Favor Tumor Growth Research Articles

ISY-29-1 - Scientific basis of new targeted therapies for myeloma

Based on positive clinical benefits with new agents, patients with multiple myeloma (MM) should receive the most effective therapy based on proteasome inhibitors and/or immunomodulatory drugs (IMiDs) as backbone medication to achieve CR with the goal of extended survival. Proteasome inhibitors perturb protein handling pathways to elicit ER stress and disrupt survival signaling. Panobinostat, a pan-deacetylase inhibitor, synergistically augments the interference with protein degradation and thereby MM cell death with proteasome inhibitors, while altering gene expression. IMiDs have multiple proposed mechanisms of action, including degradation of IKZF1 and IKZF3 to decrease IRF4 and cMyc, pro-survival mediators, in MM cells. MM cells alter their surrounding microenvironment through bone destruction, angiogenesis and immune paresis in bone marrow where they colonize, which in turn favors tumor growth and survival, thereby forming a progressive vicious cycle between tumor progression and the microenvironmental alteration. These new agents also target the tumor microenvironment to disrupt the vicious cycle. Besides, proteasome inhibitors and panobinostat exert a bone anabolic effect via suppression of osteoclast activity and stimulation of osteoblast differentiation. New generation IMiDs, lenalidomide and pomalidomide, have immunomodulatory activity to improve immune function and thereby MM cell killing. Of note, these IMiDs enhance ADCC activity against MM cells with anti-CS1 and anti-CD38 therapeutic monoclonal antibodies, and checkpoint blockade-mediated MM cytotoxicity with anti-PD-1 antibody, yielding better outcomes. Due to the incurable nature of MM, however, the development of newer agents is ongoing. Upcoming novel molecular targeting agents with different modes of action and immunotherapeutic agents will be integrated into MM treatment regimens with a great therapeutic impact, and further evolution of the treatment paradigm for MM is eagerly anticipated.

32P - BMP-4 production by bladder cancer cells favors tumor progression and promotes the development of a pro-tumoral immune environment

Aim/Background: Bladder cancer (BC) is a current clinical and social problem. At diagnosis, most patients present non-muscle invasive tumor, characterized by high recurrence rate which may progress to muscle invasive disease and metastatic spreading. BMP-dependent signaling arising from stromal bladder tissue mediates urothelial homeostasis by promoting urothelial cell differentiation. However, the possible role of BMP ligands in BC is still unclear. Methods: We have measure BMP4 expression by bladder cancer cell lines and further characterize the impact of that BMP4 expression upon monocyte/macrophage polarization by in vitro experiments. Results: Using a series of human BC cell lines and patient samples, we observed increased expression of BMP4 in advanced stages and undifferentiated tumors in close correlation with epithelial-mesenchymal transition (EMT). In agreement, the p-Smad 1,5,8 staining in tumors showing EMT signs was reduced in parallel with increased miR-21 and reduced BMPR2 expression. Remarkably, BMP4 expression also correlates with markers of type-2 macrophage differentiation. In vitro experiments shows that both recombinant BMP4 and BMP4-containing conditioned media from BC cell lines favors monocytes differentiation towards macrophages with a M2 phenotype as shown by the expression and secretion of IL-10, together with immunomodulatory effector functions that contributes to create a tumor promoting environment. Conclusions: All these findings suggest that BMP-4 secretion by BC cells provide the M2 signal necessary for pro-tumoral immune environment. In addition, the repression of BMPR2 by miR-21 makes the tumor cells refractory to the pro-differentiating actions mediated by BMP ligands favoring tumor growth. Collectively our data highlight the importance of BMP signaling in bladder tumorigenesis and suggest new potential therapeutic approaches for managing BC. Legal entity responsible for the study: Centro de Investigaciones Energéticas Medioambientales y Tecnológicas (CIEMAT) Funding: MINECO grants SAF2012-34378 and SAF2015-66015-R, Comunidad Autónoma de Madrid grant S2010/BMD-2470 (Oncocycle Program), MSyC grant ISCIII-RETIC RD12/0036/0009 and PIE 15/00076 to JMP Disclosure: All authors have declared no conflicts of interest.

Growth and metastasis of lung adenocarcinoma is potentiated by BMP4-mediated immunosuppression

ABSTRACTCancer cells modulate the recruitment and function of inflammatory cells to create an immunosuppressive microenvironment that favors tumor growth and metastasis. However, the tumor-derived regulatory programs that promote intratumoral immunosuppression remain poorly defined. Here, we show in a KrasLA1/+p53R172HΔg/+-based mouse model that bone morphogenetic protein-4 (BMP4) augments the expression of the T cell co-inhibitory receptor ligand PD-L1 in the mesenchymal subset of lung cancer cells, leading to profound CD8+ T cell-mediated immunosuppression, producing tumor growth and metastasis. We previously reported in this model that BMP4 functions as a pro-tumorigenic factor regulated by miR-200 via GATA4/6. Thus, BMP4‐mediated immunosuppression is part of a larger miR‐200‐directed gene expression program in tumors that promotes tumor progression, which could have important implications for cancer treatment.

24-Hydroxycholesterol participates in pancreatic neuroendocrine tumor development

Cells in the tumor microenvironment may be reprogrammed by tumor-derived metabolites. Cholesterol-oxidized products, namely oxysterols, have been shown to favor tumor growth directly by promoting tumor cell growth and indirectly by dampening antitumor immune responses. However, the cellular and molecular mechanisms governing oxysterol generation within tumor microenvironments remain elusive. We recently showed that tumor-derived oxysterols recruit neutrophils endowed with protumoral activities, such as neoangiogenesis. Here, we show that hypoxia inducible factor-1a (HIF-1α) controls the overexpression of the enzyme Cyp46a1, which generates the oxysterol 24-hydroxycholesterol (24S-HC) in a pancreatic neuroendocrine tumor (pNET) model commonly used to study neoangiogenesis. The activation of the HIF-1α-24S-HC axis ultimately leads to the induction of the angiogenic switch through the positioning of proangiogenic neutrophils in proximity to Cyp46a1+ islets. Pharmacologic blockade or genetic inactivation of oxysterols controls pNET tumorigenesis by dampening the 24S-HC-neutrophil axis. Finally, we show that in some human pNET samples Cyp46a1 transcripts are overexpressed, which correlate with the HIF-1α target VEGF and with tumor diameter. This study reveals a layer in the angiogenic switch of pNETs and identifies a therapeutic target for pNET patients.

Dabigatran antagonizes growth, cell-cycle progression, migration, and endothelial tube formation induced by thrombin in breast and glioblastoma cell lines.

Thrombin activates its G‐coupled seven transmembrane protease‐activated receptor (PAR‐1) by cleaving the receptor's N‐terminal end. In several human cancers, PAR1 expression and activation correlates with tumor progression and metastatization. This provides compelling evidence for the effectiveness of an appropriate antithrombin agent for the adjuvant treatment of patients with cancer. Dabigatran is a selective direct thrombin inhibitor that reversibly binds to thrombin. In this study, we aimed to explore if dabigatran may affect mechanisms favoring tumor growth by interfering with thrombin‐induced PAR‐1 activation.We confirmed that exposure of tumor cells to thrombin significantly increased cell proliferation and this was coupled with downregulation of p27 and concomitant induction of cyclin D1. Dabigatran was consistently effective in antagonizing thrombin‐induced proliferation as well as it restored the baseline pattern of cell cycle protein expression. Thrombin significantly upregulated the expression of proangiogenetic proteins like Twist and GRO‐α in human umbilical vascular endothelial cells (HUVEC) cells and their expression was significantly brought down to control levels when dabigatran was added to culture. We also found that the chemoattractant effect of thrombin on tumor cells was lost in the presence of dabigatran, and that the thrombin antagonist was effective in dampening vascular tube formation induced by thrombin. Our data support a role of thrombin in inducing the proliferation, migration, and proangiogenetic effects of tumor cells in vitro. Dabigatran has activity in antagonizing all these effects, thereby impairing tumor growth and progression. In vivo models may help to understand the relevance of this pathway.

Mesenchymal stromal cells (MSCs) and colorectal cancer: a troublesome twosome for the anti-tumour immune response?

The tumour microenvironment (TME) is an important factor in determining the growth and metastasis of colorectal cancer, and can aid tumours by both establishing an immunosuppressive milieu, allowing the tumour avoid immune clearance, and by hampering the efficacy of various therapeutic regimens. The tumour microenvironment is composed of many cell types including tumour, stromal, endothelial and immune cell populations. It is widely accepted that cells present in the TME acquire distinct functional phenotypes that promote tumorigenesis. One such cell type is the mesenchymal stromal cell (MSC). Evidence suggests that MSCs exert effects in the colorectal tumour microenvironment including the promotion of angiogenesis, invasion and metastasis. MSCs immunomodulatory capacity may represent another largely unexplored central feature of MSCs tumour promoting capacity. There is considerable evidence to suggest that MSCs and their secreted factors can influence the innate and adaptive immune responses. MSC-immune cell interactions can skew the proliferation and functional activity of T-cells, dendritic cells, natural killer cells and macrophages, which could favour tumour growth and enable tumours to evade immune cell clearance. A better understanding of the interactions between the malignant cancer cell and stromal components of the TME is key to the development of more specific and efficacious therapies for colorectal cancer. Here, we review and explore MSC- mediated mechanisms of suppressing anti-tumour immune responses in the colon tumour microenvironment. Elucidation of the precise mechanism of immunomodulation exerted by tumour-educated MSCs is critical to inhibiting immunosuppression and immune evasion established by the TME, thus providing an opportunity for targeted and efficacious immunotherapy for colorectal cancer growth and metastasis.

The administration of drugs inhibiting cholesterol/oxysterol synthesis is safe and increases the efficacy of immunotherapeutic regimens in tumor-bearing mice.

Tumor-derived metabolites dampen tumor-infiltrating immune cells and antitumor immune responses. Among the various metabolites produced by tumors, we recently showed that cholesterol oxidized products, namely oxysterols, favor tumor growth through the inhibition of DC migration toward lymphoid organs and by promoting the recruitment of pro-tumor neutrophils within the tumor microenvironment. Here, we tested different drugs capable of blocking cholesterol/oxysterol formation. In particular, we tested efficacy and safety of different administration schedules, and of immunotherapy-based combination of a class of compounds, namely zaragozic acids, which inhibit cholesterol pathway downstream of mevalonate formation, thus leaving intact the formation of the isoprenoids, which are required for the maturation of proteins involved in the immune cell function. We show that zaragozic acids inhibit the in vivo growth of the RMA lymphoma and the Lewis lung carcinoma (LLC) without inducing side effects. Tumor growth inhibition requires an intact immune system, as immunodeficient tumor-bearing mice do not respond to zaragozic acid treatment. Of note, the effect of zaragozic acids is accompanied by a marked reduction in the LXR target genes Abcg1, Mertk, Scd1 and Srebp-1c in the tumor microenvironment. On the other hand, zoledronate, which blocks also isoprenoid formation, did not control the LLC tumor growth. Finally, we show that zaragozic acids potentiate the antitumor effects of active and adoptive immunotherapy, significantly prolonging the overall survival of tumor-bearing mice treated with the combo zaragozic acids and TAA-loaded DCs. This study identifies zaragozic acids as new antitumor compounds exploitable for the treatment of cancer patients.

Culture supernatants of cervical cancer cells induce an M2 phenotypic profile in THP-1 macrophages

Patients with cervical cancer (CxCa) typically present an infiltrate of tumor-associated macrophages, which is associated with a poor prognosis. We found that CxCa cell lines (HeLa, SiHa, and C-33A) secreted factors involved in regulating tumor growth including IL-6, IL-4, PDGFAA, HGF, VEGF, ANG-2, and TGF-β3. We assessed the effects of culture supernatants from these cell lines on macrophages derived from the THP-1 cell line. Macrophages treated with culture supernatants from CxCa cells developed an M2-like phenotype with expression of CD163, low nitric oxide release, and high secretion of IL-6, PDGFAA, HGF, ANG-2, and VEGF. The macrophages continued to produce PDGFAA, PDGFBB, and VEGF 48h after the CxCa cell culture supernatants were removed. The induction of M2 macrophages in vivo favors tumor growth, angiogenesis, tissue remodeling, and metastasis. These results demonstrated that factors secreted by CxCa cells induced a stable M2 phenotype in THP-1 macrophages.

Abstract 4037: Enhancement of efferocytosis by rhoA kinase inhibitor following doxorubicin treatment synergistically reduces tumor growth

Abstract Massive dying of tumor cells after chemotherapy may promote chronic inflammation which favors tumor growth by uncleared secondary necrotic cells. Given that defects in apoptotic cell clearance have been linked with various inflammatory diseases and autoimmunity, the prompt removal of apoptotic cells by phagocytes is important for anti-tumor immunity. To improve the anti-tumor effect of doxorubicin, we investigated the role of RhoA-kinase (ROCK) inhibitors on efferocytosis by macrophages, which is important for anti-tumor immunity and tissue regeneration. Treatment of bone marrow-derived macrophages (BMDMs) with ROCK inhibitor increased efferocytosis of dying tumor cells by doxorubicin in vitro. ROCK-blockade after doxorubicin treatment group showed effective inhibition of tumor growth and also T cell-mediated elimination of immunogenic tumors more than the doxorubicin monotherapy group in established tumor models. Furthermore, we also confirmed that macrophage depletion abrogated the therapeutic effect of ROCK inhibitor after doxorubicin treatment on tumor growth. Taken together, our findings indicate that ROCK inhibitor following doxorubicin treatment reduced tumor growth via efferocytosis enhancement. Citation Format: Gihoon Nam, In-San Kim. Enhancement of efferocytosis by rhoA kinase inhibitor following doxorubicin treatment synergistically reduces tumor growth. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4037.

Abstract 2452: Exosomes as mediators of paracrine signalling that promote invasive behavior in rhabdomyosarcoma

Abstract Exosomes are small membranous vesicles secreted into body fluids by multiple cell types, including tumor cells. Tumor exosomes contain intact and functional protein, mRNA and miRNA that may alter the cellular environment to favor tumor growth. Rhabdomyosarcoma (RMS) is an aggressive childhood soft tissue tumor, with two distinct subtypes, alveolar (ARMS) and embryonal (ERMS) histology. ARMS tumors are characterized in the majority of cases by a fusion oncoprotein PAX3-FOXO1, thought to be an initiating factor in tumorigenesis. To date, no studies have been performed to investigate the possible role or content of exosomes in paracrine signaling in RMS. In the present study, we isolated and characterized exosomes secreted from RMS cells, using 3 ERMS (fusion oncoprotein negative) and 2 ARMS (fusion oncoprotein positive) cell lines. RNA analysis showed that small RNA were enriched in RMS-derived exosomes, therefore we focused on evaluating the miRNA content. Array expression analysis showed that exosome miRNA clustered together well, and to a higher extent than cellular miRNA, in ARMS cell lines. Similarly, in the 2 ERMS cell lines that had mutant p53, exosome miRNA clustered together and the pattern of expression was very different from the corresponding cellular miRNA. ARMS and ERMS exosome-enriched miRNA were different, and only 2 miRNA were found to be common among both; putative targets are implicated in cancer and inflammation. Functionally, using in vitro assays, we found that both ERMS- and ARMS-derived exosomes significantly increased the cellular migration and invasion of normal human fibroblasts, and had a positive effect on viability and proliferation of both fibroblasts and RMS cells. In vivo, Matrigel plug assay showed that RMS-derived exosomes had a positive effect on the migration and invasion of stromal cells, suggesting a positive role on angiogenesis. We conclude that RMS-derived exosomes can exert specific paracrine effects on recipient cells, enhancing cell viability as well as invasive properties of fibroblasts and stromal cells, and therefore is likely to play a role in promoting RMS angiogenesis and metastasis. Ongoing studies are aimed at directly assessing the role of RMS-derived exosomes in clinical angiogenesis and metastasis, using in vivo murine models, in addition to dissecting the role of specific enriched miRNA on recipient cell biology. Citation Format: Sandra E. Ghayad, Ghina Rammal, Farah Ghamloush, Hussein Basma, Raya Saab. Exosomes as mediators of paracrine signalling that promote invasive behavior in rhabdomyosarcoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 2452.

Sepsis-induced expansion of granulocytic myeloid-derived suppressor cells promotes tumour growth through Toll-like receptor 4.

Severe sepsis remains a frequent and dreaded complication in cancer patients. Beyond the often fatal short-term outcome, the long-term sequelae of severe sepsis may also impact directly on the prognosis of the underlying malignancy in survivors. The immune system is involved in all stages of tumour development, in the detection of transforming and dying cells and in the prevention of tumour growth and dissemination. In fact, the profound and sustained immune defects induced by sepsis may constitute a privileged environment likely to favour tumour growth. We investigated the impact of sepsis on malignant tumour growth in a double-hit animal model of polymicrobial peritonitis, followed by subcutaneous inoculation of MCA205 fibrosarcoma cells. As compared to their sham-operated counterparts, post-septic mice exhibited accelerated tumour growth. This was associated with intratumoural accumulation of CD11b(+) Ly6G(high) polymorphonuclear cells (PMNs) that could be characterized as granulocytic myeloid-derived suppressor cells (G-MDSCs). Depletion of granulocytic cells in post-septic mice inhibited the sepsis-enhanced tumour growth. Toll-like receptor (TLR)-4 (Tlr4) and Myd88 deficiencies prevented sepsis-induced expansion of G-MDSCs and tumour growth. Our results demonstrate that the myelosuppressive environment induced by severe bacterial infections promotes malignant tumour growth, and highlight a critical role of CD11b(+) Ly6G(high) G-MDSCs under the control of TLR-dependent signalling. Copyright © 2016 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Impaired lymphatic function accelerates cancer growth.

Increased lymphangiogenesis is a common feature of cancer development and progression, yet the influence of impaired lymphangiogenesis on tumor growth is elusive. C3HBA breast cancer and KHT-1 sarcoma cell lines were implanted orthotopically in Chy mice, harboring a heterozygous inactivating mutation of vascular endothelial growth factor receptor-3, resulting in impaired dermal lymphangiogenesis. Accelerated tumor growth was observed in both cancer models in Chy mice, coinciding with reduced peritumoral lymphangiogenesis. An impaired lymphatic washout was observed from the peritumoral area in Chy mice with C3HBA tumors, and the number of macrophages was significantly reduced. While fewer macrophages were detected, the fraction of CD163+ M2 macrophages remained constant, causing a shift towards a higher M2/M1 ratio in Chy mice. No difference in adaptive immune cells was observed between wt and Chy mice. Interestingly, levels of pro- and anti-inflammatory macrophage-associated cytokines were reduced in C3HBA tumors, pointing to an impaired innate immune response. However, IL-6 was profoundly elevated in the C3HBA tumor interstitial fluid, and treatment with the anti-IL-6 receptor antibody tocilizumab inhibited breast cancer growth. Collectively, our data indicate that impaired lymphangiogenesis weakens anti-tumor immunity and favors tumor growth at an early stage of cancer development.

MiR-146a Exerts Differential Effects on Melanoma Growth and Metastatization.

Malignant melanoma is the most aggressive form of skin cancer; therefore, it is crucial to disclose its underlying molecular mechanisms. MicroRNAs (miRNAs) are small endogenous noncoding RNAs able to posttranscriptionally downregulate the expression of direct target genes. Using a melanoma progression model, miR-146a was identified as a key double-acting player in melanoma malignancy. In fact, miR-146a is able to enhance tumor growth, while it suppresses dissemination. It was determined that miR-146a coordinated melanoma cell growth by its direct targets lunatic fringe (LFNG) and NUMB, which operate on the NOTCH/PTEN/Akt pathway; while inhibition of metastasis formation was linked to decreased expression of ITGAV and ROCK1. Relevantly, miR-146a expression correlated with melanoma recurrence and was enriched in both patient-derived melanoma and cutaneous metastasis specimens, while its direct targets were depleted. However, miR-146a levels drop in circulating tumor cells (CTCs), suggesting the necessity for miR-146a expression to fluctuate during tumor progression in order to favor tumor growth and allow dissemination. This study reconciles the contradictory biologic functions of miR-146a in melanoma progression and unravels distinct molecular mechanisms that need to be considered for therapeutic interventions. miR-146a controls melanoma progression in a dual way, promoting growth and inhibiting dissemination; however, it is poorly expressed in CTCs, resulting in overall tumor spreading and distant-site colonization. Mol Cancer Res; 14(6); 548-62. ©2016 AACR.

Changing perspective on oncometabolites: from metabolic signature of cancer to tumorigenic and immunosuppressive agents.

During tumorigenesis, the shift from oxidative phosphorylation to glycolysis in ATP production accounts for the dramatic change in the cellular metabolism and represents one of the major steps leading to tumour formation. The so-called Warburg effect is currently considered something more than a mere modification in the cellular metabolism. The paradox that during cancer cell proliferation the increase in energy need is supplied by glycolysis can be only explained by taking into account the many roles that intermediates of glycolysis or TCA cycle play in cellular physiology, besides energy production. Recent studies have shown that metabolic intermediates induce changes in chromatin structure or drive neo-angiogenesis. In this review, we present some of the latest findings in the study of cancer metabolism with particular attention to how tumour metabolism and its microenvironment can favour tumour growth and aggressiveness, by hijacking and dampening the anti-tumoral immune response.

Priming of neutrophils toward NETosis promotes tumor growth

ABSTRACTNeutrophils play a major role in cancer biology and both pro- and antitumoral functions of tumor-infiltrating neutrophils have been described. We have shown that tumors, by releasing G-CSF into the bloodstream, prime circulating neutrophils to form neutrophil extracellular traps (NETs) and we have detected the presence of NETs within the tumor microenvironment. Here, we report, using PAD4-deficient mice with a defect in neutrophil chromatin decondensation and NET formation, that the priming of neutrophils toward NETosis favors tumor growth. Interestingly, in a tumor model that does not release G-CSF and in which neutrophils are not primed for NETosis, PAD4-deficiency did not reduce tumor growth. However, supplying exogenous G-CSF to the wild-type (WT) host promoted intratumoral NETosis and tumor growth. Taken together, our results suggest that the priming of neutrophils for NETosis by the tumor or its environment leads to the accumulation of intratumoral NETs and a growth advantage to the tumor. Our work unveiled a pro-tumoral role for NETs which strengthens their potential as a new target in the fight against cancer.

The sonic hedgehog signaling pathway contributes to the development of salivary gland neoplasms regardless of perineural infiltration.

The pleomorphic adenoma (PA), mucoepidermoid carcinoma (MEC), and adenoid cystic carcinoma (ACC) are common tumors arising from salivary glands whose histopathology is heterogeneous. The sonic hedgehog signaling pathway (Hh) and signal transducer and activator of transcription 3 (STAT3) play important roles in cell proliferation, favoring tumor growth. The aim of this investigation was to study components of the Hh pathway, as well as STAT3 in salivary gland neoplasms in an attempt to add information about the biological characteristics of these neoplasms. We used 9 cases of PA, 17 cases of ACC, and 20 cases of MEC. Using immunohistochemistry, SHH, GLI1, SUFU, HHIP, and STAT3 were investigated. For comparative purposes, MCM3 (cellular proliferation marker) was also included. In PA, there was high expression of cytoplasmic SHH and SUFU and low expression of STAT3 and MCM3. In the ACC, there was high expression of GLI1, HHIP, and STAT3 and low expression of SHH, SUFU, and MCM3. In the MEC, we observed high expression of SHH, GLI1, SUFU, and HHIP and low expression of STAT3 and MCM3. There was a statistically significant difference between SHH (p = 0.0064), STAT3 (p = 0.0003), and MCM3 (p = 0.0257) when all tumors were compared and a higher expression in parenchyma for all tumors when stroma and parenchyma were compared (p < 0.05). These findings suggests a possible role of Hh pathway in the development and maintenance of the cytoarchitectural pattern of PA, ACC, and MEC, as well as the participation of STAT3 in the development of ACC, irrespective perineural infiltration.

Tumor-Associated Macrophages and Neutrophils in Tumor Microenvironment.

Distinct tumor microenvironment forms in each progression step of cancer and has diverse capacities to induce both adverse and beneficial consequences for tumorigenesis. It is now known that immune cells can be activated to favor tumor growth and progression, most probably influenced by the tumor microenvironment. Tumor-associated macrophages and tumor-associated neutrophils can exert protumoral functions, enhancing tumor cell invasion and metastasis, angiogenesis, and extracellular matrix remodeling, while inhibiting the antitumoral immune surveillance. Considering that neutrophils in inflammatory environments recruit macrophages and that recruited macrophages affect neutrophil functions, there may be various degrees of interaction between tumor-associated macrophages and tumor-associated neutrophils. Platelets also play an important role in the recruitment and regulation of monocytic and granulocytic cells in the tumor tissues, suggesting that platelet function may be essential for generation of tumor-associated macrophages and tumor-associated neutrophils. In this review, we will explore the biology of tumor-associated macrophages and tumor-associated neutrophils and their possible interactions in the tumor microenvironment. Special attention will be given to the recruitment and activation of these tumor-associated cells and to the roles they play in maintenance of the tumor microenvironment and progression of tumors.

CD90(+) stromal cells are the major source of IL-6, which supports cancer stem-like cells and inflammation in colorectal cancer.

IL-6 is a pleiotropic cytokine increased in CRC and known to directly promote tumor growth. Colonic myofibroblasts/fibroblasts (CMFs or stromal cells) are CD90(+) innate immune cells representing up to 30% of normal colonic mucosal lamina propria cells. They are expanded in CRC tumor stroma, where they also known as a cancer associated fibroblasts (CAFs). Cells of mesenchymal origin, such as normal myofibroblasts/fibroblasts, are known to secrete IL-6; however, their contribution to the increase in IL-6 in CRC and to tumor-promoting inflammation is not well defined. Using in situ, ex vivo and coculture analyses we have demonstrated that the number of IL-6 producing CMFs is increased in CRC (C-CMFs) and they represent the major source of IL-6 in T2-T3 CRC tumors. Activity/expression of stem cell markers-aldehyde dehydrogenase and LGR5- was significantly up-regulated in colon cancer cells (SW480, Caco-2 or HT29) cultured in the presence of conditioned medium from tumor isolated C-CMFs in an IL-6 dependent manner. C-CMF and its derived condition medium, but not normal CMF isolated from syngeneic normal colons, induced differentiation of tumor promoting inflammatory T helper 17 cells (Th17) cell responses in an IL-6 dependent manner. Our study suggests that CD90(+) fibroblasts/myofibroblasts may be the major source of IL-6 in T2-T3 CRC tumors, which supports the stemness of tumor cells and induces an immune adaptive inflammatory response (a.k.a. Th17) favoring tumor growth. Taken together our data supports the notion that IL-6 producing CAFs (a.k.a. C-CMFs) may provide a useful target for treating or preventing CRCs.

Mesenchymal STEM CELLS Favor Tumor Growth By Generating Granulocyte-like Myeloid Derived Suppressor CELLS in CML Patients

INTRODUCTION. The complex interplay between cancer cells and immune system allows neoplastic cells to evade immune surveillance and expand. Recently, our and another group have demonstrated that a subpopulation of myeloid cells, defined as "granulocytic myeloid-derived suppressor cells" (G-MDSC), plays an important role for immune escape in chronic myeloid leukemia (CML) patients by reducing T cell activation. The aim of this study was to evaluate the influence of Mesenchymal stem cells (MSC) on generation of MDSCs by comparing CML MSCs (n=10) with healthy donors (HD) MSC (n=8).METHODS. G-MDSC (CD11b+CD33+CD14-HLADR- cells) were analyzed in peripheral blood (PB) of 20 healthy donors (HD) and 30 CML patients at diagnosis by cytofluorimetric analysis. Immuno-suppressive activity was tested through incubation of G-MDSC with autologous CFSE-labeled T cells and stimulation with phytohaemagglutinin (PHA). Controls included a positive T cell proliferation control (T cells plus PHA) and a negative one (T cells only). After three days, T cell proliferation was analyzed by flow cytometry. For G-MDSC generation, human peripheral blood mononucleated cells (PBMC) from HD were cultured alone and with MSC of CML (n=10) or HD (n=8) (1:100 ratio). After one week, G-MDSC were isolated using anti-CD66b magnetic microbeads and the phenotype was confirmed by cytofluorimetric analysis. Expression of ARG1, NOS2, PTGS2, TNFα, TGFβ, IL6, IL10, IL1β was also evaluated using real time PCR.RESULTS. Percentage of cells with a G-MDSC phenotype was greater in PB obtained from CML patients than HD (82.5±9.6% vs 56,2±5.4%, p<0.0001). G-MDSC were able to inhibit T cell proliferation compared to positive control (25±5% vs 48±7.6%, p=0.0057). To investigate if CML MSC may be involved in G-MDSC generation, we incubated HD PBMC with CML or HD MSC for one week. After magnetic isolation, we found that only CML MSC-educated G-MDSC acquired immune-suppressive ability, inhibiting T cell proliferation compared to G-MDSCs control (isolated from PBMC cultured in medium alone) (32±12% vs 63±5.9%, p=0.003). On the contrary, HD MSC-educated G-MDSC did not show any suppressive effect. We also found that CML MSC-educated G-MDSC expressed higher level of the following immune modulatory factors: TNFα (20.8±19.3, p=0.006), IL1β (47.3±25.2, p=0.001), PTGS2 (20.7±10.9, p=0.002) and IL6 (33.8±13.9, p=0.004) compared to HD MSC-educated G-MDSCs (arbitrarily 2-ΔΔCt value: 1). MSC WE also observed ane an up-regulation of PTGS2 (19±4.4, p=0.04), TGFβ (6±3, p=0.01) and IL6 (5±2.8, p=0.04) in CML MSCs at time 0 with a great variability among the patients (calculated value of 2-ΔΔCt in HD MSC was 1). After 48 h of co-culture with PBMC, CML MSC showed statistically significant up-regulation of ARG1 (23.5±11.9, p=0.02), TGFβ (4.8±3, p=0.04), IL10 (5.6±2.8, p=0.03) and IL6 (54.3±23, p=0.02) expression, suggesting that multiple mechanisms are involved in MDSC induction by CML MSC.CONCLUSION. Our work demonstrates that CML MSCs are able to activate MDSCs favoring cancer immune evasion in CML patients. DisclosuresPalumbo:Novartis: Honoraria, Other: Advisory Board.

Abstract B202: Combined therapy of temozolomide and PF-562271, a PYK2 inhibitor, reduces glioma tumor growth and dispersal compare to temozolomide monotherapy

Abstract Microglia infiltrate majority of gliomas and release factors, which favor tumor growth and dispersal. Previously we demonstrated that microglia residing within the tumor stimulate glioma cell dispersal through the proline rich tyrosine kinase 2 (Pyk2) signaling cascade (Rolon-Reyes et al. 2015). We hypothesize that the use of pharmacological inhibitors of Pyk2 in applied therapy can significantly reduce the dispersal of tumor cells and improve the outcome of treatment. In the present study, we investigated both in vitro and in vivo, the effect of the combined treatment of glioblastoma with temozolomide (TMZ) together with PF-562271 (a Pyk2 and FAK inhibitor), vs. TMZ monotherapy. Proliferation and migration assays were performed for a number of primary human glioma cell lines, as well as U87 and HS683cell lines in the presence and absence of microglia. Tumor size and invasion area were evaluated in brains of GL261 tumor bearing C57BL/6 mice for the assessment of the effectiveness of treatment. Our data demonstrate that PF-562271 (16nM) and TMZ (100μM) given alone reduced in vitro migration and proliferation of glioma cells in the presence of microglia. PF-562271 (16nM) given in combination with TMZ at a concentration of just 10μM provided the same effect as TMZ 100 μM along, without reduction of efficacy. In vivo experiments demonstrated that oral administration of PF-562271 (twice/daily, 25 mg/kg, orally) reduced invasion of glioma cells at the tumor edge, while TMZ (50 mg/kg, once/day, orally) reduced the tumor growth. Combined treatment revealed significantly more prominent effect on both tumor growth and invasion compare to monotherapy. These data indicate that the combination of TMZ and PF-562271 reduces both tumor growth and dispersal of tumor cells to surrounding areas. Moreover, in vitro experiments revealed a complex effect of these drugs on glioma cell proliferation and migration, allowing the reduction of the applied concentration of TMZ without the loss of its therapeutic effect. This research was made possible by NIH grant numbers: 1SC2GM102040-01A1, 8G12MD007583-27, Title V PPOHA grant number P031M105050, NIH NIMH R25 MH055929 and RO1-CA18382701A1. Citation Format: Kimberleve Rolon-Reyes, Luis A. Cubano, Alfredo Quiñones-Hinojosa, Lilia Kucheryavykh. Combined therapy of temozolomide and PF-562271, a PYK2 inhibitor, reduces glioma tumor growth and dispersal compare to temozolomide monotherapy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B202.