Macrophages Promote Tumor Growth Research Articles

The Effects of Hypoxia on the Immune-Metabolic Interplay in Liver Cancer.

M2-like macrophages promote tumor growth and cancer immune evasion. This study used an in vitro model to investigate how hypoxia and tumor metabolism affect macrophage polarization. Liver cancer cells (HepG2 and VX2) and macrophages (THP1) were cultured under hypoxic (0.1% O2) and normoxic (21% O2) conditions with varying glucose levels (2 g/L or 4.5 g/L). Viability assays and extracellular pH (pHe) measurements were conducted over 96 hours. Macrophages were exposed to the tumor-conditioned medium (TCM) from the cancer cells, and polarization was assessed using arginase and nitrite assays. GC-MS-based metabolic profiling quantified TCM meta-bolites and correlated them with M2 polarization. The results showed that pHe in TCMs decreased more under hypoxia than normoxia (p < 0.0001), independent of glucose levels. The arginase assay showed hypoxia significantly induced the M2 polarization of macrophages (control group: p = 0.0120,0.1%VX2-TCM group: p = 0.0149, 0.1%HepG2-TCM group: p < 0.0001, 0.1%VX2-TCMHG group: p = 0.0001, and 0.1%HepG2-TCMHG group: p < 0.0001). TCMs also induced M2 polarization under normoxic conditions, but the strongest M2 polarization occurred when both tumor cells and macrophages were incubated under hypoxia with high glucose levels. Metabolomics revealed that several metabolites, particularly lactate, were correlated with hypoxia and M2 polarization. Under normoxia, elevated 2-amino-butanoic acid (2A-BA) strongly correlated with M2 polarization. These findings suggest that targeting tumor hypoxia could mitigate immune evasion in liver tumors. Lactate drives acidity in hypoxic tumors, while 2A-BA could be a therapeutic target for overcoming immunosuppression in normoxic conditions.

Abstract 5365: Characterizing signaling in macrophage-GBM cell coculture

Abstract Macrophages are a type of immune cell that play a crucial role in the tumor microenvironment. In tumors, macrophages can be polarized towards a pro-inflammatory phenotype, referred to as M1, or towards an anti-inflammatory and tumor-promoting phenotype, referred to as M2. M2 macrophages promote tumor growth by secreting cytokines that increase tumor proliferation, angiogenesis, and dampen the immune response from other immune cells such as T cells and dendritic cells. The balance between M1 and M2 macrophages in the tumor microenvironment is critical in determining the outcome of cancer progression and response to therapy. By understanding the pathways that lead to macrophage polarization, we can develop therapeutics that inhibit this process and support an anti-tumor macrophage response. Co-culture is a powerful model to study interactions between different cell types. We created co-cultures with macrophages and glioblastoma multiforme (GBM) cells to understand how these cell-cell interactions can lead to altered cell phenotype. Several studies have looked at the interaction of two populations using a transwell system where one cell population is placed in a well with a permeable membrane and the other population is placed below, allowing for the exchange of soluble factors. However, this does not account for an important mode of cell-cell interaction, which is physical contact. Therefore, we tested how macrophages derived from primary monocytes polarized in the context of conditioned media (supernatant), cell lysate, and direct co-culture. We used U87-MG as a model GBM cell line. We assayed the M2 phenotype by using flow cytometry with CD163 as a marker. Our findings show that direct co-culture is crucial for promoting macrophage M2 polarization. Using our in vitro model for tumor-induced macrophage polarization, we conducted phosphoproteomic analysis to characterize signals associated with this phenotypic change. To study how co-culture induced differential signaling in tumor populations and macrophages, we devised a method to fix cells to preserve cell signaling, followed by separation using flow cytometry and LC-MS/MS analysis of individual cell populations. We created co-cultures of U87-MG tumor cells and macrophage-differentiated THP-1 cells, a monocyte cell line. We then tested whether our method could reliably separate the different populations, whether we could detect altered signals due to co-culture, and showcase how separating cell populations yields information that could not be gathered from bulk analysis of co-cultures. Our data shows that the analysis was highly reproducible and that the signaling from cells separated from co-culture was highly similar to signaling in cells that were grown individually. However, some phosphorylation residues were uniquely upregulated in the co-culture condition, pointing to signaling that may occur in response to tumor interaction. Citation Format: Alicia D. D'Souza, Rachit Mukkamala, Ryuhjin Ahn, Forest M. White. Characterizing signaling in macrophage-GBM cell coculture [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5365.

Curcumin Reprograms TAMs from a Protumor Phenotype towards an Antitumor Phenotype via Inhibiting MAO-A/STAT6 Pathway.

M1 phenotype macrophages have anticancer characteristics, whereas M2 phenotype macrophages promote tumor growth and metastasis. A higher M1/M2 ratio, therefore, has a beneficial effect on the tumor immune microenvironment, thereby inhibiting tumor growth. The natural alkaloid curcumin is found to have anticancer properties. However, the mechanism remains unclear. In this study, a cell co-culture system and M2 macrophage model were used to evaluate the effects of curcumin on tumor-associated macrophage (TAM) phenotypes. Our results demonstrate that curcumin reprogrammed the M2 macrophages by reducing the level of anti-inflammatory cytokines (TGF-β, Arg-1, and IL-10) and an M2 surface marker (CD206) induced by Cal27 cells or IL-4, as well as upregulating proinflammatory cytokines (TNF-α, iNOS, and IL-6) and an M1 surface marker (CD86). The in vitro assays suggested that curcumin treatment suppressed the migration and invasion of the Cal27 cells induced by the M2-like macrophages. Mechanistically, the repolarization of TAMs may be attributed to the inhibition of monoamine oxidase A (MAO-A)/STAT6 signaling after curcumin treatment. Collectively, our results show that the anticancer effects of curcumin could be explained by reprogramming TAMs from a protumor phenotype towards an antitumor phenotype.

Identification of tumor-associated macrophage subsets that are associated with breast cancer prognosis.

BackgroundBreast cancer is the leading cause of cancer‐related deaths in women, demanding new treatment options. With the advent of immune checkpoint blockade, immunotherapy emerged as a treatment option. In addition to lymphocytes, tumor‐associated macrophages exert a significant, albeit controversial, impact on tumor development. Pro‐inflammatory macrophages are thought to hinder, whereas anti‐inflammatory macrophages promote tumor growth. However, molecular markers to identify prognostic macrophage populations remain elusive.MethodsWe isolated two macrophage subsets, from 48 primary human breast tumors, distinguished by the expression of CD206. Their transcriptomes were analyzed via RNA‐Seq, and potential prognostic macrophage markers were validated by PhenOptics in tissue microarrays of patients with invasive breast cancer.ResultsNormal human breast tissue contained mainly CD206+ macrophages, while increased relative amounts of CD206− macrophages were observed in tumors. The presence of CD206+ macrophages correlated with a pronounced lymphocyte infiltrate and subsets of CD206+ macrophages, expressing SERPINH1 and collagen 1, or MORC4, were unexpectedly associated with improved survival of breast cancer patients. In contrast, MHCIIhi CD206− macrophages were linked with a poor survival prognosis.ConclusionOur data highlight the heterogeneity of tumor‐infiltrating macrophages and suggest the use of multiple phenotypic markers to predict the impact of macrophage subpopulations on cancer prognosis. We identified novel macrophage markers that correlate with the survival of patients with invasive mammary carcinoma.

High Expression of Angiopoietin-like Protein 4 in Advanced Colorectal Cancer and its Association with Regulatory T Cells and M2 Macrophages.

Colorectal cancer (CRC) is one of the most aggressive tumours in the human digestive system. Most CRC patients have poor prognosis due to metastasis and recurrence. Angiopoietin-like 4 (ANGPTL4) is involved in tumour development. Regulatory T (Treg) cells and M2 macrophages promote tumour growth and metastasis. Herein, we explored the changes of ANGPTL4 expression in CRC patients at different stages and observed whether in situ tumour-Treg and -M2 macrophages are correlated with ANGPTL4 expression. Serum ANGPTL4 (sANGPTL4) levels of 70 CRC patients and 10 healthy controls were detected by ELISA. ANGPTL4, Foxp3 and CD163 expression levels in CRC tissues were measured by immunohistochemistry. Recombinant ANGPTL4 (rANGPTL4) proteins were further added into cell-culture systems for induction of Treg cells and M2 macrophages. The results showed both sANGPTL4 and in situ tumour-ANGPTL4 expression levels increased in Dukes C-D stage CRC patients. Foxp3+ and CD163+ cells in tumour tissue sections were also more intensive in Dukes C-D stage patients than in Dukes A-B stage patients. Foxp3+ and CD163+ cells in tumour tissues were positively correlated with both tissue and sANGPTL4 expression (P < 0.01). Recombinant ANGPTL4 promoted the induction of murine Treg cells and M2 macrophages ex vivo. Therefore, elevated ANGPTL4 expression could be a marker for advanced CRC. Treg cell and M2 macrophage induction could be one of the mechanisms of tumour promotion mediated by ANGPTL4.

Abstract 1742: Dual inhibitor of immunokinase and pan-RAF for the treatment of KRAS-mutated cancers

Abstract Tumor-associated macrophages (TAMs) represent most of the white blood cell population in solid tumors. TAMs usually display an M2-like phenotype due to signals from the tumor microenvironment, such as IL-10, VEGFA and CSF-1/M-CSF. These anti-inflammatory and pro-tumorigenic macrophages promote tumor growth and metastasis. The presence of TAMs is usually correlated with a poor clinical outcome in cancer patients. Modulation of the immunosuppressive factors within the tumor microenvironment is a key issue in tumor immunology. TAMs subvert the anti-cancer function of tumor-infiltrating T lymphocytes. TAMs also modify the immune cell population within the tumor microenvironment to decrease anti-tumor immune cells while simultaneously increase the immunosuppressive cell population to promote tumorigenesis. As a result, targeting of TAMs may be a promising new approach to cancer treatment. CSF-1 is the crucial growth and differentiation factor for macrophage and CSF-1R is exclusively expressed by cells of the monocyte lineage, suggesting that CSF-1R is an attractive therapeutic target to enable interference with TAMs. In preclinical models, macrophage depletion by CSF-1R inhibitor increased the antitumor effects of VEGF-targeted therapies. Furthermore, failure of antiangiogenic therapy is caused by the pro-angiogenic Tie2-expressing monocytes infiltrating tumor tissues. These data provide the rationale for the combination of antiangiogenic drugs with macrophage targeting strategies to increase the therapeutic efficacy and to prevent drug resistance. Therefore, we tried to find inhibitors of immunokinase using structure-based molecular modeling and identified a selective inhibitor, SJP-1601. SJP-1601 potentially inhibited CSF-1R, Tie2, and VEGFR2 in vitro kinase assay. Interestingly, SJP-1601 also inhibited B-RAF, C-RAF and B-RAF V600E in kinase panel assay. We orally administered SJP-1601 in colorectal cancer xenografts with mutant K-RAS and observed the dose-dependent reduction of tumor volume. In addition, oral administration of SJP-1601 attenuated tumor growth correlated with enriched CD8+ T cells and decreased regulatory T cells in tumor stroma in BALB/c mice bearing 4T1 tumor cells or C57BL/6 mice bearing MC38 tumor cells. These data confirmed the preclinical merit of CSF1-CSF1R signaling blockade alone or as a part of combinatorial therapies to offer synergistic immunotherapeutic effects in treatments of human cancer. In this study, we discovered an orally active dual inhibitor of immunokinase &amp; pan-RAF. SJP-1601 not only improved the efficacy of adoptive T cell therapy through inhibition of immunosuppressive macrophage recruitment and activation in immunocompetent mice but also potentiated the response of xenograft with mutant KRAS by preventing pan-RAF. Our findings suggest that SJP-1601 could be an excellent preclinical candidate for the treatment of cancer with mutant K-RAS. Citation Format: Sungpyo Hong, Soon Kil Ahn, Ho-Seok Kwon, Yongbin Park, Min-Hyo Ki, Hee Jong Shin. Dual inhibitor of immunokinase and pan-RAF for the treatment of KRAS-mutated cancers [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 1742.

Abstract IA21: Immune mechanisms in gastrointestinal stromal tumor

Abstract Gastrointestinal stromal tumor (GIST) is the most common sarcoma subtype and typically contains an activating mutation in KIT or, less frequently, PDGFRA. Imatinib mesylate inhibits the KIT and PDGFRA oncoproteins and achieves a partial response or stable disease in over 80% of patients with advanced GIST. Unfortunately, disease progression typically occurs within 2 years, usually due to a secondary KIT mutation. Moreover, while adjuvant imatinib prolongs recurrence-free survival, it does not appear to increase the chance of cure following surgery. Thus, novel therapeutic strategies are needed for GIST, which has led us over the last 8 years to investigate the immune response. In a genetically engineered mouse model of GIST with a Kit deletion, we discovered that imatinib works partly by inhibiting tumor cell production of an immunosuppressive enzyme called indoleamine 2,3-dioxygenase (IDO), which catabolizes L-tryptophan. The downstream tryptophan metabolites are immunosuppressive and promote the development and activation of regulatory T cells (Treg) and suppress effector T cells. Consequently, by decreasing IDO production, imatinib can enhance the antitumor immune response. IDO expression was also found in human GIST specimens. While imatinib alone increased the T-cell immune response, we studied the effects of additional T-cell activation. Anti-CTLA-4 increased the antitumor efficacy of imatinib in the mouse model, which led to an NCI trial (NCT01643278) that tested the combination of ipilimumab and dasatanib in advanced, refractory GIST. Disappointingly, there were no responses by RECIST criteria, although dasatinib has limited activity in resistant GIST and concomitant tyrosine kinase inhibition was necessary in the mouse model. Nevertheless, the importance of intratumoral T cells was also shown by Zitvogel, who found that intratumoral CD8 T cells detected by immunohistochemistry correlated with survival in human GIST. Recently, we analyzed 106 human GISTs and found that intratumoral T cells often had a high-level expression of programmed death 1 (PD-1), and there was tumor expression of its ligand PD-L1. In the mouse model, anti-PD-1 or anti-PD-L1 alone lacked efficacy, but each increased the antitumor effects of imatinib. We also performed a detailed analysis of tumor-associated macrophages (TAMs) in 57 human GISTs. Macrophages are generally categorized as classically (M1) or alternatively (M2) activated, although these terms really just define the extremes of a spectrum. M1 macrophages can lyse tumor cells, secrete IL-12 and TNF, and present antigen to T cells. Meanwhile, M2 macrophages promote tumor growth by secreting IL-10 and supporting angiogenesis. Surprisingly, we found that TAMs in GIST were M1 at baseline, became M2 after imatinib therapy, and reverted to M1 after imatinib resistance developed. Accordingly, transcriptome analysis of purified TAMs revealed that there were no genes expressed more than two-fold between untreated and resistant GISTs. Because the TAMs were M1, it may be preferable to pursue strategies to activate TAMs in GIST, instead of depleting them. Recently, we have found in the GIST mouse model that anti-CD40 therapy increased the antitumor response to imatinib. It is now clear that there is a natural antitumor immune response to GIST, given the presence of M1 TAMs and intratumoral T cells expressing PD-1. However, other aspects of the immune response to GIST remain undefined. Manipulating the immune environment in GIST has potential therapeutic benefit. Several immunotherapy trials are opening in GIST, including those incorporating IDO inhibition and anti-PD-1. Citation Format: Ronald DeMatteo. Immune mechanisms in gastrointestinal stromal tumor [abstract]. In: Proceedings of the AACR Conference on Advances in Sarcomas: From Basic Science to Clinical Translation; May 16-19, 2017; Philadelphia, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(2_Suppl):Abstract nr IA21.

Immunotherapy in lymphoma.

Immunotherapy in lymphoma.

IMPS-33THE EXTRACELLULAR MATRIX OF GLIOMA TUMORS INFLUENCES MACROPHAGE ACTIVITY

Glioblastoma (GB) is the most common and aggressive form of malignant primary brain tumors in adults. Immunotherapeutics are a promising avenue of treatment, but their effectiveness can improve if we further understand the immune system's role in tumor progression. The tumor extracellular matrix (ECM) and tumor-associated macrophages promote tumor growth and invasion, but the effect of the tumor ECM on macrophage activity is unknown. We hypothesize that the physical and biological properties of the tumor ECM drive macrophage phenotypes that promote tumor growth. In this study, we investigated the ECM-macrophage interface from both a biological and physical perspective. We found that tumor ECM stiffness and composition promoted the proinflammatory activity of macrophages in short-term cultures. This is surprising considering most investigators have found that macrophages have anti-inflammatory activity in mouse tumors. We also examined whether proinflammatory mediators were present in GB tumors. We found that TNF-alpha, a proinflammatory cytokine, was present in 7 out of 7 tumors of patients with GB. Also, we found that IL-10, an anti-inflammatory cytokine, was present in 6 out of 6 tumors of patients with GB. Our results show a need to clinically define the inflammatory activity in the tumor microenvironment and identify the mechanisms that drive inflammation.

Re-Educating Myeloma Associated Macrophages with Lenalidomide

Introduction: The bone marrow niche plays a critical role in determining the fate of malignant plasma cells in multiple myeloma (MM). Macrophages are an abundant component of the stromal cell compartment and are believed to support proliferation, survival, and drug resistance of MM cells. Conversely, macrophages can directly kill tumor cells and participate in antitumor immune responses as effector cells. Moreover, macrophages are key immune effector cells for the therapeutic effect of monoclonal antibodies. Lenalidomide, an immunomodulatory drug (IMiD®) is used for the treatment of MM, also in the combination with therapeutic antibodies. Lenalidomide is thought to target the stromal support, but its precise influence on the phenotype or the effector functions of macrophages is still unclear.Methods: To investigate the effect of lenalidomide on the interaction between macrophages and malignant plasma cells in vitro, we coincubated lenalidomide pretreated macrophages with several MM cell lines, and analysed the viability, proliferation and phenotype. For in vivo studies we utilized 5TMM mice, a suitable animal model for MM. Animals were treated with lenalidomide (50 mg/kg 5days/week) for 3 weeks, and the effector functions and phenotype of isolated bone marrow macrophages were analyzed. In addition, macrophages in the bone marrow of MM patients treated with lenalidomide were characterized by immunohistochemistry and flow cytometry.Results: We showed, that infiltrating macrophages in the bone marrow of MM patients display an anti-inflammatory M2-like phenotype characterized by the expression of surface marker CD163, CD206, PD-L1 and cytokine/chemokine secretion (e.g. IL10, CXCL10, APRIL, BAFF and RANKL). Incubation of macrophages with lenalidomide in vitro, substantially changed their transcriptional program (e.g. downregulation of IRF4 and upregulation of IRF5) and their phenotype (e.g. downregulation of the surfaces marker CD163, CD206, and upregulation of CD16, CD64, CD40 and CD86). Furthermore, we show that lenalidomide treatment decreases the expression of RANKL, BAFF and APRIL, while tumoricidal effector molecules (e.g. TRAIL, cathelicidine, Granzyme B) were increased. When lenalidomide treated macrophages were cocultured with MM cells significant cytotoxicity was detected, for all MM cell lines tested. In contrast, untreated macrophages promote tumor growth and viability of MM cells.Conclusion: Lenalidomide in vitro influences macrophages by reverting an anti-inflammatory M2 like profile to a more immunogenic phenotype. In addition it impacts on the support function by decreasing the secretion of important growth factors for B-cells. Similar results were observed in first in vivo studies. Taken together our results imply that lenalidomide interrupts an important stromal cell function thereby influencing survival of MM cells. DisclosuresNo relevant conflicts of interest to declare.

Alternatively activated (M2) macrophages promote tumour growth and invasiveness in hepatocellular carcinoma

The roles of alternatively activated (M2) macrophages on pro-tumour phenotypes have been well documented in many cancers except hepatocellular carcinoma (HCC). Considering their close relationship with chronic tissue injuries as well as enhanced tumour invasiveness and growth, we aimed to investigate the direct effects of M2 macrophages on HCC. M2 macrophages in 95 HCC clinical specimens were quantified using immunohistochemistry and quantitative PCR. The pro-tumour functions and the underlying molecular mechanisms of M2 macrophages in HCC were investigated in vivo and in an in vitro co-culture system. In the clinical study, high M2-specific CD163 (hazard ratio=2.693; p=0.043) and scavenger receptor A (hazard ratio=3.563; p=0.044) levels indicated poor prognosis and correlated with increased tumour nodules and venous infiltration in HCC patients. In an orthotopic model, the liver tumour volume was increased 3.26-fold (1.27 cm3±0.36) after M2 macrophage injection compared with the control (0.39 cm3±0.05) (p=0.032). An increased rate of lung metastasis was also found in the treatment group. In vitro, co-cultivation with M2 macrophages elevated the number of HCC cells (MHCC97L) and migration events by 1.3-fold and 3.2-fold, respectively (p<0.05). Strongly induced by MHCC97L, M2 macrophage-derived CCL22 was proven to enhance tumour migration capacities and correlate with venous infiltration in HCC patients. Increased epithelial-mesenchymal transition (EMT) via Snail activation in MHCC97L was found to be promoted by M2 macrophages and CCL22. M2 macrophages contribute to poor prognosis in HCC and promote tumour invasiveness through CCL22-induced EMT.

Interferon-γ and celecoxib inhibit lung-tumor growth through modulating M2/M1 macrophage ratio in the tumor microenvironment.

Tumor-associated macrophages play an important role in tumor growth and progression. These macrophages are heterogeneous with diverse functions, eg, M1 macrophages inhibit tumor growth, whereas M2 macrophages promote tumor growth. In this study, we found that IFNγ and/or celecoxib (cyclooxygenase-2 inhibitor) treatment consistently inhibited tumor growth in a mouse lung cancer model. IFNγ alone and celecoxib alone increased the percentage of M1 macrophages but decreased the percentage of M2 macrophages in the tumors, and thus the M2/M1 macrophage ratio was reduced to 1.1 and 1.7 by IFNγ alone and celecoxib alone, respectively, compared to the M2/M1 macrophage ratio of 4.4 in the control group. A combination of IFNγ and celecoxib treatment reduced the M2/M1 macrophage ratio to 0.8. Furthermore, IFNγ and/or celecoxib treatment decreased expression of matrix metalloproteinase (MMP)-2, MMP-9, and VEGF, as well as the density of microvessels in the tumors, compared to the control group. This study provides the proof of principle that IFNγ and/or celecoxib treatment may inhibit lung-tumor growth through modulating the M2/M1 macrophage ratio in the tumor microenvironment, suggesting that IFNγ and celecoxib have potential to be further optimized into a new anticancer therapy.

GATA-3 Expression Promotes IL-10 Production, Alternative Macrophage Polarization, and Identifies a Subset Of High-Risk PTCL, NOS

BackgroundIL-10 polarized macrophages promote tumor growth and survival. The T-cell transcription factor GATA binding protein-3 (GATA3) epigenetically regulates IL10 expression. Therefore, we investigated the extent to which GATA-3 regulates IL-10 production and macrophage polarization in T-cell lymphomas. MethodsMonocyte-derived macrophages were generated and polarized with T-cell lymphoma conditioned media ((TCL-CM). Immunophenotypic and functional characteristics associated with IL10-polarized macrophages were examined. GATA3-dependent cytokine production was evaluated in T-cell lymphoma lines by shRNA knockdown. Alternatively polarized macrophages and GATA3 expression were examined in a cohort of patients with peripheral T cell lymphoma, not otherwise specified (PTCL, NOS) (n=76). Comparisons among groups were evaluated using a Student's t test. Progression-free and overall survival (PFS & OS) was estimated using the Kaplan-Meier method and two-tailed log-rank test based on Cox proportional hazards model. ResultsTo investigate the ability of T-cell lymphomas to alternatively polarize macrophages, monocytes from healthy donors were cocultured with TCL-CM. Macrophages were shown to have immunophenotypic characteristics of IL10-polarized macrophages (pSTAT3+/CD16hi/CD163hi/HLA-DR-/lo) by flow cytometry, which was abrogated by IL10, but not IL6, neutralization. TCL-CM polarized macrophages produced abundant IL-10 and were impaired in their ability to stimulate T-cell proliferation. Collectively, these results demonstrate that T-cell lymphomas promote alternative macrophage polarization in an IL-10 dependent manner, and both CD163 and pSTAT3 may aid in their identification in clinical T-cell lymphoma specimens. Therefore, CD163+/pSTAT3+ macrophages were visualized by immunofluorescence in PTCL, NOS specimens. IL-10 polarized macrophages were not identified in normal tissues, but were abundant constituents of the tumor microenvironment in 68% of the PTCL, NOS specimens examined (n=31). STAT3 phosphorylation in response to IL-10 is dependent upon the Janus kinases (JAK) JAK1 and TYK2, both of which are inhibited at nanomolar (and clinically achievable) concentrations by the “JAK2” inhibitor ruxolitinib. Therefore, we sought to determine whether ruxolitinib may prevent IL-10-induced macrophage polarization. As expected, ruxolitinib inhibited IL-10-induced STAT3 phosphorylation. More importantly, macrophages polarized in the presence of ruxolitinib lost the immunophenotype characteristics of IL-10 polarized macrophages, and were CD16-/loCD163-/loHLA-DRhi. Furthermore, IL-10 production was significantly impaired in macrophages polarized in the presence of ruxolitinib, while their ability to stimulate T-cell proliferation was significantly increased. In a separate initiative, we identified GATA3 as the key regulator of IL-10 and Th2 cytokine (IL-4, IL-13 and IL-5) expression in T-cell lymphoma lines by shRNA knockdown assays. Therefore, we examined GATA3 expression by immunohistochemistry in PTCL, NOS specimens. GATA-3 expression was observed in most of the T-cell lymphoma lines and in 47% of PTCL, NOS specimens (n=76). In the cohort of the 76 PTCL, NOS patients, the median PFS and OS observed in GATA3+ PTCL, NOS was 5 months (4-8 months, 95% CI) and 8 months (5-14 months, 95% CI), respectively. In contrast, the median PFS and OS in GATA3- PTCL, NOS was 1.3 years (0.6-1.6, 95% CI) and 1.6 years (0.7-6.9 95% CI), respectively. ConclusionsAlternatively polarized macrophages are abundant in T-cell lymphomas and are generated in an IL-10- and STAT3-dependent manner. GATA-3 regulates IL-10 production in these lymphomas, and its expression identified a high-risk subset of PTCL, NOS with distinct clinicopathological features. As IL-10-dependent STAT3 phosphorylation and macrophage polarization were impaired by ruxolitinib, the JAK2 inhibitors may warrant closer scrutiny as immunomodulatory agents. [Display omitted] Disclosures:Off Label Use: Ruxolitinib, a JAK2 inhibitor, may warrant closer scrutiny as an immunomodulatory agent in TCL therapeutics.

Macrophages promote tumour growth and liver metastasis in an orthotopic syngeneic mouse model of colon cancer

Tumour-associated macrophages have been shown to promote proliferation, angiogenesis and metastasis in several carcinomas. The effect on colon cancer has not yet been clarified. Furthermore, Kupffer cells in the liver might initiate the formation of metastases by directly binding tumour cells. An orthotopic syngeneic mouse model of colon cancer as well as a liver metastases model has been studied, using murine CT-26 colon cancer cells in Balb/c-mice. Macrophages were depleted in both models by clodronate liposomes. Tumour sizes and metastases were determined using 7-Tesla MRI. The macrophage and vascular density in the orthotopic tumours as well as the Kupffer cell density in the livers were evaluated using immunohistochemistry. Animals in the macrophage-depleted group displayed significantly smaller primary tumours (37 ± 20mm(3)) compared to the control group (683 ± 389mm(3), p = 0.0072). None of the mice in the depleted group showed liver or peritoneal metastases, whereas four of six control mice displayed liver and five out of six mice peritoneal metastases. The vascular density was significantly lower in the macrophage-depleted group (p = 0.0043). In the liver metastases model, animals of the Kupffer cell-depleted group (14.3 ± 7.7) showed significantly less liver metastases than mice of the two control groups (PBS liposomes, 118.5 ± 28.2, p = 0.0117; NaCl, 81.7 ± 23.2, p = 0.0266). The number of liver metastases correlated directly with the Kupffer cell density (p = 0.0221). Macrophages promote tumour growth, angiogenesis and metastases in this orthotopic syngeneic mouse model. Kupffer cells enhance the formation of metastases in the liver.

Interleukin-17 and Prostaglandin E2 Are Involved in Formation of an M2 Macrophage-Dominant Microenvironment in Lung Cancer

Tumor-associated macrophages (TAMs) are divided into M1 and M2 macrophages. M1 macrophages inhibit tumor growth, whereas M2 macrophages promote tumor growth and metastasis. The aim of this study was to examine the possible causes leading to the formation of an M2-macrophage-dominant tumor microenvironment in non-small-cell lung cancer. Forty-eight archived lung tumor samples were examined for the expression of interleukin-17 (IL-17) receptors, IL-17 receptor A (IL-17RA) and IL-17 receptor C (IL-17RC), and the number of TAMs using immunohistochemical staining. Twenty fresh lung tumors and matched normal lung tissues were examined for expression of IL-17, cyclooxygenase-2, and prostaglandin E2 (PGE2), using enzyme-linked immunosorbent assay and Western blot analysis. Macrophage-migration assays were performed using fresh lung tumor tissues and IL-17 as chemoattractants. Induction of M2-macrophage differentiation was analyzed using real-time quantitative polymerase chain reaction. TAMs expressed IL-17RA and IL-17RC. Lung tumors expressed higher levels of IL-17, cyclooxygenase-2, and PGE2, compared with normal lung tissues. Lung tumor tissues attracted migration of mouse RAW264.7 macrophages and primary peritoneal macrophages through IL-17, which was mediated by IL-17RA and IL-17RC. IL-17 did not induce either M1- or M2-macrophage differentiation. However, human lung cancer A549 cells strongly induced M2-macrophage differentiation of RAW264.7 macrophages when the two cell lines were cocultured. The inductive factor secreted by A549 cells was identified to be PGE2. IL-17 recruits macrophages, and PGE2 induces M2-macrophage differentiation, hence the increased levels of IL-17 and PGE2 in lung cancer contribute to the formation of an M2-macrophage-dominant tumor microenvironment.

Abstract 1690: Tumor-educated macrophages promote tumor growth and peritoneal metastasis in an orthotopic nude mouse model of human pancreatic cancer

Abstract There is a strong association between poor survival and increased macrophage density in many cancers. In the current study, we determined whether macrophages from tumor-bearing mice (tumor-educated macrophages) had greater tumor-promoting capability than macrophages from non-tumor-bearing mice. Ten transgenic nude mice ubiquitously-expressing GFP were injected subcutaneously with the human pancreatic cancer cell line, BXPC3 stably expressing RFP. GFP-expressing macrophages from tumor-bearing transgenic GFP mice were harvested and defined as “tumor-educated macrophages”. Macrophages were also harvested from transgenic GFP mice (n=10) without subcutaneous tumors and identified as “naïve macrophages.” Three groups of mice were studied: 1) A control group without addition of macrophages (n=10); 2) A naïve group, with weekly intraperitoneal (ip) injection of 106 naïve macrophages per mouse (n=10); and 3) A tumor-educated group, with weekly ip injection of 106 tumor-educated macrophages per mouse (n=10). The study ended with termination of the tumor-educated group after 8 weeks due to a pre-morbid state identified in all mice. At this time, all mice in each of the three study arms were terminated, imaging was performed, and total tumor weight was obtained. In the control group, the average primary tumor weighed 668 mg; only three mice (30%) developed peritoneal metastases with an average weight of 241 mg. The naïve-macrophage group had an average tumor weight of 823 mg (p=0.51); 50% developed peritoneal metastases with an average weight of 975 mg (p=0.029). The tumor-educated-macrophage group had an average primary tumor weight of 2095 mg (p=0.001); 75% of mice developed peritoneal metastases with an average weight of 2135 mg (p=0.008). When comparing naïve- to tumor-educated- groups, primary tumor weight was significantly greater in the tumor-educated group (p=0.003), and the average weight of metastasis was 2.2 times greater in the tumor educated group, however this did not reach statistical significance (p = 0.17). When comparing naïve- to tumor-educated-groups, there was no statistically significant difference with regard to body weight (p=0.87), or weight of metastasis (p=0.68); however primary tumor weight was significantly greater in the tumor-educated group (p=0.013). Tumor-educated-macrophages specifically promote tumor growth progression in an orthotopic nude mouse model of pancreatic cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1690. doi:1538-7445.AM2012-1690

Combined Blockade of Integrin-α4β1 Plus Cytokines SDF-1α or IL-1β Potently Inhibits Tumor Inflammation and Growth

Tumor-associated macrophages promote tumor growth by stimulating angiogenesis and suppressing antitumor immunity. Thus, therapeutics that inhibit macrophage recruitment to tumors may provide new avenues for cancer therapy. In this study, we showed how chemoattractants stromal cell-derived growth factor 1 alpha (SDF-1α) and interleukin 1 beta (IL-1β) collaborate with myeloid cell integrin-α4β1 to promote tumor inflammation and growth. We found that SDF-1α and IL-1β are highly expressed in the microenvironments of murine lung, pancreatic, and breast tumors; surprisingly, SDF-1α was expressed only by tumor cells, whereas IL-1β was produced only by tumor-derived granulocytes and macrophages. In vivo, both factors directly recruited proangiogenic macrophages to tissues, whereas antagonists of both factors suppressed tumor inflammation, angiogenesis, and growth. Signals induced by IL-1β and SDF-1α promoted the interaction of talin and paxillin with the cytoplasmic tails of integrin-α4β1, thereby stimulating myeloid cell adhesion to endothelium in vitro and in vivo. Inhibition of integrin-α4β1, SDF-1α, or IL-1β was sufficient to block tumor inflammation and growth, and the combined blockade of these molecules greatly accentuated these effects. Furthermore, antagonists of integrin-α4β1 inhibited chemotherapy-induced tumor inflammation and acted synergistically with chemotherapeutic agents to suppress tumor inflammation and growth. These results show that targeting myeloid cell recruitment mechanisms can be an effective approach to suppress tumor progression.

Targeted imaging of tumor-associated M2 macrophages using a macromolecular contrast agent PG-Gd-NIR813

Tumor-associated macrophages (TAMs) are diverse population containing multiple subtypes. M2 macrophages promote tumor growth and metastasis, in part by secreting a wide range of proangiogenic factors and growth factors. Selective depletion of M2 macrophages has been evaluated as a novel approach to anti-cancer therapy. In this study, a dual magneto-optical imaging probe, PG-Gd-NIR813 was synthesized and evaluated for non-invasive assessment of TAMs after intravenous injection. PG-Gd-NIR813 injected in nude rats bearing C6 tumors showed high uptake of the polymeric contrast agent in the tumor at 1 and 48 h after injection both in vivo and ex vivo optical imaging. T1-weighted MR imaging results showed accumulation of PG-Gd-NIR813 into the tumor necrotic area, which was confirmed by TUNEL staining of resected tumors. The uptake of PG-Gd-NIR813 within tumor necrosis decreased after animals were treated by the macrophage-depleting agent. Immunohistochemical staining demonstrated that PG-Gd-NIR813 colocalized with CD68 (marker for macrophages) and CD169 (marker for activated macrophages), but not with CD163 (residential macrophages). Using combined near-infrared fluorescence imaging and magnetic resonance imaging (MRI), we demonstrated that the accumulation of PG-Gd-NIR813 in tumors was mediated through M2 TAMs. Therefore, poly(l-glutamic acid) based reagents could be potentially used to image response to antitumor therapies targeted at M2 TAMs. Furthermore, poly(l-glutamic acid) is a promising carrier for candidate immunotherapeutics targeting M2 TAMs.