Role Of Tumor-associated Macrophages Research Articles

The distribution of intratumoral macrophages correlates with molecular phenotypes and impacts prognosis in colorectal carcinoma.

The role of tumour-associated macrophages (TAMs) in colorectal cancer (CRC) remains elusive. In this study, we aimed to examine the correlation between TAMs, clinicopathological features, tumour-infiltrating lymphocytes (TILs) and prognosis in CRC by the use of image analysis. Immunohistochemical staining for CD68 and CD163 was performed as pan-macrophage and M2-macrophage markers, respectively. Each marker was analysed separately for intra-epithelial and stromal area densities. All four macrophage densities showed a significant correlation with one another (P=0.001). Intra-epithelial CD68+ macrophage densities showed a correlation with pTNM stage (P=0.008), microsatellite instability (MSI) (P<0.001), CpG island methylator phenotype (CIMP) (P<0.001) and TIL densities (P<0.001). Intra-epithelial CD163+ macrophage densities wereassociated with perineural invasion, MSI, CIMPandTIL densities (P<0.001). Stromal CD68+ and CD163+ macrophage densities had a significantrelationship with intra-epithelial and stromal CD3+ (P=0.001 and P<0.001, respectively) and CD8+ (P<0.001) T cells. High intra-epithelial CD68+ macrophage density was associated with worse overall survival (HR=1.386, 95% CI=1.043-1.843, P=0.025) and progression-free survival (HR=1.522, 95% CI=1.146-2.020, P=0.004). Intra-epithelial CD68+ macrophage density was also an independent prognostic factor of the progression-free survival (HR=1.447, 95% CI=1.076-1.947, P=0.015) of CRC patients regardless of pTNM stage, lymphatic, venous, and perineural invasions and TIL densities. Our results indicate that the density of intratumoural macrophages is a useful prognostic indicator for further stratifying T cell populations in CRC.

Abstract 4742: Role of tumor-associated macrophages in esophageal cancer: PD-L1 expression and prognosis

Abstract Introduction: It has become clear that macrophages are important cells forming cancer microenvironment and tumor-associated macrophages (TAM) promote their proliferation in various cancer types. In particularly, M2-type macrophages have been reported to promote the production of anti-inflammatory factors and the infiltration of regulatory T cells and suppress anti-tumor immunity, and new antitumor therapies that control tumor growth by controlling TAM have been proposed. In addition, "anti-PD-1 antibody" has attracted attention also in esophageal cancer, but the relationship between TAM and PD-L1 expression has not been clarified. Methods: A retrospective study was performed on 301 patients who underwent esophagectomy for esophageal cancer between March 2005 and June 2013. We performed immunohistochemistry and evaluation of macrophage markers (CD163 and CD204) and PD-L1. In addition, we performed in vitro experiments to clarify the cellular malignancy and relationship with PD-L1 expression. Results: Out of 301 cases of esophageal cancer, 156 cases showed CD163 high expression (51.8%) while 146 cases showed CD204 high expression (48.5%). We divided into three groups of TAM status based on CD204 and CD163 expression (CD163 and CD204 both positive: TAM status high (n=100), either one positive: moderate (n=102), both negative: low (n=99)). In TAM high group, there were more advanced cancers and adenocarcinoma cases (P&amp;lt;0.0001, P=0.042, respectively). Interestingly, TAM status correlated with PD-L1 expression (P=0.0005). We found that there was a significant difference in overall survival, cancer specific survival and disease-free survival among these three groups (log rank P = 0.0062, P= 0.0012, P= 0.0006, respectively), and TAM status correlated with poor prognosis. We performed double immunostaining of PD-L1 and CD163, there is a part where PD-L1 is expressed around TAM, suggesting that the expression of PD-L1 and the presence of TAM are correlated. We performed co-culture of esophageal cancer cell lines (TE-8 and KYSE30) and activated macrophage. In real time PCR PD-L1 expression were elevated at co-culture with activated macrophage. Fluorescence activated cell sorting (FACS) revealed PD-L1 expression positive cells were increased at co-culture with activated macrophage. In invasion assay, when co-cultured with activated macrophages, the number of infiltrating cells were significantly increasing (P=0.00021). In timelapse imaging showed increased cell activity by co-culture with activated macrophage. Conclusion: This study suggested that TAM correlated with poor prognosis in patients with esophageal cancer. In addition, TAM correlated with invasive capacity and migratory capacity. Importantly, TAM correlated PD-L1 expression in esophageal cancer cell lines. Citation Format: Taisuke Yagi, Yoshifumi Baba, Yuki Koga, Tomoyuki Uchihara, Yuki Kiyozumi, Hiroshi Sawayama, Yukiharu Hiyoshi, Masaaki Iwatsuki, Yuji Miyamoto, Naoya Yoshida, Hideo Baba. Role of tumor-associated macrophages in esophageal cancer: PD-L1 expression and prognosis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4742.

PO-285 Role of tumour associated macrophages (TAMs) in regulation of cancer stem cell (CSCs) enrichment in breast cancer

IntroductionStromal cells present in the tumour microenvironment play an important role in tumour growth and progression. Studies have shown increased infiltration of tumour associated macrophages (TAMs) correlated with increased malignancy of cancer. In our study, we have explored the macrophage and cancer cell interaction, in polarisation of macrophages and their effect on cancer stem cell (CSC) enrichment in breast cancer.Material and methodsWe treated macrophages (Raw 264.7) with conditioned medium (CM) of mice breast cancer cells (4 T1) and observed for the polarisation by analysing M2 macrophage marker CD206 through flow cytometry and immunofluorescence. Further, we studied the enrichment of breast CSCs in response to activated/polarised macrophage CM by analysing CSC specific markers ALDH1 activity and Sca-1 expression by flow cytometry and expression of CSC specific transcription factors sox-2, oct-3/4 and nanog using immunoblotting as well as immunofluorescence. Moreover, breast tumour growth in response to activated macrophage CM was assessed by developing orthotopic breast cancer models in Balb/c mice.Results and discussionsWe have shown that treatment with CM of breast cancer cells polarises macrophages into M2 type which is a prevalent phenotype in TAM population. Interestingly, treatment with CM of tumour cell activated macrophages in breast cancer cells has shown increased ALDH1 activity and Sca-1 expression along with CSC associated transcription factors Sox-2, Oct3/4 and Nanog, indicating the role of TAMs in CSC enrichment. Jak-Stat pathway has also been upregulated in TAM CM treated breast cancer cells and inhibition of Stat-3 by its specific inhibitor resulted in abrogation of CSC enrichment. Further, intra-tumoral injection of activated macrophage CM enhanced the breast tumour growth in Balb/c mice depicting the role of tumour activated macrophage in breast tumour promotion.ConclusionWe observed that breast cancer cells polarise macrophages towards TAM phenotype that further promotes breast tumour progression by enhancing CSC phenotype.

The role of tumor-associated macrophages on tumor progression in non-small cell lung carcinoma

Amaç: Akciğer kanseri tüm dünyada kanserden ölümlerin en sık sebebidir. Küçük&#x0D; hücreli dışı akciğer karsinomları (KHDAK) da en sık görülen akciğer kanser&#x0D; tipidir. Bu çalışmada küçük hücreli dışı akciğer karsinomlarında, tümör&#x0D; ilişkili makrofajların (TAM) tümör progresyonu ve metastaz üzerindeki etkileri&#x0D; araştırıldı.&#x0D; &#x0D; Gereç ve&#x0D; Yöntem: Ocak 2011 ile Mayıs 2016 tarihleri arasında&#x0D; Başkent Üniversitesi Tıp Fakültesi Patoloji Anabilim Dalında KHDAK tanısı alan&#x0D; 80 olgu çalışmaya dahil edildi. Olguların klinik takip bulguları kaydedildi ve&#x0D; tüm olgulara immünhistokimyasal olarak CD 68 antikoru uygulandı.&#x0D; &#x0D; Bulgular: Olguların 36’sı (%45) skuamöz hücreli karsinom (SHK), 36’sı (%45)&#x0D; adenokarsinom, 6’sı (%7.5) büyük hücreli karsinom ve 2’si de (%%2.5)&#x0D; adenoskuamöz karsinom idi. Yapılan immünhistokimyasal çalışma sonucunda, 28&#x0D; olgunun (%35) tümör dokusunda 1+, 21 olgunun (%26.3) tümör dokusunda 2+ ve 31&#x0D; olgunun (%38.8) tümör dokusunda ise 3+ TAM yoğunluğu izlendi. Evre 1 olan 32&#x0D; hastanın yalnızca 1 tanesi (%3.1) 3+ TAM yoğunluğuna sahipken, evre 2 olan 32&#x0D; hastanın 17’sinde (%53.1) ve evre 3+4 olan 16 hastanın ise 13’ünde (%81.3) 3+&#x0D; TAM yoğunluğu saptandı. TAM yoğunluğu 1+ olan hastaların ortalama yaşam ömrü 39.7±17.4&#x0D; ay iken, TAM yoğunluğu 2+ olan hastaların 36.6±17.1 ay ve TAM yoğunluğu 3+ olan&#x0D; hastaların ise 27.3±18.4 aydı (p&amp;lt;0.05).&#x0D; &#x0D; Sonuç: Bu çalışmada, patolojik T evresi ve TNM evresi yüksek olan KHDAK’da TAM&#x0D; sayısının daha yüksek olduğu görüldü ve yüksek TAM sayısı olan hastaların&#x0D; ortalama sağkalım süresinin de, düşük TAM sayısı olan hastaların sağkalım&#x0D; süresinden kısa olduğu saptandı.

Integrated microRNA and mRNA expression profile analysis of tumor-associated macrophages after exposure to single-dose irradiation

BackgroundRadiotherapy (RT) is a common approach that accounts for nearly 50% of cancer patient treatment and has the potential for long-term tumor control. Recently, we published a research article on gene expression profiling of tumor-associated macrophages (TAM) that were exposed to ionizing radiation (IR). Single-dose irradiation of tumors could initiate differentially expressed genes in TAM as a time series from irradiated tumors that are associated with the immune response. It is also well known that human cancers are associated with microRNA (miRNA) alterations that are involved in cancer progression. However, the role of miRNA on TAM after exposure to irradiation remains unclear. ResultsIn this study, miRNA expression profiles from microarrays were used to identify the key miRNAs and correlating pathways involved in the role of TAMs in tumor progression and recurrence after RT. Using a mouse tumor model, we identified miRNA pattern changes over time in response to irradiation. Based on our results, we hypothesize that miRNA expression in the irradiated tumor may be used as a distinguishing marker to indicate the best time for therapeutic intervention to prevent tumor recurrence after RT. ConclusionsWe established a murine model irradiated with a single dose of 25 Gy that could initiate temporal changes in the expression of miRNAs associated with cell proliferation and the immune response, as evidenced by macrophages directly extracted from irradiated tumors after two weeks of IR. Statistical analyses were conducted by comparing the miRNA expression in macrophages from non-irradiated versus irradiated tumors. Thus, our study could lead to a better understanding of the function of miRNA expressions, which changed temporally in an irradiated tumor microenvironment.

Tumor-Associated Macrophages: Therapeutic Targets for Skin Cancer.

Tumor-associated macrophages (TAMs) and regulatory T cells (Tregs) are significant components of the microenvironment of solid tumors in the majority of cancers. TAMs sequentially develop from monocytes into functional macrophages. In each differentiation stage, TAMs obtain various immunosuppressive functions to maintain the tumor microenvironment (e.g., expression of immune checkpoint molecules, production of Treg-related chemokines and cytokines, production of arginase I). Although the main population of TAMs is immunosuppressive M2 macrophages, TAMs can be modulated into M1-type macrophages in each differential stage, leading to the suppression of tumor growth. Because the administration of certain drugs or stromal factors can stimulate TAMs to produce specific chemokines, leading to the recruitment of various tumor-infiltrating lymphocytes, TAMs can serve as targets for cancer immunotherapy. In this review, we discuss the differentiation, activation, and immunosuppressive function of TAMs, as well as their benefits in cancer immunotherapy.

Folate receptor-α targeted near-infrared fluorescence imaging in high-risk endometrial cancer patients: a tissue microarray and clinical feasibility study.

ObjectiveDetection and resection of all malignant lesions is pivotal in staging and cytoreductive surgery (CRS) of endometrial cancer (EC). Intraoperative EC detection could be enhanced using OTL-38, a fluorescent-labelled folate receptor-α (FRα) targeted imaging agent. The objectives of this study were to investigate which subgroups of high-risk EC patients express FRα and assess feasibility of intraoperative EC detection using OTL-38.ResultsFRα expression on TMA was significantly correlated with tumor type (p < 0.01). Eighty-two percent of serous and clear cell carcinomas showed FRα expression. Four patients were enrolled in the clinical study. Using fluorescence imaging all omental (n = 3) and lymph node (LN) metastases (n = 16) could be clearly identified, including one otherwise undetected omental metastasis. However, false-positive fluorescence was identified in 17/50 non-metastatic LNs, caused by OTL-38 targeting of FRβ, expressed by tumor-associated activated macrophages.ConclusionsThis study describes high FRα expression in serous and clear cell EC and demonstrates the first experience of intraoperative FRα-targeted tumor detection in patients with these subtypes of EC. Although all metastases could be clearly identified using OTL-38, the role of tumor-associated macrophages should be further evaluated.MethodsImmunohistochemical (IHC) staining of FRα expression was performed on tissue micro arrays (TMA) of 116 patients with high-risk EC features. Patients with either serous or clear cell EC, planned for staging or CRS, were eligible for inclusion in the clinical study and received an intravenous dose of 0.0125 mg/kg OTL-38, 2-3 hours prior to surgery. Resected lesions, identified by standard-of-care and/or fluorescence imaging, were histopathologically assessed for FRα and tumor status.

Role of tumor-associated macrophages in hematological malignancies

Macrophages constitute an important component of the microenvironment. They exhibit a remarkable plasticity in their phenotypes and functions. Recent studies have not only revealed the morphological, phenotypic, and functional characteristics of different macrophage subpopulations under physiological conditions but also clarified the mechanisms of alteration in macrophages under pathological conditions. Macrophages present in the tumor microenvironment are known as tumor-associated macrophages (TAMs), which are involved in tumor proliferation, angiogenesis, invasion, metastasis, and chemotherapy resistance. In hematological malignancies, macrophages infiltrate into the tissues that are invaded by myeloma, lymphoma, leukemia, and other malignant cells, acquire a specific activated phenotype, and participate in the progression of the disease. The term TAMs is generally used in myeloma and lymphoma, and in leukemia, it is modified as leukemia-associated macrophages (LAMs), acute leukemia-associated macrophages (AAMs), or “nurse-like cells” (NLCs). This review summarizes the knowledge regarding the progression of macrophages in hematological malignancies.

Tumor-associated macrophages promote neuroblastoma via STAT3 phosphorylation and up-regulation of c-MYC.

Tumor-associated macrophages (TAMs) are strongly associated with poor survival in neuroblastomas that lack MYCN amplification. To study TAM action in neuroblastomas, we used a novel murine model of spontaneous neuroblastoma lacking MYCN amplification, and observed recruitment and polarization of TAMs, which in turn enhanced neuroblastoma proliferation and growth. In both murine and human neuroblastoma cells, we found that TAMs increased STAT3 activation in neuroblastoma cells and transcriptionally up-regulated the MYC oncogene. Analysis of human neuroblastoma tumor specimens revealed that MYC up-regulation correlates with markers of TAM infiltration. In an IL6ko neuroblastoma model, the absence of IL-6 protein had no effect on tumor development and prevented neither STAT3 activation nor MYC up-regulation. In contrast, inhibition of JAK-STAT activation using AZD1480 or the clinically admissible inhibitor ruxolitinib significantly reduced TAM-mediated growth of neuroblastomas implanted subcutaneously in NOD scid gamma mice. Our results point to a unique mechanism in which TAMs promote tumor cells that lack amplification of an oncogene common to the malignancy by up-regulating transcriptional expression of a distinct oncogene from the same gene family, and underscore the role of IL-6-independent activation of STAT3 in this mechanism. Amplification of MYCN or constitutive up-regulation of MYC protein is observed in approximately half of high-risk tumors; our findings indicate a novel role of TAMs as inducers of MYC expression in neuroblastomas lacking independent oncogene activation.

Macrophages/microglia in glioblastoma: a Zelig-like story of changing phenotypes

In the framework of an increasing number of human cancers being effectively tackled by novel and innovative therapeutic approaches, high-grade glioblastoma (GBM) multiforme stands as a most difficult task for our hopes of effective therapies, or cures. GBM usually localizes at the level of subcortical white matter, in the cerebral hemispheres. Probably because the tumor arises within an immunological sanctuary, immune responses elicited by GBM appear to involve mostly the innate immune system: up to 50% of human GBM tumor mass is represented by tumor-associated macrophages (TAMs), which include both resident microglia and bone marrow-derived macrophages. The source of TAMs, the relative contribution of resident microglia and systemic macrophages, as well as the putative role of TAMs in tumor growth are currently a matter of thorough investigation and dispute.

Gene expression profiling of tumor-associated macrophages after exposure to single-dose irradiation

Radiotherapy (RT) is a common cancer treatment approach that accounts for nearly 50% of patient treatment; however, tumor relapse after radiotherapy is still a major issue. To study the crucial role of tumor-associated macrophages (TAMs) in the regulation of tumor progression post-RT, microarray experiments comparing TAM gene expression profiles between unirradiated and irradiated tumors were conducted to discover possible roles of TAMs in initiation or contribution to tumor recurrence following RT, taking into account the relationships among gene expression, tumor microenvironment, and immunology. A single dose of 25Gy was given to TRAMP C-1 prostate tumors established in C57/B6 mice. CD11b-positive macrophages were extracted from the tumors at one, two and three weeks post-RT. Gene ontology (GO) term analysis using the DAVID database revealed that genes that were differentially expressed at one and two weeks after irradiation were associated with biological processes such as morphogenesis of a branching structure, tube development, and cell proliferation. Analysis using Short Time-Series Expression Miner (STEM) revealed the temporal gene expression profiles and identified 13 significant patterns in four main groups of profiles. The genes in the upregulated temporal profile have diverse functions involved in the intracellular signaling cascade, cell proliferation, and cytokine-mediated signaling pathway. We show that tumor irradiation with a single 25-Gy dose can initiate a time-series of differentially expressed genes in TAMs, which are associated with the immune response, DNA repair, cell cycle arrest, and apoptosis. Our study helps to improve our understanding of the function of the group of genes whose expression changes temporally in an irradiated tumor microenvironment.

Tumor-associated macrophages promote Ezrin phosphorylation-mediated epithelial-mesenchymal transition in lung adenocarcinoma through FUT4/LeY up-regulation.

Tumor-associated macrophages (TAMs) are key components of tumor microenvironment (TME) during tumorigenesis and progression. However, the role of TAMs in lung adenocarcinoma is still unclear. In this study, we aimed to clarify the mechanism underlying the crosstalk between TAMs and epithelial-mesenchymal transition (EMT) of lung adenocarcinoma. Fucosyltransferase IV (FUT4) and its synthetic cancer sugar antigen Lewis Y (LeY) was aberrantly elevated in various solid tumors, it plays critical role in the invasion and metastasis. Here, we found that in lung adenocarcinoma samples, the density of TAMs correlates with E-cadherin level and LeY level. In vitro assays, M2 macrophages promoted FUT4/LeY expression through the transforming growth factor-β1(TGF-β1)/Smad2/3 signaling pathway. FUT4/LeY was indispensable in M2 macrophages-mediated cytoskeletal remodeling and EMT. Furthermore, fucosylation of Ezrin mediated by FUT4/LeY can promote the phosphorylation of Ezrin, which was the critical mechanism of M2 macrophages-induced EMT. In vivo assays confirmed that M2 macrophages promoted EMT through the up-regulation of LeY and phosphorylated Ezrin. Together, our results revealed that TAMs promote Ezrin phosphorylation-mediated EMT in lung adenocarcinoma through FUT4/LeY- mediated fucosylation. Targeting this newly identified signaling may offer new possibilities for immunotherapy in lung adenocarcinoma.

Role of tumor-associated macrophages in renal cell carcinoma pathogenesis

The role of tumor stroma in malignant tumor pathogenesis cannot be disputed. Macrophages are one of the crucial elements of tumor stroma. Tumor-associated macrophages (TAMs) are type 2-activated macrophages (M2). They were first described in 1992. They carry CD206, CD163, FXIIIa, βIG-H3, stabilin 1, YKL-39, SI-CLP, tenascin С, LOX-1, fibronectin, MARCO, interleukin 1 receptor antagonist (IL-1RA) and other markers. Unlike proinflammatory macrophages (M1), М2 display high anti-inflammatory activity and are responsible for inflammation reaction suppression and tissue recovery in inflamed area. TAMs significantly contribute to tumor progression by stimulating cell proliferation, angiogenesis, and suppression of antitumor immune response. Identification of macrophages in renal tumors involves a limited number of markers, which doesn’t allow making a conclusive answer about their function. However, a correlation between TAMs content and a negative disease prognosis can be considered proven. Studies of M1 and M2 using different markers have shown that renal tumors contain high levels of TAMs with mixed M1/M2 phenotype. TAMs in renal tumors are highly proangiogenic and immunosuppressive. TAMs density can be used as a prognostic marker, but development of an effective treatment strategy aimed at inhibition of TAMs antitumor activity requires systemic research involving a wide panel of M1 and M2 macrophage markers.

High-Throughput Characterization and New Insight into the Role of Tumor Associated Macrophages (TAMs) in Multiple Myeloma (MM)

The advent of immunotherapy in MM urges the need for in-depth knowledge about immune cells towards improved characterization of patients' immune profiles. Based on few studies, TAMs were suggested to be abundant in the MM tumor microenvironment where they promote cell growth and chemoresistance. However, further investigation about TAMs is warranted because i) their potential to induce chemoresistance was not determined tacking into account the effect of bone marrow (BM) stroma; ii) their chemoprotective role may be altered by the introduction of monoclonal antibodies (mAb) that target MM cells through ADCP; and iii) TAMs have been characterized with low-throughput flow cytometry, and recent studies in other clinical settings have suggested that novel markers may be more informative to characterize TAMs.Here, we started by producing human M2-like macrophages (ie. phenotypically similar to TAMs in MM) and investigated their contribution to chemoresistance, with or without MM patients’ derived BM stroma (Panels A&B). In contrast to BM stroma, human derived M2-like macrophages were not capable to protect MM (RPMI-8226 and MM1S) cells against bortezomib or dexamethasone. Furthermore, whereas macrophages showed a trend (2.4 fold change, P=.06) to increase baseline MM cell growth (ie. Control in Panels A&B), it became irrelevant upon adding BM stroma to the culture (1.1 fold change, P=.52). No significant differences were observed in the presence of daratumumab or isatuximab, including ADCP that could have been potentially induced by the two anti-CD38 mAbs.Afterwards, based on novel 8-color multidimensional flow cytometry mAb combinations and automated maturation tools, we characterized with high-throughput resolution the BM monocytic/macrophage system by identifying up to 7 different maturation related cell subsets: monoblasts I, II and III, promonocytes, classic and intermediate monocytes, and three novel macrophages subsets identified according to SLAN expression, amongst others (Panels C&D). Accordingly, upon sensitive FACS-sorting we demonstrated that the 3 macrophage subsets had unique transcriptomes (Panel E) consistent with lower immune suppressive signatures from SLAN- into SLAN+ macrophages (eg. down-regulation of VCAN, ENTPD1 and STAB1).We then investigated whether the monocytic/macrophage BM system was truly altered in newly-diagnosed MM patients (n=30) by comparing it to that of healthy donors (HD; n=15). Our results show that total monocytic cells were increased in MM (although the distribution of different monocytic subsets was identical to HD); total intermediate monocytes were also increased in MM (Panel F). By contrast, the percentage of macrophages was identical between HD and MM patients but their subset distribution was significantly altered in MM, and was consistent with an accumulation of more immune suppressive SLAN- TAMs. Next, we investigated whether treatment altered TAMs composition by analyzing 10 patient longitudinal samples collected at diagnosis, after VRD induction, and after HDT/ASCT. Interestingly, we observed that while TAMs remained unaltered after VRD induction, there was a marked shift in their distribution after HDT/ASCT with an even greater accumulation of immune suppressive SLAN- TAMs (P=.08), at expenses of reduced numbers of SLAN+ TAMs (P=.01). Furthermore, chemoresistant MRD-positive patients (n=7/10) after HDT/ASCT showed a 2-fold increment in immune suppressive SLAN- TAMs vs MRD-negative cases.In conclusion, by taking into account the effect of MM patients' BM stroma we showed that TAMs do not induce significant tumor growth. TAMs do not protect MM cells from classic drugs, and MM cells with normal CD38 levels (ie. non lentiviral-transduced to express abnormally high levels of CD38) were not targeted by anti-CD38 mAbs via ADCP. We also showed that there is an expansion of classic and intermediate monocytes but not TAMs in MM; however, using multidimensional flow cytometry to identify novel TAM subsets we revealed that newly diagnosed patients are enriched with immune suppressive TAMs that become further expanded after therapy. Thus, improved monitoring of TAMs could help identifying patients less susceptible to benefit from immunotherapy due to an immune suppressive tumor microenvironment. [Display omitted] DisclosuresMateos:Takeda: Honoraria; Amgen: Honoraria; Celgene: Honoraria; Janssen: Honoraria. Paiva:Celgene: Honoraria, Research Funding; Janssen: Honoraria; Takeda: Honoraria, Research Funding; Sanofi: Consultancy, Research Funding; EngMab: Research Funding; Amgen: Honoraria; Binding Site: Research Funding.

Comprehensive Proteomics Analysis Reveals Metabolic Reprogramming of Tumor-Associated Macrophages Stimulated by the Tumor Microenvironment.

Tumor-associated macrophages (TAMs) are major components of the tumor microenvironment. Although a role for TAMs in promoting tumor progression has been revealed, the differentiation mechanisms and intrinsic signals of TAMs regulated by the tumor microenvironment remain unclear. Here we constructed an in vitro TAMs cell model, TES-TAMs, which is from tumor-extract-stimulated bone-marrow-derived macrophages. We performed a comparative proteomics analysis of bone-marrow-derived macrophages and TES-TAMs, which indicated that TES-TAMs possessed characteristic molecular expression of TAMs. Intriguingly, the signal pathways enriched in up-regulated differentially expressed proteins of TAMs demonstrated that glycolysis metabolism reprogramming may play an important role in TAM differentiation. We found that hexokinase-2, a key mediator of aerobic glycolysis, and the downstream proteins PFKL and ENO1 were remarkably increased in both TES-TAMs and primary TAMs from our MMTV-PyMT mice model. This phenomenon was then verified in human THP-1 cell lines stimulated by tumor extract solution from breast cancer patient. Taken together, our study provides insight into the induction of TAM differentiation by the tumor microenvironment through metabolic reprogramming.

The Role of Tumor Associated Macrophages in the Tumor Microenvironment: Mechanism and Functions.

During tumor progression, several types of inflammatory cells are recruited into the tumor site, where they participate in tumor-associated inflammation, such as fibroblasts, granulocytes, lymphocytes and macrophages. Macrophages infiltrating into the tumor microenvironment are termed tumorassociated macrophages (TAMs). In response to multiple stimuli, macrophages undergo recruitment and polarization, and play an important role in further tumor development. In this article, we present a review of the mechanism of macrophage' recruitment, and the role that TAMs play in promoting immunosuppression, tumor progress and metastasis, as well as chemoresistance and future therapeutic strategies.

The impact of hypoxia on tumor-associated macrophages.

The role of tumor-associated macrophages (TAMs) in cancer is often correlated with poor prognosis, even though this statement should be interpreted with care, as the effects of macrophages primarily depend on their localization within the tumor. This versatile cell type orchestrates a broad spectrum of biological functions and exerts very complex and even opposing functions on cell death, immune stimulation or suppression, and angiogenesis, resulting in an overall pro- or antitumoral effect. We are only beginning to understand the environmental cues that contribute to transient retention of macrophages in a specific phenotype. It has become clear that hypoxia shapes and induces specific macrophage phenotypes that serve tumor malignancy, as hypoxia promotes immune evasion, angiogenesis, tumor cell survival, and metastatic dissemination. Additionally, TAMs in the hypoxic niches within the tumor are known to mediate resistance to several anticancer treatments and to promote cancer relapse. Thus, a careful characterization and understanding of this macrophage differentiation state is needed in order to efficiently tailor cancer therapy.

Abstract 1578: Exploring the role of tumor-conditioned macrophage metabolism on extravasation of pancreatic ductal adenocarcinoma cells

Abstract A persistently low survival rate and more than 80% of patients experiencing relapse after surgery combine to make pancreatic ductal adenocarcinoma (PDAC) the 4th leading cause of death from cancer. Recent studies showed that the presence of tumor-associated macrophages (TAMs) may support cancer cells in each step of the metastatic cascade: growth at the primary site followed by epithelial-mesenchymal transition (EMT), intravasation into lymph and blood vessels, circulation to a distant site, stoppage due to adhesion or size restriction, extravasation into tissue, and colonization in this secondary organ. However, the role of TAMs and their metabolic profile in tumor progression need further investigation. The objective of our studies was to examine the potential for macrophages to switch from oxidative phosphorylation to aerobic glycolysis (the Warburg effect), similar to what is observed in cancer cells, and how this metabolic conversion may influence their pro-metastatic activity. To address this question we differentiated human peripheral blood monocytes with non tumor- and tumor-conditioned media to obtain macrophages and TAMs, respectively. First, we showed in a live glycolytic stress test that TAMs had a greater glycolytic capacity compared to non tumor-conditioned macrophages. Next, a 3D in-vitro microfluidic extravasation assay consisting of an endothelial monolayer was used to assess the effect of macrophages on PDAC cell extravasation across the endothelial wall into an extracellular matrix-like hydrogel. We observed that the extravasation propensity of PDAC cells was increased by the presence of TAMs but was not significantly affected by non tumor-conditioned macrophages. This suggests that the tumor-conditioned media fostered monocyte differentiation toward pro-metastatic macrophages. To assess if the macrophage metabolic switch toward glycolysis was the key factor promoting the increment in cancer cell extravasation, we treated the TAMs with 2-Deoxyglucose (2-DG), a competitive inhibitor of glycolysis at the level of hexokinase. Indeed, we observed a significant reduction in the rate of PDAC cell extravasation when TAMs were treated with 2-DG compared to untreated TAMs. Our results confirmed that the glycolytic metabolism was involved in modulating pro-tumoural macrophage function. Taken together, our data suggest that the macrophage metabolic pathway is a potential therapeutic target for controlling immune cell contributions to tumor progression and that patient responses to current treatments might be improved by inhibition of macrophage glycolysis. Citation Format: Giulia Adriani, Hweixian L. Penny, Je Lin Sieow, Siew Cheng Wong, Roger D. Kamm. Exploring the role of tumor-conditioned macrophage metabolism on extravasation of pancreatic ductal adenocarcinoma cells. [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 1578.

Abstract LB-346: Dual inhibition of Ang-2 and VEGF receptors normalizes tumor vasculature and prolongs survival in glioblastoma by altering macrophages

Abstract OBJECTIVE: Here we aimed to overcome resistance to anti-VEGF therapy in glioblastoma (GBM), the most common and aggressive adult primary brain tumor. INTRODUCTION: Current standard of care, including chemo-radiation, confers modest overall survival benefits of less than 1.5 years in patients. GBMs are highly dependent on angiogenesis, and anti-VEGF therapy is a promising approach to prolong survival. While anti-VEGF therapy prolongs progression-free survival in GBM, patients rapidly become refractory and overall survival is not increased. We previously demonstrated that angiopoietin-2 (Ang-2) may confer resistance to anti-VEGF receptor (VEGFR) treatment, as ectopic overexpression of Ang-2 compromises the benefits of VEGFR inhibition in murine GBM. Additionally, circulating Ang-2 levels in GBM patients rebound after an initial decrease following administration of cediranib, a pan-VEGFR tyrosine kinase inhibitor, suggesting Ang-2 may mediate resistance to anti-VEGF pathway inhibition. EXPERIMENTAL DESIGN: In two orthotopic models of GBM, Gl261 (murine) and U87 (human), we used MRI and Optical Frequency Domain Imaging (OFDI) to measure whether dual inhibition of Ang-2 and VEGFR reduces tumor burden and improves survival. We examined morphological changes in the tumor vasculature by immunohistochemistry, and phenotypical changes in tumor associated macrophages (TAMs) by flow cytometry. RESULTS: Dual therapy with cediranib (a pan-VEGFRs inhibitor) and MEDI3617 (an Ang-2 neutralizing antibody) improved survival over each therapy alone by delaying Gl261 growth. In U87 tumors, combined treatment increased tumor necrosis. Dual therapy enhanced morphological normalization of vessels and led to changes along the M1/M2 spectrum of CD45+CD11b+F4/80+ TAMs. We analyzed the functional role of TAMs by inhibiting TAM recruitment with an anti-colony stimulating factor-1 antibody (anti-CSF-1) in combination with dual therapy. Depletion of TAMs compromised the survival benefit of anti-Ang-2/VEGFR therapy, suggesting that TAMs mediate the survival benefits of dual therapy. CONCLUSION: Here we successfully demonstrated that dual inhibition of Ang-2 and VEGFRs prolongs survival in preclinical GBM models by reducing the viable tumor burden, improving vascular normalization, and reshaping the tumor immune-microenvironment mediated by TAMs. This combination may represent a potential therapeutic strategy to overcome the limitations of anti-VEGF monotherapy in GBM patients by integrating the complementary effects of anti-Ang2 treatment on vessels and immune cells. Citation Format: Zohreh Amoozgar, Teresa E. Peterson, Nathaniel D. Kirkpatrick, Yuhui Huang, Christian T. Farrar, Koen A. Marijt, Jonas Kloepper, Meenal Datta, Giorgio Seano, Keehoon Jung, Walid S. Kamoun, Trupti Vardam, Matija Snuderl, Jermaine Goveia, Sampurna Chatterjee, Ana Batista, Alona Muzikansky, Ching Ching Leow, Lei Xu, Tracy T. Batchelor, Dan G. Duda, Dai Fukumura, Jain K. Jain. Dual inhibition of Ang-2 and VEGF receptors normalizes tumor vasculature and prolongs survival in glioblastoma by altering macrophages. [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 LB-346.

Abstract 4156: Functional analysis of tumor-associated macrophage utilizing virus-guided fluorescent imaging of pancreatic cancer cells in the peritoneal cavity

Abstract Objectives: Postoperative recurrence occurs in approximately 80% of pancreatic cancer, and the peritoneum is the second most frequent metastatic site next to the liver. Recent evidence suggests that cancer microenvironment plays key roles in metastasis. To investigate mechanism of intraperitoneal metastasis in pancreatic cancer, we tried to detect cancer cell in the peritoneal wash from pancreatic cancer patients and analyze intraperitoneal cancer microenvironment. A genetically engineered adenovirus, TelomeScan, which replicates and expresses GFP only in telomerase-active cancer cells, was employed to detect cancer cells, and it enabled us to distinguish cancer cell from co-existence cells in the abdominal cavity. We explored the role of tumor-associated macrophages (TAMs) by using this virus-guided fluorescent imaging system. Methods: Peritoneal wash was obtained from 20 pancreatic cancer patients during operation. The cells in the wash were infected with TelomeScan for 24 hours. Samples from cases with TelomeScan-expressing GFP-positive cells were further subjected to immunofluorescence assay for analysis of microenvironment. The antibodies against CD45, CD14, and CD204 were used in immunostaining as markers of leukocytes, monocytes and TAMs, respectively. To investigate the effect of TAMs on pancreatic cancer, Panc1 and BxPC-3 cell lines were co-cultured with PMA (phorbol 12-myristate 13-acetate)-treated monocytes and analyzed their changes. Results: The three out of 20 cases were positive in cytology, and GFP positive cells were detected after TelomeScan infection in those cases. In the wash, pancreatic cancer cells existed together with many leukocytes including macrophages. Further analysis demonstrated that those macrophages were TAMs. After Panc1 or BxPC-3 cells were co-cultured with TAMs, E-cadherin was decreased in Panc1, α-SMA and Vimentin was increased in BxPC-3. These results suggested that Epithelial to Mesenchymal Transition (EMT) was induced in pancreatic cancer cells by TAMs. Conclusion: Pancreatic cancer cells and TAMs were detected in the peritoneal wash using TelomeScan and immunostaining. The results suggested that EMT induced by TAMs may promote intraperitoneal metastasis of pancreatic cancer. Citation Format: Kazuya Kuwada, Shunsuke Kagawa, Megumi Watanabe, Shuichi Sakamoto, Satoru Kikuchi, Shinji Kuroda, Ryuichi Yoshida, Hiroshi Tazawa, Tetuya Kagawa, Toshiyoshi Fujiwara. Functional analysis of tumor-associated macrophage utilizing virus-guided fluorescent imaging of pancreatic cancer cells in the peritoneal cavity. [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 4156.