophils in human hepatocellular carcinoma

Neutrophils are one of the most abundant components in human hepatocellular carcinoma (HCC) and have been shown to play important roles in regulating disease progression. However, neutrophils are very short-lived cells in circulation, and mechanisms regulating their accumulation and functions in HCC are not yet fully understood. Monocytes were purified from non-tumor or paired tumor tissues of patients with HCC, and their production of neutrophil-attracting chemokines was evaluated. Mechanisms regulating the expression of CXCL2/8 by tumor monocytes, and the role of tumor monocyte-derived chemokines and cytokines in modulating neutrophil accumulation and functions were studied with both exvivo analyses and invitro experiments. Monocyte-derived CXCL2 and CXCL8 were major factors in regulating the recruitment of neutrophils into tumor milieus. These chemokines, in addition to tumor-derived soluble factors, could inhibit apoptosis and sustain survival of neutrophils, thus leading to neutrophil accumulation in tumor tissues. Moreover, monocyte-derived TNF-α acted synergistically with tumor-derived soluble factors to induce the production of the pro-metastasis factor OSM by neutrophils. Further, the glycolytic switch in tumor-infiltrating monocytes mediated their production of CXCL2 and CXCL8 via the PFKFB3-NF-κB signaling pathway. Accordingly, levels of PFKFB3, CXCL2/CXCL8 production in monocytes and infiltration of OSM-producing neutrophils were positively correlated in human HCC tissues. Our results unveiled a previously unappreciated link between monocytes and neutrophils in human HCC, identifying possible targets that could be therapeutically exploited in the future. Neutrophils constitute a major but poorly understood component of human hepatocellular carcinoma (HCC). Herein, we unveil a novel mechanism by which metabolic switching in monocytes promotes the accumulation of neutrophilsin the tumors of patients with HCC. Both monocyte-produced chemokines and signals from the tumor microenvironment promote the production of the pro-metastatic factor OSM by neutrophils. These data identify potential targets for immune-based anticancer therapies for HCC.

The Engagement Between MDSCs and Metastases: Partners in Crime.

Tumor metastases represent the major cause of cancer-related mortality, confirming the urgent need to identify key molecular pathways and cell-associated networks during the early phases of the metastatic process to develop new strategies to either prevent or control distal cancer spread. Several data revealed the ability of cancer cells to establish a favorable microenvironment, before their arrival in distant organs, by manipulating the cell composition and function of the new host tissue where cancer cells can survive and outgrow. This predetermined environment is termed “pre-metastatic niche” (pMN). pMN development requires that tumor-derived soluble factors, like cytokines, growth-factors and extracellular vesicles, genetically and epigenetically re-program not only resident cells (i.e., fibroblasts) but also non-resident cells such as bone marrow-derived cells. Indeed, by promoting an “emergency” myelopoiesis, cancer cells switch the steady state production of blood cells toward the generation of pro-tumor circulating myeloid cells defined as myeloid-derived suppressor cells (MDSCs) able to sustain tumor growth and dissemination. MDSCs are a heterogeneous subset of myeloid cells with immunosuppressive properties that sustain metastatic process. In this review, we discuss current understandings of how MDSCs shape and promote metastatic dissemination acting in each fundamental steps of cancer progression from primary tumor to metastatic disease.

Enhanced expression of CD39 and CD73 on T cells in the regulation of anti-tumor immune responses

ABSTRACT Synthesis of extracellular adenosine by the ectonucleotidases CD39 and CD73 represents an important pathway of immune suppression in the tumor microenvironment. Using two mouse models (RET transgenic melanoma and Panc02 orthotopic pancreatic adenocarcinoma), we identified an elevated frequency of ectonucleotidase-expressing T cells in tumors and spleens. Importantly, these ectonucleotidase-positive T cells also showed a pronounced expression of PD-1. Conversely, the PD-1+ T cell subsets in tumors contained substantially larger proportions of ectonucleotidase-expressing cells compared to their counterparts lacking PD-1 expression. Our in vitro experiments showed that the activation of normal T cells resulted in an increase in the CD39 expression. CD39+ and CD73+ T cells displayed effector or memory phenotypes and produced IFN-γ, thereby linking ectonucleotidase expression to T cell effector functions. An accumulation of conventional and regulatory T cells expressing CD39 and/or CD73 was also detected in the peripheral blood of patients with melanoma and pancreatic cancer. Moreover, we demonstrated a significant association between low frequencies of circulating CD73+CD8+ T cells and CD73+CD4+ regulatory T cells and better overall survival of melanoma patients. Tumor-derived soluble factors (in particular, TGF-β) significantly enhanced the frequencies of ectonucleotidase-expressing cells in mice. Our findings suggest that the upregulation of ectonucleotidase expression in T cells promotes extracellular adenosine accumulation and represents an important mechanism of homeostatic immune auto-regulation, which could be hijacked by tumors to evade anti-cancer immunity. Targeting CD39 and CD73 can open new avenues for cancer immunotherapy.

Induction of tumor-specific CD8+ cytotoxic T lymphocytes from na\xefve human T cells by using Mycobacterium-derived mycolic acid and lipoarabinomannan-stimulated dendritic cells

The main effectors in tumor control are the class I MHC molecule-restricted CD8+ cytotoxic T lymphocytes (CTLs). Tumor-specific CTL induction can be regulated by dendritic cells (DCs) expressing both tumor-derived epitopes and co-stimulatory molecules. Immunosuppressive tolerogenic DCs, having down-regulated co-stimulatory molecules, are seen within the tumor mass and can suppress tumor-specific CTL induction. The tolerogenic DCs expressing down-regulated XCR1+CD141+ appear to be induced by tumor-derived soluble factors or dexamethasone, while the immunogenic DCs usually express XCR1+CD141+ molecules with a cross-presentation function in humans. Thus, if tolerogenic DCs can be reactivated into immunogenic DCs with sufficient co-stimulatory molecules, tumor-specific CD8+ CTLs can be primed and activated in vivo. In the present study, we converted human tolerogenic CD141+ DCs with enhanced co-stimulatory molecule expression of CD40, CD80, and CD86 through stimulation with non-toxic mycobacterial lipids such as mycolic acid (MA) and lipoarabinomannan (LAM), which synergistically enhanced both co-stimulatory molecule expression and interleukin (IL)-12 secretion by XCR1+CD141+ DCs. Moreover, MA and LAM-stimulated DCs captured tumor antigens and presented tumor epitope(s) in association with class I MHCs and sufficient upregulated co-stimulatory molecules to prime naïve CD3+ T cells to become CD8+ tumor-specific CTLs. Repeat CD141+ DC stimulation with MA and LAM augmented the secretion of IL-12. These findings provide us a new method for altering the tumor environment by converting tolerogenic DCs to immunogenic DCs with MA and LAM from Mycobacterium tuberculosis.

The Endless Saga of Monocyte Diversity.

Cancer immunotherapy relies on either restoring or activating the function of adaptive immune cells, mainly CD8+ T lymphocytes. Despite impressive clinical success, cancer immunotherapy remains ineffective in many patients due to the establishment of tumor resistance, largely dependent on the nature of tumor microenvironment. There are several cellular and molecular mechanisms at play, and the goal is to identify those that are clinically significant. Among the hematopoietic-derived cells, monocytes are endowed with high plasticity, responsible for their pro- and anti-tumoral function. Indeed, monocytes are involved in several cancer-associated processes such as immune-tolerance, metastatic spread, neoangiogenesis, and chemotherapy resistance; on the other hand, by presenting cancer-associated antigens, they can also promote and sustain anti-tumoral T cell response. Recently, by high throughput technologies, new findings have revealed previously underappreciated, profound transcriptional, epigenetic, and metabolic differences among monocyte subsets, which complement and expand our knowledge on the monocyte ontogeny, recruitment during steady state, and emergency hematopoiesis, as seen in cancer. The subdivision into discrete monocytes subsets, both in mice and humans, appears an oversimplification, whereas continuum subsets development is best for depicting the real condition. In this review, we examine the evidences sustaining the existence of a monocyte heterogeneity along with functional activities, at the primary tumor and at the metastatic niche. In particular, we describe how tumor-derived soluble factors and cell-cell contact reprogram monocyte function. Finally, we point out the role of monocytes in preparing and shaping the metastatic niche and describe relevant targetable molecules altering monocyte activities. We think that exploiting monocyte complexity can help identifying key pathways important for the treatment of cancer and several conditions where these cells are involved.

Glycolytic activation of peritumoral monocytes fosters immune privilege via the PFKFB3-PD-L1 axis in human hepatocellular carcinoma

Programmed cell death 1 ligand 1 (PD-L1) expression on antigen-presenting cells is essential for T cell impairment, and PD-L1-expressing macrophages may mechanistically shape and therapeutically predict the clinical efficacy of PD-L1 or programmed cell death 1 blockade. We aimed to elucidate the mechanisms underlying PD-L1 upregulation in human tumor microenvironments, which remain poorly understood despite the clinical success of immune checkpoint inhibitors. Monocytes/macrophages were purified from peripheral blood, non-tumor, or paired tumor tissues of patients with hepatocellular carcinoma (HCC), and their possible glycolytic switch was evaluated. The underlying regulatory mechanisms and clinical significance of metabolic switching were studied with both ex vivo analyses and in vitro experiments. We found that monocytes significantly enhanced the levels of glycolysis at the peritumoral region of human HCC. The activation of glycolysis induced PD-L1 expression on these cells and subsequently attenuated cytotoxic T lymphocyte responses in tumor tissues. Mechanistically, tumor-derived soluble factors, including hyaluronan fragments, induced the upregulation of a key glycolytic enzyme, PFKFB3, in tumor-associated monocytes. This enzyme not only modulated the cellular metabolic switch but also mediated the increased expression of PD-L1 by activating the nuclear factor kappa B signaling pathway in these cells. Consistently, the levels of PFKFB3+CD68+ cell infiltration in peritumoral tissues were negatively correlated with overall survival and could serve as an independent prognostic factor for survival in patients with HCC. Our results reveal a mechanism by which the cellular metabolic switch regulates the pro-tumor functions of monocytes in a specific human tumor microenvironment. PFKFB3 in both cancer cells and tumor-associated monocytes is a potential therapeutic target in human HCC. Programmed cell death 1 ligand 1 (PD-L1) expressed on antigen-presenting cells, rather than tumor cells, has been reported to play an essential role in checkpoint blockade therapy. A fundamental understanding of mechanisms that regulate the expression of PD-L1 on tumor-infiltrating monocytes/macrophages will undoubtedly lead to the possibility of developing novel PD-L1 blockade strategies with high specificity and efficiency. The current study unveils a novel mechanism by which metabolic switching links immune activation responses to immune tolerance in the tumor milieu, identifying potential targets for future immune-based anti-cancer therapies.

Gemcitabine promotes tumor cell-derived inflammatory responses leading to immunosuppression

Abstract Some chemotherapeutic agents can selectively deplete MDSC in vivo. However, recent studies suggest that Gemcitabine (GEM) and 5-Fluorouracil (5-FU) treatment decrease the anticancer efficacy by activating the inflammasome pathway in MDSC. In addition, chemotherapy can modify the tumor microenvironment by induction of chronic inflammation. Therefore, regulations of MDSC by chemotherapy still remain elusive. Here, we explored the mechanisms by which multi-dose clinical regimen of GEM treatment induce an ongoing inflammation leading to the enhanced immunosuppressive activity of monocytic MDSC (M-MDSC). We found that repeated GEM treatment promoted the expansion and differentiation of immunosuppressive M-MDSC although tumor development was delayed in E0771 tumor-bearing mice. Accordingly, IFN-γ-producing CD4 and CD8 T cells were significantly decreased in the TME of GEM-treated mice. The conditioned medium of GEM-treated tumor cells (GEM-CM) promoted the differentiation of immunosuppressive mouse BM Ly6Chigh cells. Tumor-derived soluble factors, such as GM-CSF and soluble ICAM-1(sICAM-1), were up-regulated upon chemo-drug treatment. Blocking of GM-CSF or ICAM-1 with neutralizing Abs could partially reduce immunosuppression of Ly6Chigh cells induced by GEM-CM. Enhanced production of reactive oxygen species (ROS) and activation of NF-κB were observed in GEM-treated tumor cells. Treatment with the inhibitor of NF-κB signaling could abrogate GEM-induced hyperexpression of GM-CSF in E0771 cells. Together, our results define an undesired effect of GEM-induced ROS which leads to NF-κB activation, up-regulation of GM-CSF, and subsequent differentiation of immunosuppressive myeloid cells.

Paget's "Seed and Soil" Theory of Cancer Metastasis: An Idea Whose Time has Come.

The concept that the pattern of metastatic spread of cancer is not random and that cancer cells exhibit preferences when metastasizing to organs, dates back to 1889 when Steven Paget published his "seed and soil" hypothesis. He proposed that the spread of tumor cells is governed by interaction and cooperation between the cancer cells (seed) and the host organ (soil). Extensive studies during the last several decades have provided a better understanding of the process of metastatic spread of cancer and several stages such as intravasation, extravasation, tumor latency, and development of micrometastasis and macrometastasis have been defined. Furthermore, recent studies have shown that the target organs may be prepared for metastatic deposits by the development of premetastatic niches. This specialized microenvironment is involved in promoting tumor cell homing, colonization, and subsequent growth at the target organ. The premetastatic niche consists of accumulation of aberrant immune cells and extracellular matrix proteins in target organs. The primary tumor plays a key role in the development of premetastatic niches by producing tumor-derived soluble factors which mobilize bone marrow-derived hematopoietic cells to the premetastatic niche. Exosomes-derived from the primary tumor also contribute to cancer-favorable microenvironment in the premetastatic niches. These changes prime the initially healthy organ microenvironment and render it amenable for subsequent metastatic cell colonization.

MiR-130a and miR-145 reprogram Gr-1+CD11b+ myeloid cells and inhibit tumor metastasis through improved host immunity

Tumor-derived soluble factors promote the production of Gr-1+CD11b+ immature myeloid cells, and TGFβ signaling is critical in their immune suppressive function. Here, we report that miR-130a and miR-145 directly target TGFβ receptor II (TβRII) and are down-regulated in these myeloid cells, leading to increased TβRII. Ectopic expression of miR-130a and miR-145 in the myeloid cells decreased tumor metastasis. This is mediated through a downregulation of type 2 cytokines in myeloid cells and an increase in IFNγ-producing cytotoxic CD8 T lymphocytes. miR-130a- and miR-145-targeted molecular networks including TGFβ and IGF1R pathways were correlated with higher tumor stages in cancer patients. Lastly, miR-130a and miR-145 mimics, as well as IGF1R inhibitor NT157 improved anti-tumor immunity and inhibited metastasis in preclinical mouse models. These results demonstrated that miR-130a and miR-145 can reprogram tumor-associated myeloid cells by altering the cytokine milieu and metastatic microenvironment, thus enhancing host antitumor immunity.

Expression of Indoleamine 2,3-Dioxygenase Induced by IFN-γ and TNF-α as Potential Biomarker of Prostate Cancer Progression.

Inflammation has been suggested to play an important role in onset and progression of prostate cancer (PCa). Histological analysis of prostatectomy specimens has revealed focal inflammation in early stage lesions of this malignancy. We addressed the role of inflammatory stimuli in the release of PCa-specific, tumor-derived soluble factors (PCa-TDSFs) already reported to be mediators of PCa morbidity, such as indoleamine 2,3-dioxygenase (IDO) and interleukin (IL)-6. Inflammation-driven production and functions of PCa-TDFSs were tested “in vitro” by stimulating established cell lines (CA-HPV-10 and PC3) with IFN-γ or TNF-α. Expression of genes encoding IDO, IL-6, IFN-γ, TNF-α, and their receptors was investigated in tumor tissues of PCa patients undergoing radical prostatectomy, in comparison with benign prostatic hyperplasia (BPH) specimens. IFN-γ and TNF-α-treatment resulted in the induction of IDO and IL-6 gene expression and release in established cell lines, suggesting that the elicitation of PCa-TDSFs by these cytokines might contribute to progression of cancer into an untreatable phenotype. An analysis based on timing of biochemical recurrence revealed the prognostic value of IDO but not IL-6 gene expression in predicting recurrence-free survival in patients (RFS) with PCa. In addition, a urine-based mRNA biomarker study revealed the diagnostic potential of IDO gene expression in urines of men at risk of PCa development.

The roles of microglia/macrophages in tumor progression of brain cancer and metastatic disease.

Malignant brain tumors and brain metastases are highly aggressive diseases that are often resistant to treatment. Consequently, the current prognosis of patients with brain tumors and metastases is dismal. Activated microglia and macrophages are often observed in close proximity to or within the malignant tumor masses, suggesting that microglia/macrophages play an important role in brain tumor progression. Microglia, being resident macrophages of the central nervous system, form a major component of the brain immune system. They exhibit anti-tumor functions by phagocytosis and the release of cytotoxic factors. However, these microglia/macrophages can be polarized into becoming tumor-supportive and immunosuppressive cells by certain tumor-derived soluble factors, thereby promoting tumor maintenance and progression. The activated microglia/macrophages also participate in the process of tumor angiogenesis, metastasis, dormancy, and relapse. In this review, we discuss the recent literature on the dual roles of microglia/macrophages in brain tumor progression. We have also reviewed the effect of several well-known microglia/macrophages-derived molecules and signals on brain tumor progression and further discussed the potential therapeutic strategies for targeting the pro-tumor and metastatic functions of microglia/macrophages.

SC06.04 Immunotherapy of Malignant Pleural Mesothelioma and Thymic Malignancies: The End of the Beginning?

SC06.04 Immunotherapy of Malignant Pleural Mesothelioma and Thymic Malignancies: The End of the Beginning?

Overview of the Soluble and Membrane-bound Tumor Factors Limiting NKmediated Immune Surveillance

Many evidences suggest that NK cells are effective in patrolling for and eliminating tumors in their onset phase, but hardly limit the progression of large established solid tumors. Beside the transition of tumor cells towards a more aggressive phenotype, the NK cell efficacy might be limited by a complex immunosuppressive milieu present in the tumor microenvironment. Indeed, different mechanisms damping NK cell function have been shown in these last years. These include a plethora of tumor-derived immunomodulatory soluble factors (TGF-β, MIF, adenosine, L-Kynurenin, PGE2) as well as soluble ligands (MICA, ULBP-2, PVR, B7-H6) that compete with membrane-bound tumor ligands for binding to activating NK receptors. During NK-tumor cell contact the NK cell function can also be inhibited by the engagement on NK cells of different inhibitory receptors. The specific ligands might be either constitutively expressed at the tumor cell surface (HLA-I, B7-H3, PVR) or de novo induced/up-regulated (PD-Ls) by immunostimulatory factors (IFN-γ, TNF-α). These are largely released during the active phases of the immune responses and exert an unwonted side effect called “tumor adaptive immune resistance”. This review aims to summarize the best-known molecular mechanisms that, at various times and in different ways, can limit the efficacy of the NK-mediated immune surveillance of tumors.

Abstract C36: Tumor-associated macrophages enhance tumor invasiveness and promote CD4+ T-cell recruitment in Gastric Cancer

Abstract Macrophages are one of the major populations of tumor-infiltrating immune cells. To date, several studies have shown that high tumor-associated-macrophages (TAMs) count (CD68high) or high CD68/CD3 ratio in gastric cancer patients is associated to poor surgical outcome or shorter overall survival. Other than macrophages, high CD4+ T cells (i.e. T helper 2 cells and regulatory T cells) are also associated to poor prognosis. Nevertheless, it is still unclear how these immune cells are recruited to the gastric tumor microenvironment and contributing to cancer progression. We hypothesize that gastric tumor secretes soluble factors to recruit monocytes to the lesion and drives their differentiation into tumor macrophages. These TAMs in turn assist pro-tumor immune-cell infiltration and tumor progression. Gastric cancer (GC) cell lines representing intestinal (OCUM-1, NCI-N87, MKN28, MKN45, SNU-5) and diffuse (AZ521, TMK-1, IST-1, CLS145 and YCC18) subtypes were used. CD14+ monocytes, CD4+CD45RA+ and CD4+CD45RO+ T cells were isolated from healthy blood donors and used to examine the gastric tumor ability to recruit these cells. Normal macrophages (MΦ) and GC-macrophages (GC-MΦ) were derived from CD14+ monocytes respectively with macrophage colony-stimulating factor and various gastric tumor-derived soluble factors. Phenotypes of the derived macrophages were examined by cytokine profiling and cell surface marker expression using flow cytometry. Various GC-MΦ secreted factors were also utilized to study the recruitment of CD4+CD45RA+ and CD4+CD45RO+ T cells. To examine the effect of the GC-MΦ on tumor proliferation and invasiveness, gastric tumor cells were conditioned with various GC-MΦ secreted factors prior to MTS assay and transwell assay. Indeed, we observed a significant 2-fold increase in the migration of CD14+ monocytes towards the various gastric tumor-derived soluble factors. We also observed that gastric tumor-derived soluble factors conditioned monocytes towards M2 phenotype macrophages (CD68+/CD206+/CD163+/IL10high/IL12low). Importantly, soluble factors derived from NCI-N87 and TMK-1-conditioned MΦ, but not normal MΦ, significantly increased the proliferation of NCI-N87 and TMK-1 gastric tumor cells by 26.7±10.4% and 14.5±4.0% respectively. In addition, transmigration of these gastric tumor cells were increased following exposure to their respective GC-MΦ derived soluble factors. Furthermore, we observed that the soluble factors derived from GC-MΦ, but not MΦ, were capable of enhancing the recruitment of both CD4+CD45RO+ (memory) and CD4+CD45RA+ (naïve) T cells. Notably, the memory T cells were more responsive towards the GC-MΦ-derived soluble factors compared to naïve T cells. Taken together, our findings suggest that gastric tumor secretes factors that attract CD14+ monocytes into the tumor lesion and subsequently changing them to M2-like macrophages with pro-tumor properties. These TAMs in turn enhance CD4+ T cell recruitment as well as gastric tumor cell invasion and proliferation. This, therefore, indicates a gastric tumor-dependent influence on immune cells to assist gastric tumor progression. Citation Format: Pin Yan Low, Yaw Chyn Lim, Wei Peng Yong, Bok Yan, Jimmy So. Tumor-associated macrophages enhance tumor invasiveness and promote CD4+ T-cell recruitment in Gastric Cancer. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr C36.

Three-Dimensional Mechanical Loading Modulates the Osteogenic Response of Mesenchymal Stem Cells to Tumor-Derived Soluble Signals.

Dynamic mechanical loading is a strong anabolic signal in the skeleton, increasing osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BM-MSCs) and increasing the bone-forming activity of osteoblasts, but its role in bone metastatic cancer is relatively unknown. In this study, we integrated a hydroxyapatite-containing three-dimensional (3D) scaffold platform with controlled mechanical stimulation to investigate the effects of cyclic compression on the interplay between breast cancer cells and BM-MSCs as it pertains to bone metastasis. BM-MSCs cultured within mineral-containing 3D poly(lactide-co-glycolide) (PLG) scaffolds differentiated into mature osteoblasts, and exposure to tumor-derived soluble factors promoted this process. When BM-MSCs undergoing osteogenic differentiation were exposed to conditioned media collected from mechanically loaded breast cancer cells, their gene expression of osteopontin was increased. This was further enhanced when mechanical compression was simultaneously applied to BM-MSCs, leading to more uniformly deposited osteopontin within scaffold pores. These results suggest that mechanical loading of 3D scaffold-based culture models may be utilized to evaluate the role of physiologically relevant physical cues on bone metastatic breast cancer. Furthermore, our data imply that cyclic mechanical stimuli within the bone microenvironment modulate interactions between tumor cells and BM-MSCs that are relevant to bone metastasis.

Activation of NK and CD8+ T-Cells with a Novel IL-15 and TGF-Beta Receptor Fusion Protein Confers Anti-Tumor Immunity

The use of cytokines as agents to augment immune responses against malignancies have been dealt setbacks due to immune selection of tumors, resulting in subpopulations that elaborate tumor-derived soluble factors, such as transforming growth factor-beta (TGF-β), which suppress immune effector functions. TGF-β is overexpressed by many solid and hematological malignancies and is well known to inhibit the proliferation and anti-tumor functions of lymphomyeloid cells.In order to maximize cytokine-based immunotherapy against tumors, we have designed a novel fusion protein consisting of proinflammatory murine interleukin-15 (IL-15) linked to the sushi domain of the IL-15Rα chain (IL15Rαsushi +IL15) fused in frame to the C'-terminus of a dimeric murine TGF-β-receptor (type II, TβRII) ectodomain-based ligand trap, termed FIST-15 (Fusion of Interleukin 15 with Sushi to TGF-β receptor). The rationale for the design of this protein is to prevent tumor-derived TGF-β from suppressing the immune response via the TGF-β ligand trap moiety, while simultaneously providing a potent stimulus for the activation of anti-tumor responses by an IL-15R agonist (IL-15Rαsushi +IL15).FIST-15 can neutralize TGF-β induced Smad signaling, and induce STAT3 and STAT5 phosphorylation by immunoblot and intracellular flow cytometric analysis of lymphocytes, suggesting that both protein domains are biochemically active. Functionally, FIST-15 is able to induce CD8+ T-cell proliferation at rates greater than IL-15 alone (CD8+ T-cell replicative index or fold-expansion of responding cells: 40, FIST-15, vs. 10, IL-15; p-value of unpaired T-test <0.05). The mitogenic effects of IL-15 are abrogated in CD8+ T-cells and NK cells in the presence of TGF-β. However, FIST-15 can overcome TGF-β mediated inhibition in both these cellular subsets (CD8+ T-cell replicative index: 20, FIST-15, vs. 5, IL-15, and NK cell replicative index: 40, FIST-15 vs. 5, IL-15; p-value <0.05).Rapid proliferation of the CD8+ central memory phenotype (CD62L+, CD44+) T-cells are seen with FIST-15 treatment. Compared to IL-15 expanded CD8+ T-cells, FIST-15 treatment also produced more IFN-γ, TNF-α, and IL-2 secreting CD8+ T-cells upon PMA/ionomycin stimulation. In addition to cytokines, production of anti-tumor effector molecules such as granzyme B is known to be inhibited by TGF-β. FIST-15 treated NK cells were superior to IL-15 treated NK cells in granzyme B production, even in the presence of TGF-β, as assayed by flow cytometric analysis (86.8% vs. 30.7% granzyme B expressing cells). Functionally, FIST-15 treated NK cells were also significantly more cytolytic against TGF-β secreting B16 murine melanoma cells in vitro compared to IL-15 treated NK cells (83.5% killing, FIST-15, vs. 24.4% killing, IL-15).C57Bl/6 mice with pre-established, syngeneic B16 melanoma tumors were treated with FIST-15 to assay the anti-tumor effects of the fusion protein in vivo. Mice receiving FIST-15 showed a significant delay in tumor growth (mean tumor volume: 345mm3) compared to control mice receiving conditioned media (mean tumor volume: 814.12mm3; p-value of paired T-test = 0.02) by day 21 post-tumor implantation. Furthermore, FIST-15 treated mice showed a significant survival advantage compared to control treated mice (80% vs 0%; p-value of log rank test = 0.0019) by day 27 post-tumor implantation. Mice immunized with B16 tumors transduced to express FIST-15 were also protected against subsequent wildtype B16 tumor challenge, suggesting that FIST-15 can trigger an adaptive immune response against tumor.Ongoing work utilizing FIST-15 in murine models of hematological malignancies, such as EL-4 lymphoma and C1498 AML, is currently underway. These models were selected due to their known overexpression of TGF-β isoforms that systemically inhibit endogenous anti-tumor responses, as well as the efficacy of immunotherapeutic agents. Indeed, many hematological malignancies acquire mutations that render them insensitive to the growth-inhibitory effects of TGF-β, where it may then be overexpressed as an oncogene to promote further tumor growth by inhibiting the immune system's anti-tumor capabilities (Dong et al Blood 2006). FIST-15 may present a viable immunotherapeutic strategy for hematological malignancies by combining the immune activating effects of IL-15 with the neutralization of immunosuppressive TGF-β. DisclosuresNg:Emory University: Patents & Royalties. Galipeau:Emory University: Patents & Royalties.